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Proteolytic activity in plant tissue and cell suspension culture Nilsson, E. Kristina 1982

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PROTEOLYTIC ACTIVITY IN PLANT TISSUE AND CELL SUSPENSION CULTURE  by  E. KRISTINA NILSSON B. Sc., The University of Western Ontario, 1  t  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Food Science Department)  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1982 E. K r i s t i n a Nilsson, 1982  IE-6  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head of  representatives.  my  It is  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department of  F~~ooE>  AJOic  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  n/R-n  O^-  5  / ? f i  written  ABSTRACT P r o t e o l y t i c enzymes a r e common i n p l a n t s b u t a r e u s u a l l y to endogenous p r o t e i n .  specifi  P l a n t proteases w i t h s p e c i f i c i t i e s a p p l i c a b l e to  the food i n d u s t r y i n c l u d e p a p a i n , f i c i n and b r o m e l a i n .  Other p l a n t s have  been used i n t r a d i t i o n a l methods of food p r e p a r a t i o n f o r t h e i r a c t i o n on food components.  proteolytic  The f o l l o w i n g s p e c i e s were i n v e s t i g a t e d f o r  p r o p a g a t i o n i n t i s s u e c u l t u r e : • C a r i c a papaya, F i c u s c a r i c a , Cynara c a r d u n c u l u s , Galium verum, C i r c i u m a r v e n s e , D i e f f e n b a c h i a amoena, D. p i c t a and Ananas comosus.  T i s s u e s of the f i r s t f i v e of these  demonstrated  p r o t e o l y t i c a c t i v i t y by c l e a r i n g of m i l k t u r b i d i t y i n agar medium. Commercial p a p a i n and f i c i n p r e p a r a t i o n s a r e c u r r e n t l y o b t a i n e d from l a t e x of immature papaya and f i g f r u i t , r e s p e c t i v e l y . tion  was conducted,  i n p a r t , t o determine  the f e a s i b i l i t y of p r o d u c i n g  these two enzymes by the i n v i t r o c e l l c u l t u r e t e c h n i q u e . of  This investiga  Standard method  a s e p t i c seed g e r m i n a t i o n and l e a f t i s s u e e x c i s i o n were employed f o r  callus initiation.  C e l l s u s p e n s i o n c u l t u r e s d e r i v e d from c a l l u s were  m a i n t a i n e d i n B5 medium a t 28 °C i n d a r k n e s s . determined  Proteolytic activity  by a m o d i f i c a t i o n of the Food Chemicals  and p r o t e i n c o n t e n t was determined  was  Codex method f o r p a p a i n  by B r a d f o r d ' s d y e - b i n d i n g , method.  P r o d u c t i o n of p r o t e i n and p r o t e a s e v a r i e d among c e l l  cultures,  but c o u l d be i n f l u e n c e d by changes t o some n u t r i t i o n a l f a c t o r s .  Fig cells  were grown i n medium supplemented w i t h s i n g l e amino a c i d s i n the presence of  e i t h e r n i t r a t e o r ammonia as a source of i n o r g a n i c n i t r o g e n . A l l  n i t r a t e - b a s e d media produced ammonia-based media.  h i g h e r y i e l d s of c e l l d r y weight  than  G l u t a m i c and a s p a r t i c a c i d s were most s t i m u l a t o r y  iii  growth, p r o t e i n a c c u m u l a t i o n and m i l k , added at 3% resulted  a highly  i n h i g h e r p r o t e i n and  c a s e i n and milk.  ( v / v ) , was  p r o t e a s e a c t i v i t y of f i g c e l l s . e f f e c t i v e growth s t i m u l a n t ,  Skimmed and  p r o t e a s e l e v e l s than the amino a c i d s .  Fresh  whey, added i n d i v i d u a l l y , produced s i m i l a r r e s u l t s to skimmed  C i t r i c a c i d , added at the  l e v e l found i n the  3% m i l k supplement, a l s o  caused s t i m u l a t i o n of f i g c e l l growth, p r o t e i n s y n t h e s i s  and  a c t i v i t y not  I t appears  nitrogen  also  s i g n i f i c a n t l y d i f f e r e n t from skimmed m i l k .  accumulation and  reduction  an energy requirement which c o u l d be  i n f i g c e l l s may  protease  have been l i m i t e d by  s a t i s f i e d w i t h the a d d i t i o n of  a c i d or m i l k whey to the b a s a l medium.  that  citric  iv  CONTENTS  page  Abstract  i i  T a b l e of Contents List  of T a b l e s  List  of F i g u r e s  iv v vi  Acknowledgements  v i i  I.  Introduction  II.  L i t e r a t u r e Review 1. P l a n t p r o t e a s e s : D e s c r i p t i o n and use 2. P l a n t t i s s u e and c e l l c u l t u r e  III.  Materials 1. 2. 3. 4.  IV.  C a l l u s c u l t u r e of s e l e c t e d s p e c i e s Suspension c u l t u r e o f f i g and papaya Assay methods Medium supplementation  18 23 24 30  Results 33 40 53 57 58  Discussion 1. 2. 3. 4. 5. 6.  VI.  6 14  and Methods  1. C u l t u r e o f p l a n t t i s s u e s 2. Assessment of assay methods f o r biomass, p r o t e i n and protease 3. M i l k c l o t t i n g a c t i v i t y 4. E l e c t r o p h o r e s i s 5. Medium supplementation V.  1  Tissue d e d i f f e r e n t i a t i o n C e l l suspension c u l t u r e s D e t e r m i n a t i o n of biomass, p r o t e i n and p r o t e a s e Nitrogen n u t r i t i o n i n f i g c e l l cultures S t i m u l a t o r y e f f e c t s of skimmed m i l k and m i l k components A p p l i c a t i o n s , problems and p o t e n t i a l of p l a n t c e l l cultures  Summary  References Biographical  73 75 76 86 93 95 101 104  information  114  V  LIST OF TABLES page I.  Products detected  i n plant tissue culture  5  II.  C h a r a c t e r i s t i c s of the t h r e e major p l a n t p r o t e a s e s  III.  C o m p o s i t i o n of B5 medium  IV.  S u r v i v a l of p l a n t s p e c i e s  V.  D r y i n g methods f o r h a r v e s t e d  VI.  C e l l e x t r a c t i o n methods  46  VII.  M i l k c l o t t i n g a c t i v i t y o f papaya and f i g c e l l e x t r a c t s  56  VIII.  I n f l u e n c i n g p r o t e i n and p r o t e a s e s y n t h e s i s w i t h a n t i b i o t i c s , d e t e r g e n t and s u l f u r compounds  70  7 19  i n tissue culture f i g and papaya c e l l s  34 42  vi  LIST OF FIGURES page  1.  S u b s t r a t e r e s i d u e s i n papain a c t i v e s i t e  10  2.  Papain:  11  3.  E s t a b l i s h m e n t of papaya t i s s u e and c e l l  4.  Papaya c a l l u s and c e l l  5.  F i g e x p l a n t s and c a l l u s  36  6.  Bedstraw t i s s u e on agar and i n l i q u i d medium  37  7.  Protein  39  8.  H e r i t a b i l i t y of p r o t e i n  9.  S e t t l e d c e l l volume as an i n d i c a t o r of h a r v e s t weight  44  10.  Growth of papaya and f i g i n B5: biomass and pH changes  45  11.  Bradford's p r o t e i n  48  12.  FCC p r o t e a s e assay:  13.  E f f e c t o f pH and temperature  14.  I n f l u e n c e of some o r g a n i c compounds on p r o t e o l y t i c a c t i v i t y  55  15.  Inorganic nitrogen:  59  16.  Amino a c i d supplements: e f f e c t on biomass  17.  Amino a c i d supplements: e f f e c t on p r o t e i n activity  t h i o l - d i s u l f i d e interchange  suspension  and p r o t e a s e v a r i a b i l i t y  culture  20  culture  35  among papaya and f i g  and p r o t e a s e p r o d u c t i v i t y  assay: standard curve standard curve  41  52  on p r o t e o l y t i c a c t i v i t y  e f f e c t on biomass, p r o t e i n  and p r o t e a s e  54  61 and p r o t e a s e  18.  Proteins  19.  Changes i n p r o t e i n and p r o t e a s e of papaya and f i g c e l l suspension c u l t u r e s over 3 weeks  65  20.  Skimmed m i l k and components: e f f e c t on biomass  66  21.  Skimmed m i l k and components: e f f e c t on p r o t e i n and protease a c t i v i t y  68  I n t r a - and e x t r a - c e l l u l a r p r o t e i n c e l l suspension c u l t u r e s  72  22.  and p e p t i d e s : e f f e c t on p r o t e a s e a c t i v i t y  62 64  and p r o t e a s e i n f i g  vii  Acknowledgements  The  author wishes t o express g r a t e f u l acknowledgement t o  the many f r i e n d s and a c q u a i n t a n c e s who have d i r e c t l y o r i n d i r e c t l y helped  i n the p l a n n i n g , e x p e r i m e n t a l , and w r i t i n g stages o f t h i s work. Drs. P.M. Townsley and G.G. J a c o l i a r e r e s p o n s i b l e f o r f o s t e r -  i n g an i n t e r e s t  i n , and enthusiasm  Drs. S. Nakai and  f o r , p l a n t t i s s u e and c e l l  culture.  I.E.P. T a y l o r were generous w i t h t h e i r time t o h e l p  w i t h q u e s t i o n s i n b i o c h e m i s t r y and p l a n t metabolism.  Dr. B . J . Skura  has h e l p f u l l y s u p p l i e d i d e a s and new p e r s p e c t i v e s . A s s i s t a n c e of the f o l l o w i n g people was much a p p r e c i a t e d : Mr.  T. Kuwata and Mr. R. Yada f o r h e l p w i t h e l e c t r o p h o r e s i s , Mr. D. Arm-  s t r o n g of the UBC greenhouses f o r a q u i s i t i o n and i d e n t i f i c a t i o n o f p l a n t m a t e r i a l , Miss L. Robinson f o r g u i d i n g me through S. Yee f o r g e n e r a l t e c h n i c a l and moral I will  d a t a a n a l y s i s , and Mr.  support.  always owe a g r e a t d e a l t o my p a r e n t s , A i n o and  B j o r n N i l s s o n , f o r t e a c h i n g the v a l u e of e d u c a t i o n w i t h o u t ation.  I am a l s o i n d e b t e d t o my husband, Robert  Shadwick, who was  l a r g e l y r e s p o n s i b l e f o r my i n i t i a t i n g and completing has  misrepresent-  this project.  He  t i r e l e s s l y p r o v i d e d a s s i s t a n c e , c o n s o l a t i o n and d i s c i p l i n e as  required. F i n a n c i a l a s s i s t a n c e from an NSERC p o s t - g r a d u a t e s c h o l a r s h i p , a UBC summer f e l l o w s h i p and the J.F. Morgan award was welcome, and a l l are g r a t e f u l l y  acknowledged.  1  I.  INTRODUCTION  Many of the substances used i n the p h a r m a c e u t i c a l , food, f l a v o u r and perfume i n d u s t r i e s o r i g i n a t e from p l a n t s , and a l t h o u g h the trend over the l a s t f i f t y years has been towards c h e m i c a l s y n t h e s i s , p l a n t s s t i l l remain an important source of many of these compounds, f o r a number of r e a s o n s . (1)  The  compounds are d i f f i c u l t  and/or  c o s t l y to s y n t h e s i z e .  (2)  Complex m i x t u r e s , eg. rose o i l , s u c c e s s f u l l y by man.  (3)  I s o l a t i o n from a n a t u r a l source circumvents many of the r e g u l a t i o n s which must be s a t i s f i e d b e f o r e a c h e m i c a l l y s y n t h e t i c compound can be used commercially as a food o r drink a d d i t i v e .  (4)  Chemical s y n t h e s i s may, depending on the compound, r e s u l t i n a mixture of isomers which cannot be s e p a r a t e d on a commercial s c a l e . T h i s i s of obvious importance when the major d e s i r a b l e p r o p e r t y r e s i d e s with one isomer.  cannot be  constituted  The m a j o r i t y of commercially u s e f u l substances o r i g i n a t e from p l a n t s grown i n t r o p i c a l and s u b - t r o p i c a l r e g i o n s of the world and the a v a i l a b i l i t y and c o s t of these m a t e r i a l s i s f r e q u e n t l y a f f e c t e d by p o l i t i c a l and economic c o n s i d e r a t i o n s i n the c o u n t r i e s of o r i g i n .  These comments from a p u b l i c a t i o n by Yeoman e t a l . (1980) c o n c i s e l y summarize the reasons t i s s u e and c e l l c u l t u r e .  We  f o r and  j u s t i f i c a t i o n of r e s e a r c h i n p l a n t  have a dependence on p l a n t s  much more than energy and n u t r i e n t s .  to p r o v i d e us w i t h  Most food systems are complex,  i n c l u d i n g such minor components as pigments,  gums, enzymes, e s s e n t i a l  oils  2  and  other  f l a v o u r compounds, most of which are d e r i v e d  from p l a n t s .  With the g o a l of s e l f - s u f f i c i e n c y , p l a n t b r e e d i n g programs aimed a t a d a p t a t i o n ( B o z z i n i , 1980). hybrids  was  of p l a n t s  The  to temperate c l i m a t e s  time r e q u i r e d  d r a s t i c a l l y reduced by  f o r t h i s approach had  already  or greenhouse growth  f o r development of new t i s s u e and  been l a i d by  are  c u l t i v a r s and  organ c u l t u r e .  researchers  The  groundwork  interested in rapid  methods of p l a n t p r o p a g a t i o n , h y b r i d i z a t i o n , e l i m i n a t i o n of or r e s i s t a n c e pathogens ( R e i n e r t & B a j a j , 1977; i m p o r t a n t compendia, c o n t a i n i n g  Ingram & Helgeson, 1980). articles  Several  d e a l i n g w i t h these i s s u e s , have  been e d i t e d by Barz e t a l . (1977), R e i n e r t Sharp e t a l . (1979) and  and  Bajaj  (1977), Thorpe  (1978),  S a l a e t a l . (1980).  From p l a n t development v i a t i s s u e c u l t u r e , the next l o g i c a l is  to circumvent the  t r a d i t i o n a l p l a n t form and  the compounds of i n t e r e s t from p l a n t c e l l s Commercial use o n l y one  food  grown i n v i t r o  research  and  biochemical  research  and  the food  cosmetics,  waste t r e a t m e n t .  development i n the enzyme f i e l d has  and  than development of new  harvested  f o r other  reasons.  i n d u s t r i e s underlines methods of p r o d u c t i o n .  the  be  recovered The  great  been l a r g e l y  In  one  the  plants  importance of enzymes to some  There i s a p l e t h o r a  market, even a s e l e c t i o n to perform any  already  microbial i n o r i g i n .  from by-products of  need f o r r e s e a r c h  a  of annual s a l e s of enzymes  from p l a n t s , the remainder being  P l a n t enzymes may  As  enzyme sources or p r o c e s s e s .  beverage i n d u s t r y , a p p r o x i m a t e l y 12%  are p r o t e a s e s d e r i v e d  isolate  industry i s  to improvements i n c o s t or e f f i c i e n c y of enzyme p r o c e s s e s rather  step  ( K l e i n , 1960).  among many which a l s o i n c l u d e photochemicals, tanning,  directed i n use,  proceed d i r e c t l y to  of enzymes i s widespread:  p h a r m a c e u t i c a l s , surgery, result,  to  i n t o a l t e r n a t e sources of m i c r o b i a l enzymes on function.  food  or the  Most, i f not a l l ,  of  3  these  a r e s y n t h e s i z e d by s e l e c t e d s t r a i n s of micro-organisms grown i n l a r g e  fermentation acidity,  v e s s e l s under c o n t r o l l e d c o n d i t i o n s of temperature, a e r a t i o n ,  n u t r i t i o n and  agitation.  appears r a t h e r p r i m i t i v e , having e x t r a c t i o n , d r y i n g and  By c o n t r a s t , p l a n t enzyme  progressed  little  improvement of s t o r a g e  production  beyond p r i m a r y  stability  (Ortiz et a l . ,  1980). P l a n t t i s s u e and i n d u s t r i a l production  c e l l c u l t u r e h o l d g r e a t promise w i t h r e s p e c t  of p l a n t p r o d u c t s .  began about t h i r t y years  ago,  and  w i t h r e s p e c t to n u t r i t i o n a l and physiology  and  c e l l metabolic  each p l a n t product harnessing  has  The  a r t of p l a n t t i s s u e c u l t u r e  s i n c e progressed  biochemical  activity  of i n t e r e s t , s i m i l a r  aspects.  i s necessary  to a young s c i e n c e , A knowledge of  the  f o r each s p e c i e s  and  t o , but more complex  c l a s s e s , primary or secondary m e t a b o l i t e s . precursors,  intermediates  and  i n t o one  Primary m e t a b o l i t e s  two  endproducts of metabolism i n a c t i v e l y - g r o w i n g  to be s u b j e c t to c e r t a i n s t e a d y - s t a t e  t h a t an i n c r e a s e i n r e c o v e r y  of  include  These would be more c o r r e c t l y v i s u a l i z e d as i n t r a c e l l u l a r  Such m a t e r i a l s are l i k e l y  pools.  levels,  so  of a c e r t a i n compound c o u l d o n l y be achieved  means of an i n c r e a s e d c e l l h a r v e s t . lead  than,  micro-organisms. U s e f u l compounds of p l a n t o r i g i n g e n e r a l l y f a l l  cells.  to  Improvements i n c e l l  growth would  by  then  to i n c r e a s e d t o t a l p r o d u c t i v i t y . The  pharmaceutical  i n d u s t r i e s have c o n t r i b u t e d most to our  knowledge of p l a n t secondary metabolism.  Many drugs and  cosmetics  present  are  d e r i v e d from r a r e p l a n t s , or s y n t h e s i z e d ,by common p l a n t s i n such s m a l l quantities  that large harvests  P l a n t t i s s u e s and  cells  and  complex e x t r a c t i o n schemes are  required.  i n c u l t u r e have o f t e n been found to s y n t h e s i z e  d e s i r e d m a t e r i a l s , a l b e i t g e n e r a l l y i n very s m a l l amounts.  Because  the  4  secondary m e t a b o l i t e s mature and provides  such as a l k a l o i d s are accumulated, e s p e c i a l l y i n  even senescent t i s s u e , t h e i r presence i n p l a n t t i s s u e c u l t u r e s  impetus f o r improvements i n methodology. Aharonowitz and  between primary and multiple  functions  Demain (1980) argued a g a i n s t  secondary m e t a b o l i t e s , and  concurrent  p o i n t i n g out  production.  to c e r t a i n r e g u l a t o r y mechanisms.  regulatory points  i n m e t a b o l i c pathways may  of any  compounds.  p u b l i c a t i o n , Plant Tissue Culture  The  be  r e c e n t CRC  the p o s s i b i l i t y  (Chemical Rubber  as a Source o f B i o c h e m i c a l s ,  ample evidence f o r i n t e r e s t and  field.  many p l a n t p r o d u c t s i d e n t i f i e d  of  they  An u n d e r s t a n d i n g of  used to i n f l u e n c e  Staba (1980) p r o v i d e s Table I l i s t s  distinction  A l l p l a n t metabolism,  state, i s subject  or c a t a b o l i s m  the  synthesis Co.)  e d i t e d by  J.  potential i n this in cultures:  a s t e r i s k e d items have been produced i n p l a n t c u l t u r e s i n q u a n t i t i e s a t l e a s t e q u a l to the p a r e n t p l a n t s  (Zenk, 1978).  Table  I  Products d e t e c t e d i n p l a n t c u l t u r e s . (*) compounds produced i n q u a n t i t i e s a t l e a s t equal to i n t a c t p l a n t s (dry weight b a s i s ) References: Campbell, e t a l . (1965); H e i n s t e i n & E l - S h a g i (1981); Khanna & Staba (1968); Misawa (1977); N i c k e l l (1980); Tabata, e t a l . ( 1 9 7 8 ) ; Townsley (1974); T u r n b u l l , e t a l . (1980); Zenk (1978)..  Class  Examples  alkaloids  a j m a l i c i n e * , a t r o p i n e , c a f f e i n e * , codeine, g l y c o a l k a l o i d s , i n d o l e a l k a l o i d s , morphine, n i c o t i n e , s e r p e n t i n e * , tropane a l k a l o i d s , v i n d o l i n e  antileukemic/antitumor antimicrobial  agents  agents  campothecin, e l e p h a n t i n , maytansine, h a r r i n g t o n e n e ,  lolamarine, v i n c r i s t i n e  plumbagin, and u n i d e n t i f i e d compounds i n c u l t u r e s of p o p l a r , avocado, l e t t u c e , c a u l i f l o w e r and marijuana  benzo-compounds  coumarin, g e n t i s i c a c i d , t o c o p h e r o l , ubiquinone*,  carbohydrates  agar,  cardiac glycosides  c u l t u r e s of  enzymes  amylases, c a t a l a s e , dehydrogenases, i n v e r t a s e , k i n a s e s , myrosinase, phosphatase, proteases, ribonuclease  ethylene  c u l t u r e s of mung bean, soybean, r o s e , f l a x , wheat, r i c e  foods;  c u l t u r e s of c a r r o t , grape, tomato; aroma/flavours l i c o r i c e , cocoa, c o f f e e ; m i r a l i n , s t e v i o s i d e  f l a v o u r s ; sweeteners  geranial,  furano-compounds  visnagin*,  lipids/oils  mint medicinals  Digitalis  anthraquinones*,  plasmin  i n h i b i t o r , ginsengoside*,  phenolies  r o s m a r i n i c a c i d * , hydrangenol,  pigments  anthocyanins,  vitamins  onion,  rutamarin  a s c o r b i c , c h l o r o g e n i c , cinnamic,  saponins  i n c u l t u r e s of  citronellol  organic acids  steroids,  acid  sugars, p o l y s a c c h a r i d e s , s t a r c h , l i g n i n  fragrances/perfumes  miscellaneous  vanillic  campestrol,  citric,  L-dopa*  fumaric, o x a l i c , shikimic, v a n i l l i c  p u t r e s c i n e (under study)  b e t a n i n , c a r o t e n o i d s , c h l o r o p h y l l , f l a v o n o i d s , gossypol  cholesterol, diosgenin*, lanosterol, s i t o s t e r o l ,  a s c o r b i c a c i d , thiamine, v i t a m i n K  squalene  6  II.  1.  LITERATURE REVIEW  Plant proteases:  D e s c r i p t i o n and  uses  P r o t e o l y t i c enzymes f i n d uses i n the f o l l o w i n g food i n d u s t r y sectors:  baking,  meat p r o c e s s i n g . treatment  brewing, p r o t e i n h y d r o l y s a t e p r o d u c t i o n , cheese-making There i s a l s o some i n t e r e s t i n t h e i r use i n waste  or r e d u c t i o n , such as h y d r o l y s i s of s c l e r o p r o t e i n s (Jones  M e r c i e r , 1974;  and  Yamamoto, 1975).  Criteria  p a r t i c u l a r purpose i n c l u d e s p e c i f i c i t y ,  &  f o r enzyme s e l e c t i o n f o r a  h e a t - s t a b i l i t y , pH optimum and  p o s s i b l e presence of i n h i b i t o r s i n the enzyme p r e p a r a t i o n or the  the  intended  substrate. The and 1980  ficin,  major p l a n t p r o t e a s e s  estimated  (Wolnak, 1980).  c u r r e n t l y employed are papain,  to t o t a l over $16 Papain  i s i n use  d e s c r i b e d by Jones and M e r c i e r  (1974).  crude enzyme p r e p a r a t i o n s  (dried  low-temperature s t o r a g e .  Appropriate  bromelain  m i l l i o n i n international sales i n i n many food and non-food  industries,  They p o i n t e d out the i n s t a b i l i t y  l a t e x ) and  the n e c e s s i t y f o r r e f i n e m e n t  facilities  are g e n e r a l l y c o n f i n e d  l a r g e enzyme companies l o c a t e d i n North America, Europe o r Japan, w h i l e crude p a p a i n  i s purchased mainly  investigated  the s t o r a g e and  from t r o p i c a l n a t i o n s .  drying characteristics  r e p o r t e d an optimum d r y i n g temperature of 50-55°C. declined l a t e x had  of and to the  O r t i z e t a l . (1980)  of papaya l a t e x . Proteolytic  They  activity  i n the presence of sodium c h l o r i d e w h i l e a d d i t i o n of EDTA to f r e s h a p r e s e r v a t i v e e f f e c t on  activity.  T a b l e I I summarizes p r o p e r t i e s of the three major p l a n t w i t h most i n f o r m a t i o n a v a i l a b l e on p a p a i n . c o n t a i n i n g a t l e a s t one be e n z y m a t i c a l l y a c t i v e .  proteases,  A l l three enzymes are monomers  c y s t e i n e r e s i d u e which must be i n a reduced  form to  B a s i c a l l y , c a t a l y s i s depends on the f o r m a t i o n  of  Table I I  C h a r a c t e r i s t i c s of the t h r e e major p l a n t p r o t e a s e s c u r r e n t l y used i n the food  industry.  Data f o r t h i s t a b l e came from the f o l l o w i n g  references:  (1) Bergmeyer, 1974  (2) M u r a c h i ,  1970  (4) Arnon, 1970  (5) Yamamoto, 1975  (6) Gould, 1975  (8) Tang, 1974  (9) K u n i m i t s u & Yasunobu, 1970  (7) Englund, e t a l . ,  1968  (10) S g a r b i e r i , e t a l . ,  Similarities  1964  i n primary s t r u c t u r e near the r e a c t i v e papain ficin bromelain  (3) L i e n e r & F r i e d e n s e n , 1970  cysteine*  Pro-Val-Lys-Asn-Gln-Gly-Ser-Cys-Gly-Ser-Cys*-Trp Pro-Ile-Arg-Gln-Gln-Gly-Gln-Cys-Gly-Ser-Cys*-Trp Ser-Val-Lys-Asn-Gln-Asn-Pro-Cys-Gly-Ala-Cys*-Trp  l a t e x of papaya  source  m o l e c u l a r weight  terminal residues  N C  21,000 23,000  bromelain  f icin  papain  property  fruit  f r u i t or stem f l u i d pineapple  l a t e x of f i g f r u i t  33,000  24-27,000 (3) 25,500 (7)  (5,6) (4)  proteins, peptides,  esters  proteins, peptides,  esters  Phe  group  b a s i c or aromatic c a r b o n y l r e s i d u e s : Arg, Phe, Tyr  substrates  proteins, peptides,  specificity  basic carbonyl Arg, Lys, Phe  temperature  most a c t i v e @ 50-60 °C, s t a b l e 30 min, 70 °C (5)  most a c t i v e @ 50-60 °C  most a c t i v e @ 50-60 °C  pH  optimum pH 5-7; s t a b l e pH 3-11(5), 3-9 (6)  optimum pH 6-8; pH 3.5-9 (5)  optimum pH 6-8, except t i n (optimum pH 5)  Pi  8.75  9.0  heavy metals, o x i d a n t s , s o r b i c a c i d (5)  heavy metals, o x i d a n t s , s o r b i c a c i d (5)  inhibitors  heavy m e t a l s , o x i d a n t s , s o r b i c a c i d (5) isothiocyanates (8) cleft Asp  cleft (3)  c l e f t involving cysteine and h i s t i d i n e (2)  stability  active site associated proteases notes  esters  (1,2)  valine glycine  leucine alanine  isoleucine asparagine  of  residues:  (4)  involving Cys„ , and s IS) s  H  1 5 g  i  1  5  9  chymopapain A & B lysozyme  (4,5,9)  synthetic a c t i v i t y i n p l a s t e i n r e a c t i o n s (5)  or Tyr carbonyl  stable  9.55  (3)  involving  cysteine  up to 10 a c t i v e components (3, 10)  gela-  (2)  5 isozymes  (2)  g l y c o p r o t e i n : 1.5-2.5% carbohydrate, bonded to Asn  9  an  intermediate  cysteine  complex of the s u b s t r a t e  residue.  T h i s has  been e l a b o r a t e d  e x p l o r a t i o n as reviewed by P o l g a r case, the enzyme c l e f t of  ( F i g . 1).  p r o b a b l y coupled w i t h the substrate  attack.  overcome by peptide by  (1977) w i t h r e s p e c t  these i s the d i s c r i m i n a t i n g r e s i d u e ,  The  The  imidazole  linear separation  interchange  Brocklehurst  and  Kierstan  They gave evidence  e f f e c t s the c o n v e r s i o n The  residues, involved  (Englund e t a l . , 1968).  