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In vitro culture of four Rubus species Donnelly, Danielle J. 1980

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C.I IN VITRO CULTURE OF FOUR RUBUS SPECIES  by  Danielle J u l i e Donnelly B.Sc.  (Agr.), Macdonald College, M c G i l l University, 1976  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (The Department of Plant Science)  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1980 (c)  Danielle J u l i e Donnelly  6  In  presenting  an  advanced  the I  for  thesis  degree  Library  further  this  shall  agree  scholarly  at the University make  that  p u r p o s e s may  h i s representatives.  of  this  written  of  University  Date  gain  April  Science  1980  Columbia  Columbia,  f o r reference copying  by t h e Head  I t i s understood  of British  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  of British  f o rextensive  be g r a n t e d  f o r financial  Plant  f u l f i l m e n t o f the requirements f o r  available  permission.  Department  The  i tfreely  permission  by  thesis  in partial  shall  that  n o t be a l l o w e d  and  of this  o f my  copying  I agree  that  study. thesis  Department  or  or publication without  my  A b s t r a c t  M e r i s t e m g a t i o n  of  d u c i n g  and  T h i s a l  c u l t u r e d mass  A  of  a  and  i n c u b a t i o n red  p r i o r red  to  for  m i c r o p r o p a -  t e c h n i q u e s e l e c t  determine  developed  were  f o r  p r o -  germplasm.  the  the  c u l t u r e  u s i n g  b l a c k  p l a n t s  c u l t u r e .  Some  n u t r i t i o n of  red  b l a c k  h y b r i d .  B a i l e y ) ,  S a t i s f a c t o r y i n o s i t o l ,  e s t a b l i s h e d  g i v e n  f o r  were  l e v e l s  hormones, red  r a s p b e r r y  d e t e r m i n e d .  e t i o l a t i o n  r a s p b e r r y ,  g i v e n  r a s p b e r r y  loganobaccus  p h o t o p e r i o d were  were  (R.  v i t a m i n s ,  then  and  r a s p b e r r y  p l a n t s  v i t r o  L.).  elements,  temperature  r a s p b e r r y  to  l o g a n b e r r y  pH  i n  d i s e a s e - f r e e  l o g a n b e r r y - l i k e  and  by  v a l u a b l e  i n i t i a t e d  f i r s t  L.),  minor  c a r b o h y d r a t e s ,  a  requirements  was  o c c i d e n t a l i s  Some  was  idaeus  procedure  major  i s  p r o p a g a t i n g  (Rubus  t a y b e r r y ,  f o l l o w e d  p l a n t s  e n v i r o n m e n t a l  r a s p b e r r y  and  c u l t u r e  i n v e s t i g a t i o n  and  (R.  t i p  p r e - t r e a t m e n t  l o g a n b e r r y ,  h e a t - t r e a t m e n t  and  p r i o r  to  r o o t i n g  i n  c u l t u r e . E x p l a n t v i t r o . tem  A p i c a l  t i p s  o n l y  i n f l u e n c e i n c r e a s e was  not  source the  a f f e c t e d  meristem i n  some  s u r v i v a l v i g o u r  i n  i n  p l a n t s  p r i o r  s p e c i e s .  r a t e  were  were  media.  i n c r e a s e d ,  of  and  to  t i p  of  l a t e r a l  e x c i s i o n  S p r i n g - t i m e  e f f e c t s  b l a c k  not  s u p e r i o r  p l a n t s .  meristem  With  but  Season  c u l t u r e .  The to  s u r v i v a l ,  t i p s  c u l t u r e d  advantageous.  Rubus  growth  s i z e  d i d  m e r i s not  d i d  appear  to  E t i o l a t i n g  source  p l a n t s  h e a t - t r e a t m e n t  c u l t u r e  r a s p b e r r y , r o o t i n g  v a r i e d  s u r v i v a l hastened.  of w i t h and  iii. Heat-treatment of on  loganberry  meristem t i p s .  slightly  With red  increased;  The  i n succession  shoot p r o l i f e r a t i o n ,  b a s a l medium u s e d was  Effect  of d i l u t i n g  was  not  bud  proliferation.  ing.  dramatic.  E f f e c t s of  the  with  IBA.  and  not  affected.  rooting,  mg/L  (BAP)  (NAA)  caused  root  callus  formation  of  pH  and  t e m p e r a t u r e u s e d was  i t was  i n t e n s i t y , and  four  the  use  of  and  27°C.  raspberry  reports.  