b a s i c scheme f o r t h i s  but  d i s u l f i d e bonds, s i n c e the the  (Kramer & Whitaker, 1964;  seven  Englund and  preparations  are  S g a r b i e r i e t a l . , 1964).  For  in  cysteine  other  is eight. very  Two  but o n l y one  appears to have  t o t a l h a l f - c y s t i n e value  a c t i v i t y of commercial p r e p a r a t i o n s predicted.  Papain c o n t a i n s  Similarly, f i c i n  l i t e r a t u r e i n d i c a t e that f i c i n  recent  s i m i l a r to t h a t of p a p a i n .  of which i s a r e a c t i v e s u l f h y d r y l , the  i n d i s u l f i d e bonds.  suggesting  2.  i n d i c a t e a mechanism very  o n l y one  (1973)  of i n a c t i v e  s u l f h y d r y l groups have been demonstrated i n denatured f i c i n , active f i c i n  is  p o s s i b l y spanned  Less i s known of the p r o t e o l y t i c a c t i o n of f i c i n , investigations  One  nucleophile  catalytic cleft,  p r o p a p a i n to the a c t i v e enzyme c o n f i g u r a t i o n . i n Figure  this  of these amino a c i d s i s  proposed a zymogen-like mechanism f o r p a p a i n .  t r a n s i t i o n i s given  group of H i s ^ g  2  residue.  In  seven amino a c i d r e s i d u e s .  t h i o l of C y s 5 forming the a c t i v e  substrate  that a t h i o l - d i s u l f i d e  to p a p a i n .  second i n the N - t e r m i n a l d i r e c t i o n  f o l d i n g to produce the  the d i s c r i m i n a t o r y  active  upon v i a s e v e r a l avenues of  i s capable of b i n d i n g  t o the p o i n t of cleavage  for  a c y l moiety w i t h the  six  being  three Reports i n  heterogeneous t h i s reason,  precise  i n complex systems cannot r e a d i l y be  co-workers (1968) demonstrated t h a t the  multiplicity  of a c t i v e f r a c t i o n s c o u l d be a t t r i b u t e d to a u t o l y t i c a c t i o n which had i n t e r f e r e d with p r o t e o l y t i c c a p a c i t y of the  enzyme.  not  H  0  H  I  II  I  N -C - C I  r 0  '  N -C I  i  j  H !  H  0  j  nil  I  CfN-C-Cj-N-C  0  H  0  H  II  I  II  I  C-N  C -C  R  ,R DISCRIMINATING RESIDUE  R  II  0 I  II  N - C - C - N - C - C  1  SITE OF CLEAVAGE  S u b s t r a t e fragment accommodated by the papain a c t i v e s i t e . are h e l d i n the enzyme c l e f t . p o i n t of c l e a v a g e ,  O H  I  ,R  i g u r e 1.  H  The  Seven amino a c i d  second r e s i d u e i n the N-terminal  residues  d i r e c t i o n from  the " d i s c r i m i n a t i o n r e s i d u e " , i s p r e f e r e n t i a l l y hydrophobic.  From L. P o l g a r , 1977,  J . Biochem. 8:  171-176.  the »o  11  C  yS[63U V 25, — V — T C  S  H  'Sl59  PROPAPAIN  V  S  H  '  R- S  S  (22)  e  cysK ° y  or  s  v-r-  25^  CN  '  1221  (63U  Cys V J  ' 25 : 3  His  S  (22)  R-S  159  ' 159  (63)  Cys  Cys  H  e  or  CN  e  PAPAIN  F i g u r e 2.  T r a n s i t i o n of propapain interchange  to p a p a i n :  thiol-disulfide  as the method of zymogen a c t i v a t i o n .  From K. B r o c k l e h u r s t & M.P.J. K i e r s t a n , 1973 Nature, New B i o l o g y 242:167-170  12  Most c u r r e n t t e c h n i c a l i n f o r m a t i o n on the bromelains Table I I .  Although  stem and  f r u i t bromelains  are a l s o  i s outlined i n  sulfhydryl  p r o t e i n a s e s , they a r e g l y c o p r o t e i n s and d i f f e r from f i c i n and p a p a i n i n o t h e r respects.  E s t e r a s e and amidase a c t i v i t i e s  fundamental d i f f e r e n c e :  both papain and  of the three enzymes i n d i c a t e  ficin  show a p p r o x i m a t e l y  a  equal  a f f i n i t y f o r c o r r e s p o n d i n g s y n t h e t i c e s t e r and amide s u b s t r a t e s while stem b r o m e l a i n has times  a k  f o r BAEE ( o C - N - b e n z o y l - L - a r g i n i n e  c a t  as l a r g e as the k  (Murachi,1970). between 5 and  c a t  Stem and  f o r BAA  (oc-N-benzoyl-L-arginine  f r u i t bromelains  11, with o n l y one  ethyl ester)  140  amide),  have h a l f - c y s t i n e values r e p o r t e d  reactive cysteine residue.  Meat p r o c e s s i n g i s the major a p p l i c a t i o n of p l a n t p r o t e a s e s . P o l y n e s i a n s and Hawaiians have t r a d i t i o n a l l y  t r e a t e d meats with papaya o r  p i n e a p p l e , presumably f o r the improved t e x t u r e , though f l a v o u r improvement i s no doubt a l s o i m p o r t a n t . or i t was has  marinated  Meat was  rubbed, o r even b o i l e d , with the raw  by s t o r i n g wrapped i n papaya l e a v e s .  used  immediately  p r e - o r p o s t - s l a u g h t e r and  p r e p a r a t i o n f o r cooking or canning meat and American product  f i s h products.  t e n d e r i z e d by antemortem i n j e c t i o n of p a p a i n .  thorough  North  T h i s has  was  been c o n s i d e r e d  by the meat i n d u s t r y i n t h e i r quest f o r an  inexpensive  means of meeting the consumers' demand f o r tender, yet l e a n ,  meat (Goeser,  1961).  Muscle f i b r e p r o t e i n s and  collagen, including  w a l l s , are h y d r o l y z e d to some e x t e n t i n v i v o , and cooking  The  upon  r e t a i l e d under the name of ProTen, no l o n g e r a v a i l a b l e ,  a g r e a t breakthrough and  meat i n d u s t r y  a p p l i e d and e l a b o r a t e d on these a n c i e n t r e c i p e s f o r meat t e n d e r i z a t i o n .  T e n d e r i z e r s are now  beef  The  fruit  (Kang & Warner, 1974).  Organ t i s s u e s  capillary  f u r t h e r degraded upon  (kidneys, l i v e r , heart, etc.)  r e c e i v e h i g h e r doses of the enzyme u n l e s s s l a u g h t e r r a p i d l y f o l l o w s injection.  O r s i and Major (1973) developed  an assay procedure  for routine  13  use  i n the Hungarian meat i n d u s t r y .  plant proteases ( f i c i n ,  papain and  By  t h i s method, a c t i v i t y of a l l t h r e e  b r o m e l a i n ) was  w i t h tenderness r a t i n g s of a p a n e l of  found to c o r r e l a t e  judges.  P l a n t p r o t e a s e s have a l s o been i n v e s t i g a t e d a p p l i c a t i o n i n the d a i r y i n d u s t r y ,  Sardinas  for potential  p r i m a r i l y f o r rennet s u b s t i t u t i o n i n  cheese manufacture ( B a l l s & Hoover, 1937; (1973) and  Cooke & C a y g i l l ,  (1976) have both l i s t e d  1974).  the  discussed.  functions  must perform:  coagulation  out  the  two  distinct  of the m i l k , a l l o w i n g  r e t e n t i o n of o t h e r c o n s t i t u e n t s ,  and  curd  three  expression  hydrolysis  already the  F i c i n and  proteolytic, 1974;  papain p r e p a r a t i o n s  according  t o S c o t t and  for further  Kosikowski, 1977).  expression  by  the  This  f i r m curd  Furthermore, b i t t e r  as w e l l as  papain:  converted  to p a r a c a s e i n ,  l o s s e s due  to c a s e i n  added to milk but,  they are  then b a c t e r i a l c u l t u r e s are p e p t i d e f o r m a t i o n might be  inactivated  microbial  too  and  highly  Cooke & C a y g i l l , to whey hydrolysis.  A c e r t a i n degree of roquefort.  of b r o m e l a i n ,  when c a s e i n has (eg., by peroxide  added f o r r i p e n i n g . the  use  but  flavour  though, i n such cheeses as  patented methods f o r the  the enzymes are  1972;  develop upon r i p e n i n g .  b i t t e r n e s s i s sometimes d e s i r a b l e ,  and  (Sardinas,  r e s u l t s i n lower y i e l d s due  f l a v o u r s may  S a r d i n a s (1976) e x p l a i n e d  used to make cheese are others  enzyme  of whey  or enzymic a c t i o n i n aging, to produce c h a r a c t e r i s t i c t e x t u r e , aroma.  Scott  a number of p l a n t sources of  c o a g u l a n t s , among them pumpkin, cardoon, sundew and Scott also pointed  well  ficin  been treatment);  Another s o l u t i o n to  i n c l u s i o n , i n r i p e n i n g c u l t u r e s , of  bitter  bacteria  t h a t w i l l m e t a b o l i z e these p r o d u c t s . The preparation  e x t r a c t of cardoon i s r e p o r t e d l y  still  of an unripened cheese ( V i e i r a de Sa  K o s i k o w s k i , 1977).  V i e i r a de Sa and  i n use  in Iberia for  & Barbosa, 1970b;  Barbosa (1972) made Edam, S e r r a  and  14  R o q u e f o r t cheeses cardunculus).  u s i n g an e x t r a c t from cardoon  They found the maximum c l o t t i n g a c t i v i t y a t 70°C but most  s i m i l a r to the c l o t t i n g  time of animal rennet a t 32°C.  o n l y o n e - t h i r d as much e x t r a c t was curd firmness. manufacture to  In sheep's  of s o f t - b o d i e d cheeses  milk,  r e q u i r e d as i n cow's milk f o r s u i t a b l e  They concluded t h a t the cardoon e x t r a c t was  suitable for  l i k e S e r r a , not as good f o r R o q u e f o r t  (Galium verum) has been e x t r a c t e d f o r use i n  t r a d i t i o n a l cheese-making i n the Middle E a s t . C h e s h i r e , England 1973).  i n the l a t e  18  t h  and e a r l y 1 9  I t was  centuries  t h  Sardinas (1976) c i t e d a r e p o r t of cheese  a l s o i n use i n  manufacture  W i t h a n i a coagulans, h i s t o r i c a l l y used i n I n d i a .  (Scott, using berries  In summary, i t appears  t h a t t h e r e are numerous p l a n t s known to have m i l k - c l o t t i n g p r o p e r t i e s , of  due  decreased y i e l d s , and u n s u i t a b l e f o r Edam p r o d u c t i o n . L a d i e s ' bedstraw  of  f l o w e r s (Cynara  which have been i n v e s t i g a t e d  few  f o r p r a c t i c a l a p p l i c a t i o n or f o r  i d e n t i f i c a t i o n of the c o a g u l a t i n g p r i n c i p l e s . I f p l a n t p r o t e a s e s are to f i n d use i n t h i s a s p e c t of the  dairy  i n d u s t r y , i t i s e s s e n t i a l to develop c l o s e c o n t r o l s over enzyme a c t i v i t y . Because of t h i s requirement, many r e s e a r c h e r s have turned to work w i t h i m m o b i l i z e d enzymes and have not produced of  "cold-renneting" techniques.  an e f f e c t i v e means of r e p l a c i n g rennet i n the  t r a d i t i o n a l cheeses.  2.  manufacture  M i c r o b i a l rennets and p e p s i n are commonly used,  do not g e n e r a l l y e q u a l the r e n n e t - c o a g u l a t e d cheeses ( S a r d i n a s , 1972;  To d a t e , these methods  Sternberg,  P l a n t t i s s u e and c e l l  i n sensory  but  properties  1976).  culture  P l a n t t i s s u e c u l t u r e s are commonly d e r i v e d from e x c i s e d l e a f , fruit,  s e e d l i n g or bud  tissue.  These t i s s u e s are i n c u b a t e d a s e p t i c a l l y on a  15  n u t r i e n t agar medium, s e v e r a l of which have been developed f o r g e n e r a l or s p e c i a l purposes (Murashige & Skoog, 1962; 1968).  Under a p p r o p r i a t e  conditions  undifferentiated plant c e l l s , is  rapid u n t i l  (every  10-40  c u l t u r e s can callus  c a l l u s t i s s u e , c o n s i s t i n g of masses of  forms.  In a d e v e l o p i n g  the c a l l u s must be  days, depending on  the  sometimes be e s t a b l i s h e d  depends upon both growth c o n d i t i o n s  tissue).  concise research  and  very  Thorpe, 1978,  i n p l a n t t i s s u e and  knowledge r e g a r d i n g subject  cell  suspension  on  the  culture.  senesence.  New  and,  of n u t r i e n t s .  There i s a  Dougall  (1980) has  c u l t u r e s are i n i t i a t e d  i f adequate, are  Much l i k e  the development of  carbohydrate and  outstanding  the  nitrogen  f u n c t i o n s , p l a n t hormone  by  and  supplementation or  d e d i c a t i o n and  the c o n t r i b u t i o n s  of O.L.  &  s t a t u s of n u t r i t i o n  i n media proven s u c c e s s f u l f o r  then m o d i f i e d  Such work r e q u i r e s  large  presented a  the r e l a t i o n s h i p of n u t r i t i o n to d i f f e r e n t i a t i o n  years of r e s e a r c h , (1977) b e i n g  requirements and  and  f a c t o r s (see R e i n e r t  current  of numerous ongoing i n v e s t i g a t i o n s :  a c t i v i t i e s and  the  cultures  m i c r o b i a l metabolism, p l a n t c e l l c u l t u r e s are  metabolism, m i c r o n u t r i e n t  species  cell  suspension  (media, temperature, a e r a t i o n )  f o r example).  readable synopsis  To  agitation.  l i t e r a t u r e on m a n i p u l a t i o n of these two and  division  transferred  Plant c e l l  (species, plasmids, n u c l e i c a c i d r e p l i c a t i o n ) .  B a j a j , 1977,  cell  d i s s e c t e d and  Success or f a i l u r e of p l a n t t i s s u e and  body of  callus,  from c a l l u s by d i s p e r s i o n of  i n l i q u i d medium w i t h a p p r o p r i a t e  genetics  Gamborg e t a l . ,  the c e l l mass begins to r e s t r i c t n u t r i e n t uptake.  encourage c e l l p r o l i f e r a t i o n , often  White, 1943;  use  the  other  omission  investment of many  Gamborg (1975) and  H.B.  Street  examples.  P l a n t t i s s u e c u l t u r e has endeavours (Boxus & D r u a r t ,  1980;  been q u i t e s u c c e s s f u l i n h o r t i c u l t u r a l Murashige, 1978).  As d i s c u s s e d  i n the  16  i n t r o d u c t o r y chapter, t h i s has become a p o p u l a r and expanding pharmaceutical  r e s e a r c h as w e l l .  plant tissues,  there have been a few attempts  vitro.  The f i r s t  composition al.,  of axenic c u l t u r e o f  to grow papaya t i s s u e i n  r e p o r t of papaya c a l l u s c u l t u r e s was p r o b a b l y t h a t of  Medora e t a l . (1973), these c u l t u r e s .  S i n c e the advent  field i n  i n which they gave evidence  They have subsequently  and s u b s t r a t e s p e c i f i c i t i e s  1979; M e l l e t a l . ,  1975, 1979).  of p r o t e o l y t i c a c t i v i t y i n  p u b l i s h e d i n f o r m a t i o n about medium (Bilderback e t a l . ,  1976; Medora e t  Enzyme a c t i v i t y was f i r s t  assessed  u s i n g a c a s e i n s u b s t r a t e to which b u f f e r e d e x t r a c t s of mortar-ground, lyophilized  t i s s u e was added  (Medora e t a l . ,  1973).  a z o c a s e i n and c a s e i n y e l l o w were b e t t e r s u i t e d  L a t e r work showed t h a t  f o r assay of crude  extracts,  w h i l e hemoglobin, a z o c o l l , and h i d e powder azure were a l l inadequate. and  Hogan (1976, 1979) have a l s o propagated  p l a n t l e t s from embryoids.  A r o r a and S i n g h  Medhi  papaya c a l l u s and produced (1978) found t h a t the most  e f f e c t i v e growth hormones f o r papaya c a l l u s development were NAA (naphthalene  a c e t i c a c i d , 1.0 mg/1), k i n e t i n  (0.5 mg/1) and g i b b e r e l l i c  (1.0 mg/1) a c i d .  P r o p a g a t i o n of papaya p l a n t s v i a t i s s u e c u l t u r e , however,  r e q u i r e d changing  of hormone c o n c e n t r a t i o n s d u r i n g p l a n t l e t development  ( L i t z & Conover, 1978). Apte e t a l . (1979) r e p o r t e d p r o t e o l y t i c a c t i v i t y i n t i s s u e c u l t u r e s of p i n e a p p l e . the presence milk.  Callus,  initiated  from  l a t e r a l buds, was grown i n  o f NAA (10 mg/1), c a s e i n h y d r o l y s a t e (0.4 g/1) and 15% coconut  T h i s group e x t r a c t e d the enzyme(s) by acetone  blendor-homogenized t i s s u e . found g r e a t f l u c t u a t i o n s  Using a c a s e i n h y d r o l y s i s assay method,  i n activity  was  lower  one  has yet» r e p o r t e d attempts  f r a c t i o n a t i o n of they  over a 50-day p e r i o d , but p r o t e o l y s i s  i n c a l l i and regenerated p l a n t l e t s  than i n the mature p l a n t .  to improve p r o t e a s e y i e l d s o r to produce  No  17  active c e l l  suspension  c u l t u r e s from papaya o r p i n e a p p l e .  Other s p e c i e s i n v e s t i g a t e d h e r e i n have not p r e v i o u s l y been s t u d i e d as u n d i f f e r e n t i a t e d t i s s u e .  L i t z and Conover (1977) propagated  D i e f f e n b a c h i a from e x c i s e d l a t e r a l buds, with a minimum of c a l l u s formation.  There appear to be no r e p o r t s i n the l i t e r a t u r e r e g a r d i n g i n  v i t r o propagation The  of f i g .  f o c a l p o i n t of t h i s r e s e a r c h p r o j e c t was to a s s e s s  enzyme a c t i v i t y i n c e l l produce p r o t e a s e s .  suspension  c u l t u r e s d e r i v e d from p l a n t s known to  P r e l i m i n a r y work t h e r e f o r e r e q u i r e d p r e p a r a t i o n of  c a l l u s c u l t u r e s from a number of p l a n t sources: cardoon, D i e f f e n b a c h i a and bedstraw. develop c e l l led  suspension  cultures.  to an e v a l u a t i o n of protease  papaya, f i g , p i n e a p p l e ,  From t h i s stage,  i t was necessary  Successful establishment  activity.  s t i m u l a t i o n of p r o t e o l y t i c enzyme p r o d u c t i o n c u l t u r e s , w i t h concomitant o b s e r v a t i o n s production  proteolytic  under the t e s t c o n d i t i o n s .  of these  to  then  A l l subsequent work was aimed a t and a c t i v i t y i n c e l l  suspension  on growth and t o t a l p r o t e i n  18  III.  1.  MATERIALS  and METHODS  C a l l u s c u l t u r e of s e l e c t e d  species  G e n e r a l methods and c o n d i t i o n s :  The f o l l o w i n g seven p l a n t s were used as  t i s s u e sources f o r c a l l u s c u l t u r e : p i n e a p p l e , bedstraw and t h i s t l e . sterilization,  papaya,  f i g , cardoon,  dumbcane,  A s i m i l a r procedure of e x c i s i o n ,  trimming and p l a t i n g was f o l l o w e d f o r a l l t i s s u e s , w i t h  d e t a i l e d methods g i v e n below. E x p l a n t s and c a l l i aluminum f o i l exposure.  were i n c u b a t e d i n p l a s t i c  b a s i n s covered w i t h  to m a i n t a i n h u m i d i t y and minimize c o n t a m i n a t i o n and  light  I n c u b a t i o n was a t a temperature of 28 ± 3°C and i n t o t a l  ness, so t h a t c u l t u r e s were exposed  to l i g h t and temperature  dark-  fluctuations  o n l y f o r b r i e f p e r i o d s , no l o n g e r than one hour, d u r i n g o b s e r v a t i o n and transfer.  A sterile  type was used  t r a n s f e r c a b i n e t of the h o r i z o n t a l laminar a i r f l o w  ( E n v i r c o , Becton D i c k i n s o n Co., USA).  d i s h e s were used throughout. (Gamborg e t a l . ,  1968),  a t 0.6%  the medium most commonly used:  (w/v) f o r c a l l u s  tissues.  protease a c t i v i t y by v i s i b l e  skimmed milk,  papaya  ( D i f c o ) was  Developing c a l l i  i n an  were a s s e s s e d  c l e a r i n g of B5 agar media c o n t a i n i n g 3%  Papaya was propagated from e x p l a n t s of germinated seeds as  i n F i g u r e 3.  and the a r i l s  two r i n s e s of s t e r i l e dry  Bacto-agar  (Murashige  B5-M.  C a r i c a papaya; outlined  MS medium  Media were s t e r i l i z e d  a u t o c l a v e a t 15 p s i and 121 °C f o r 15 min. for  petri  T a b l e I I I g i v e s the c o m p o s i t i o n of B5 medium  & Skoog, 1962) was used f o r some young t i s s u e s . added  D i s p o s a b l e 5 cm  with s t e r i l e  The seeds were a s e p t i c a l l y removed from a market p e e l e d o f f each seed. distilled  filter  paper.  The seeds were then soaked i n  water f o r about 5 minutes each then b l o t t e d A t t h i s p o i n t , about h a l f  the seeds so p r e -  B5 Medium and P l a n t Hormones  Table I I I  From  Gamborg, M i l l e r and Ojima (1968):  Salts  Exp. C e l l Res. 50: 151-1  NaH_P0.' H 0 2 4 KN0„ '3 (NH ) S0 2  h 4  2  4  150  mg/1  2500  mg/1  134  mg/1  MgSO  7H 0  250  mg/1  CaCl,  2H 0  150  mg/1  2  2  KI  0.75 mg/1  Fe  (Sequestrene 330-Iron)  Micronutrients stock s o l u t i o n  1.0 g  H3BO3  0.3 g  2  ZnSO,' 7H 0 4  2H 0 2  CuSO, CoCl,  6H 0 2  25  mg  25  mg  25  mg  Vitamins stock s o l u t i o n  thiamine  10  mg  100  mg  10  mg  pyridoxine myo-inositol sucrose  P l a n t Hormones  IAA  ml/1  10  ml/1  20  g/1  ( i n 100 ml)  nicotinic acid  Carbohydrate  1.0  0.3 g  2  2  mg/1  ( i n 100 ml)  MnSO^' H 0  Na Mo0 *  28  1  g  (indole-3-acetic acid)  1.0 mg/1  2,4-D ( d i c h l o r o p h e n o x y a c e t i c a c i d ) o r p-cpa ( p a r a c h l o r o p h e n o x y a c e t i c  1.0 mg/1  acid)  1.0 mg/1  o r 2,4,5-T ( t r i c h l o r o p h e n o x y a c e t i c a c i d ) 1.0 mg/1 kinetin Adjuncts  (optional)  (6-furfurylaminopurine)  0.1 mg/1  agar  6.0 g/1  skimmed m i l k  30  casein  hydrolysate  yeast extract  ml/1  0.5-2.0 g/1 0.5-2.0 g/1  20  papaya fruit  callus dispersed  fpZ5\ in small /Ml  seeds  volume liquid  B5  seed arils removed  germinated on water agar  callus  from  seedling explants I B5 agar)  transfers small  gure 3.  multiple transfers with  multiple of  decreasing inoculu  fragments  P r e p a r a t i o n o f papaya f o r t i s s u e and c e l l seeds, c a l l u s and c e l l a t 28 °C i n d a r k n e s s .  suspension c u l t u r e s  culture. A l l were m a i n t a i n e d  21  pared were m e c h a n i c a l l y damaged so as to break the tough seed o t h e r h a l f were l e f t i n t a c t .  coat:  A l l seeds were p l a c e d on s t e r i l e  distilled  water-agar (WA) i n p e t r i d i s h e s w i t h a minimum s u r f a c e a r e a of 5 cm seed.  explanted  and segments no l o n g e r than 2 cm t r a n s f e r r e d to n u t r i e n t media.  (dichlorophenoxyacetic acid, mg/l) and k i n e t i n  1.0 mg/1  2,4-D  ), IAA ( i n d o l e - 3 - a c e t i c a c i d ,  (6-furfurfyrlaminopurine,  0.1 mg/1).  f o r m a t i o n i n 2-5 weeks. o r i g i n a l explant s i z e the p a r e n t  t i s s u e was approximately  for callus five  times the  ( e s t i m a t e d t i s s u e volume), i t was d i s s e c t e d away from  t i s s u e and t r a n s f e r r e d to f r e s h agar  medium.  Such passages were  a minimum of three times, w i t h a t l e a s t two weeks' growth each  time, p r i o r inoculum  When c a l l u s  1.0  Plates containing  e x p l a n t s were i n c u b a t e d under the same c o n d i t i o n s and observed  repeated  About  the new s e e d l i n g s were a s e p t i c a l l y  These were based on B5 o r MS and g e n e r a l l y c o n t a i n e d  to f u r t h e r experiments or p r o p a g a t i o n  i n l i q u i d media.  The  s e l e c t e d f o r t r a n s f e r was always near the f r i n g e s of the c a l l u s so  to take o n l y v e r y young, r a p i d l y - g r o w i n g  Ficus c a r i c a : trees.  per  2  P l a t e s were i n c u b a t e d as d e s c r i b e d above u n t i l g e r m i n a t i o n .  the f o u r t h day a f t e r g e r m i n a t i o n ,  as  the  F i g t i s s u e c u l t u r e s were i n i t i a t e d  The leaves were prepared  p i e c e s of both  tissue. from leaves of f i g  f o r e x p l a n t a t i o n by g e n t l y washing l a r g e  laminae and p e t i o l e s under running water then  soaking  minutes i n two washes of 10% commercial b l e a c h , f o l l o w e d by r i n s i n g changes of s t e r i l e d i s t i l l e d water, 10 minutes each. aseptically  trimmed, exposing  e x p l a n t fragments a v e r a g i n g a r e a f o r laminae.  5-10 i n three  Cut edges were  f r e s h s u r f a c e s , and these p i e c e s c u t i n t o  1 cm i n l e n g t h f o r p e t i o l e s and 1-2 cm  2  in  These e x p l a n t s , b l o t t e d d r y , were t r a n s f e r r e d to B5 o r MS  agar p l a t e s and i n c u b a t e d under c o n d i t i o n s d e s c r i b e d above. a l l were t r a n s f e r r e d to f r e s h agar  A f t e r one week,  then i n c u b a t e d a g a i n u n t i l c a l l u s  was  22  observed.  C a l l u s t i s s u e a t l e a s t e q u a l to the o r i g i n a l e x p l a n t i n s i z e  d i s s e c t e d f r e e and t r a n s f e r r e d t o f r e s h a g a r . young c a l l u s  t i s s u e preceded  Cynara c a r d u n c u l u s : Nursery,  Albany,  A t l e a s t t h r e e passages of  attempts a t l i q u i d  culture.  Seeds of cardoon were a g i f t of N i c h o l s Garden  Oregon, USA.  The seeds were s t e r i l i z e d  commercial b l e a c h ( 2 x 1 0 min) and r i n s i n g i n s t e r i l e 10 m i n ) .  Some were c r a c k e d , o t h e r s  d e s c r i b e d f o r papaya. handled  l e f t intact,  from the UBC P l a n t S c i e n c e Department greenhouses.  a c c o r d i n g to the method g i v e n above f o r f i g . The h i g h  Otherwise,  sterilization  the c u l t u r e method was  a u x i n , 2,4-D, was r e p l a c e d w i t h 2,4,5-T  Ananas comosus:  Pineapple  the tops of p i n e a p p l e s were prepared explants  l e s s than 1 cm^ i n s i z e .  contamination  protocol:  procedure  under-ripe f r u i t .  min  totalling  (trichlorophenoxyacetic acid).  retail.  the market  Basal leaf explants  as d e s c r i b e d f o r f i g , u s i n g  from  final  C r o s s - s e c t i o n s of v e g e t a t i v e leaves as d e s c r i b e d f o r dumbcane.  was a l s o a p p l i e d to e x p l a n t s  Standard  1x10  the same as g i v e n f o r f i g .  i n c l u d i n g base and l e a f margins were s t e r i l i z e d latter  were  They were p r e -  t i s s u e was d e r i v e d from two s o u r c e s ,  f r u i t and v e g e t a t i v e p l a n t , both purchased  of  on WA as  1 x 20 min i n 15% b l e a c h , f o l l o w e d by 3 s t e r i l e water r i n s e s  30-40 min.  The  then germinated  Leaves of two s p e c i e s of dumbcane  r a t e , however, r e q u i r e d much more severe  The  d i s t i l l e d water (3 x  as d e s c r i b e d above.  obtained  and  by soaking i n 15%  E x p l a n t s of s e e d l i n g s and r e s u l t i n g c a l l u s were  D i e f f e n b a c h i a amoena & D. p i e t a :  pared  was  from the f l e s h of  media (B5 and MS) were used, except  slightly  f o r the use  2,4,5-T.  Galium verum;  C u l t u r e s of l a d i e s ' bedstraw o r i g i n a t e d from  freshly-  i harvested cardoon,  seeds.  These were s t e r i l i z e d  a c c o r d i n g to the method g i v e n f o r  but with the a d d i t i o n of a w e t t i n g agent to the f i r s t b l e a c h soak  23  (2 drops P a l m o l i v e d e t e r g e n t per 100 ml b l e a c h s o l u t i o n ) .  Bedstraw seeds  c a r r y dense s u r f a c e d i s p e r s a l appendages which would otherwise w e t t i n g and e f f e c t i v e s t e r i l i z a t i o n . were 0.5-1 .0 cm i n l e n g t h .  E x p l a n t s of the germinated  These m o d i f i c a t i o n s i n c l u d e d a l l the hormones l i s t e d i n  T a b l e I I I , 0.05-0.5% c a s e i n , 1-5% e x t r a c t and 4 mM  thiourea.  v i s u a l examination  excision.  Leaves and  of  surface hairs,  rinsed i n s t e r i l e  Suspension  media and  viable cultures in liqiud  2 mg/1).  Cell  media.  by  fresh  prior  tissues  5-10  min  soakings  i n 15% b l e a c h  Trimmed e x p l a n t dimensions  proliferated  B5 was  were  used.  r a p i d l y enough to e s t a b l i s h  the b a s a l medium with the f o l l o w i n g  (1 mg/1)  and  p-cpa  (p-chlorophenoxyacetic  c u l t u r e s began with d i s p e r s a l ,  in  r e p r e s e n t i n g a t l e a s t 10% of the  F l a s k s of more than double  the r e q u i s i t e  c o t t o n and  final  necks with e l a s t i c  28 ± 3°C, dark,  110  so as  They were  f u r t h e r p r o t e c t e d with  l a y e r s of paper t o w e l l i n g secured about the f l a s k I n c u b a t i o n c o n d i t i o n s were c o n s t a n t :  liquid  c a p a c i t y were used  to p r o v i d e a l a r g e s u r f a c e area f o r a e r a t i o n of the medium. stoppered with cheesecloth-wrapped  and  papaya  mg/1), IAA  suspension  medium, of l a r g e c a l l u s volume.  i n two  papaya c a l l i  (0.1  collected  i n c u b a t i o n c o n d i t i o n s were  c u l t u r e of f i g and  kinetin  yeast  stems were s u r f a c e - w e t t e d w i t h 70% e t h a n o l because  water (3 x 10 m i n ) .  Only f i g and  hormones:  ( v a r . horridum) was  then s t e r i l i z e d  Standard  m i l k , 0.2%  initiation.  Wild t h i s t l e  to  0.5-2.0 cm.  skimmed m i l k , 10% coconut  E f f e c t s of these m o d i f i c a t i o n s were assessed  for callus  C i r c i u m arvense:  acid,  to encourage  f o r m a t i o n , r a t h e r than the f i n e r o o t - l i k e p r o l i f e r a t i v e s t r u c t u r e s  which developed.  2.  seedlings  I n c u b a t i o n c o n d i t i o n s were as d e s c r i b e d above.  Numerous m o d i f i c a t i o n s of B5 medium were made i n attempts callus  prevent  two bands.  rpm on r o t a r y  New  24  Brunswick  shakers.  A f t e r 1 - 4 weeks' growth, depending  c a l l u s sloughed c e l l s and  readily  the suspension t h i c k e n e d , these primary  s u s p e n s i o n c u l t u r e s were t r a n s f e r r e d  volume.  to as " c e l l s u s p e n s i o n c u l t u r e s " o r " c e l l  a c c o r d i n g to t r a d i t i o n a l t e r m i n o l o g y i n the l i t e r a t u r e . such c o n s e c u t i v e t r a n s f e r s preceded  the  cell  to f r e s h media u s i n g a l a r g e - b o r e  p i p e t t e and an inoculum s i z e o f 10-15% of the f i n a l w i l l be r e f e r r e d  on how  H e r e i n , these cultures",  A minimum of t h r e e  further experimentation i n c e l l  line  s e l e c t i o n , p r o t e a s e assay or medium c o m p o s i t i o n as d e s c r i b e d below.  3.  Assay methods  Biomass:  C e l l dry weight o b t a i n e d from i n i t i a l  ( i e . , medium + inoculum)were  cell  s u s p e n s i o n volumes  c o n v e r t e d to y i e l d s based on one  weights were determined by three methods:  to c o n s t a n t weight  litre.  Dry  in a  c o n v e c t i o n oven a t 60 °C, i n a vacuum oven a t 60°C, or i n a V i r t i s freeze-dryer  (low heat, condenser  temperature  C e l l s were h a r v e s t e d by f i l t r a t i o n NJ, USA)  then t r a n s f e r r e d  -60°C, l e s s than 1 mm  through M i r a c l o t h  to p r e - d r i e d , pre-weighed  Hg).  (Chicopee M i l l s ,  Inc.,  aluminum d i s h e s .  After  e s t a b l i s h m e n t of the r e l a t i o n s h i p between r e s u l t s of these methods, a l l h a r v e s t weights were d e r i v e d from l y o p h i l i z e d samples s i n c e these were then used  f o r enzyme a s s a y s . A r a p i d method of a s s e s s i n g c e l l  volume" ( N i c k e l l & M a r e t z k i , 1969), (1975).  T h i s method was  v a l i d i t y because upon h a r v e s t . min.  a l s o employed by Behrend and  compared to c e l l dry weights  of the advantage  decanted  to e v a l u a t e i t s  t h a t r e s u l t s c o u l d be o b t a i n e d  immediately 30  through M i r a c l o t h to t r a p f l o a t i n g c e l l s  suspensions poured  into  cell  Mateles  C e l l suspension c u l t u r e s were allowed to s e t t l e about  The medium was  the t h i c k c e l l  growth i s based on " s e t t l e d  l a r g e graduated c e n t r i f u g e  and  tubes.  25  C e l l s c o l l e c t e d on the f i l t e r were a l s o added, by s c r a p i n g of the f i l t e r a flat cell dry  surface.  Tubes were c e n t r i f u g e d 3-5 min a t 200 xg and the  volume was  read d i r e c t l y .  The c e l l s were then l y o p h i l i z e d  on  settled  to o b t a i n  weights.  C e l l extraction:  Three methods were compared f o r e x t r a c t i o n of  i n t r a c e l l u l a r m a t e r i a l to determine p r o t e i n s and,  i n particular,  accomplished by s o n i c a t i o n homogenizing  (1-4 min,  the most e f f e c t i v e one  active proteases.  (80 W,  f o r r e l e a s e of  C e l l disruption  30 & 120 s e c ; Braunsonic  was  1510),  by  P o l y t r o n K i n e m a t i c a PCU-1, Brinkmann Instruments)  and  by g r i n d i n g i n a mortar w i t h o r w i t h o u t washed sand as an a b r a s i v e (45-90 sec  a t an average  r a t e of 60-80 s t r o k e s / m i n ) .  s t a n d a r d method f o r f u r t h e r s t u d i e s . i c e - c h i l l e d e x t r a c t i o n b u f f e r , 0.1 throughout.  These  The  l a s t of these became the  In a l l c a s e s , c e l l s were suspended  M phosphate  a t pH 6.0,  crude e x t r a c t s were f i l t e r e d  and kept on i c e  through M i r a c l o t h to remove  c e l l d e b r i s then assayed f o r p r o t e i n c o n t e n t and p r o t e o l y t i c P r o t e i n content:  Brilliant  used r o u t i n e l y due Blue-G, Sigma) and  The d y e - b i n d i n g method of B r a d f o r d  to the s i m p l i c i t y rapidity  (10 min).  p r o t e i n d e t e r m i n a t i o n s by three o t h e r methods. at  280 nm  (Beckman DB  without f i l t r a t i o n  T h i s method was Ultraviolet light  compared to absorbance accurately  and high-speed c e n t r i f u g a t i o n of the e x t r a c t s due  the p r o t e i n , i t was  considered unsuitable.  was  tested.  N i t r o g e n c o n t e n t of c e l l s  (AOAC, 1975),  Technicon Autoanalyzer I I nitrogen a n a l y z e r . 1951)  to  Since t h i s step would a l s o p e l l e t some  determined by the m i c r o - K j e l d a h l method  (Lowry e t a l . ,  (one reagent, Coomassie  spectrophotometer) c o u l d not be determined  i n t e r f e r e n c e caused by c l o u d i n e s s . of  activity.  Crude c e l l e x t r a c t s were kept on i c e and assayed f o r  p r o t e i n content w i t h i n three hours. (1976) was  in  Finally,  was  and q u a n t i t a t e d by a the Lowry procedure  A d i l u t i o n s e r i e s of bovine serum albumin  26  ( F r a c t i o n V,  Sigma) was  i n c l u d e d as the standard  p r o t e i n f o r Bradford's  and  Lowry's me t h o d s . Protease a c t i v i t y :  The  method of the U.S.  N a t i o n a l Research  Food Chemicals Codex (1966), h e r e a f t e r r e f e r r e d to as selected.  T h i s procedure was  substrate concentration described)  was  was  0.2  used i n p l a c e  extended to 2 h r , and  modified  as  g/100  a t 0.4  mM  ml;  extraction buffer  of f o u r tubes;  dithiothreitol  ( D t t , Sigma, 2 mM  f i n a l concentration)  and  two  (8,000 xg,  the absorbance a t 280 two  incubated  post-incubation,  was (BDH)  (as p r e v i o u s l y  of a c t i v a t i o n b u f f e r ; the i n c u b a t i o n p e r i o d  Samples were both c e n t r i f u g e d p r i o r to r e a d i n g  method,  f o l l o w s : Hammarsten c a s e i n  r e p l a c e d c y s t e i n e as the a c t i v a t o r , added w i t h Ohio, USA,  the FCC  Council's  incubated  final  10 min,  nm.  concentration)  EDTA (G. F r e d e r i c k  immediately p r i o r  to  S o r v a l l RC-2)  Smith,  incubation. and  filtered  Each sample e v a l u a t i o n  sample/substrate/activator  consisted  r e a c t i o n mixtures  s u b s t r a t e / a c t i v a t o r mixtures to which sample was a l o n g w i t h TCA  was  added  (trichloroacetic acid, Fisher).  R e s u l t s of t h i s method were compared to t r i a l s of seven  others:  (a) d i g e s t i o n of Hide Powder Azure (a d y e - l a b e l l e d c o l l a g e n , Sigma) according 5.0 (b)  to Savage and  ml i n p l a c e esterase  according  t o A r a k i and the  Abe  extending  time to 1 hr; diffusion ethyl  substrate; the a g a r - d i f f u s i o n method of Holmes  and  (1973);  c a s e i n o l y t i c a c t i v i t y using  (1978);  the i n c u b a t i o n  of e x t r a c t d i l u t e d to  (1980), u s i n g BAEE ( N - b e n z o y l - L - a r g i n i n e  c a s e i n o l y t i c a c t i v i t y by  Ernstrom (d)  of beer, and  ml  a c t i v i t y by pH-dependent i n d i c a t i o n of r a d i a l  e s t e r , Sigma) as (c)  Thompson (1970), u s i n g 0.5  the Bio-Rad "Protease d e t e c t i o n k i t "  27  (e)  g e l a t i n d i g e s t i o n from f i l m , by  (1961), u s i n g 0.5  the method of G l e n i s t e r and  ml e x t r a c t d i l u t e d to 5.0  Becker  ml i n s t e a d of beer, and  Kodak  Panatomic-X f i l m ; (f)  fluorescence  l o s s of ANS  Sigma) a c c o r d i n g and  (1-anilino-8-naphthalenesulfonate,  to Spencer and  a c t i v a t e d (Dtt and  Spencer (1974), u s i n g  EDTA) crude c e l l e x t r a c t s  spectrophoto-fluorometer;  e x c i t a t i o n and  emission  Mg  the c a s e i n  salt,  substrate  (Aminco-Bowman wavelengths, 370  and  460nm, r e s p e c t i v e l y ) ; (g)  development of f l u o r e s c e n c e  by r e a c t i o n of f l u o r e s c a m i n e  TCA-soluble c a s e i n d i g e s t i o n products, al.  (1979), u s i n g 0.2%  according  emission  In a l l cases,  wavelengths', 390  and  ground i n a c o l d mortar w i t h  described  Appropriate  Calbiochem) or p u r i f i e d referred  to as The  ficin  "standard" FCC  to p r e c i p i t a t i o n and  the samples t e s t e d were f i l t e r e d  concentrations  incubated  reading  extracts  of  i c e - c h i l l e d buffer  as  of p u r i f i e d papain ( A f r i c a n ,  (Sigma) enzyme standards were used, and  Supernatant absorbance a t 280  s e l e c t e d f o r r o u t i n e protease nm  i s dependent on  determinations.  the q u a n t i t y o f  10%  T C A - s o l u b l e aromatic amino a c i d s r e l e a s e d from the c a s e i n s u b s t r a t e Hence, a s e r i e s of s t a n d a r d s c o n t a i n i n g  (Merck) i n q u a n t i t i e s of 0-300 ug per r e a c t i o n volume (2.1 c o n t a i n i n g 3.6  mg  representative  of d i g e s t i o n p r o d u c t s as w e l l as a c c o u n t i n g  c a s e i n ) was  i n c l u d e d i n each a s s a y .  spontaneous c a s e i n h y d r o l y s i s , and activity.  One  are  throughout.  method was  the i n c u b a t i o n p e r i o d .  with  475nm, r e s p e c t i v e l y ) .  lyophilized cells above.  to the method of Chism e t  Hammarsten c a s e i n without sodium a z i d e  0.025-0.5 ml crude c e l l e x t r a c t f o r 1 hr p r i o r ( e x c i t a t i o n and  (Sigma) w i t h  thus was  This  ml  during  L-tyrosine  already was  for  any  used to q u a n t i t a t e p r o t e o l y t i c  u n i t of p r o t e o l y t i c a c t i v i t y was  d e f i n e d as  t h a t amount of  28  a c t i v e enzyme(s) e x t r a c t a b l e will  from one l i t r e of c e l l s u s p e n s i o n c u l t u r e  r e l e a s e , from c a s e i n , TCA-soluble m a t e r i a l e q u i v a l e n t  t y r o s i n e under r e a c t i o n c o n d i t i o n s Conditions  defined  temperature of i n c u b a t i o n .  The m o d i f i e d  FCC p r o t e a s e  f o r v a r i a t i o n w i t h r e a c t i o n pH and  One e x t r a c t from a l a r g e c e l l s u s p e n s i o n  of f i g was used f o r a l l t e s t c o n d i t i o n s , s e r i e s and the temperature s e r i e s .  stored  culture  f r o z e n 2 days" between the pH  The i n f l u e n c e of pH was t e s t e d  b u f f e r s a t pH 5.5, 6.0, 7.0 and 8.4. were t e s t e d :  t o 100 ug  above.  i n f l u e n c i n g protease a c t i v i t y :  d e t e c t i o n method was e v a l u a t e d  that  A t pH 6.0, the f o l l o w i n g  27, 38, 47, 54 and 67 °C. A p p r o p r i a t e  using  temperatures  s e r i e s of s t a n d a r d  f i c i n and t y r o s i n e were i n c l u d e d w i t h each s e t of c o n d i t i o n s . A number of reagents which c o u l d p o t e n t i a l l y a c t i v a t e t h e extracted  enzyme(s) were t e s t e d under standard  w i t h three  controls.  These c o n t r o l s were t r y p s i n i n h i b i t o r  mg/ml r e a c t i o n mixture, sodium t e t r a t h i o n a t e concentration, follows:  and no a d j u n c t .  L-cysteine-HCl  2 mM, g l u t a t h i o n e at  6 mM, c a l c i u m  c o n d i t i o n s , 40 °C and pH 6.0,  (ICN) a t 6 mM  (Sigma) a t 0.4 final  The p o t e n t i a l a c t i v a t o r s t e s t e d were as  (MCB) a t 1.4 mM, d i t h i o t h r e i t o l a t 2.0 mM,  (MCB) a t 6 mM, thiocyanate  SDDC (sodium d i e t h y l d i t h i o c a r b a m i d e ,  (Anachemia Chemicals, Ltd.)  r e a c t i o n mixture, and t h i o u r e a  EDTA a t MCB)  a t 2.5 u l / m l  ( M a l l i n k r o d t ) a t 6 mM f i n a l  concentration.  Both papain and f i c i n were t e s t e d i n the presence of these reagents, and compared to the behaviour of the f i g c e l l Milk clotting a c t i v i t y : harvested  by f i l t r a t i o n  Papaya and f i g c e l l s u s p e n s i o n c u l t u r e s were through M i r a c l o t h .  l y o p h i l i z e d , weighed and e x t r a c t e d above.  extract.  according  C o l l e c t e d c e l l s were to the methods  E x t r a c t s were k e p t on i c e and used w i t h i n 2 h r .  described  The milk  clotting  assay of B a l l s and Hoover (1937) was used, w i t h no r e s u l t s , so the f o l l o w i n g  29  method was d e v i s e d .  Skimmed milk was d i l u t e d  to a s o l i d s content of 5% and  the pH a d j u s t e d t o 5.6 w i t h 2 M H3PO4 then a u t o c l a v e d i n 25-ml f l a s k s c o n t a i n i n g 3 ml each.  Erlenmeyer  C e l l e x t r a c t , 0.3 ml, was added to each of  t h r e e f l a s k s and these were i n c u b a t e d i n a water bath shaker a t 35°C up t o 24 h r . C l o t t i n g  times were compared to h i g h l y d i l u t e d  standard papain, a t a  maximum c o n c e n t r a t i o n of 0.3 mg/ml. Electrophoresis:  E l e c t r o p h o r e s i s of f i c i n - l i k e p r o t e i n s i n crude  e x t r a c t s i n acrylamide  g e l was attempted.  cell  The method of M e l a c h o u r i s  as m o d i f i e d by Mr. T. Kuwata ( u n p u b l i s h e d ) , was a p p l i e d t o a v e r t i c a l g e l apparatus.  The s e p a r a t i o n g e l s t r i e d were 9-10% a c r y l a m i d e  (1968), slab  (Bio-Rad)  p o l y m e r i z e d w i t h 0.24-0.28% b i s - a c r y l a m i d e ; the c o n c e n t r a t i o n g e l s were 3-3.6% a c r y l a m i d e ; 8.9.  the e l e c t r o p h o r e t i c b u f f e r was T r i s - T E M E D - g l y c i n e  a t pH  A f t e r e l e c t r o p h o r e s i s (4.5-5 h r 100 V ) , g e l s were f i x e d i n  TCA-isopropanol-water  (15% TCA and 25% i s o p r o p a n o l i n d i s t i l l e d  water) f o r  40 min, s t a i n e d with amido b l a c k 10B (0.025% w/v i n 4:5:1 H2O:methanol:acetic a c i d ) 30-50 min, and d e s t a i n e d 1 .5-2 days i n 3 changes of d e s t a i n i n g s o l u t i o n The  (9.71:3.57:1 H2O: m e t h a n o l : a c e t i c  d i s c g e l method of Weber e t a l . (1972) was a l s o employed,  u s i n g 7.5-9.3% a c r y l a m i d e . buffer  acid).  E l e c t r o p h o r e s i s was conducted  (pH 7.2) w i t h 0.1% SDS (sodium  i n 0.1 M phosphate  dodecylsulfate, Fisher).  s u b j e c t e d to 2 mA p e r g e l 20-30 min then 5 mA per g e l u n t i l b l u e marker dye approached the d i s t a l end.  Gels were  the bromophenol  Gels d i s l o d g e d from g l a s s  tubes  were s t a i n e d up to 2 h r w i t h Coomassie B r i l l i a n t Blue-G (0.25%, w/v, i n 1:1:0.2 H20:methanol:acetic H20:acetic  a c i d ) and d e s t a i n e d 1.5-2 days i n 35:3:2  acid:methanol. Samples were prepared  f o r e l e c t r o p h o r e s i s i n a v a r i e t y of ways i n  s e a r c h of one t h a t would e f f e c t i v e l y demonstrate p r o t e i n s p r e s e n t i n c e l l  30  extracts.  For e l e c t r o p h o r e s i s without SDS,  T r i t o n X-100 in  SDS,  i n Carbowax 20M  4.  ( A p p l i e d S c i e n c e ) 2-6  hr.  8 M urea, and  tubing  used were f i c i n and  electrophoretic  trials  trypsin  (Sigma).  were mixed 4:1  buffer (Fisher),  For e l e c t r o p h o r e s i s p-mercaptoethanol  i n stoppered tubes a c c o r d i n g to the method o f Deutch  Standards  to  to the e x t r a c t i o n  Many samples were c o n c e n t r a t e d i n d i a l y s i s  samples were mixed w i t h SDS,  5 min  all  were added, alone or i n combination,  some t r i a l s .  packed  sodium t e t r a t h i o n a t e , SDDC and  with  and  boiled  (1976).  Samples and  standards f o r  with g l y c e r o l p r i o r  to a p p l i c a t i o n  gels.  Medium  supplementation F i v e groups of n u t r i e n t supplements were i n v e s t i g a t e d with r e s p e c t  to  enhancement of p r o t e a s e p r o d u c t i o n of a c t i v i t y  in cell  extracts:  i n o r g a n i c n i t r o g e n , amino a c i d s , p r o t e i n s , milk and i t s v a r i o u s components, and m i s c e l l a n e o u s o r g a n i c compounds, i n c l u d i n g two r e a g e n t s and SDDC, an oxidase  1 mM). or  3  (2.5 g/1,  two  thiol  inhibitor.  I n o r g a n i c n i t r o g e n and amino a c i d s : B5 medium as KN0  antibiotics,  N i t r o g e n i s p r e s e n t i n the standard  or 25 mM)  and  (NH ) S0 4  2  4  (0.134 g/1,  or  Media were prepared o m i t t i n g e i t h e r of these, but s u b s t i t u t i n g  Na S0 2  4  as a p p r o p r i a t e to m a i n t a i n the non-nitrogenous  M a t e l e s , 1975).  To each of these was  ions (Behrend  added one of the f o l l o w i n g :  KC1 &  alanine,  a r g i n i n e , a s p a r t i c a c i d , c y s t e i n e , g l u t a m i c a c i d , g l y c i n e or p r o l i n e ( a l l L-isomers  from Sigma) a t 5 mM  purposes,  a medium c o n t a i n i n g o n e - h a l f the s t a n d a r d n i t r a t e  prepared, and 3%  final  concentration.  For  comparative  (v/v) skimmed milk added to a p o r t i o n of  nitrate-ammonium c o m b i n a t i o n . s i m u l t a n e o u s l y from one  (12.5 mM)  was  each  Small f l a s k s of these media were i n o c u l a t e d  s u s p e n s i o n c u l t u r e of F i c u s c a r i c a grown i n a  31  l o w - n i t r o g e n medium, 20% of the normal B5 l e v e l . i n c u b a t i o n these c o n d i t i o n e d c u l t u r e s were used the same media f o r 14 days'  F o l l o w i n g 9 days' to i n o c u l a t e another  i n c u b a t i o n p r i o r to assessment.  s e t of  C e l l s were  h a r v e s t e d and both c e l l s and media l y o p h i l i z e d and a n a l y z e d f o r p r o t e i n c o n t e n t and p r o t e a s e a c t i v i t y , P r o t e i n s and p e p t i d e s : p r o t e i n s and  as d e s c r i b e d above.  B5 medium was  supplemented w i t h a s e l e c t i o n of  r e l a t e d m a t e r i a l s to i n v e s t i g a t e t h e i r p o s s i b l e s t i m u l a t i o n of  protease a c t i v i t y .  These were as f o l l o w s :  (w/v), soy p r o t e i n i s o l a t e  ("Farefax",  c a s e i n powder ( F i s h e r ) a t  N u - L i f e N u t r i t i o n ) a t 0.3%  wheat g l u t e n ("WhetPro", I n d u s t r i a l G r a i n Products) a t 0.3% extract  ( D i f c o ) a t 0.3%  albumen a t 1%  ( v / v ) , enzymatic  skimmed milk a t 3% a t 0.3% days.  (w/v), g e l a t i n  (v/v).  Two  (MCB)  a t 0.3%  (w/v),  (w/v), beef  (w/v), f r e s h  c a s e i n h y d r o l y s a t e (ICN) a t 0.3%  egg  (w/v),  and  c o n t r o l s i n c l u d e d were s o l u b l e s t a r c h ( D i f c o )  (w/v), and no supplements.  A l l f l a s k s were h a r v e s t e d a f t e r  Both media and c e l l s were a n a l y z e d f o r protease a c t i v i t y and  v a l u e s summed f o r t o t a l  0.3%  18 these  protease.  M i l k and i t s components:  Skimmed m i l k was  f o r s u s p e n s i o n - c u l t u r e d c e l l s of papaya and  used  to supplement B5 medium  f i g . T h i s e n r i c h e d medium  compared to s t a n d a r d B5 with r e s p e c t to biomass, p r o t e i n and  was  protease  a c t i v i t y over a growing p e r i o d of 23 days under standard c o n d i t i o n s of incubation.  Assay procedures  The  e f f e c t of s e v e r a l components of skimmed milk on protease  a c t i v i t y of f i g c e l l s was the medium.  assessed by simple or complex supplementation  of  S i n g l e supplements, a l l reagent grade, were added a t l e v e l s  approximating citric  are d e s c r i b e d i n p r e c e d i n g s e c t i o n s .  acid  t h a t which 3% skimmed milk would p r o v i d e f o r B5 medium: (49.8 mg/1), r i b o f l a v i n  as CaCl3*2H 0, (1.336 g/1 2  (53 ug/1), n i a c i n  (24 ug/1)  and  calcium,  i n a d d i t i o n to t h a t a l r e a d y p r e s e n t i n B5).  32  Lactose replaced  20% o f sucrose i n one t e s t medium, t h a t i s 4 g/1.  Commercial c a s e i n powder ( F i s h e r ) was r e h y d r a t e d , p r e c i p i t a t e d and c o l l e c t e d by the  centrifugation. same l e v e l .  casein  Added a t 0.3%  These c a s e i n s  (w/v), i t was compared to f r e s h c a s e i n a t  were prepared by c o a g u l a t i o n  s o l u t i o n or f r e s h l i q u i d  skimmed m i l k w i t h slow a d d i t i o n of  p h o s p h o r i c a c i d a t room temperature u n t i l continued s t i r r i n g , for  the pH reached 5.2.  pH t o 5.6 w i t h NaOH, most of the whey was f i l t e r e d paper and s i n t e r e d g l a s s  of 2.7% ( v / v ) .  t o accommodate  to u l t r a f i l t r a t i o n  f o r 10,000 d a l t o n s .  Both f i l t r a t e and  A l l media c o n t a i n i n g medium c o n t a i n i n g  casein  combination  o r whey prepared i n  15% l e s s i n i t i a l  phosphate,  the a d d i t i o n a l phosphate from the c a s e i n p r e c i p i t a t i o n s t e p .  were i n o c u l a t e d  from p r e - c o n d i t i o n e d  20 ml of each r e s p e c t i v e  t e s t medium.  h a r v e s t e d and both c e l l s  and medium  protease  through Whatman No. 5  were used as B5 supplements, i n d i v i d u a l l y and i n a 1:1  t h i s manner began with a b a s a l  Flasks  A f t e r adjustment of the  (10-15 um), then s u b j e c t e d  (Pellicon, Millipore) with a f i l t e r  to a t o t a l  A f t e r 20 min  the c a s e i n was c o l l e c t e d by c e n t r i f u g a t i o n a t 10,000 xg  15 min and the whey (supernatant) decanted o f f .  retentate  of a g i t a t e d  c e l l suspensions grown 7 days i n  A f t e r 14 days' i n c u b a t i o n ,  cells  were  f r a c t i o n s a n a l y z e d f o r p r o t e i n and  activity.  S t a t i s t i c a l analysis:  Results  of major experiments i n medium  s u p p l e m e n t a t i o n were p r o c e s s e d by a n a l y s i s p r e p a r e d f o r the U n i v e r s i t y 1980) and G e n l i n  (Grieg  of B r i t i s h  & Bjerring,  treatments, Duncan's m u l t i p l e of 1 and 5%.  of v a r i a n c e  using  two programs  Columbia computing c e n t r e ,  1980).  MFAV (Le,  To determine d i f f e r e n c e s  range t e s t was a p p l i e d  Data were grouped a c c o r d i n g to these  among  at probability levels  ranges.  33  IV.  1.  RESULTS  C u l t u r e of p l a n t  tissues  Papaya t i s s u e s , C. papaya, were s u c c e s s f u l l y c u l t u r e d  from e x c i s e d  segments of r a d i c l e s and h y p o c o t y l s of a x e n i c a l l y - g e r m i n a t e d seeds.  Early  trials  first  indicated  t h a t seeds germinated  removed and seeds r i n s e d .  The a r i l s  germination (Gherardi & V a l i o , papaya c u l t u r e s  very p o o r l y u n l e s s a r i l s were  c o n t a i n substances  1976).  i n h i b i t o r y to seed  T a b l e IV i n c l u d e s s u r v i v a l  l e v e l s of  to both c a l l u s and c e l l s u s p e n s i o n stages ( F i g u r e 4a & b ) .  C a l l i were p a l e , s o f t and r e a d i l y d i s p e r s a b l e i n l i q u i d  medium.  F i g t i s s u e s , F. c a r i c a , were s u c c e s s f u l l y propagated  from  leaf  segments to c a l l u s and suspension stages a t the remarkably h i g h s u r v i v a l l e v e l of 95% (Table I V ) .  F i g s u s p e n s i o n c u l t u r e s were r e a d i l y  and maintained, and were thus used n u t r i t i o n and enzyme p r o d u c t i o n .  established  f o r experimental studies i n c e l l The appearance  of f i g c a l l u s i s  i l l u s t r a t e d i n F i g u r e 5 (a&b); c e l l s u s p e n s i o n c u l t u r e s were i d e n t i c a l t o those of papaya, c o n s i s t i n g of a p a l e y e l l o w s l u r r y of c e l l s . Seeds of cardoon, C. c a r d u n c u l u s , showed a 14% g e r m i n a t i o n r a t e , on average, and t i s s u e s were e x c i s e d from h y p o c o t y l s and r a d i c l e s f o r further propagation. effective  S t e r i l i z a t i o n procedures  used were not s u f f i c i e n t l y  f o r these hard seedcoats and a h i g h r a t e of endogenous  contamination  resulted.  Galium,  l a d i e s ' bedstraw,  seeds, but r a t h e r developed  d i d not form c a l l u s from  as a mass of r o o t - l i k e s t r u c t u r e s  In attempts  to bypass  transferred  to l i q u i d medium.  germinated (Figure 6a).  the stage of c a l l u s f o r m a t i o n , germinated Under these c o n d i t i o n s ,  tissue  was  the r o o t s c o n t i n u e d  34  S u r v i v a l of P l a n t T i s s u e  T a b l e IV  plant  source  Ananas comosus  % survival (2 mos.)  explants type & number  fruit, 223  0  100  Circium  leaf, petiole, 110  stem  leaf, petiole, 222  stem  Cynara c a r d u n c u l u s  form i n 6 mos.  l e a f , stem  C a r i c a papaya  arvense  Cultures  seeds  22  callus & 11 s u s p e n s i o n s  11  callus & 2 suspensions  0  72  seeds  22  callus  40  seedlings  23  callus  Dieffenbachia p i c t a & amoena Ficus  carica  254  petiole  leaf, petiole, 133 150  Galium verum  leaf &  81  stem  19  seeds  callus  stem  seeds  77 l e a f &  16 little ( a l l D.amoena) 95  callus & suspensions  0 petiole  11  roots?  63  roots?  0  35  F i g u r e 4.  Dark-grown papaya c a l l u s and c e l l  suspension  (a)  Papaya c a l l u s on B5 agar, 22 days a f t e r  (b)  Papaya c e l l  suspension c u l t u r e ,  cultures.  transfer.  18 days a f t e r  transfer.  F i g u r e 5.  F i g c a l l u s development.  (a)  F i g c a l l u s f o r m a t i o n on p r i m a r y e x p l a n t s , 12 days a f t e r e x c i s i o n from l e a f laminae and p e t i o l e s .  (b)  Dark-grown f i g c a l l u s a f t e r 4 g e n e r a t i o n s  (transfers).  37  F i g u r e 6.  Dark-grown bedstraw t i s s u e on agar and i n l i q u i d  B5-M.  (a)  Bedstraw growth on B5 agar medium, 35 days a f t e r t r a n s f e r ,  (b)  Growth o f n o n - c a l l u s bedstraw t i s s u e i n l i q u i d B5 medium c o n t a i n i n g 3% skimmed m i l k . F l a s k on r i g h t i s u n i n o c u l a t e d B5-M.  38  t o develop, forming l a r g e t a n g l e d (Figure 6b).  Milk-containing  masses as a r e s u l t of r o t a r y shaker a c t i o n  medium i l l u s t r a t e d p r o t e o l y t i c a c t i v i t y .  medium was checked m i c r o s c o p i c a l l y used f o r s i n g l e c e l l not  f o r sloughed c e l l s which may  have been  s u s p e n s i o n , but these were very few i n number.  p r o l i f e r a t e when t r a n s f e r r e d  to f r e s h medium,  The  They d i d  s u g g e s t i n g t h a t they were  a c t u a l l y dead c e l l s , p o s s i b l y sloughed from the r o o t c a p area of d e v e l o p i n g roots.  The s u r v i v a l r a t e of c u l t u r e s Tissues  of dumbcane  (Circium) a l l suffered high trials for  (Table  i s given  (Dieffenbachia),  pineapple  (Ananas) and t h i s t l e  r a t e s of m i c r o b i a l c o n t a m i n a t i o n i n s e v e r a l  I V ) . T h i s t l e e x p l a n t s formed some c a l l u s which was propagated  many months but grew s l o w l y  suspensions.  and would not grow as s i n g l e  cell  Dumbcane and p i n e a p p l e e x p l a n t s c a l l u s e d very p o o r l y and  n e i t h e r formed s u f f i c i e n t  t i s s u e to attempt c e l l s u s p e n s i o n c u l t u r e s .  V a r i a b i l i t y among c e l l s u s p e n s i o n c u l t u r e s : cell  i n T a b l e IV.  F i g and papaya c e l l s  grown i n  s u s p e n s i o n e x h i b i t e d d i f f e r e n c e s not n e c e s s a r i l y r e l a t e d to  environmental c o n d i t i o n s . growth and p r o d u c t i o n 1978).  Figure  It is likely  that genetic  of p r o t e i n and p r o t e a s e  factors  influenced  (Mandels, 1972; S k i r v i n ,  7 (a&b) shows y i e l d s of p r o t e i n and p r o t e a s e i n c e l l  extracts  from f o u r papaya and f i g batch c u l t u r e s grown two weeks i n B5-M medium p r i o r to h a r v e s t . and  There was a p p r o x i m a t e l y a 3 - f o l d d i f f e r e n c e i n p r o t e i n  a 4 - f o l d d i f f e r e n c e i n protease a c t i v i t y among these four papaya  cultures:  i n the four f i g c e l l c u l t u r e s .  have been c o r r e l a t e d with p r o t e i n p r o d u c t i o n  Although protease a c t i v i t y of papaya and f i g c e l l s ,  #1 of each, f o r example, t h i s was not n e c e s s a r i l y true  of  This  cell  the c o r r e s p o n d i n g d i f f e r e n c e s were 4 - f o l d and ' . 5 - f o l d ,  respectively,  #2).  content  v a r i a b i l i t y existed despite  the same medium and i n c u b a t i o n  (e.g.,  papaya  may  culture  culture  s i m i l a r p r o p a g a t i o n h i s t o r i e s and use  conditions.  Unfortunately,  such  Figure 7.  Protein-and protease v a r i a b i l i t y among c e l l extracts from four papaya and four f i g c e l l suspension cultures.  A l l samples were grown 13-17 days i n B5 medium with 3% skimmed milk.  40  d i f f e r e n c e s d i d not appear to be p e r s i s t e n t .  F i g u r e 8 i s a c o m p i l a t i o n of  p r o t e i n and enzyme a c t i v i t y d a t a from some papaya and f i g c e l l c u l t u r e s , but does not r e p r e s e n t a s p e c i f i c experiment  suspension  in itself.  Protein  and enzyme p r o d u c t i v i t y d i d not appear to be c o r r e l a t e d with number of transfers,  s i n c e some c o n s e c u t i v e c e l l  while others d e c l i n e d .  showed an improvement  Again, p r o t e i n s y n t h e s i s and enzyme a c t i v i t y d i d n o t  n e c e s s a r i l y change t o g e t h e r . p r o d u c t i v i t y , i t was  suspensions  With t h i s degree  e s s e n t i a l to use one  of v a r i a b i l i t y  inoculum  i n culture  of a s t a n d a r d volume f o r  each e x p e r i m e n t a l s e t , as a c o n t r o l a g a i n s t changes with number of and inoculum age. to  J u d g i n g from  the v a r i a t i o n s apparent  the next, h e r e d i t y alone was  from one  p r o b a b l y a minor f a c t o r .  generation  Other g e n e t i c  f a c t o r s such as n u c l e i c a c i d s y n t h e s i s , r e p a i r , r e g u l a t i o n and inclusions  transfers  foreign  l i k e plasmids or v i r u s e s were p r o b a b l y r e s p o n s i b l e f o r much of  the v a r i a b i l i t y n o t e d .  An i n v e s t i g a t i o n of g e n e t i c c o n t r o l s  c e l l growth and p r o d u c t i o n of p r o t e i n and p r o t e a s e i n c e l l was  beyond the scope  2.  Assessment of assay methods  suspension  of the p r e s e n t work.  C e l l growth and biomass p r o d u c t i o n : harvested c e l l s ,  influencing  Growth, as i n d i c a t e d by weight of  i s the s i m p l e s t i n d i c a t o r of a p p r o p r i a t e n u t r i t i o n .  Since  there are s e v e r a l methods of a s s e s s i n g growth, some of these were compared. T a b l e V g i v e s h a r v e s t weight d a t a from both papaya and determined  by f r e e z e - d r y i n g , oven-drying and  were r e p r o d u c i b l e f o r each method and  vacuum-oven d r y i n g .  10% more and  lyophilized  f i g cells  as  Results  i n d i c a t e d , f u r t h e r , t h a t the two  methods were the same (Students's t-test,oc=0.05), samples weighing  f i g suspensions  with l y o p h i l i z e d  oven  papaya  weighing 6% more ( i e . ,  c o n t a i n i n g t h a t much more water a f t e r r e a c h i n g a c o n s t a n t w e i g h t ) .  These  41  C- papaya I  C- papaya II  F- carica  callus  callus  callus  F i g u r e 8.  The r o l e of h e r e d i t y i n v a r i a b i l i t y city:  of p r o d u c t i v e  capa-  a c o m p i l a t i o n of data from one f i g and two papaya  c e l l cultures.  Git = g e n e r a t i o n number i n suspension  c u l t u r e ; pn = p r o t e i n content ps = t o t a l  protease  (units/1).  ( c e l l s + medium), (mg/1);  Table V  A comparison of d r y i n g methods f o r papaya and f i g c e l l s from s u s p e n s i o n c u l t u r e s . wet weight,  Each was i n i t i a l l y  1.800 g b l o t t e d  n = number o f samples; s = s t a n d a r d  d e v i a t i o n of  the mean.  d r y i n g method  papaya  fig mean d r y weight (mg)  mean d r y weight (mg)  s  c o n v e c t i o n oven 60 °C (n=8)  17.7  0.46  14.9  0.58  vacuum oven 60 °C (n=8)  17.1  2.07  15.3  0.46  19.2  0.75  16.0  0.45  freeze  dryer (n=6)  43  d i f f e r e n c e s must be r e c a l l e d i n c o n s i d e r i n g  p r o t e i n and enzyme d a t a , as  t i s s u e s were always l y o p h i l i z e d upon h a r v e s t denaturation  or hydrolysis  Settled c e l l  settled  cell  problems of enzyme  of endogenous p r o t e i n .  volumes c o r r e l a t e d w e l l with c e l l d r y weights up t o  about 30 ml c e l l s , o b t a i n e d from 50-ml c e l l Above t h i s ,  to a v o i d  the curve reached a p l a t e a u  suspension cultures  i n d i c a t i n g that  large d i f f e r e n c e s i n  volumes were not n e c e s s a r i l y w e i g h t - r e l a t e d .  T h i s may have  been due t o d i f f i c u l t y of c e l l p a c k i n g i n the r e s t r i c t i v e 50-ml c e n t r i f u g e  (Figure 9 ) .  vessels  used,  tubes.  T y p i c a l growth c u r v e s , on the b a s i s  of l y o p h i l i z e d weights, f o r  papaya and f i g c e l l c u l t u r e s grown i n B5 medium a r e p r e s e n t e d i n F i g u r e 10. The  logarithmic  growth phase began w i t h i n  maximum i n 17-21 d a y s . began t o c l i m b initial  f o u r days, with biomass r e a c h i n g  There was an i n i t i a l  a f t e r the f o u r t h day, r e a c h i n g  medium.  drop i n medium pH, which  a  then  v a l u e s more b a s i c than the  With f i g and papaya c u l t u r e s , a p p r o x i m a t e l y  equivalent  biomass i n B5 medium, about 3 g/1, were produced. C e l l extraction:  Table VI g i v e s p r o t e i n y i e l d s and enzyme a c t i v i t y of  extracts  a Polytron  and  made u s i n g  pestle.  homogenizer, a Braun s o n i c a t o r  No d i s t i n c t d i f f e r e n c e s were e v i d e n t  o r a mortar  i n protein yields or  enzyme a c t i v i t y of homogenized o r mortar-ground samples.  Sonication  d i d not  appear to be as e f f e c t i v e i n p r o t e i n e x t r a c t i o n , and thus a l s o showed lower protease a c t i v i t y .  Only a s i n g l e sample was prepared by each method, though  assayed i n d u p l i c a t e , s i n c e a l a r g e volume of c e l l s was r e q u i r e d  i n order to  do  o n l y to  the comparison on a s i n g l e c u l t u r e .  speculate  on the d i f f e r e n c e s  enzyme a c t i v i t y  seen:  than the s h o r t e r  I t i s therefore  possible  150 sec homogenizing r e s u l t e d i n lower  time, s o n i c a t i o n showed lower p r o t e i n  e x t r a c t i o n and p r o t e a s e a c t i v i t i e s  than other methods.  Where c e l l y i e l d s ,  600  • 400 CT)  200  10  F i g u r e 9.  Settled  settled  c e l l volume as an  determine the r e l a t i o n s h i p  20  cell  volume  30,  (nrA)  i n d i c a t o r of h a r v e s t weight.  AO  50  Harvested f i g c e l l s were used to  of s e t t l e d c e l l volume to c e l l d r y weight ( l y o p h i l i z e d ) .  F i g u r e 10.  Growth of papaya and f i g c e l l open symbols, f i g ;  suspension c u l t u r e s i n B5 medium:  c l o s e d symbols, papaya;  biomass and pH  dry weight from l y o p h i l i z e d  cells.  changes,  46  T a b l e VI  L i b e r a t i o n o f p r o t e i n and a c t i v e p r o t e a s e from f i g c e l l s by d i f f e r e n t e x t r a c t i o n 0.545 g c e l l s  methods.  Each sample c o n s i s t e d o f  ( d r y w e i g h t ) , and a l l were ground w i t h 0.1 M  phosphate b u f f e r , pH 6.0.  extraction method  extraction time ( s e c )  protein (mg/g d r y c e l l s )  protease a c t i v i t y (mg/g d r y c e l l s ) ( m g / g p r o t e i n )  mortar & p e s t l e  90  15.5  6.3  0.50  mortar & p e s t l e w i t h sand  45  11.4  7.1  0.62  90  14.2  6.7  0.47  120  10.4  7.1  0.68  240  10.7  5.7  0.54  30  9.5  4.8  0.51  120  11.1  4.7  0.42  Polytron homogenizer  Braun sonicator  means o f d u p l i c a t e d e t e r m i n a t i o n s u s i n g p r o t e i n assay.  Bradford's  means o f d u p l i c a t e d e t e r m i n a t i o n s u s i n g t h e m o d i f i e d FCC method f o r p r o t e a s e . TCA-soluble d i g e s t i o n products were equated w i t h q u a n t i t i e s o f L - t y r o s i n e prepared i n the same manner.  47  and  thus e x t r a c t i o n volumes, were s m a l l , the P o l y t r o n homogenizer and  Braun  s o n i c a t o r both r e s u l t i n r a p i d h e a t i n g of the s o l u t i o n , even with the use an i c e b a t h .  of  T h i s c o u l d a c t i v a t e enzymes e a r l i e r than d e s i r e d , or cause  enzyme d e n a t u r a t i o n . a low temperature further c e l l  Because i t i s e a s i e r  u s i n g a mortar,  t h i s was  e x t r a c t i o n s were conducted  to g r i n d c e l l s while m a i n t a i n i n g the p r e f e r r e d method.  with i g n i t e d  sand  All  i n a mortar k e p t  on i c e , u s i n g c h i l l e d b u f f e r (4-8 ° C ) . Protein quantitation: and  simple.  Bradford's  method of p r o t e i n q u a n t i t a t i o n i s r a p i d  F i g u r e 11 shows a s t a n d a r d curve of absorbance a t 595  p l o t t e d a g a i n s t p r o t e i n c o n t e n t , u s i n g bovine s t a n d a r d curve was  produced  linear regression.  nm  serum albumen (BSA).  f o r each p r o t e i n assay and  T h i s p r e c a u t i o n circumvented  r e s u l t s determined  prepared  sensitivity. range of 2.5  by  possible v a r i a b i l i t y  i n t r o d u c e d by any change i n the reagent or e x p e r i m e n t a l c o n d i t i o n s . reagent was  A  Fresh  f o r t n i g h t l y as l o n g e r s t o r a g e r e s u l t e d i n a l o s s of  The p r o t e i n c o n t e n t of c e l l e x t r a c t s was t o 7% of dry  found  to be i n the  weight.  Other methods of p r o t e i n assay were l e s s e f f e c t i v e and/or more cumbersome. protein  The Lowry procedure  (BSA)  and  thus, was  r e q u i r e d h i g h e r c o n c e n t r a t i o n s of s t a n d a r d  not s e n s i t i v e enough f o r d e t e c t i o n of p r o t e i n i n  e x t r a c t s from s m a l l c e l l h a r v e s t s . compounds p r e s e n t i n crude 280  nm  l i g h t cannot  method was  Because of t u r b i d i t y and  the spectrum  c e l l e x t r a c t s , simple absorbance readings  be assumed to r e f l e c t p r o t e i n c o n t e n t alone, so  of  with this  rejected. N i t r o g e n c o n t e n t has  o f t e n been used  as a measure of p r o t e i n .  K j e l d a h l d i g e s t i o n gave r e s u l t s r a n g i n g from 27.4 weight of d r i e d f i g c e l l s ,  a v e r a g i n g 3.58%  T h i s would i n d i c a t e an average  to 46.5  for c e l l s  mg  n i t r o g e n per g  grown i n B5 medium.  t o t a l p r o t e i n c o n t e n t of 22.4%  (n=20, s=2.98)  F i g u r e 11:  B r a d f o r d ' s p r o t e i n assay: This plot  fits  t y p i c a l standard curve u s i n g bovine serum albumin  the r e g r e s s i o n e q u a t i o n  y = O.Olx + 0.02  (r  2  = 0.99).  (BSA).  co  49  if  the average p r o t e i n n i t r o g e n  nitrogen  f a c t o r of 6.25  was  applied.  However, these  d e t e r m i n a t i o n s were based on d i g e s t i o n of e n t i r e c e l l s ,  p r o t e a s e assays were conducted on c e l l e x t r a c t s . inappropriate involved  and  for routine  use  T h i s method was  deemed  i n e x t r a c t d i g e s t i o n because of the  the p o s s i b i l i t y of v a r i a t i o n i n the  i n t r a c e l l u l a r proteins  whereas  or amino a c i d pools d u r i n g  time  r a t i o of n u c l e i c a c i d s  to  development of i n d i v i d u a l  cells. As contained  determined by B r a d f o r d ' s method, e x t r a c t s  an average of 5.56%  suspension c u l t u r e s  two  protein  (n=28, s=1 .65)  weeks a f t e r t r a n s f e r .  At  of f i g c e l l s  i n rapidly-growing  the same time, i n  B5  medium w i t h o u t milk, the average p r o t e i n c o n t e n t of f i g c e l l e x t r a c t s 3.53%  (n=14, s=1.33).  These two  by K j e l d a h l d i g e s t i o n s disrupted  c e l l s , and  since  f i g u r e s are much lower than t h a t  t h i s method omits i n s o l u b l e p r o t e i n s  does not encompass n u c l e i c a c i d s and  cell  was  determined in  othernon-protein  nitrogen. Protease quantitation:  The  c l e a r i n g of m i l k - c o n t a i n i n g  e a r l i e s t i n d i c a t i o n of p r o t e o l y t i c a c t i v i t y i n p l a n t was  evident  i n e x p l a n t s and  and  bedstraw.  agar was  tissue cultures.  c a l l u s t i s s u e s of papaya, f i g , cardoon,  C e l l suspension c u l t u r e s  of papaya and  f i g , and medium.  evidently  used as an  a suitable substrate,  so  s e l e c t i o n of assay methods.  t h i s phenomenon was  T h i s d e c i s i o n tended to lead  This  thistle  liquid-grown  bedstraw a l s o demonstrated c l e a r i n g of m i l k - c o n t a i n i n g  i n the  the  Casein  was  indicator into  p o t e n t i a l d a i r y a p p l i c a t i o n s , whereas a m e a t - d i g e s t i o n assay method such that devised  by O r s i and  Major (1973) would have l e d to an  as  i n v e s t i g a t i o n of  meat-industry a p p l i c a t i o n s . Gel d i f f u s i o n methods of enzyme d e t e c t i o n c e l l extracts.  A r a k i and  Abe's method  (1980) r e l i e s  proved i n e f f e c t i v e f o r on a l o c a l i z e d zone of  50  c o l o u r change i n the agar medium c o n t a i n i n g BAEE s u b s t r a t e . i n d i c a t i n g e s t e r o l y t i c a c t i v i t y c o u l d be d e t e c t e d c o n t a i n i n g c e l l e x t r a c t , though papain was The  No  surrounding  detectable  zones  wells  down to 0.1  mg/ml.  BioRad g e l d i f f u s i o n t e s t i s a l s o performed i n agar, but c o n t a i n s  as the s u b s t r a t e .  The  protease-containing evident  d i f f u s i o n zone i s seen as a c l e a r a r e a  wells.  surrounding  Again, no zones around c e l l e x t r a c t s were  under the s p e c i f i e d assay c o n d i t i o n s , a l t h o u g h standard  detected  as  low as  ficin  10 ug/ml i n the presence of d i t h i o t h r e i t o l .  d i f f u s i o n method of Holmes and  Ernstrom  and  agar  for their d i f f u s i o n  but  r e s u l t s were s i m i l a r to  I t would appear t h a t , i f p r o t e a s e  these t e s t s were not o p t i m a l  The  was  (1973) i s a l s o based on c a s e i n ,  i s performed i n tubes ( v e r t i c a l d i f f u s i o n ) , BioRad method.  casein  was  present,  the  conditions  of  through or a c t i v i t y i n an  agar environment. Another p r o t e i n s u b s t r a t e , c o l l a g e n , was methods.  The  azure-bound hide  powder (Savage & Thompson, 1970)  d i g e s t e d by an e x t r a c t of f i g c e l l s , c e l l dry weight e x t r a c t e d .  i n v e s t i g a t e d by  equivalent  ficin  extracted  concentrations  These are both a t the  detectable  low end  is,  of a standard  unsatisfactory results.  also  range of  proteases  curve f o r each enzyme of i n t e r e s t .  methods of p r o t e a s e The  mg  activity.  f i g c e l l s would r e q u i r e a s i m i l a r s e r i e s of the f i c i n  Fluorometric  was  of the  each assay of papaya c e l l s would r e q u i r e a d i l u t i o n s e r i e s of  p a p a i n , and  per  to 0.059 ug  None of the above methods permits comparison w i t h other without preparation  slightly  ficin  F i l m g e l a t i n ( G l e n i s t e r & Becker, 1961)  cells.  with  was  t o 0.03-0.04 ug  d i g e s t e d by enzymes i n the crude f i g c e l l e x t r a c t , e q u i v a l e n t f i c i n per mg  two  That  standard standards.  d e t e c t i o n t e s t e d gave  method of Spencer and Spencer (1974) appeared  to work w e l l w i t h chymotrypsin, but was  s u b j e c t to c o m p l i c a t i o n s  when used  51  with s u l f h y d r y l proteases.  ANS f l u o r e s c e n c e  increased  s h a r p l y upon a d d i t i o n  of the c e l l e x t r a c t s , p o s s i b l y due t o i n t e r a c t i o n of -SH r e a c t i v e groups w i t h the c a s e i n s u b s t r a t e . This complication, activity  in  Fluorescence  and the l a c k of any evidence i n d i c a t i n g p r o t e o l y t i c  crude c e l l e x t r a c t s , d i s c o u r a g e d  f l u o r i m e t r i c methods. pre-incubation  The procedure g i v e n  addition.  among samples c o n t a i n i n g  The  f u r t h e r use of d i r e c t  by Chism e t a l . (1979) c a l l s f o r  of the sample with the c a s e i n s u b s t r a t e ,  p r e c i p i t a t i o n and f l u o r e s c a m i n e variability  d i d not subsequently d e c r e a s e .  p r i o r to TCA  There was a great  l e s s than 1.0 u n i t / m l  d e a l of  of p a p a i n .  FCC method f o r papain was adopted f o r r o u t i n e q u a n t i t a t i o n of  protease a c t i v i t y , modified  as d e s c r i b e d  i n section III-3.  T h i s method  proved t o be the most r e l i a b l e and s e n s i t i v e one t h a t c o u l d c o n v e n i e n t l y a p p l i e d on a r o u t i n e b a s i s . data are derived related  One advantage of the FCC method i s t h a t  from absorbance r e a d i n g s  i n the l i t e r a t u r e ,  curve f o r each assay, as i l l u s t r a t e d contained equivalent  Tyrosine  i s most  so i t was used to prepare a i n Figure  12.  p r o t e o l y t i c a c t i o n on the c a s e i n s u b s t r a t e  standard  C e l l extracts  NPN s o l u b l e i n 10% TCA which produced absorbance t o as much t y r o s i n e as 1 mg/ml.  readings  was based on s u b t r a c t i o n of samples.  By l i n e a r  regression  these d i f f e r e n c e s were r e l a t e d to the standards to g i v e  equivalent  amounts of t y r o s i n e which would have been r e l e a s e d  phenylalanine  often  Thus assessment of true  absorbances of c o n t r o l s from absorbances of incubated  proteolytic activity.  final  a t 280 nm and can r e a d i l y be  to q u a n t i t i e s of a s i n g l e aromatic amino a c i d .  commonly r e p o r t e d  be  from c a s e i n by  I f aromatic amino a c i d s , tryptophan, t y r o s i n e and  comprise a p p r o x i m a t e l y 15% o f c a s e i n , then r e s u l t s of 80 ug of  t y r o s i n e r e l e a s e d would i n d i c a t e 1.2 mg of amino a c i d s r e l e a s e d assuming a l l amino a c i d s a r e r e l e a s e d  equally.  from  casein,  040  Figure  12.  T y p i c a l p r o t e a s e assay equation  s t a n d a r d curve u s i n g L - t y r o s i n e .  y = 0.0018x - 0.01  (r  2  = 0.99).  This plot f i t s  the r e g r e s s i o n  53  Factors  i n f l u e n c i n g protease a c t i v i t y :  The e f f e c t of pH on p r o t e a s e  a c t i v i t y a t 38 °C of f i c i n and f i g c e l l e x t r a c t i s i l l u s t r a t e d 13a.  Although f i c i n  constant  activity at this  relatively  over the pH range 5.5 t o 8.4, the f i g c e l l e x t r a c t demonstrated a  c l e a r pH optimum about 6.0. standard  temperature remained  i n Figure  f i c i n preparation  Temperature a l s o had l i t t l e  e f f e c t on t h e  over the range of 28-67 °C, whereas the f i g c e l l  e x t r a c t showed a temperature optimum around 47 °C ( F i g . 13b). The  i n f l u e n c e of s e v e r a l compounds on f i c i n a c t i v i t y  shown i n F i g u r e compound.  14.  Two c o n c e n t r a t i o n s  of f i c i n were t e s t e d w i t h each  Only c y s t e i n e and d i t h i o t h r e i t o l  (with o r w i t h o u t EDTA) had  pronounced s t i m u l a t o r y e f f e c t s , while t h i o c y a n a t e , t r y p s i n i n h i b i t o r were i n h i b i t o r y . resulted i n a s l i g h t increase contained  no f i c i n .  a t 38 °C i s  Glutathione,  sodium t e t r a t h i o n a t e and  EDTA a l o n e and SDDC  i n absorbance over the base l e v e l s ,  which  Protease a c t i v i t y of the f i g c e l l e x t r a c t was not  a f f e c t e d g r e a t l y by any of the t e s t compounds, but was s t i m u l a t e d by D t t , w i t h o r w i t h o u t EDTA.  slightly  SDDC a l s o appeared to be s t i m u l a t o r y , b u t  there was i n t e r f e r e n c e by c o l o u r development i n those r e a c t i o n  tubes.  T h i o c y a n a t e , sodium t e t r a t h i o n a t e and t r y p s i n i n h i b i t o r had e s s e n t i a l l y no e f f e c t on assay r e s u l t s .  3. M i l k c l o t t i n g  activity  Clotting  times of c e l l e x t r a c t s were determined, a c c o r d i n g  procedure i n s e c t i o n I I I - 3 , f o r c e l l and  one of f i g .  Results,  was 5.5 h r (papaya #4). c l o t milk  casein.  Also  given  from f i v e c u l t u r e s of papaya  i n T a b l e V I I , show the f a s t e s t c l o t t i n g  The o n l y included  extracts  to the  fig cell  extract tested required  i n Table V I I a r e c l o t t i n g  times  time  12 hr t o  required  54  £  8 c  CM II  0150  0, 0100 o  c o  _Q  °  0O50l  XI  a  6-5  5.5  pH  ft5  7-5  E c CM 0-240 II  0> 0-180t u  c o  I  0-120  o &060T  '0 V>-+26  F i g u r e 13.  Effect cell  — 34  42  58  temperature  66  (°C)  o f pH and temperature on a c t i v i t y o f f i c i n and f i g  extract.  Protease  a c t i v i t y was determined by the modi-  f i e d FCC method (see t e x t ) . (X).  50  F i c i n was used a t 0.42 mg/ml  F i g c e l l s were e x t r a c t e d by g r i n d i n g w i t h  0.1 M  phosphate b u f f e r , (a)  C a s e i n s u b s t r a t e prepared i n b u f f e r s o f d i f f e r e n t samples incubated a t 40 °C.  (b)  Casein  s u b s t r a t e , pH 6.0, samples incubated  pH, a l l  a t 27-57 °C.  55  DITHIOTHREITOL (Dtt) CYSTEINE  -x-o  Dtt • EDTA EDTA GLUTATHIONE  • XO-  .124%  SDDC TRYPSIN INHIBITOR Dtt • TRYPSIN INHIBITOR Ca-THIOCYANATE Na-TETRATHIONATE THIOUREA CONTROL 40  %  F i g u r e 14.  activity  80  I n f l u e n c e of some a c t i v a t o r s and i n h i b i t o r s on p r o t e o l y t i c activity.  Papain  (100 ug) , c y s t e i n e d e s i g n a t e d as  of  activity  (—o-);  ficin  of  activity  (-¥r);  f i g cell  100% of a c t i v i t y carbamate.  (-*-).  100%  (21 ug) , D t t d e s i g n a t e d as  100%  e x t r a c t , D t t + EDTA s e t as  SDDC i s sodium  diethyldithio-  56  Table VII  M i l k c l o t t i n g a c t i v i t y of p l a n t c e l l comparison to standard cell  papain.  c u l t u r e s and one f i g c e l l  onding to d a t a i n F i g u r e  Five different culture  clotting (hr)  (a)  cell  extracts  papaya  1  13.0  papaya  2  12.5  papaya  3  15.0  papaya  4  5.5  papaya  5  18.0  fig  12.0  4  papain standards  (ug/ml)  16.3  3.5  8.2  5.0  5.4  10.8  4.1  14.0  0  means of d u p l i c a t e  (numbers  7) were e x t r a c t e d  f o r c l o t t i n g a c t i v i t y as d e s c r i b e d  test material  extracts i n  >24  determinations  and  i n the t e x t .  time  papaya corresptested  57  for  a d i l u t i o n s e r i e s of crude p a p a i n .  These standards i n d i c a t e t h a t  e x t r a c t with the s h o r t e s t c l o t t i n g time possesses c l o t t i n g equivalent  to about 8 ug/ml p a p a i n , by  even l e s s a c t i v e .  d a t a are a v a i l a b l e r e g a r d i n g  4.  other  activity  linear interpolation.  M i l k c l o t t i n g a c t i v i t y was cell  the  Others were  not a s s e s s e d r o u t i n e l y so  c u l t u r e s such as  later  no  generations.  Electrophoresis The  proteins  i n c e l l e x t r a c t s was  p r o t e i n and cells  g r e a t e s t d i f f i c u l t y encountered i n attempts a t s e p a r a t i o n concentration.  E x t r a c t s n o r m a l l y used f o r  p r o t e a s e d e t e r m i n a t i o n s were o b t a i n e d  ( i n the form of a spongy mat)  b u f f e r to wet  the t i s s u e , and  the  by  one  grinding l y o p h i l i z e d  i n a mortar w i t h s u f f i c i e n t  t h i c k s l u r r i e s were then  E x t r a c t s were g e n e r a l l y found to c o n t a i n 0.2-1.2 mg bands were d e t e c t a b l e  phosphate  filtered.  protein/ml.  by e l e c t r o p h o r e s i s of these c e l l  sample through a s m a l l Sephadex G-25  of  No  extracts.  protein  Passage of  column, e q u i l i b r a t e d w i t h  e x t r a c t i o n b u f f e r , to remove compounds p o s s i b l y i n t e r f e r i n g with p r o t e i n m o b i l i t y d i d not appear to h e l p . b o i l i n g with urea and standard  sample was  c o n t a i n i n g 0.5 t h e r e was  mg  p-mercaptoethanol was  of 4-5  p r o t e i n bands i n the p u r i f i e d  showed three Only one  No  Over a p e r i o d  twice  hr  electrophoresis,  was  ficin.  four,  T r y p s i n was  applied  found to migrate more r e a d i l y though i t ,  bands. attempt was  f i n i s h e d , unfixed  c l e a r i n g was  and  The  recrystallized,  s u f f i c i e n t movement to i n d i c a t e t h r e e , p o s s i b l y  to the g e l as a standard  of the  of samples prepared  no more s u c c e s s f u l .  a Sigma f i c i n p r e p a r a t i o n ,  protein/ml.  poorly-separated  too,  SDS-electrophoresis  visible  made to l o c a t e p r o t e o l y t i c bands by  g e l with Bio-Rad c a s e i n protease d e t e c t i o n i n 8 hr a t 28  °C.  Concentration  of samples  contact agar. by  by  58  long-term  c o n t a c t w i t h Carbowax 20 M ( A p p l i e d S c i e n c e , Pa.), 5-8  samples being c o n t a i n e d i n d i a l y s i s  t u b i n g , was  a l s o inadequate.  c a s e s , f l o c c u l a t i o n of the c o n c e n t r a t e o c c u r r e d , and mobility.  hr a t 6  °C,  In many  none showed improved  I t i s p o s t u l a t e d t h a t p r o t e i n s i n c e l l e x t r a c t s were p h y s i c a l l y  bound to o t h e r e x t r a c t components, or to the p o l y a c r y l a m i d e g e l i t s e l f ,  thus  p r e v e n t i n g t h e i r m o b i l i t y i n a g e l environment while c a u s i n g no i n t e r f e r e n c e w i t h p r o t e i n or p r o t e a s e a s s a y s , conducted s u g g e s t i o n would be supported agar-based  5.  protease  Medium  by  in fluid  environments.  the n e g a t i v e r e s u l t s o b t a i n e d  This  with  assays.  supplementation Four groups of n u t r i e n t supplements were i n v e s t i g a t e d with r e s p e c t  to  enhancement of p r o t e a s e p r o d u c t i o n and  activity  i n c e l l extracts:  low  molecular weight n i t r o g e n s o u r c e s , p r o t e i n s , skimmed milk and i t s components, and  m i s c e l l a n e o u s o r g a n i c compounds i n c l u d i n g  Each group of m a t e r i a l s comprised-a c u l t u r e s handled suspension.  s e p a r a t e s e r i e s of c e l l  Only f i g c e l l  c u l t u r e s were used  half  n i t r o g e n sources  i n B5 medium was  The  relative  determined  ammonium ( n i t r a t e o n l y ) .  with skimmed m i l k (3%, v / v ) . t h a t n i t r a t e was p r o t e i n and nitrate.  The  the most important  importance  u s i n g complete B5, nitrate  Each of these was  results,  cell  f o r these s t u d i e s .  the normal n i t r a t e c o n c e n t r a t i o n , B5 without  and B5 w i t h o u t  antibiotics.  suspension  u n i f o r m l y and each i n o c u l a t e d from a s i n g l e  Nitrogen nutrition i n f i g c e l l cultures: two  two  of  the  B5 w i t h  (ammonium o n l y ) also  prepared  p r e s e n t e d i n F i g u r e 15,  of the n i t r o g e n s o u r c e s .  indicate  Biomass,  total  t o t a l p r o t e a s e a c t i v i t y were a l l lowest i n the absence of  Although  milk went a long way  toward c o r r e c t i n g t h i s d e f i c i e n c y i n  terms of biomass, i t d i d not p r o v i d e a s i g n i f i c a n t improvement i n p r o t e i n o r p r o t e a s e y i e l d over the non-milk media where n i t r a t e was  absent.  12 T  [N0 +MILKl3  800  10t  [%*,ti<4 N  H  'c  [NOyMILKl t  N  »  3  600  H  16-0  14-0  NH ] t  MILK 6f 5 b,  o E  II  in ai o z < cr  .2 4 -O  INH •MILKkJ t 4  E  CO?  LU . II  400  <°-  ex.  c a> .*-» o  *>  tr  30  INtyNH^  o a  o a»  u. Q. 200  O — I  a.  I N 0 ]—3  INH^MILKK^  10  [ F i g u r e 15.  I n o r g a n i c n i t r o g e n supply i n f i g c e l l  c u l t u r e s , and i n t e r a c t i o n w i t h m i l k .  (a) E f f e c t on biomass. Shaded bars g i v e c e l l weights from the same medium w i t h the a d d i t i o n of 3% skimmed m i l k . (b) E f f e c t on p r o t e i n and p r o t e a s e a c t i v i t y . Range spans encompass groups o f s i g n i f i c a n t l y d i f f e r e n t media, a t t h e 1% l e v e l , u s i n g Duncan's m u l t i p l e range t e s t .  £  60  S e v e r a l amino a c i d s were added to incomplete B5 medium n i t r a t e or ammonium) i n search utilized  to s t i m u l a t e  c e l l harvests  of keys to n i t r o g e n  production  ( F i g u r e 16)  of n i t r o g e n metabolism.  of p r o t e i n and  underlined  (only  metabolism t h a t may  active protease.  Results  the importance of n i t r a t e as the  In i t s presence, s e v e r a l amino a c i d s  be of  base  stimulated  growth w h i l e i t s absence produced g e n e r a l l y poor growth i r r e s p e c t i v e of other  supplements w i t h the s o l e e x c e p t i o n Glutamate a c t u a l l y s t i m u l a t e d  medium to a l e v e l e q u i v a l e n t  of  milk.  protein synthesis  in  non-nitrate  to unsupplemented n i t r a t e medium, an adequate  replacement f o r n i t r a t e ( F i g u r e  17a).  Stimulation  most obvious w i t h the a d d i t i o n of a s p a r t a t e with glutamate, c y s t e i n e and  of p r o t e a s e a c t i v i t y  to n i t r a t e - c o n t a i n i n g medium,  p o s s i b l y arginine also causing  improvement i n p r o t e a s e a c t i v i t y ,  relative  to complete B5  With the ammonium-based medium, there were no d i f f e r e n c e s among supplements w i t h r e s p e c t  outstanding  a  significant  (Figure  a r g i n i n e and  alanine,  Only m i l k a l l others  and  17b).  significant  to protease a c t i v i t y .  Duncan's  m u l t i p l e range t e s t a t the 5% p r o b a b i l i t y l e v e l gave o v e r l a p p i n g the e n t i r e d a t a base.  ranges over  c y s t e i n e were s i g n i f i c a n t l y b e t t e r  falling  was  between and  overlapping  than  these  extremes. These data i n d i c a t e t h a t the amino a c i d s capable of protein synthesis  were a l s o apt  a d d i t i o n a l case of c y s t e i n e ,  the  i n d i c a t i n g high p r o t e i n l e v e l s . of enzymes n o r m a l l y produced by synthesis.  It i s doubtful  to i n c r e a s e  protease a c t i v i t y .  i n v e r s e was Cysteine  stimulating With  also true, high protease l e v e l s  a t 5mM  may  have caused a c t i v a t i o n  the c e l l s , o r a c t u a l l y s t i m u l a t e d  t h a t glutamate and  aspartate  serve  enzyme  only  enzyme-related f u n c t i o n s , s i n c e they caused s t i m u l a t i o n of t o t a l synthesis  the  as w e l l as an improvement i n t o t a l p r o t e o l y t i c a c t i v i t y  protein of  61  ALANINE  Cr  ARGININE  rzr  ASPARTIC ACID  CYSTEINE  GLUTAMIC ACID  GLYCINE  PROLINE  •V/ MILK I H 2  Figure  16.  Amino a c i d in f i g c e l l  cell  4  biomass (g/l)  supplements and skimmed m i l k : suspension c u l t u r e s .  weights from NO^-based  / / — ( 11  1 2  e f f e c t on biomass  Shaded b a r s g i v e  media, open bars from NH^  cell  media.  150  900  NO  NH,  '145 •MILK .  • MILK  4  NH 0  'c  850 250  cn cr  •  3  ASP-  E 30  GLU-  UJ O  z  O  •  ASP 150  •  • GLU f—* MILK  ALA-  •  CYS-  50 ControlPRO-  a. —  o a* to a a>  ^ 1 • CYS — • ARG —  ARG-  •  •  <  o II  •  .•MILK • CYS  ALA-'  Control-rtt • PRO—^1  yGLU /•ASP ALA ARG ^Control -•CYS PRO  Figure  17.  Amino a c i d supplements and m i l k : cultures deprived  e f f e c t on p r o t e i n and protease  of e i t h e r n i t r a t e or ammonia.  activity  in f i g cell  N i t r a t e - b a s e d media are on the l e f t  ammonia-based media on the r i g h t s i d e o f both (a) p r o t e i n , and (b) p r o t e a s e . contained  o n l y NO^  or NH^  but no amino a c i d s .  and  The c o n t r o l  Range spans encompass groups of a l l media  d i f f e r i n g from each o t h e r a t the 5% l e v e l , u s i n g Duncan's m u l t i p l e range  test.  63  suspension-cultured f i g c e l l s . E f f e c t of p r o t e i n s and p e p t i d e s on f i g c e l l s : c e l l c u l t u r e s was tested.  Protease a c t i v i t y of f i g  not s t i m u l a t e d by most of the p r o t e i n a c e o u s m a t e r i a l s  R e s u l t s are g i v e n i n F i g u r e 18, i n c l u d i n g two  w i t h no supplementation d i f f e r e n t chemistry. activity  and one  Only milk and egg albumen r e s u l t e d i n p r o t e a s e  l e v e l s s i g n i f i c a n t l y h i g h e r than B5 medium a l o n e .  also i n h i b i t o r y .  On  the c o n t r a r y ,  casein hydrolysate  s i g n i f i c a n t l y lower p r o t e a s e a c t i v i t y i n f i g c e l l  it  one  c o n t a i n i n g s o l u b l e s t a r c h , a polymer of very  beef e x t r a c t , c a s e i n , wheat g l u t e n , g e l a t i n and  was  c o n t r o l media,  produced  c u l t u r e s (p=0.01).  Starch  Causes of enzyme i n h i b i t i o n were not examined, though  i s s p e c u l a t e d t h a t a d s o r p t i o n of p r o t e o l y t i c enzymes or a s s o c i a t e d ions  by polymers c o u l d lead to i n t e r f e r e n c e with a c t i v i t y . e a s i e r to work w i t h i n media than egg albumen, was  Skimmed m i l k , b e i n g  selected for further  s t u d y with r e s p e c t to i d e n t i f i c a t i o n of s t i m u l a t o r y f a c t o r s . E f f e c t of skimmed m i l k and  i t s components on f i g c e l l s :  3% skimmed milk to B5 medium had papaya and  fig cells.  i n F i g u r e 19.  a profound  T y p i c a l p r o t e i n and  s t i m u l a t o r y e f f e c t on growth of p r o t e a s e p r o d u c t i v i t i e s are shown  from skimmed milk as d e s c r i b e d i n s e c t i o n I I I - 4 .  were pure compounds added a t l e v e l s a p p r o x i m a t e l y  yields  from  The  r e s u l t s , F i g u r e 20,  obtained with milk.  C a s e i n and  i n e f f e c t i v e growth promoters.  Others  e q u i v a l e n t to q u a n t i t i e s  showed a 1 0 - f o l d spread i n biomass  t h i s range of supplements. Whey and  u l t r a f i l t e r e d whey both produced  produced  a d d i t i o n of  Some of the supplements added to B5 medium i n t h i s study were  derived d i r e c t l y  found i n m i l k .  The  the r e t a i n e d p o r t i o n of  growth which was  a t l e a s t equal to t h a t  especially ultrafiltered  whey were  Of the simple supplements, o n l y c i t r i c  a good growth response,  approaching  t h a t of milk  itself.  acid  64  5  d> n  ,UJ  lo z < SKIMMED  EGG  MILK  ALBUMEN  SOY  PROTEIN ISOLATE CONTROL  CASEIN HYDROLYSATE BEEF EXTRACT WHEAT GLUTEN GELATIN  CASEIN POWDER STARCH  p r o t e a s e  Figure 18.  Proteins and peptides: c e l l cultures.  a c f i v i t y  (units/1)  2  0  effect on protease a c t i v i t y i n f i g  Two controls were included, starch and  unsupplemented B5 medium.  Range spans encompass groups of  d i f f e r i n g at the 1% p r o b a b i l i t y level (Duncan's multiple range t e s t ) .  F i g u r e 19.  P r o t e i n and p r o t e a s e a c t i v i t y open symbols, f i g ;  i n f i g and papaya c e l l  c l o s e d symbols, papaya  suspension c u l t u r e s over 3 weeks,  66  ID O  WHEY CASEIN • WHEY RETENTATE MILK CITRIC ACID UF RECOMBINED CASEIN CASEIN POWDER CONTROL LACTOSE  NIACIN UF FILTRATE CALCIUM RIBOFLAVIN  2 Figure  20.  cell  4 dry  6 weight  8 (g/l)  10  Skimmed m i l k  and i t s components as supplements:  on biomass.  F i g c e l l c u l t u r e s were grown i n B5  the above supplements.  12  effect containing  UF r e f e r s to u l t r a f i l t r a t i o n p r o d -  u c t s , r e t e n t a t e , f i l t r a t e o r these recombined  (1:1).  Range spans encompass groups d i f f e r i n g from each u s i n g Duncan's m u l t i p l e range t e s t a t the 5%  other,  level.  67  P r o t e i n y i e l d was  h i g h e s t w i t h skimmed m i l k , whey and  (recombined),  f o l l o w e d by u l t r a f i l t r a t i o n  powdered) and  citric  acid  ( F i g . 21a).  p r o t e i n s y n t h e s i s as whole milk, and this.  Proteolytic activity,  milk u l t r a f i l t r a t i o n whey-containing  Citric  acid  the other supplements f e l l was  added to the medium.  than other supplements.  achieved u s i n g u l t r a f i l t r a t i o n  The  r e t e n t a t e was  In e f f e c t ,  f a r s h o r t of  g r e a t e s t when the Milk, significantly  unusually high due  protease  to i t s a d d i t i o n a t  the same l e v e l as whole whey, although i t a c t u a l l y comprised t w o - t h i r d s of the t o t a l whey volume.  and  l e d to o n l y 57% as much  media, f r e s h c a s e i n and c i t r a t e a l l produced  g r e a t e r enzyme a c t i v i t y activity  retentate, casein (fresh  as shown i n F i g u r e 21b,  r e t e n t a t e was  casein+whey  about  this resulted  i n the  a d d i t i o n of 50% more of these components than are n o r m a l l y added i n the skimmed milk supplement. 10,000-dalton  filter,  The  a c t i v e component of whey was  r e t a i n e d by  so the u l t r a f i l t r a t e had no s t i m u l a t o r y e f f e c t  the  on  y i e l d s of biomass, p r o t e i n or p r o t e a s e .  Recombined w i t h r e t e n t a t e ,  i n t e r m e d i a t e r e s u l t s were o b t a i n e d .  e f f e c t i v e n e s s of the r e t a i n e d  f r a c t i o n of whey was  The  most n o t a b l e i n t o t a l p r o t e a s e a c t i v i t y , where i t  y i e l d e d 38% h i g h e r a c t i v i t y than skimmed m i l k . r e c o n s t i t u t e d c a s e i n powder produced g/1 half  respectively,  f i g cells  the p r o t e a s e a c t i v i t y  produced  Although  f r e s h c a s e i n and  s i m i l a r c e l l h a r v e s t s , 6.57  grown with the  5.26  l a t t e r demonstrated o n l y about  of those grown w i t h f r e s h c a s e i n .  Citric  a s i g n i f i c a n t improvement i n p r o t e a s e a c t i v i t y r e l a t i v e  unsupplemented B5 medium (p=0.05).  and  Enzyme a c t i v i t y  acid  to  i n the  c i t r a t e - s u p p l e m e n t e d f i g c e l l c u l t u r e r e l e a s e d the e q u i v a l e n t of approximately  400 mg  of t y r o s i n e per l i t r e of 14-day c e l l s ,  s i g n i f i c a n t l y d i f f e r e n t from whey.  a level  not  t h a t o b t a i n e d with skimmed milk, c a s e i n o r  68  8-0 UF RETENTATE  J-700  MILK CASEIN •WHEY  OO WHEY  ~+60  MILK  UF RETENTATE,  UF  E  UF RECOMBINED  RECOMBINED,  500  CASEIN CASEIN •WHEY  E  CASEIN.  c  WHEY  CASEIN POWDER'  o  CITRIC ACID.  CITRIC ACID-  CL  k-o  O  in cn O + 300  CONTROL UF-FILTRATE NIACIN  OJ  §3  LACTOSE  >»  •4—•  s e in  CASEIN POWDER  Q.  f  +2 0  LACTOSE  —X  CONTROL  CALCIUM  NIACIN  RIBOFLAVI/ I N ^ l  UF-FILTRATE  100  CALCIUM RIBOFLAVIN  F i g u r e 21.  E f f e c t of skimmed (b)  m i l k components on (a) p r o t e i n , and  protease a c t i v i t y ,  in f i gcell  suspension c u l t u r e s .  UF r e f e r s t o u l t r a f i l t r a t i o n p r o d u c t s , r e t e n t a t e , or  these recombined ( 1 : 1 ) .  of  media p r o d u c i n g s i g n i f i c a n t l y d i f f e r e n t  filtrate,  Range spans encompass groups  (Duncan's m u l t i p l e range t e s t ,  p=0.05).  results  69  I n f l u e n c i n g s y n t h e s i s and r e l e a s e of p r o t e i n and p r o t e a s e : i n c l u d e s d a t a from  two experiments  to i n f l u e n c e p r o t e i n and  p r o d u c t i v i t y by m a t e r i a l s not d i r e c t l y r e l a t e d s u l f u r - c o n t a i n i n g compounds, g l u t a t h i o n e and medium a l o n e . protease  G l u t a t h i o n e produced  suggested  (biomass,  by Erez  to c e l l  nutrition.  The  t h i o u r e a , were compared to B5  Thiourea resulted  i n a decrease  and  in a l l  p r o t e i n and p r o t e a s e ) , not a growth s t i m u l a n t as  (1978).  Chloramphenicol  and  c y c l o h e x i m i d e both had n e g a t i v e e f f e c t s  biomass, p r o t e i n c o n t e n t and p r o t e a s e a c t i v i t y Chloramphenicol  protease  a 40% h i g h e r biomass, but p r o t e i n  l e v e l s were not improved.  three f a c t o r s  Table V I I I  in f i g cell  on  cultures.  r e s u l t e d i n 83% of the c e l l h a r v e s t weight o b t a i n e d i n the  c o n t r o l , w h i l e c y c l o h e x i m i d e produced  o n l y 32% as much biomass.  e f f e c t of c y c l o h e x i m i d e  to i t s mechanism of a c t i o n ,  i s attributed  The  drastic  i n t e r f e r e n c e w i t h the r o l e of t-RNA i n p e p t i d e bond f o r m a t i o n a t the ribosomal subunit.  Chloramphenicol  a l s o i n t e r f e r e s with p r o t e i n s y n t h e s i s ,  but o n l y on m i t o c h o n d r i a l ribosomes. medium p r o t e a s e The  There was  presence  of the d e t e r g e n t Span-80, d i d not s t i m u l a t e p r o t e a s e a c t i v i t y was  t h i s sample than i n the c o n t r o l , 3.5  W i t h i n each t e s t s e t , biomass was proteolytic  no s i g n i f i c a n t d i f f e r e n c e i n  l e v e l s among samples.  p r o d u c t i o n of p r o t e i n s i n g e n e r a l , and in  80S  and  3.9  somewhat  lower  units/1 respectively.  p a r a l l e l l e d by both p r o t e i n s y n t h e s i s and  activity.  I t d i d not appear t h a t p r o t e o l y t i c a c t i v i t y was dying c e l l s , Kende, 1979).  releasing intracellular  due  (eg., v a c u o l a r ) p r o t e a s e s  to rupture of (Boiler &  C e l l s exposed to c y c l o h e x i m i d e would have been expected  s u f f e r damage, y e t d i d not i n d i c a t e h i g h e r enzyme l e v e l s  than the  to  control.  Table V I I I  E f f e c t o f m i s c e l l a n e o u s o r g a n i c m a t e r i a l s on growth of f i g c e l l s and p r o d u c t i o n o f p r o t e i n and p r o t e a s e . presented: See  (a)  i n suspension  culture,  Data from two d i f f e r e n t experiments a r e  based on B5 medium, and (b)  based on B5 w i t h 3% skimmed m i l k .  t e x t f o r c o n c e n t r a t i o n s of t e s t m a t e r i a l s . R e l a t i v e v a l u e s a r e based on the  control.  n= number o f d e t e r m i n a t i o n s of biomass, p r o t e i n , p r o t e a s e . s= s t a n d a r d d e v i a t i o n of the mean  medium  (a)  (b)  biomass (g/D  relative  protein (mg/1)  relative  protease a c t i v i t y (units/1) relative  B5  0.86 n=4, s=0.07  1.00  39.3 n=2  1.00  1.12 n=2  1.00  B5 + thiourea  0.75 n=4, s=0.10  0.87  22.2 n=2  0.57  0.54 n-2.  0.48  B5 + glutathione  1.20 n=2  1.39  27.0 n=2  0.69  0.94 n=2  0.83  B5-M  6.47 n=3, s=0.41  1.00  407.9 n=3, s=6.57  1.00  3.91 n=3, s=0.12  1.00  B5-M + chloramphenicol  4.37 n=3, s=1.02  0.68  185.4 n=2  0.45  3.24 n=2  0.83  B5-M + cycloheximide  1.80 n=3, s=0.31  0.28  162.2 n=3, s=3.63  0.40  1.60 n=3, s=0.30  0.41  B5-M + Span-80  7.33 n=3, s=1.85  1.13  425.8 n=3, s=5.35  1.04  3.507 n=3, s=0.18  0.90  71  Furthermore, f i g c e l l s  grown i n B5 p l u s skimmed milk g e n e r a l l y gave  evidence  of c a s e i n h y d r o l y s i s , by c l e a r i n g of the medium, w i t h i n a week of s u b c u l t u r e . O c c a s i o n a l l y , c l e a r i n g c o u l d be d e t e c t e d activity,  then,  o c c u r r e d w e l l before  a s s o c i a t e d w i t h mass c e l l d e a t h . p r o t e o l y t i c a c t i v i t y detected  i n f o u r days.  mid-log phase and  was  As shown i n F i g u r e 19,  decreased  after  the  This  not l i k e l y  the  to be  l e v e l of  t h i r d week, d u r i n g  s t a t i o n a r y phase, when c e l l death became an important  the  f a c t o r i n the  population. A comparison of r e s u l t s of f o u r experiments i n v o l v i n g assessment of protease activities  a c t i v i t y i n f i g c e l l s i s g i v e n i n F i g u r e 22 are presented  ( e x t r a c e l l u l a r ) and the a d d i t i o n of m i l k was  contained  and  0.80  in their  i n the c e l l  two  (a&b).  component f r a c t i o n s ,  extracts ( i n t r a c e l l u l a r ) .  The  enzyme  i n the medium With or  without  to B5 medium, a much l a r g e r p r o p o r t i o n of t o t a l p r o t e i n  i n the c e l l s  than i n the medium:  i n c e l l s grown i n B5 and  B5-M,  average p r o p o r t i o n s  respectively.  Protease  medium, average p r o p o r t i o n s  c e l l s being 0.44  media, r e s p e c t i v e l y .  there was  0.49  i n B5 and  B5-M  a wide s c a t t e r i n the d i s t r i b u t i o n  proportion i n c e l l s  ranging  so i t was  in  However,  of enzyme a c t i v i t y ,  to 0.7,  was  the  not p o s s i b l e to  assay o n l y one  of these  constant.  i n c l u s i o n of the above t e s t compounds i n media d i d not appear  to  The  two  from about 0.3  0.95  activity  d i v i d e d almost e q u a l l y between c e l l s and and  of  f r a c t i o n s then m u l t i p l y by a p r o p o r t i o n a l i t y  i n f l u e n c e the p r o p o r t i o n of e x t r a c e l l u l a r enzyme  activity.  8  ~ 'c  6  3  o O 0»  <U 2  8 CL  A  B  B5 re 22.  A  B  A  C  A  B  I n t r a - o r e x t r a c e l l u l a r l o c a t i o n of p r o t e i n and protease a c t i v i t y cultures.  Both  media and c e l l  e x t r a c t s were assayed.  (A,B,C) and f o u r B5-M-grown c u l t u r e s r e f e r only  t o d i f f e r e n t experiments.  C  B5-M  B5  B5-M  B  in fig cell  Data from three B5-grown c u l t u r e s  (A,B,C,D) a r e presented, where the l e t t e r Solid line, cell  suspension  extract;  dotted  line,  codes medium.  73  V.  DISCUSSION  1.  Tissue d e d i f f e r e n t i a t i o n Successful  u t i l i z a t i o n of p l a n t t i s s u e and  dependent upon means of i n f l u e n c i n g c e l l growth and c u l t u r e s c o n s i s t of immature and l a r g e l y from cambial and is,  of n e c e s s i t y ,  semi-solid part,  media are  to the  medium.  The  subject  intensive. to v a r y i n g  growth, g e n e t i c  s t a t u s and  Several  This  derived  P r o p a g a t i o n of such t i s s u e s  Furthermore, t i s s u e s grown  on  degrees of morphogenesis due,  s i t u a t i o n requires  with  in  the  are maintained  cell  been d e r i v e d ,  to  systems.  plant species  were s e l e c t e d  f o r the purpose of  suspension c u l t u r e s .  F i c u s c a r i c a , Ananas comosus, D i e f f e n b a c h i a Cynara c a r d u n c u l u s and  Circium  arvense.  producing  These were C a r i c a papaya,  amoena, D.  p i c t a , Galium verum,'  Hundreds of p l a n t  c a l l u s f o r a v a r i e t y of p u r p o s e s .  species  have been  Those t h a t have not,  either  been i n v e s t i g a t e d , or have p a r t i c u l a r environmental or n u t r i t i o n a l  requirements t h a t have not  yet been i d e n t i f i e d or accommodated.  f o r t h i s study were handled by  e x c i s i o n and today, MS  incubation  on  two  of the  the  The  t r a d i t i o n a l methods of  species explant  most common t i s s u e c u l t u r e media i n  (Murashige & Skoog, 1962)  and  B5  (Gamborg e t a l . , 1968).  D e v e l o p i n g c a l l i were i n c u b a t e d under a s i n g l e e n v i r o n m e n t a l regime: in  in  as  a knowledge of  metabolism, much of which has  proteolytically active c e l l  use  Callus  p r e f e r a b l y w i t h o u t f l u c t u a t i o n over time, such  d a t e , from batch c u l t u r e  selected  cells  d e s i r a b l e g o a l i s a system i n which a l l c e l l s  continuous-feed fermentors.  have not  development.  s p a t i a l a s s o c i a t i o n of c e l l s with each other and  i d e n t i c a l conditions,  propagated as  cultures i s  r e l a t i v e l y unspecialized  parenchymatous c e l l s .  very labour  cell  t o t a l darkness.  Over the  f i r s t four  to f i v e months of e s t a b l i s h i n g  28°C  74  c u l t u r e s , o n l y a few v a r i a b l e s were e x e r c i s e d . (IAA, NAA,  These were the p l a n t auxins  2,4-D, 2,4,5-T, and p - c p a ) , t r a n s f e r time ( a t c a l l u s  a f t e r 2 weeks, a f t e r 5 weeks), medium supplements  inoculum s i z e  (2-15 mm  initiation,  i n d i a m e t e r ) , and  (yeast e x t r a c t , c a s e i n h y d r o l y s a t e , and skimmed  milk).  V i s u a l examination of growing t i s s u e s determined the most s u i t a b l e c o n d i t i o n s f o r those s p e c i e s which d i d c a l l u s and d e v e l o p i n v i t r o . C a l l u s c u l t u r e s were o b t a i n e d from papaya, and t h i s t l e .  The f i r s t  f i g , dumbcane, cardoon  two of these showed the f a s t e s t growth.  Explants  from p i n e a p p l e , dumbcane and mature cardoon were a l l s u b j e c t to heavy endogenous c o n t a m i n a t i o n , and no p i n e a p p l e e x p l a n t s s u r v i v e d more than t h r e e weeks.  Bedstraw p r e s e n t e d an unusual r e a c t i o n , i n t h a t seeds developed no  c a l l u s but stem e x p l a n t s r e s u l t e d i n a n o n - c a l l u s outgrowth  to produce a  r o o t - l i k e mass. F i g and papaya t i s s u e s responded e q u a l l y w e l l t o MS and B5 Of the a u x i n s , IAA a l o n e was  less e f f e c t i v e  than the remainder, but c o u l d be  used i n combination w i t h the p h e n o x y a c e t i c a c i d s , adopted.  so p-cpa was  arbitrarily  The b e s t t r a n s f e r i n t e r v a l v a r i e d from one c a l l u s to another, but  appeared to be s u i t a b l e about one week a f t e r r a p i d outgrowth of callus.  media.  This generally resulted  friable  i n t r a n s f e r s a f t e r 20-30 days, w i t h the  minimum fragment t r a n s f e r r e d s u c c e s s f u l l y  b e i n g about 5 mm  i n diameter.  Both y e a s t e x t r a c t and c a s e i n h y d r o l y s a t e i n h i b i t e d c a l l u s development, l a t t e r r e s u l t i n g i n browning of t i s s u e s . growth s t i m u l a n t .  an  effective  T h i s supplement had been i n use p r e v i o u s l y as an  i n d i c a t o r of p r o t e o l y t i c a c t i v i t y unpublished).  Skimmed milk was  the  i n papaya  callus cultures  (Townsley,  Thus, skimmed m i l k i n agar media p r o v i d e d an e f f e c t i v e  method f o r d e t e r m i n a t i o n of p r o t e o l y t i c enzyme p r o d u c t i o n .  rapid  75  Callus  initiation  from papaya e x p l a n t s has  o t h e r l a b o r a t o r i e s (Medora e t a l . , 1973; Conover, 1978;  Medhi & Hogan, 1976).  a l s o been r e p o r t e d from  Arora & Singh,  1978;  P l a n t p r o p a g a t i o n was  and Conover, w h i l e Medora's group r e p o r t e d the presence t h e i r papaya c a l l u s c u l t u r e s (Medora e t a l . , 1973; M e l l e t a l . , 1979).  P i n e a p p l e has  Litz & the g o a l of L i t z  of proteases  B i l d e r b a c k e t a l . , 1976;  a l s o been used f o r m i c r o - p r o p a g a t i o n ,  t h i s work bypasses c a l l u s f o r m a t i o n by use  of a p i c a l and  a x i l l a r y buds,  r e q u i r i n g o n l y a c o n t i n u a t i o n of the n a t u r a l morphogenetic sequence et  a l . , 1976).  previously  2.  in  Development of c a l l u s c u l t u r e s from f i g has  but thus  (Mathews  not been  reported.  C e l l suspension c u l t u r e s For economic reasons,  the e s t a b l i s h m e n t  of c e l l  suspension  c u l t u r e s i s r e a l l y the f i r s t s t e p towards l a r g e - s c a l e p r o d u c t i o n of p l a n t c e l l s or t h e i r m e t a b o l i t e s c u l t u r e systems i n c l u d e : and  inoculum,  medium and  ( D o u g a l l , 1980).  The  advantages of  suspension  f a s t e r growth r a t e , u n i f o r m i t y of c e l l  s i m p l i c i t y of m i c r o s c o p i c examination,  the p o s s i b i l i t y of d i r e c t p l a t i n g  ready  environment  a l t e r a t i o n of  f o r c l o n i n g purposes  the  (Widholm,  1980). Fragments of the r o o t - l i k e outgrowth from bedstraw e x p l a n t s transferred  to l i q u i d medium c o n t i n u e d  to develop  form, r e s u l t i n g i n l a r g e t a n g l e d masses. tendency to c a l l u s of  f o r m a t i o n and  little  medium c o n t a i n i n g o n l y f r e e c e l l s  i n v e s t i g a t i o n of these c u l t u r e s was  M i c r o s c o p i c examination root-cap c e l l sloughing.  r e s u l t e d i n no  culture.  Calli  showed  no  Transfer  f u r t h e r growth,  so  discontinued.  C a l l u s c u l t u r e s of f i g , papaya and attempt suspension  i n the same d i f f e r e n t i a t e d  thistle  were d i s s e c t e d and  grew w e l l enough t o t r a n s f e r r e d to  liquid  76  medium of the same c o m p o s i t i o n and maintained on r o t a r y shakers, a g a i n a t 28°C i n t o t a l d a r k n e s s . ing  Because t h i s t l e  grew p o o r l y , even i n m i l k - c o n t a i n -  medium," s u s p e n s i o n c u l t u r e of t h i s s p e c i e s was not pursued  further.  Papaya c a l l u s broke up s l o w l y , forming a f i n e c e l l s u s p e n s i o n by the t h i r d t r a n s f e r , w h i l e f i g c e l l s r e a d i l y d i s p e r s e d w i t h i n two weeks of i n t r o d u c t i o n to  l i q u i d medium.  aggregation.  N e i t h e r s p e c i e s p r e s e n t e d the common problem  M i c r o s c o p i c examination r e v e a l e d s i n g l e c e l l s  of c e l l  and s m a l l clumps  up to about 20-30 c e l l s i n s u s p e n s i o n c u l t u r e s beyond the t h i r d  transfer.  When skimmed milk was p r e s e n t i n the medium, c l e a r i n g of the milk t u r b i d i t y c o u l d be d e t e c t e d i n 5-10 days and t h i c k c e l l in  14-20 days.  s l u r r i e s were  formed  I t was apparent t h a t Gamborg's B5 medium, p a r t i c u l a r l y w i t h  the a d d i t i o n of skimmed m i l k ( a t a r a t e o f 3%,v/v), was n u t r i t i o n a l l y adequate was  f o r growth.  In terms of d r y weight,  n o t unusual over a two-week p e r i o d .  a 50-65% c o n v e r s i o n of sucrose  Papaya and f i g c e l l  suspension  c u l t u r e s have not p r e v i o u s l y been r e p o r t e d i n the l i t e r a t u r e , and the r a p i d , l u x u r i a n t growth a t t a i n e d by both was remarkable.  3.  D e t e r m i n a t i o n of biomass,  p r o t e i n and p r o t e a s e  C e l l h a r v e s t y i e l d i s the most common method of a s s e s s i n g growth i n p l a n t c e l l c u l t u r e s (Rose & M a r t i n , 1974). determined,  I f d r y weights a r e  t h i s i s a l s o the most a c c u r a t e measure a v a i l a b l e .  Settled  cell  volumes and f r e s h weight y i e l d s have a l s o been used as growth i n d i c a t o r s ( N i c k e l l & Maretzki,1969; harvested c e l l s highly variable.  Byrne & Koch 1962).  isdifficult  Because water c o n t e n t of  to s t a n d a r d i z e , f r e s h weight values can be  S e t t l e d c e l l volumes from f i g c u l t u r e s were found to  a c c u r a t e l y r e f l e c t d r y weight d u r i n g the e a r l y phases l a t t e r method was s e l e c t e d f o r i t s r e l i a b i l i t y  of growth, but t h e  and g e n e r a l i z a b i l i t y .  Papaya  77  and  f i g c e l l s were found  to be very d i f f e r e n t i n s i z e and  was  supposed t h a t they would pack d i f f e r e n t l y .  c e l l s dried  18-20  and Asakura  (1981) are among those who  the p o s s i b i l i t y proteolysis,  s t r u c t u r e , so i t  Gamborg e t a l . (1968) used  h i n a vacuum oven a t 60°C, as others have s i n c e . have used l y o p h i l i z e d c e l l s .  of heat l a b i l e p r o t e a s e s and  o v e n - d r i e d weights  These weights  Due  to o b t a i n biomass data  bore a c o n s t a n t r e l a t i o n s h i p to  from both papaya and  fig cell  suspensions.  P r o t e i n s are c o n s i d e r e d primary m e t a b o l i t e s of p l a n t c e l l s c u l t u r e and  t h e i r p r o d u c t i o n i s thus more c l o s e l y  n u t r i t i o n a l s t a t u s and metabolism. non-storage  i n s i t u contain approximately  organs (Thomas & Davey, 1975).  found  secondary  5% p r o t e i n  In seeds and  c o n t e n t i s more commonly 10-20% of the dry weight. and papaya c e l l s  in  l i n k e d to g e n e r a l  r a p i d growth than a r e p r o d u c t s of  Plant c e l l s  to  the p o t e n t i a l f o r endogenous  a l l h a r v e s t e d c e l l s were f r e e z e - d r i e d  p r i o r to e x t r a c t i o n .  Kato  (db) i n  tubers, p r o t e i n  Suspension-cultured f i g  grown i n B5 medium w i t h or w i t h o u t milk were g e n e r a l l y  to c o n t a i n 3.5-7.0% p r o t e i n  (x=4.55, n=45) i n crude e x t r a c t s . T h i s  range i s comparable to t h a t r e p o r t e d by Gamborg and F i n l a y s o n (1969),  a  range of 1.4-8.7% p r o t e i n i n f o u r t e e n s p e c i e s c u l t u r e d i n v i t r o . I f K j e l d a h l n i t r o g e n values o b t a i n e d f o r f i g c e l l s t a t i v e , a v e r a g i n g 3.58% approximately  of the dry weight,  then i t must be surmised  80% of the n i t r o g e n i s e i t h e r n o n - e x t r a c t a b l e or  proteinaceous.  I t c o u l d be bound i n the c e l l w a l l and  as n u c l e i c a c i d s o r o t h e r n o n - p r o t e i n n i t r o g e n (NPN) absorbance values of u n i n c u b a t e d  TCA-soluble  assays were an i n d i c a t i o n of h i g h NPN possibility  were r e p r e s e n -  was  that  non-  membranes, or  present  i n the e x t r a c t s .  r e a c t i o n mixtures  c o n t e n t of c e l l e x t r a c t s .  High  i n protease This  not c o n s i d e r e d i n c a l c u l a t i o n of p r o t e i n from K j e l d a h l  n i t r o g e n by Gamborg and F i n l a y s o n (1969).  The  range of n i t r o g e n c o n t e n t s  78  in  f i g c e l l s was  not p a r t i c u l a r l y h i g h i n comparison to o t h e r  v a l u e s , lower than and  8.3%  the r e p o r t e d 5-7%  i n rice cells  (Cifferi  that suspension-cultured  i n tobacco  e t a l . , 1980).  cells The  (Kato  literature  & Asakura,  l a t t e r authors  reported  r i c e c e l l s c o n t a i n 46% p r o t e i n (db), a very  f i g u r e i n comparison to f i g c e l l s  and  1981)  even to r i c e g r a i n , which i s  high 6-8%  protein. S e v e r a l methods of p r o t e i n assay were c o n s i d e r e d in  this project.  Bradford for  (1976).  The  method of c h o i c e was  I t was  found  the d y e - b i n d i n g  for routine technique  to be s u f f i c i e n t l y r e p r o d u c i b l e and  t h i s work, but i s e s p e c i a l l y  use  of sensitive  to be p r a i s e d f o r i t s s i m p l i c i t y  and  rapidity. Most work d e t a i l i n g enzyme s y n t h e s i s and suspension  c u l t u r e s has  f u n c t i o n s , and  little  the U n i t e d S t a t e s — D .  study. these  Reports of p r o t e a s e s arising  B i l d e r b a c k , D.E.  Campbell.  I t i s mainly  from papaya  to t e s t f o r p r o t e a s e  B i l d e r b a c k , R. Medora, G.P.  Academy of S c i e n c e s , 1966). absorbance u n i t s , and  and  present  extracted  The  authors  g i v e n i n the Food Chemicals Codex ( N a t i o n a l  Unfortunately,  r e s u l t s were r e p o r t e d i n  thus c o u l d not r e a d i l y be compared h e r e i n .  p r e s e n t work began with an assessment of a l t e r n a t i v e  d e t e c t i o n methods. Becker  Mell, J .  t h e i r work which s t i m u l a t e d the  a c t i v i t y on a v a r i e t y of s u b s t r a t e s .  used the assay method f o r papain  callus  from a group of r e s e a r c h e r s i n  They produced papaya c a l l u s c u l t u r e s , then l y o p h i l i z e d  The  metabolic  i n f o r m a t i o n e x i s t s w i t h r e s p e c t to enzymes w i t h  t i s s u e s i n v i t r o are the e x c e p t i o n ,  and J.M.  in c e l l  d e a l t w i t h the enzymes i n v o l v e d i n normal  p o t e n t i a l commercial a p p l i c a t i o n .  Ong  activity  Adapting  protease  the g e l a t i n d i g e s t i o n method of G l e n i s t e r and  (1961) to p l a n t c e l l e x t r a c t s p r o v i d e d  the f i r s t evidence  that  p r o t e o l y t i c a c t i v i t y v i s u a l i z e d by the c l e a r i n g of milk i n l i q u i d media  was  79  e x t r a c t a b l e and  s u f f i c i e n t l y s t a b l e f o r exogenous a s s a y .  T h i s method,  however, i s not p r e c i s e enough to be u s e f u l i n comparison of c e l l grown i n media w i t h o n l y s l i g h t m o d i f i c a t i o n s . ( A r a k i & Abe,  1980;  Bio-Rad) a l l f a i l e d  Holmes & Ernstrom, 1973; to d e t e c t a c t i v i t y  Chemicals Codex method.  cultures  Three agar d i f f u s i o n methods  "Protease  d e t e c t i o n k i t " from  t h a t c o u l d be q u a n t i t a t e d by the Food  T h i s i s b e l i e v e d to be due  to the f a i l u r e  of  the  enzyme(s) t o pass through the agar medium, thus no zones of c l e a r i n g , t u r b i d i t y or c o l o u r change c o u l d be seen a t any Fluorescence  d i s t a n c e from the  d e t e c t i o n methods f o r p r o t e a s e s  have r e c e i v e d much  a t t e n t i o n i n r e c e n t years -because of t h e i r h i g h s e n s i t i v i t y al.,  1972;  Schwabe, 1973).  (1974) r e l i e s on is  The  technique  r e p o r t e d by Spencer and  Following  the r a p i d i n i t i a l  f l u o r e s c e n c e upon a d d i t i o n of the p l a n t c e l l c o u l d be seen over 30 m i n u t e s .  I t was  a c t i v a t o r , or other substances p r e s e n t t u r b i d i t y of the r e a c t i o n mixture due substrate. predicted  The  (Udenfriend  the l o s s of f l u o r e s c e n c e as the s u b s t r a t e - b o u n d  r e l e a s e d upon p r o t e o l y s i s .  origin.  Spencer ANS  e x t r a c t , no s i g n i f i c a n t d e c l i n e  postulated  t h a t the  cysteine  i n the e x t r a c t s caused an i n c r e a s e i n to a g g r e g a t i o n  of the  casein  Another c o m p l i c a t i o n which arose  o p e r a t i n g temperature of the a s s a y s . c l e a r t h a t a temperature h i g h e r  For short-time  the was  than ambient would be n e c e s s a r y .  p r e i n c u b a t i o n of the substrate-enzyme mixture,  range of 50-500 ug  peptides.  t y r o s i n e and  of the range p r e v i o u s l y employed  the  i n c u b a t i o n , i t was Another  method, d e s c r i b e d by Chism e t a l . (1979), circumvented t h i s problem  l a b e l l i n g of TCA-soluble  reagent  increase i n  c o n t r o l r e a c t i o n , u s i n g chymotrypsin, demonstrated  l o s s of f l u o r e s c e n c e .  et  f o l l o w e d by  by  fluorescamine  T h i s approach appeared to work i n the  w i t h papain  concentrations  a t the h i g h e r  (200 ug per r e a c t i o n t u b e ) .  With  the  end  80  amount o f time, l a b o u r  and c o s t i n v o l v e d ,  t h i s method h e l d no advantage over  the Food Chemicals Codex a s s a y . A few m o d i f i c a t i o n s  were made to the Food Chemicals Codex method  f o r p a p a i n f o r ease of p r e p a r a t i o n  and q u a n t i t a t i o n .  The a c t i v a t o r used,  d i t h i o t h r e i t o l r a t h e r than c y s t e i n e , was added a t the s t a r t of the incubation  period  i n s t e a d of being  simplified  advance p r e p a r a t i o n s  included  i n the e x t r a c t i o n b u f f e r .  and assured a minimum of p r o t e o l y t i c  a c t i v i t y i n the e x t r a c t s p r i o r t o d e t e r m i n a t i o n Secondly, because a c t i v i t y was very substrate  concentration  low, i t was c o n v e n i e n t to decrease the  p o s s i b l y by improving the e f f i c i e n c y of  P r o t e o l y s i s i n e x t r a c t s of l y p h i l i z e d c e l l s was low, so  the 0.2% c a s e i n s u b s t r a t e The  of p r o t e i n c o n t e n t .  from 10 t o 2 mg/ml, a s t e p which improved the  p r e c i s i o n o f absorbance r e a d i n g s , casein digestion.  This  would r a r e l y have approached complete h y d r o l y s i s .  i n c u b a t i o n p e r i o d was a l s o extended to two hours, from one, i n c r e a s i n g  the d i f f e r e n c e s between t e s t and c o n t r o l samples. more e f f e c t i v e l y by i n c r e a s i n g the i n c u b a t i o n Unfortunately,  T h i s would have been done  temperature t o 47°C.  a t the time t h a t temperature s t a b i l i t y  of the p r o t e a s e was  determined, a g r e a t d e a l o f d a t a had been c o l l e c t e d a t 40°C, as recommended i n the o r i g i n a l method. r e s u l t s obtained and was,  complicated  T h i s would not have been t r u l y comparable to  a t a higher  incubation  temperature.  the lengthy  procedure, t h i s method was adopted f o r r o u t i n e a s s a y .  i n p a r t , compensated f o r by the s i m p l i c i t y  done c o n c u r r e n t l y . ninhydrin  Despite  of p r o t e i n  It  determinations  A s i m i l a r p r o t e a s e assay method i n v o l v e d a d d i t i o n of a  reagent t o the TCA s u p e r n a t a n t s f o r absorbance readings  (Reimerdes & K l o s t e r m e y e r , 1976).  Besides being  a t 570 nm  more cumbersome i n t h a t y e t  another reagent would be r e q u i r e d and a l l s u p e r n a t a n t s would have to be pH-adjusted, there was i n t e r f e r e n c e by the enzyme a c t i v a t o r s such as cysteine or d i t h i o t h r e i t o l .  81  Milk c l o t t i n g  a c t i v i t y has  an i n d i c a t o r of p r o t e o l y t i c a c t i v i t y P o z s a r - H a j n a l & Hegedus, 1975).  sometimes been m i s t a k e n l y understood ( P o z s a r - H a j n a l e t a l . 1974;  Although  commonly p r e s e n t i n protease  p r e p a r a t i o n s , milk c l o t t i n g a c t i v i t y i s not n e c e s s a r i l y d i r e c t l y with p r o t e o l y t i c a c t i v i t y , T h i s d i s t i n c t i o n may two  activities,  and  i t c o u l d be p o s s i b l e to favour one  temperature,  Whitaker  or the other by  (1959) r e p o r t e d on  s u b s t r a t e c o n c e n t r a t i o n and  milk c l o t t i n g a c t i v i t y of f i c i n ,  f i g c e l l c u l t u r e s y i e l d e d c l o t t i n g times  reasonable  range f o r cheese manufacture. to m i l k c l o t t i n g may  cultures i n this f i e l d .  cheese-making m a t e r i a l was  f a r i n excess  A long c l o t t i n g  time may  be d e s i r a b l e i f c o n d i t i o n s  of two p l a n t s t h a t have been used  ( V i e i r a de Sa & Barbosa,  1970a&b).  I t was  i n f i g and  unfortunate  i n suspension  culture,  for traditional  I n s u f f i c i e n t young  a v a i l a b l e f o r e x t r a c t i o n to t e s t milk c l o t t i n g  Proteolytic activity  of a  y e t improve the p o t e n t i a l f o r f i g c e l l  t i s s u e s c o u l d not be r e a d i l y propagated  s i n c e t h i s i s one  E x t r a c t s of  An i n v e s t i g a t i o n of c o n d i t i o n s  p r e v e n t i n g f o r m a t i o n of b i t t e r p e p t i d e s are d e f i n e d . t h a t cardoon  f o r the manufacture  too much p r o t e o l y t i c a c t i v i t y .  papaya and  conducive  on the  To date, however, few p l a n t  s p e c i e s have p r o v i d e d s u f f i c i e n t m i l k c l o t t i n g a c t i v i t y cheese without a l s o having  inhibitors  the  f o l l o w i n g s i m i l a r methodology to t h a t  d e v i s e d by B a l l s and Hoover (1937) f o r p a p a i n .  of  papain.  t o a d i f f e r e n c e i n optimum c o n d i t i o n s f o r the  m a n i p u l a t i o n of i n c u b a t i o n c o n d i t i o n s . e f f e c t s on pH,  correlated  as demonstrated by S k e l t o n (1971) u s i n g  occur due  as  papaya suspension  activity. c u l t u r e s became  e v i d e n t i n 5 days a t the e a r l i e s t , by a l o s s of o p a c i t y i n m i l k - c o n t a i n i n g media.  Protease d e t e r m i n a t i o n s  over time e s t a b l i s h e d 14 days as the optimum  time f o r p r o d u c t i v i t y , hence, f o r h a r v e s t .  In media w i t h o u t m i l k , t h i s  was  82  s l i g h t l y delayed cultures. cultures,  i n papaya c u l t u r e s ,  to 16-18  L o c a t i o n of enzyme a c t i v i t y ,  days, but unchanged i n f i g  a s s e s s e d o n l y with f i g c e l l  showed no s i g n i f i c a n t d i f f e r e n c e between B5 and B5-M  to the p r o p o r t i o n s of i n t r a - and  extracellular activity.  the e n t i r e c u l t u r e ( c e l l s and medium) would have to be otherwise One  concentrated,  i n order to o b t a i n the f u l l  s o l u t i o n might be achieved by a l t e r i n g  suggested Only one  with respect  I t was  lyophilized,  proteolytic  that  or  activity.  the c e l l p e r m e a b i l i t y , as  by B r o d e l i u s and Mosbach (1982) and Reese and Maguire m a t e r i a l , the d e t e r g e n t Span-80, was  (1969).  tested i n t h i s regard.  s i g n i f i c a n t d i f f e r e n c e i n medium p r o t e a s e  a c t i v i t y was  Span-grown and milk-grown f i g c e l l s .  possibility  The  clear  No  d e t e c t e d between still  exists that a  r e v e r s a l of t h i s p r i n c i p l e might be a p p l i e d , i e . , a d d i t i o n to media of substances  which p r e v e n t c e l l leakage  the h a r v e s t of c e l l s and  of enzymes, thereby n e c e s s i t a t i n g o n l y  r e q u i r i n g much l e s s e f f o r t  to c o n c e n t r a t e .  appear to be no r e p o r t s i n the l i t e r a t u r e t e s t i n g t h i s Assay d i f f i c u l t i e s : i n d e a l i n g w i t h crude  hypothesis.  S e v e r a l p o t e n t i a l problem areas may e x t r a c t s of d r i e d p l a n t c e l l s  y i e l d s of s m a l l volume c u l t u r e s to l a r g e volumes. weights were determined  There  and  be  encountered  i n transforming  In t h i s study,  harvest  by c o l l e c t i o n of c e l l s on M i r a c l o t h then  l y o p h i l i z i n g the mass removed by s c r a p i n g of the f i l t e r . t o t a l h a r v e s t weight o b t a i n e d ,  the c e l l s  adsorbed  Depending upon the  i n t o the f i l t e r  may  sometimes have c o n s t i t u t e d a s i g n i f i c a n t p r o p o r t i o n of the t o t a l weight. e s t i m a t i o n from pre-weighed f i l t e r s , 5%.  A l s o with r e s p e c t to weights,  this  l o s s was  u n l i k e l y to be more than  l y o p h i l i z a t i o n was  b e t t e r p r e s e r v e endogenous p r o t e i n and protease  By  chosen i n o r d e r  activity,  although  to  other  83  methods of d r y i n g y i e l d e d h a r v e s t lower ( f i g ) . weight was volume.  used.  on the b a s i s of  done p a r t l y to a v o i d  of true dry weight from  estimation  mainly to p r o v i d e  c a p a c i t y with a view to f e r m e n t o r - s c a l e  The  i t i s p o s s i b l e t h a t t h i s may  In compensation, a g r i n d i n g  a b u f f e r : d r y weight r a t i o of about 15:1 The  protease assays.  Materials  time of 60-75 seconds  became standard  sodium t e t r a t h i o n a t e and  shown to i n h i b i t a c t i v i t y included  thiourea.  of s u l f h y d r y l enzymes, and  trypsin inhibitor,  not  have v a r i e d from  one  using  procedure.  Such compounds c o u l d have been a c t i v e  and/or p a p a i n p r e p a r a t i o n s  and  to  presence of endogenous p r o t e a s e i n h i b i t o r s i n c e l l  a real possibility.  inhibitor  or p r o t e a s e s due  possible  thoroughness of the adopted e x t r a c t i o n procedure was  i n v e s t i g a t e d , however, and sample to a n o t h e r .  production.  performed as r a p i d l y as  the c o l d to p r e v e n t l o s s of e i t h e r p r o t e i n s  proteolysis.  culture  a c l e a r e r p i c t u r e of o v e r a l l  E x t r a c t i o n of d r i e d m a t e r i a l was  was  6%  However, most d a t a are r e p o r t e d  l y o p h i l i z e d weight, but  in  lower (papaya) and  Where c a l c u l a t i o n s based on biomass were made, the l y o p h i l i z e d  T h i s was  productive  weights about 10%  cultures  during  of standard calcium  ficin  thiocyanate,  Sodium t e t r a t h i o n a t e i s a known was  included  as a c o n t r o l :  f i c i n were s t r o n g l y i n h i b i t e d i n i t s presence, as e x p e c t e d .  i n h i b i t o r y e f f e c t of t r y p s i n i n h i b i t o r was  both p a p a i n The  somewhat s u r p r i s i n g s i n c e  a c t i v i t y of p l a n t p r o t e a s e s i s not g e n e r a l l y decreased by  this  the  material  (Fossum & Whitaker, 1968). It  i s believed  t h a t most p l a n t s p r o b a b l y c o n t a i n  protease  i n h i b i t o r s of some s o r t as a mechanism f o r r e g u l a t i o n of m e t a b o l i c enzymes a s s o c i a t e d w i t h p a r t i c u l a r developmental p e r i o d s , (Ryan, 1973). herein  to be  In f a c t , papaya c o n t a i n s  such as seed  germination  endogenous i s o t h i o c y a n a t e s ,  i n h i b i t o r y to a c t i v i t y of both p a p a i n and  ficin.  shown  These have  84  not yet reported  been i n f i g (Tang, 1974;  mechanism may  r e s p o n s i b l e f o r the s t r o n g i n h i b i t i o n of p a p a i n and  reported 1970;  be  here.  Tang & Tang, 1976).  The  Cyanide i s known to be an a c t i v a t o r of both enzymes  Liener & Friedenson,  t h e i r a c t i o n . Ascorbic  1970), but  the t h i o c y a n a t e  a c i d i s present  a l s o be produced by c e l l s i n v i t r o .  Skelton  mM  a s c o r b i c a c i d was  t e s t e d f o r p o s s i b l e s t i m u l a t o r y e f f e c t s on L a s t l y , t h i o u r e a was  s u l f u r - c o n t a i n i n g compound, and  liquid  one  because of a r e p o r t by Erez  growth of p l a n t t i s s u e i n c u l t u r e .  se.  may inhibiwithout  (1959) standard  Thiourea  was  (1978) t h a t i t included i n  media i n e a r l y attempts to e s t a b l i s h c e l l s u s p e n s i o n c u l t u r e s f o r  subsequently omitted.  i t may  by Whitaker  to  i n c l u d e d both because i t i s a  t h i s reason, but because i t appeared to produce no was  and  a s c o r b i c a c i d , but  Because of t h i s r e p o r t and  enzymes or e x t r a c t s .  (Arnon,  (1968) demonstrated the  an enzyme a c t i v a t o r . not  ficin  form i s i n h i b i t o r y  i n papaya a t the 1% l e v e l ,  t i o n of p a p a i n a c t i v i t y i n the presence of 2.3  stimulates  same  improvement i n growth, i t  I f i t c o u l d s t i m u l a t e enzyme p r o d u c t i o n ,  have become a u s e f u l a d d i t i o n d e s p i t e having  no  T h i s t e s t showed i n h i b i t i o n of papain by t h i o u r e a  t e s t e d ) , and  thus dispensed  with such a t h e o r y .  e f f e c t on growth per ( f i c i n was  In c o n c l u s i o n ,  e f f e c t i v e enzyme a c t i v a t o r s were those used i n o t h e r  however,  not  the  most  reports, cysteine  and  dithiothreitol. Fig tetrathionate,  c e l l extracts indicated l i t t l e t r y p s i n i n h i b i t o r or c a l c i u m  slightly inhibitory.  This  leaves  e f f e c t of e i t h e r sodium thiocyanate,  room f o r the p o s s i b i l i t y  are a l r e a d y f u n c t i o n i n g under i n h i b i t o r y c o n d i t i o n s . stronger If  a c t i v a t o r of proteases  n a t u r a l protease  with  i n the  thiourea  that  proteases  D i t h i o t h r e i t o l was  f i g c e l l e x t r a c t than was  i n h i b i t o r s were p r e s e n t  i n the c u l t u r e s and  a  cysteine. extracts,  o  t h e i r e f f e c t s were a t l e a s t p a r t i a l l y overcome by  being  these two a c t i v a t o r s .  85  Sodium d i e t h y l d i t h i o c a r b a m a t e t h i s can be a t t r i b u t e d  showed an apparent  The  f a c t t h a t these r e a c t i o n s do not e n t i r e l y  w i t h those of s t a n d a r d f i c i n suggests  No  d i g e s t i o n was  a t l e a s t i n p a r t , to p r o t e a s e s o t h e r than  (Ryan, 1973).  though g e l a t i n  For t h i s reason, they  to the type of s t o r a g e p r o t e i n p r e s e n t i n c e l l s ,  g l o b u l i n - s p e c i f i c protease Although  on a r o u t i n e b a s i s ,  i n pumpkin seeds (Spencer  such as  & Spencer,  l a t e x f o r commercial use,  p r o t e o l y t i c a c t i v i t y detected i n p l a n t c e l l p r o t e a s e s p r e s e n t may substrates.  commercial importance  s h o u l d be used  play a role i n The  f o r more s u i t a b l e  t h a t o n l y p r o t e i n s u b s t r a t e s of  f o r assay  i f the e v e n t u a l  application  purpose.  No p r o g r e s s was cultures.  the  papain  suspension c u l t u r e s .  have shown h i g h e r a c t i v i t y  However, i t can be argued  i s intended f o r t h i s  i t may  may  1974).  t h i s s p e c i f i c i t y does not seem to apply to the f i c i n and  e x t r a c t e d from f r u i t  the  P r o t e o l y t i c enzymes i n p l a n t s o f t e n appear t o  be a s s o c i a t e d w i t h p r o t e i n t u r n o v e r be s p e c i f i c  ficin.  u s i n g Hammarsten c a s e i n as  o t h e r s u b s t r a t e s were used demonstrated.  agree  t h a t p r o t e o l y t i c a c t i v i t y of f i g c e l l  Protease assays were performed substrate.  but  to an a r t e f a c t u a l c o l o u r change a c h i e v e d upon h e a t i n g  w i t h the c e l l e x t r a c t .  e x t r a c t s i s due,  stimulatory effect,  made i n i d e n t i f i c a t i o n of p r o t e a s e s  from  E l e c t r o p h o r e t i c s e p a r a t i o n of p r o t e i n s i n crude p l a n t  e x t r a c t s s h o u l d have made an important  c o n t r i b u t i o n toward  q u e s t i o n s of i d e n t i t y and  of p r o t e o l y t i c enzymes.  assumed t h a t p r o t e a s e s  multiplicity  cell  cell  answering It  was  s y n t h e s i z e d i n c e l l c u l t u r e would be p r e s e n t i n  l a r g e r q u a n t i t y than the s t r u c t u r a l p r o t e i n s and normal m e t a b o l i c enzymes i n cells.  Although  i t was  p o s s i b l e to v i s u a l i z e p r o t e i n bands i n a  f i c i n p r e p a r a t i o n , almost no p r o t e i n m o b i l i t y was  "pure"  e v i d e n t i n crude f i g c e l l  e x t r a c t s , even when c o n c e n t r a t e d as much as f o u r times.  Mereaptoethanol  and  86  SDS  treatments d i d not  two  possible explanations  material present migration  s i g n i f i c a n t l y improve m o b i l i t y . f o r the  lack of s u c c e s s .  There are a t  There may  have been  i n the e x t r a c t s which bound p r o t e i n s , p r e v e n t i n g  i n t o the acrylamide  their  g e l , p o s s i b l y the same f o r c e s p r o h i b i t i n g use  of g e l d i f f u s i o n methods of protease  detection.  Alternatively, extracts  c o n t a i n i n g 0.3-1.2 mg/ml t o t a l p r o t e i n , as determined by Bradford's may  not c o n t a i n s u f f i c i e n t q u a n t i t i e s of any  the same s t a i n .  4.  cultures  nutrition in f i g c e l l  I t was  postulated,  on  t h a t p r o t e i n s o r other  nitrogenous  compounds were r e s p o n s i b l e  f o r i n c r e a s e d p r o d u c t i v i t y of  f i g and  approach taken to medium n i t r o g e n  was  The  the a d d i t i o n of amino a c i d s or o t h e r  B5 medium.  Growth was  supplements. growth alone  nitrogenous  suspension-cultured  of p r o t e i n and  protease  supplementation  materials  the primary i n d i c a t o r of s u i t a b i l i t y  Production  be  the b a s i s of d r a m a t i c a l l y improved growth i n  the presence of 3% skimmed milk,  papaya c e l l s .  method,  p a r t i c u l a r p r o t e i n to  v i s i b l e as a banding p a t t e r n u s i n g  Nitrogen  least  of  to the the  were a l s o monitored, s i n c e  c o u l d not ensure enzyme s y n t h e s i s .  There i s a l a r g e body of i n f o r m a t i o n s u b j e c t of n i t r o g e n n u t r i t i o n and as i n i n t a c t p l a n t s been e s t i m a t e d  (Dougall,  i n the  1977,  1980;  Hewitt & C u t t i n g , 1977).  t h a t 50-90% of t o t a l p l a n t c e l l  nitrogen  the a c t u a l p r o p o r t i o n being  and  (Hewitt  ease of metabolism  l i t e r a t u r e on  the  metabolism i n p l a n t c e l l c u l t u r e s , as  environmental sources,  n i t r o g e n metabolism r e f l e c t s  well  I t has  i s a s s i m i l a t e d from  a f f e c t e d by  availability  e t a l . , 1977).  P r e f e r e n t i a l sources of i n o r g a n i c n i t r o g e n vary among s p e c i e s ,  1980).  basal  the a v a i l a b l e s o u r c e s  Reports i n d i c a t e t h a t the m a j o r i t y  and  ( T i s c h n e r & Lorenzen,  of p l a n t c e l l c u l t u r e s  utilize  87  ammonium n i t r o g e n b e f o r e r e d u c t i o n of n i t r a t e Bayley e t a l . , papaya  1972;  D o u g a l l , 1980).  T h i s appeared  1972).  medium (Gamborg e t a l . ,  Gamborg p o i n t e d out, i n the same p u b l i c a t i o n ,  ions at l e v e l s  g r e a t e r than 2mM  depressed growth  1970;  to be the case i n b o t h  and f i g c e l l c u l t u r e s as w e l l , e v i d e n t i n the i n i t i a l  subsequent r i s e i n pH of the growth al.,  (Gamborg & Shyluk,  d e c l i n e and 1968;  Bayley e t  t h a t ammonium  of soybean  cells in  s u s p e n s i o n c u l t u r e , the same maximum e x p e r i e n c e d i n i n t a c t p l a n t s Lea, 1976). 1OmM,  Only one paper encountered recommended a h i g h e r ammonium  possibly related  cultures  to h i s g o a l of i n d u c i n g embryogenesis  i n carrot  level, cell  ( W e t h e r e l l & D o u g a l l , 1976). Ammonia i s a s s i m i l a t e d  i n i n t a c t p l a n t s v i a two pathways,  depending upon i t s c o n c e n t r a t i o n i n the environment: synthetase a c t i v i t y , synthase a c t i v i t y , 1976;  (Miflin &  e i t h e r by glutamine  forming glutamine from glutamate, and/or by glutamate  forming glutamate from o c - k e t o g l u t a r a t e  Koiwai & Noguchi, 1972).  on ammonium a v a i l a b i l i t y ,  The  (Rhodes e t a l . ,  r a t i o of t h e i r a c t i v i t i e s  i s dependent  the former being most i m p o r t a n t a t low ammonia  c o n c e n t r a t i o n s , but the l a t t e r becoming i n c r e a s i n g l y a c t i v e a t h i g h e r l e v e l s of  ammonia, p o s s i b l y to prevent ammonia t o x i c i t y .  suspension c u l t u r e , for  Some p l a n t c e l l s i n  such as wheat, appear to have no s p e c i f i c  ammonia, w h i l e soybean  c e l l s are an example of a s p e c i e s which  very p o o r l y w i t h o u t i t ( B a y l e y e t a l . , r e p o r t e d t h a t soybean c e l l s would  1972).  grow q u i t e w e l l on ammonium s a l t s as the supplied.  The medium used h e r e i n , B5, c o n t a i n e d 25 mM ammonium ions  [1 mM  grows  Gamborg and Shyluk (1970)  s o l e n i t r o g e n source i f Krebs c y c l e a c i d s were a l s o  2 mM  requirement  (N^^SOg)].  n i t r a t e i n addition to  Ojima and O h i r a (1978) s t a t e d  that residual nitrate i s usually s u f f i c i e n t  f o r growth even a f t e r a l l the  carbohydrate has been m e t a b o l i z e d , a l t h o u g h they a l s o recommend 40  mM  88  nitrate  to enhance growth of s u s p e n s i o n - c u l t u r e d  rice c e l l s .  n i t r a t e r e q u i r e s a c t i v a t i o n of n i t r a t e r e d u c t a s e , adequate medium molybdenum and  calcium,  and  a n i t r a t e - s p e c i f i c permease (Oaks, 1977). i n the c y t o s o l and  medium i s p r o b a b l y  suggested  The  I f amino a c i d s are  (1978), c o u l d be  N i t r a t e may  be reduced to  nitrite  potassium n i t r a t e s u p p l i e d i n  carbohydrate d e f i c i e n c y . can be  without p r i o r r e d u c t i o n  to be  reducing  from  nitrate,  power. Fowler  has  from the pentose phosphate pathway of  would t h e r e f o r e be  unavailable  under c o n d i t i o n s  In the i n t a c t p l a n t , n i t r a t e need not be transported  through the xylem or s t o r e d i n  (Oaks, 1977).  processed vacuoles  Storage i n i n t r a c e l l u l a r pools  i n s i n g l e c e l l c u l t u r e as w e l l .  The  r e d u c t i o n products,  n i t r a t e p o o l a l l serve  may  ammonia  amino a c i d s , and  the c y t o p l a s m i c  n i t r a t e uptake.  Inadequate c a r b o h y d r a t e s u p p l i e s a c c o m p l i s h the same  effect  B5  potassium malate  synthesized  T h i s energy and  derived  c a r b o h y d r a t e metabolism and  occur  1976).  NADPH a r e r e q u i r e d .  immediately, but  requires  q u i t e p o s s i b l y the i n d u c t i o n of  used to produce both n i t r i t e and  (DeKock e t a l . , 1977). and  which i n t u r n  reduced f u r t h e r , to ammonia, most l i k e l y i n p l a s t i d s such  as c h l o r o p l a s t s ( M i f l i n & Lea,  both ATP  U t i l i z a t i o n of  and  to i n h i b i t f u r t h e r  ( B i d w e l l e t a l . , 1964).  E f f e c t on biomass:  I t was  evident,  from the growth of f i g c e l l s  medium l a c k i n g ammonium, t h a t n i t r a t e was the c o n t r a r y ,  ammonia was  s u r p r i s i n g r e v e l a t i o n due nitrate.  What was  the sum  growth, but  not  low  even milk,  s o l e n i t r o g e n supply,  concentration  unexpected, though, was  a c i d supplements, and Apparently,  an adequate n i t r o g e n s o u r c e .  i n s u f f i c i e n t as the to the  the  i n a l i n e a r manner.  not  .On a  i n comparison to  inability  to compensate f o r the  of n i t r o g e n p r e s e n t  i n B5  of i n d i v i d u a l amino  nitrogen  shortage.  i n the medium i s c l o s e l y l i n k e d t o  F i g c e l l growth i n the  t o t a l of 7  mM  n i t r o g e n s u p p l i e d by ammonium s u l f a t e p l u s glutamate, f o r example, exceeded  89  half  the  alone.  y i e l d from medium c o n t a i n i n g  25 mM  nitrogen  S i m i l a r l y , the combination of ammonia and  medium produced a h i g h e r yields.  The  biomass y i e l d  n i t r a t e concentration  than the  i n B5  s u s p e n s i o n c u l t u r e s , s i n c e a one-half a concomitant d e c l i n e i n y i e l d . seemed to have r e q u i r e d  i n the  n i t r a t e nitrogen  sum  i s not  in  B5  of the i n d i v i d u a l  excessive  reduction  form of n i t r a t e  for f i g c e l l  i n this nutrient resulted i n  Efficient nitrate utilization,  however,  the presence of a reduced source of n i t r o g e n ,  either  ammonia or some of the amino a c i d s . Amino a c i d metabolism i n s u s p e n s i o n - c u l t u r e d reviewed by D o u g a l l  (1980) and  Ojima and  Ohira  plant cells  (1978).  reduced form of n i t r o g e n but cannot e n t i r e l y r e p l a c e  y i e l d obtained  1968).  The  i n the presence of ammonia, f o r example  wide-ranging e f f e c t s on growth from s t r o n g l y i n h i b i t o r y to depending on  n u t r i t i o n a l h i s t o r y of (Maretzki  & Thorn, 1978;  the p l a n t s p e c i e s ,  the c u l t u r e and  inorganic  (Gamborg e t a l . ,  a s p a r a g i n e , glutamic soybean r o o t c e l l s a s p a r t i c and  a c i d or p r o l i n e  required  glutamic  (Furuhashi  the combination of  e t a l . , 1968); (c) the most u s e f u l amino a c i d s were found to be a r g i n i n e , h i s t i d i n e , 1969); (d) a l a n i n e and  i n n o x i a c e l l s w h i l e most other  nutrition,  lysine, arginine, f o r best  examples:  arginine,  & Yatazawa, 1970);  (b)  histidine,  y i e l d s (Gamborg  f o r growth of sugarcane  a s p a r t i c and  aspartic acids  tested  are a few  of a l a n i n e ,  a c i d s or p r o t e i n h y d r o l y s a t e  has  strongly  nitrogen  Following  (a) r i c e c a l l u s grew b e s t when s u p p l i e d w i t h any  Maretzki,  two-thirds  the p a r t i c u l a r amino a c i d s  M i f l i n e t a l . , 1977).  a  ammonium s a l t s .  i n c l u s i o n of amino a c i d s i n p l a n t t i s s u e c u l t u r e media  stimulatory,  been  Amino a c i d s are  Glutamine-grown soybean c e l l s were found to produce o n l y about the  has  glutamic  stimulated  acids  cells  (Nickell S  growth of Datura  amino a c i d s f e d i n d i v i d u a l l y were i n h i b i t o r y  t o growth (Fukunaga & King, 1982).  90  There are a l s o a t l e a s t two amino a c i d s being t h a t l y s i n e and reversed  by  threonine  by  others.  i n n i t r a t e medium was  strongest  reported  e t a l . (1981) found  i n h i b i t i o n of growth i n b a r l e y c u l t u r e s  was  Growth of tobacco, tomato, c a r r o t  inhibited  e f f e c t s on each s p e c i e s  p a r t i c u l a r amino a c i d supplements. 1975)  Cattoir-Reynaerts  the a d d i t i o n of a r g i n i n e .  soybean c e l l s a c i d s , with  reversed  r e p o r t s of i n h i b i t o r y e f f e c t s of some  The  and  by a common spectrum of amino being  seen with  same authors  different  (Behrend & M a t e l e s ,  the a b o l i t i o n of such i n h i b i t o r y e f f e c t s , most n o t a b l y  by  the  a d d i t i o n of a r g i n i n e or i s o l e u c i n e . A s e l e c t i o n of amino a c i d s , based on these r e p o r t s i n the l i t e r a t u r e , were added i n d i v i d u a l l y or ammonium.  In the presence of n i t r a t e ,  above the unsupplemented l e v e l , with biomass.  A s p a r t i c and  t h a t achieved  to B5 medium d e f i c i e n t i n e i t h e r n i t r a t e  with  glutamic  s i x amino a c i d s s t i m u l a t e d  o n l y g l y c i n e producing  a decrease i n  a c i d s r e s u l t e d i n y i e l d s more than double  ammonium, a l l i n the presence of n i t r a t e .  This i n d i c a t e d  a near complete u t i l i z a t i o n of the amino a c i d n i t r o g e n , presented place was  of ammonium s u l f a t e n i t r o g e n a t 2 mM.  u t i l i z e d at one-sixth,  ammonium a s s i m i l a t i o n .  and  growth  By  a t 5 mM  the same r e a s o n i n g ,  a l a n i n e a t about o n e - t h i r d  the  in  cysteine  efficiency  of  Because no combinations of amino a c i d s were t e s t e d ,  antagonism to growth i n h i b i t i o n c o u l d not be i n v e s t i g a t e d . In n i t r a t e - d e f i c i e n t media, f i g c e l l growth was w i t h o u t supplementation by e f f e c t s of glutamic  and  the t e s t amino a c i d s .  However, the  a s p a r t i c a c i d s were a g a i n noted.  ammonium media were a l l l e s s than 1 g/1  poor w i t h  or  stimulatory  Biomass y i e l d s i n  so i t would not be r e a l i s t i c  to  c a l c u l a t e percentages of growth i n h i b i t i o n below the unsupplemented ammonium medium.  Fig cells  appear to r e q u i r e both n i t r a t e and  n i t r o g e n f o r b e s t growth.  The  l a t t e r was  a source of reduced  a d e q u a t e l y s u p p l i e d by ammonium or  91  the a c i d i c amino a c i d s . While skimmed milk was  stimulatory  to growth i n both n i t r a t e  ammonium media, i t produced a g r e a t e r biomass y i e l d i n the former.  and  No  other  supplement to B5 medium t e s t e d r e s u l t e d i n e q u i v a l e n t s t i m u l a t i o n of growth.  Improved n i t r o g e n n u t r i t i o n may,  p o t e n t i a l l y enhancing p r o d u c t i o n c o u l d not p r o v i d e  activities  of c e l l s  protease:  and  amino a c i d s d i s c u s s e d  The  Two  above, and  yields  alone  p r o t e i n contents  and  protease  compared w i t h  s e r i e s of supplements were  a few  p l a n t and  respect  to  studied—the  animal p r o t e i n s .  Skimmed  i n c l u d e d i n both s e r i e s f o r comparison. Of  the amino a c i d s used to supplement B5,  a c i d s induced  the g r e a t e s t p r o t e i n p r o d u c t i o n  media, though the  p r o t e i n production, also stimulated  relative  protease  a c i d s were i n h i b i t o r y . regardless  glutamic  i n both n i t r a t e and  ammonium  c y s t e i n e were a l s o s t i m u l a t o r y  to the b a s a l l e v e l s .  activity  The  the  of  other  amino  i n ammonium-based media,  c o n s i s t e n t l y lower than i n the  amino a c i d s induced  In  same f i v e amino a c i d s  i n n i t r a t e media, while  Protease a c t i v i t y  of supplement, was  No  a s p a r t i c and  l a t t e r , as a group, gave much lower p r o t e i n y i e l d s .  n i t r a t e media, a r g i n i n e , a l a n i n e and  medium.  Cell  for f i g c e l l cultures.  media were determined and  the n i t r o g e n sources p r o v i d e d .  milk was  of p r o t e o l y t i c enzymes.  this information  E f f e c t on p r o t e i n and  however, induce p r o t e i n s y n t h e s i s ,  unsupplemented  s i g n i f i c a n t l y higher p r o t e o l y t i c a c t i v i t y  than unsupplemented ammonium medium. These r e s u l t s r e i t e r a t e d the media when enhancement of protease e f f e c t of a s p a r t i c and of a s s i m i l a t i o n and  glutamic  i n e f f e c t i v e n e s s of ammonium-based  production  a c i d s was  was  the  The  stimulatory  most l i k e l y r e l a t e d to t h e i r  metabolism i n p r o t e i n s y n t h e s i s .  reduced n i t r o g e n , carbon s k e l e t o n s  goal.  These compounds  f o r c a r b o h y d r a t e and  hydrocarbon  ease supply  92  metabolism, and a v a i l a b l e f u e l f o r ATP  production.  Some of the p r o t e i n s added t o B5 medium i n t e r f e r e d d e t e r m i n a t i o n s of h a r v e s t weights  due  to i n s o l u b i l i t y ,  becoming t r a p p e d with c e l l s upon f i l t r a t i o n . protein resulted  data f o r p r o t e a s e a c t i v i t y was improvement.  grade c a s e i n powder and enzymatic  i n h i b i t o r y to growth of c a l l u s  The  causes  tissues.  c o n t r o l f o r the e f f e c t of the presence Only  of t h e i r  the medium.  f r e s h skimmed milk and  had  egg albumen  thus c o a g u l a t e d upon  stimulated proteolytic  the activity.  complete,  ammonium-deficient media exposed a very broad  A l l m i l k - c o n t a i n i n g media y i e l d e d h i g h e s t p r o t e i n  the media without  milk.  T h i s r e s u l t was  l e v e l as n i t r a t e a l o n e .  range of yields,  not s i g n i f i c a n t l y b e t t e r than  supported  by the  d e t e r m i n a t i o n s , which c l e a r l y p l a c e d the N ^ - m i l k combination  protease a t the same  Without skimmed milk, complete B5 produced  p r o t e o l y t i c a c t i v i t y , and B5 with ammonium alone y i e l d e d the activity.  the same  albumen remained a f t e r  but ammonium medium supplemented w i t h m i l k was all  are  to be  to approximately  A comparison of the e f f e c t s of skimmed milk i n  protein levels.  to  of h i g h molecular weight m a t e r i a l ,  Much u n d i g e s t e d  i n c u b a t i o n p e r i o d , y e t i t s presence  n i t r a t e - d e f i c i e n t and  reagent  S o l u b l e s t a r c h , added o n l y as a  Note t h a t egg albumen i s not h e a t - s t a b l e , and  autoclaving  only  inhibitory action  been shown, e a r l i e r ,  showed s i g n i f i c a n t s t i m u l a t i o n of p r o t e o l y s i s , level.  For t h i s reason,  c a s e i n h y d r o l y s a t e were i n h i b i t o r y  of c u l t u r e s .  to be i n h i b i t o r y .  residual  t h a t many p r o t e i n s , i n c l u d i n g  not known, a l t h o u g h c a s e i n h y d r o l y s a t e had  a l s o proved  this  protein  e v a l u a t e d f o r the purpose of medium  T h i s data i n d i c a t e d  proteolytic activity  undigested  Similarly,  i n artefactual c e l l protein levels.  with  lowest  highest  93  5.  Stimulatory  e f f e c t s of skimmed m i l k  and  milk  components  I n c l u s i o n of skimmed m i l k i n B5 medium r e s u l t e d i n h i g h e r of biomass, p r o t e i n and other  proteolytic activity  nitrogen-containing  stimulatory  obtained  with  any  therefore postulated  that  the  to non-nitrogenous m a t e r i a l s .  The  supplements.  e f f e c t s observed were due  than c o u l d be  yields  I t was  p a r t i a l f r a c t i o n a t i o n of f r e s h skimmed milk  permitted  an e v a l u a t i o n  p o s s i b l e r e l a t i o n s h i p of the a c t i v e p r i n c i p l e ( s ) t o milk Both of these components enhanced growth, r e l a t i v e  of  c a s e i n and  the  whey.  to unsupplemented medium,  a l t h o u g h whey appeared to be more e f f e c t i v e than c a s e i n or i n t a c t m i l k . Lactose,  calcium,  yields.  The  the  n i a c i n or r i b o f l a v i n d i d n o t s i g n i f i c a n t l y a l t e r  filtrate  from u l t r a f i l t e r e d whey was  retained f r a c t i o n s i g n i f i c a n t l y stimulated The  stimulatory  C i t r a t e provides  e f f e c t of c i t r i c  also ineffective,  a c i d on growth was  c y c l e , c o n t r i b u t i n g to an improved energy p o o l f o r the  tricarboxylic acid  s y n t h e t i c pathways  I t s use  documented.  Shyluk (1970) found t h a t soybean c e l l s  the s o l e n i t r o g e n  cycle intermediates c u l t u r e s was  i n plant c e l l  striking.  a s s o c i a t e d w i t h growth.  w i t h ammonia as  not known, but  c u l t u r e media i s not  The  f u n c t i o n to these a c i d s i n  several options  were proposed:  ammonium t o x i c i t y , enhancement of ammonium t r a n s p o r t carbon requirement f o r amino a c i d s y n t h e s i s .  well  c o u l d grow  source i f Kreb's ( t r i c a r b o x y l i c  were p r o v i d e d .  while  growth of f i g c e l l s .  a v a i l a b l e carbon f o r metabolism v i a the  Gamborg and  biomass  relief  acid) cell of  or s a t i s f a c t i o n of  Ojima and  Ohira  a  (1978) a l s o  found t h a t some of the c a r b o x y l i c a c i d s , i n supplement to ammonia, promoted rice cell  growth.  One  a p p l i c a t i o n of c i t r i c  medium which i s of p a r t i c u l a r i n t e r e s t was Citrus species,  a c i d to p l a n t c e l l  reported  by E r n e r  e s p e c i a l l y orange, were known to r e q u i r e  orange j u i c e i n the growth medium.  T h i s group d i s c o v e r e d  culture  e t a l . (1975).  approximately that  the  10%  94  stimulatory acid  (2.5  e f f e c t of orange j u i c e was  g/1).  F i g s , the  f r e s h weight, but was  available.  is critical synthesize cell  no  completely reproducible  fresh f r u i t ,  information  c o n t a i n 6 meq  citric  with  a c i d per  100  on c i t r a t e c o n t e n t of the v e g e t a t i v e  to normal metabolic a c t i v i t y the r e q u i r e d  l e v e l s may  be  in fig cell.  The  capacity  l o s t over the course of  g  plant  I t i s p o s s i b l e , then, t h a t a h i g h endogenous c i t r a t e  level to  successive  t r a n s f e r s ( E r n e r e t a l . , 1975). P r o t e i n p r o d u c t i v i t y of f i g c e l l s i n media c o n t a i n i n g  or the milk  f r a c t i o n s , whey or c a s e i n , was  unsupplemented B5. production,  As u s u a l ,  with calcium,  l a c t o s e , and  A s i m i l a r p a t t e r n was a c t i v i t i e s of f i g c e l l or i t s components.  c a s e i n , milk  and  s i g n i f i c a n t l y higher  the  vitamins  biomass  resulting i n protein  alone.  s u s p e n s i o n c u l t u r e s grown i n b a s a l medium Whey, whey r e t e n t a t e  than other  media.  whereas f r e s h c a s e i n was,  containing  from u l t r a f i l t r a t i o n ,  Casein  of p r o t e a s e a c t i v i t y .  The  powder was  suggesting  fresh  responsible  ultrafiltrate  low m o l e c u l a r weight m a t e r i a l s  not s i g n i g i c a n t l y  t h a t the  the c a s e i n i t s e l f .  a r i s e s with the h y p o t h e s i s t h a t c i t r a t e was  have c o n t a i n e d  than t h a t i n  observed i n assessment of p r o t e o l y t i c  p r i n c i p l e ( s ) was/were u n l i k e l y to be  growth and  acid  c i t r i c a c i d a l l induced p r o t e o l y t i c a c t i v i t i e s  s i g n i f i c a n t l y higher stimulatory,  citric  t o t a l protein content c l o s e l y followed  l e v e l s not s i g n i f i c a n t l y d i f f e r e n t from B5  milk  citric  A  active complication  for stimulation  from whey, which  of  should  ( l e s s than 10,000 d a l t o n s ) ,  i n c l u d i n g c i t r a t e , a c t u a l l y showed no growth or p r o t e a s e enhancing e f f e c t s . The  most e f f e c t i v e milk  f r a c t i o n i n these r e s p e c t s  u l t r a f i l t r a t i o n retentate.  This discrepancy  c o - p r e c i p i t a t i o n with c a l c i u m i n whey d u r i n g  the  and  may  was  the whey  have a r i s e n as a r e s u l t of  phosphate a t n e u t r a l pH.  p r o c e s s of c a s e i n c o a g u l a t i o n ,  may  C i t r a t e , soluble  have become i n s o l u b l e  95  i n n e u t r a l i z e d whey and c o u l d not pass through the u l t r a f i l t r a t i o n membrane.  6.  Applications,  problems and p o t e n t i a l of p l a n t c e l l  cultures  A b r i e f look a t both p o s i t i v e and n e g a t i v e a s p e c t s of p l a n t c u l t u r e can h e l p applicability. multiple cells.  to put the p r e s e n t work i n t o p e r s p e c t i v e The i n t r o d u c t o r y  research  chapter p r e s e n t e d an overview of the  Yet much o f t h i s work i s s t i l l  i n the r e s e a r c h  stages,  or i s  be improved by the a d d i t i o n of whey t o  Under these c o n d i t i o n s ,  decrease the p l a n t hormone l e v e l s . c o u l d be c o n s i d e r e d  t i s s u e s and  purposes.  Growth of f i g c e l l s c o u l d b a s a l B5 medium.  i n terms of  d i r e c t i o n s taken by those p r o p a g a t i n g p l a n t  intended s o l e l y f o r research  cell  the e q u i v a l e n t  i t may be p o s s i b l e  Technically,  to omit or  the r e s u l t i n g c e l l s l u r r y  of the same s p e c i e s  i n differentiated  form. As such, d i f f i c u l t i e s  i n meeting r e g u l a t o r y  a p p r o v a l would be minimal.  The spectrum of requirements and t e s t s f o r GRAS  status  described  altogether  guidelines  f o r FDA  by Nelson (1980) and Whitaker (1980) might be a v o i d e d  f o r papaya c e l l c u l t u r e s , i f the presence of papain can be  proven, s i n c e papain has a l r e a d y s u s p e n s i o n c u l t u r e s would r e q u i r e  been d e c l a r e d  GRAS.  A p p l i c a t i o n of c e l l  development, b u t i t i s not d i f f i c u l t t o  e n v i s i o n meat marinades or y o g u r t c o n t a i n i n g  such a p u r e e .  Refrigeration  would p r e v e n t a c t i v i t y b e f o r e use o f a marinade, o r a f t e r s e t t i n g of a milk-based p r o d u c t .  In the l a t t e r case, the slow milk c l o t t i n g  activities  encountered i n papaya c e l l s u s p e n s i o n c u l t u r e s may a c t u a l l y be f a v o u r a b l e i n terms of s h e l f l i f e . immobilization  Recent advances i n techniques of whole  (Brodelius  cell  & Mosbach 1982), though p o t e n t i a l l y u s e f u l f o r  p l a n t c e l l s producing flavour c o n s t i t u e n t s , t o these p r o t e a s e p r o d u c e r s .  would n o t l i k e l y be a p p l i c a b l e  96  A p p l i c a t i o n of p l a n t t i s s u e and c e l l c u l t u r e techniques has met w i t h a few major h u r d l e s .  Underlying  many d i f f i c u l t i e s  lies  the i n f l u e n c e  of g e n e t i c s , b o t h of the p a r e n t p l a n t and of progeny c e l l s . information  i s a v a i l a b l e on c y t o g e n e t i c  been s u b s t a n t i a t e d . contain  the g e n e t i c  Plant c e l l s information  fully-differentiated plants. information  s t u d i e s but few g e n e r a l i z a t i o n s have  a r e commonly h e l d to be t o t i p o t e n t , to required  Within  t o r e g e n e r a t e new,  the mature p l a n t , e x p r e s s i o n  i s l i m i t e d by s u r r o u n d i n g c e l l s and r e g u l a t e d ,  hormones.  Such l i m i t a t i o n s to g e n e t i c  reversible  ( S t r e e t , 1977).  c u l t u r e should,  numbers of p l a n t s p e c i e s  expression  of g e n e t i c  i n p a r t , by  are believed  Undifferentiated cells  t h e r e f o r e , respond o n l y  are under e x t e r n a l c o n t r o l .  Much  to be f u l l y  growing i n s u s p e n s i o n  to environmental c o n d i t i o n s ,  which  T h i s h y p o t h e s i s has been supported by the l a r g e  t h a t have been propagated i n v i t r o then responded  to d e l i b e r a t e i n d u c t i o n of morphogenesis ( R e i n e r t & B a j a j , 1977; Murashige, 1978;  Winton, 1978).  s u c c e s s f u l l y subjected be  explained  respect  The f a c t t h a t some s p e c i e s  have not y e t been  to d e d i f f e r e n t i a t i o n f o l l o w e d  by morphogenesis  could  e i t h e r by a l a c k of t o t i p o t e n c y o r by a l a c k of knowledge w i t h  to n u t r i t i o n a l and e n v i r o n m e n t a l requirements f o r these  transformations. A c o r o l l a r y g e n e t i c phenomenon concerns m e t a b o l i c v a r i a t i o n among calli  (Townsley, 1977) and c e l l suspensions  ( S t r e e t , 1977).  Rather than the  temporal c y t o l o g i c a l changes accompanying c e l l d i f f e r e n t i a t i o n , d i f f e r e n c e s may be m a n i f e s t c o n c u r r e n t l y . and  under the same c u l t u r e c o n d i t i o n s  metabolic behaviour.  S t r e e t suggested  production  variation.  of g e n e t i c  That i s , c e l l s  metabolic  a t the same age  do not n e c e s s a r i l y e x h i b i t i d e n t i c a l t h a t one cause may be the continuous  This could  very  l i k e l y be the case i n  c a l l u s and b a t c h s u s p e n s i o n c u l t u r e s , which a r e i n a c o n t i n u a l s t a t e of  97  flux, providing  a s e l e c t i v e advantage f o r d i f f e r e n t f r a c t i o n s of the c e l l  p o p u l a t i o n s over time. ploidy  l e v e l s i n Datura and b a r l e y  falling Their has  K i b l e r and Neumann (1980) found a wide range i n  i n t o one of two g e n e t i c  cultures.  They d e s c r i b e d  c l a s s e s , m e r i s t e m a t i c or parenchymatous.  appearance was d i s t i n g u i s h a b l e  microscopically  p r o b a b l y been m i s t a k e n l y a t t r i b u t e d to c e l l  Differences  To recommended.  avoid  differences  age i n other work.  c e l l h e t e r o g e n e i t y , c e l l s e l e c t i o n has o f t e n been  The s i m p l e s t  approach i n v o l v e s  (Noguchi e t l a . , 1977).  repeated t r a n s f e r of a  those b e s t s u i t e d  not  small  to the c u l t u r e  T h i s method was a p p l i e d ,  to papaya and f i g c a l l u s and s u s p e n s i o n c u l t u r e s . a c t i v i t y varied  metabolically,  i n productivity.  inoculum o f r a p i d l y - g r o w i n g c e l l s , conditions  though t h i s d i f f e r e n c e  i n n u c l e i c a c i d c o n t e n t may then be m a n i f e s t  noted as q u a n t i t a t i v e  c e l l s as  i n t h i s study,  Although p r o t e o l y t i c  from one g e n e r a t i o n to the next, the range of v a r i a t i o n d i d  appear to exceed t h r e e -  or f o u r - f o l d .  These c e l l p o p u l a t i o n s were not  e n t i r e l y homogeneous so p r o d u c t i v i t y r e p r e s e n t e d the mean of a l l c e l l s . i s quite  likely,  though not i n v e s t i g a t e d  herein,  between i n d i v i d u a l c e l l s were much g r e a t e r , callus  to c e l l  selected  al.,  p a r t i c u l a r l y i n the e a r l i e s t  s e l e c t i o n i s another f i e l d  i n v e s t i g a t i o n — r a p i d methods of c e l l  "cell  t h a t the d i f f e r e n c e s  cultures. Related  calli.  It  for high-nicotine  identification.  producers on the b a s i s  requiring  further  Tabata e t a l . (1978)  of examination of s m a l l  They had a r a p i d method to i d e n t i f y the compounds of i n t e r e s t , the squash method" and paper chromatography f o r a l k a l o i d s  1978).  They found the s e l e c t e d  cultures  l e v e l of n i c o t i n e p r o d u c t i o n over s u c c e s s i v e available f o rdetection  to be q u i t e transfers.  of h i g h p r o t e a s e p r o d u c e r s .  (Ogino e t  stable  i n the  No such method was  Timing the c l e a r i n g of  98  milk  agar showed no d i f f e r e n c e s among c a l l i .  of i n d i c a t o r s i n agar was  also unsuccessful  Bio-Rad p r o t e a s e d e t e c t i o n method was  N o t i n g pH changes by i n c l u s i o n i n detecting differences.  a p p l i e d i n the p r e l i m i n a r y  a r a p i d d e t e c t i o n method, again w i t h o u t r e s u l t s . s e l e c t i o n method, the approach chosen was s e l e c t i o n pressures,  using only  For  search  d e n s i t i e s of 10^  t h a t dependent on endogenous  rapidly-growing  tissue.  c e l l s / m l are g e n e r a l l y r e q u i r e d  growth. For most p l a n t s p e c i e s , below which no population  growth o c c u r s .  there appears to be  of i n d i c a t o r s i n the medium. has  papaya or f i g c e l l  met  productivity in vitro:  a minimum c e l l  density,  per mm  2  not  T h i s was  cell  conducive to i n c l u s i o n difficult  not attempted  with  cultures. plant c e l l s  i n two  groups i n terms of  (a) a l l c e l l s h a v i n g s i m i l a r p r o d u c t i v e  capacity,  l e s s than the p a r e n t p l a n t , r e q u i r i n g t h a t the e n t i r e c e l l  population  be i n f l u e n c e d to i n c r e a s e  capacities differing  among c e l l s  much more p r o d u c t i v e ,  i n a population,  (b)  productive  w i t h a few in situ.  variants The  f i g c u l t u r e s suggested t h a t e i t h e r the  c e l l populations  rates included  p r o d u c t i v i t y , or  sometimes more than e v i d e n t  noted among papaya and within  clones  with l i m i t e d s u c c e s s .  suspersion  Cell  to induce  Cell plating is technically a  Zenk (1978) c l a s s i f i e d  usually  Bergmann  S u c c e s s f u l p l a t i n g r e s u l t s i n a dense  i n agar, upwards of two  p r o c e d u r e and  by  the m i c r o b i o l o g i c a l method of p l a t i n g i n agar.  to 10^  for  lack of such a  A more r e f i n e d technique f o r c e l l s e l e c t i o n , d e s c r i b e d (1977), i s based on  The  was  relatively  being  variability variability  s m a l l , or t h a t c e l l s with h i g h  growth  variants with high p r o t e o l y t i c a c t i v i t y .  Cytogenetic  instability  i s one  enountered by p l a n t t i s s u e c u l t u r i s t s . s e v e r a l s o l u t i o n s to t h i s .  Cell  of the major d i f f i c u l t i e s I t appears now  t h a t there  s e l e c t i o n , as d i s c u s s e d  are  above, i s o n l y  one  99  answer.  Others i n c l u d e methods of h a p l o i d c e l l p r o d u c t i o n  a n t h e r s or m i c r o s p o r e s , or by chromosome e l i m i n a t i o n . techniques are p r o v i d e d Culture, Results  Perspectives,  d e s i r a b l e c e l l s can  Bajaj  as pigment p r o d u c t i o n ,  D e t a i l s of  i n Plant C e l l  others  (1980).  r e a d i l y i d e n t i f i e d by  a l k a l o i d c o n t e n t and  advances have a l s o been made i n the  characteristics,  (1977), and  e d i t e d by S a l a and  sometimes be  ( W e i l l e r , 1977), and  in  theory,  and be  metabolites,  "genetic  markers"  c h a r a c t e r i s t i c s such  genetic  tailored  variability  radio-immunoassays  i s minimized.  to s p e c i f i c purposes:  biotransformation  of p r e c u r s o r  materials  each s i t u a t i o n r e q u i r e s  but  of papaya or f i g c e l l  of c e r t a i n  (Alferman & R e i n h a r d ,  Plant species  and  purposes may  be  special consideration.  suspension c u l t u r e s  p r o t e o l y t i c enzymes i s a r e a l p o s s i b i l i t y .  f o r the p r o d u c t i o n  T h i s work has  i n t r o d u c t i o n to t h i s plant-.purpose r e l a t i o n s h i p .  provided  of  only  an  Improvement of  a c l e a r d e f i n i t i o n of n u t r i t i o n a l e s s e n t i a l s , must precede  attempts a t b i o r e a c t o r - s c a l e p r o d u c t i o n .  Plant c e l l  c u l t u r e i s an  p r o p o s i t i o n , worthwhile o n l y when the c o s t of h a r v e s t i n g becomes p r o h i b i t i v e , or the p l a n t s become e x t i n c t . b r e e d e r s and  could,  or e n v i r o n m e n t a l s t r e s s ( V a s i l e t a l . , 1980),  matched and  modified,  selection is  Plant c e l l clones  production  of x e n o b i o t i c s .  p r o d u c t i v i t y , and  Recent  (see S a l a e t a l . , 1980).  autotrophy, or c a t a b o l i s m  use  Cultures:  nucleic acid content.  a p p l i c a t i o n of  Organ  i n i d e n t i f i c a t i o n of g e n e t i c a l l y l i n k e d  1980), r e s i s t a n c e to d i s e a s e  The  these  Furthermore,  With the a i d of s e n s i t i v e d e t e c t i o n methods, c e l l simplified,  ovules,  i n s e v e r a l c h a p t e r s i n P l a n t C e l l , T i s s u e and  e d i t e d by R e i n e r t and and  from  expensive  the o r i g i n a l  Horticulturists,  plant plant  p l a n t p a t h o l o g i s t s are i n t e r e s t e d i n p l a n t t i s s u e c u l t u r e as  p r o p a g a t i v e method and s p e c i a l i s s u e of  model study system, as i n d i c a t e d by  the August/82  " C a l i f o r n i a A g r i c u l t u r e " , an a p p l i c a t i o n s - o r i e n t e d  a  100  p u b l i c a t i o n of the U n i v e r s i t y of C a l f o r n i a , Department of A g r i c u l t u r e . With regard  to p l a n t p r o d u c t s y n t h e s i s , however, o n l y i n d u s t r i e s r e l a t e d t o  p h a r m a c e u t i c a l s have looked production,  despite  s e r i o u s l y a t p l a n t c e l l c u l t u r e as a means of  the c o s t s . A l a c k of knowledge and  i n d u s t r y have so f a r stymied developmental work. f o r long-term r e s e a r c h labour-intensive  and  of c u l t u r e s and  would appear t h a t the North American food need or i n c e n t i v e f o r such a committment.  the  There i s a requirement  development programs due  establishment  expertise i n  to the  slow,  cloning procedures.  i n d u s t r y does not y e t have  It the  101  VI.  SUMMARY  Tissue cardunculus),  c u l t u r e s were e s t a b l i s h e d from e x p l a n t s  thistle  (Circium arvense),  dumbcane  papaya ( C a r i c a papaya), and f i g ( F i c u s c a r i c a ) . c a l l u s ; pineapple Cultures  of cardoon  ( D i e f f e n b a c h i a amoena), Two s p e c i e s  failed  to form  (Ananas comosus) and l a d i e s *bedstraw (Galium verum).  were a l l maintained a t 28 °C i n darkness, p r e v e n t i n g  photosynthetic  (Cynara  normal  a c t i v i t y and d i f f e r e n t i a t i o n .  Papaya and f i g were s u c c e s s f u l l y m a i n t a i n e d under these c o n d i t i o n s as u n d i f f e r e n t i a t e d c e l l  s u s p e n s i o n s . Growth i n l i q u i d media  produced g r e a t e s t c e l l d r y weight i n 14-21 days, the c u l t u r e s being  faster-growing  selected for sub-culture.  P r o t e o l y t i c a c t i v i t y was apparent i n t i s s u e and c e l l c u l t u r e s by the c l e a r i n g of m i l k i n media w i t h i n a week of s u b - c u l t u r e . s e l e c t e d as the s u b s t r a t e  f o r q u a n t i t a t i o n of e x t r a c t a b l e  a c t i v i t y . Assay r e s u l t s were used to compare s t i m u l a t o r y supplements, and determine temporal p r o d u c t i v i t y . modified  form, was used r o u t i n e l y with  determined u s i n g  the d y e - b i n d i n g  was  protease e f f e c t s of medium  The FCC method, i n  t y r o s i n e content  components as the u n i t of measurement.  Casein  of the T C A - s o l u b l e  E x t r a c t a b l e p r o t e i n was r o u t i n e l y  method of B r a d f o r d .  Biomass, c e l l d r y  weight produced over the t e s t p e r i o d , was determined by f r e e z e - d r y i n g samples.  A l l data were c o n v e r t e d  to a p e r - l i t r e  b a s i s f o r c o n s i s t e n c y and  ease of comparison. B5 medium, which c o n t a i n s supply  f o r growth.  2% sucrose,  was an adequate n u t r i e n t  No m o d i f i c a t i o n s were made to the b a s a l  except where s p e c i f i e d .  ingredients  Supplements t o B5 medium were i n v e s t i g a t e d f o r  t h e i r i n f l u e n c e on growth and p r o d u c t i o n  of p r o t e i n and p r o t e a s e .  Skimmed  102  m i l k , a t 3% v/v, was c u l t u r e s produced  one  of many supplements.  fig cell  p r o t e o l y t i c enzymes t h a t would h y d r o l y s e milk c a s e i n i n  the medium w i t h i n one week. thiols,  Both papaya and  proteins, starch,  Other medium supplements i n c l u d e d amino a c i d s ,  l a c t o s e and  other milk components.  R e s u l t s showed t h a t p r o t e i n s y n t h e s i s and p r o t e o l y t i c a c t i v i t y i n e x t r a c t s of l y o p h i l i z e d production) c l o s e l y .  fig cell  c u l t u r e s f o l l o w e d growth (biomass  Those medium supplements which s t i m u l a t e d c e l l  growth, such as m i l k , l e d to h i g h e r l e v e l s of t o t a l p r o t e i n and activity.  Thus, n i t r a t e i n B5 medium was  shown to be more important,  terms of o v e r a l l growth, than ammonia as a source fig cells.  in  of i n o r g a n i c n i t r o g e n f o r  The most e f f e c t i v e amino a c i d supplements f o r s t i m u l a t i o n of  growth and p r o t e a s e p r o d u c t i o n i n f i g c e l l  c u l t u r e s were g l u t a m i c  a s p a r t i c a c i d s , w i t h c y s t e i n e a l s o improving growth.  protease  Of  p r o t e a s e a c t i v i t y , but  not  the p r o t e i n s and p e p t i d e s added t o B5 medium, o n l y milk and  albumen produced  s i g n i f i c a n t l y h i g h e r protease a c t i v i t y  D r i e d c a s e i n and  c a s e i n h y d r o l y s a t e were both i n e f f e c t i v e medium  supplements.  and  The  than the  egg  control.  cause of the s t i m u l a t o r y e f f e c t of egg albumen i s not  known. Data o b t a i n e d from  fig cell  s u s p e n s i o n c u l t u r e s grown i n B5  supplemented with milk or v a r i o u s components t h e r e o f , showed t h a t growth and p r o t e a s e a c t i v i t y were h i g h e s t w i t h whey, m i l k , acid.  Unsupplemented medium r e s u l t e d i n s i g n i f i c n a t l y  p r o t e i n and p r o t e a s e due  f r e s h c a s e i n and  to i t s c i t r i c  levels.  The  lower  citric  biomass,  b e n e f i c i a l e f f e c t of milk may  be  largely  a c i d c o n t e n t , which would have s u p p l i e d growing f i g c e l l s  w i t h and a d d i t i o n a l energy c o n t r i b u t e to i n t r a c e l l u l a r  source and  carbon  source.  It could also  l e v e l s of c i t r a t e , p o s s i b l y d r i v i n g  metabolism a t a r a t e g r e a t e r than normal.  Whether c e l l s  cell  grown i n t h i s  103  medium were normal i n s i z e o r p l o i d y was not determined. produced such a dramatic  i n c r e a s e i n biomass, although  Since whey  n o t q u i t e as h i g h as  i n t a c t milk, t h i s u n r e f i n e d m a t e r i a l c o u l d be used i n l i q u i d i n e x p e n s i v e supplement f o r growth of f i g c e l l s .  form as an  I t would a l s o be  worthwhile i n v e s t i g a t i n g f o r p r o p a g a t i o n of o t h e r p l a n t s p e c i e s i n t i s s u e or c e l l  culture. There was much v a r i a b i l i t y among papaya and f i g c e l l  c u l t u r e s , o f t e n n o t s t a b l e from one g e n e r a t i o n to the next. experiment r e q u i r e d a standard  inoculum  suspension  Each  f o r each t e s t c o n d i t i o n , and c e l l s  grown i n the same medium d i d n o t n e c e s s a r i l y produce comparable r e s u l t s i f the inoculum  differed.  I t would have been d e s i r a b l e to s e l e c t c l o n e s on  the b a s i s of h i g h p r o t e a s e p r o d u c t i v i t y , b u t no method f o r r a p i d and n o n - d e s t r u c t i v e i d e n t i f i c a t i o n was a v a i l a b l e . c u l t u r e s of papaya and f i g probably range of p r o d u c t i v e c a p a c i t i e s ,  contained  As a r e s u l t , c e l l  suspension  f a s t - g r o w i n g c e l l s with a  t o t a l data upon assay  being r e p r e s e n t a t i v e  of the average. P l a n t c e l l s propagated present-day  industrial  excellent, reliable industries.  fermentors  source  under c o n t r o l l e d c o n d i t i o n s such as or b i o r e a c t o r s , c o u l d become an  of p r o t e o l y t i c enzymes f o r the food and o t h e r  T h i s b a s i c r e s e a r c h i n d i c a t e s t h a t both papaya and f i g c e l l s  can be dark-grown as c e l l suspension proteolytic  activity.  c u l t u r e s and w i l l both  demonstrate  104  REFERENCES  Aharonowitz, Y. and A.L. Demain, 1980. B i o t e c h n o l . & B i o e n g . 22 ( s u p p l . 1):5-9  Thoughts on secondary metabolism.  A l f e r m a n , A.W. and E . Reinhard, 1980. B i o t r a n s f o r m a t i o n by p l a n t t i s s u e cultures. (pp.399-404) I n : P l a n t C e l l C u l t u r e s : R e s u l t s and P e r s p e c t i v e s F. S a l a , B. P a r i s i , R. C e l l a , O. C i f e r r i ( e d i t o r s ) . 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