normal  the  cytokinin  This  temperature  micropropagation,  tested  16  had  h o u r s was  well within plants.  the  Their  range r e p o r t e d  lights  adequate.  similar culture  micropropagation p o t e n t i a l .  f o r Rosaceous  Moderate  of c o o l white f l u o r e s c e n t  A photoperiod of  Rubus s p e c i e s  requirements are  literature  and  critical,  adequate f o r meristem t i p c u l t u r e .  were s a t i s f a c t o r y .  al  (IBA)  proliferation  c a r b o h y d r a t e s were  have been optimum f o r r e d  quirements,  axillary  medium.  Incubation  All  dilution  R o o t i n g was  i n the  light  (1962).  from l e a f p e t i o l e s ,  BAP  although  a 1:4  were c o n s i s t a n t w i t h o t h e r  e f f e c t s were r e l a t e d t o c o n c e n t r a t i o n  not  Skoog  stimulated  and  may  respectively.  hormones i n d o l e b u t y r i c a c i d  c a l l u s e d shoot bases.  Levels  only  induced meristem t i p  and  150  effect  I n o s i t o l i n t h e medium i n h i b i t e d r o o t -  induced a d v e n t i t i o u s lamina,  no  o f M u r a s h i g e and  I n o s i t o l at  Naphthalene a c e t i c a c i d  leaf  that  had  s u r v i v a l was  m a c r o e l e m e n t s , e v e n by  6-benzylaminopurine  and  raspberry,  g r o w t h r a t e was  Three media used development,  source plants  re-  nutritionin  the  IV.  Table of  Contents  Page Title  Page  Abstract  i i  Table of Contents  i  v  L i s t of F i g u r e s  vi  L i s t of Tables  ix  Acknowledgement  x  Introduction  1  M a t e r i a l s and M e t h o d s A.  B.  C.  D.  10  Black Raspberry  13  1.  C u l t u r e Media  13  2.  Source P l a n t Pre-treatment  16  3.  Cold Storage of In V i t r o P l a n t l e t s  16  Loganberry  17  1.  C u l t u r e Media  17  2.  Source P l a n t P r e - t r e a t m e n t  19  3.  Cold Storage of In V i t r o P l a n t l e t s  19  Tayberry  19  1.  C u l t u r e Media  19  Red  Raspberry  20  1.  C u l t u r e Media  20  2.  Source P l a n t Pre-treatment  23  3.  Cold Storage of In V i t r o P l a n t l e t s  24  V .  Table of Contents  (cont'd)  Page Results A.  B.  C.  D.  25 Black Raspberry  25  1.  C u l t u r e Media  25  2.  Source  3.  Cold Storage of In V i t r o P l a n t l e t s  Plant  Pre-treatment  36 3  6  Loganberry  3g  1.  C u l t u r e Media  3g  2.  Source P l a n t P r e - t r e a t m e n t  42  3.  Cold Storage of In V i t r o P l a n t l e t s  44  Tayberry  44  1.  C u l t u r e Media  44  Red  Raspberry  4g  1.  C u l t u r e Media  4g  2.  Source P l a n t P r e - t r e a t m e n t  59  3.  Cold Storage of In V i t r o P l a n t l e t s  ^2  Discussion  55  Summary  -74  Literature  Cited  Biographical Information  77  v i . L i s t  of  F i g u r e s  F i g u r e  1.  M e r i s t e m  Page  t i p s  of  b l a c k  r a s p b e r r y  months of c u l t u r e i n medium at 0.04 mg/L, showing slow, ment.  a f t e r  MS w i t h e r r a t i c  5  26  k i n e t i n d e v e l o p -  2.  M i n i a t u r e w h i t e s t r u c t u r e s formed i n some b l a c k r a s p b e r r y c u l t u r e s w h i c h were thought to be r u d i m e n t a r y f l o w e r s .  34  3.  E f f e c t of h e a t source p l a n t s t i p s i n medium 10 m g / L , BAP a mg/L, pH .5.7. from m e r i s t e m source p l a n t s ; r o o t . The r o o h e a t - t r e a t e d s from 5-8 weeks  - t r e a t i n g b l a c k r a s p b e r r y on development of m e r i s t e m MS w i t h c y s t e i n e H C l at t 0.1 mg/L, and IBA at 1.0 The r o o t e d p l a n t l e t s arose t i p s e x c i s e d from h e a t - t r e a t e d these took 4-5 weeks to t l e s s shoot was from a nonource p l a n t ; these took to r o o t .  37  Loganberry c l o n e s i l l u s t r a t i n g the e f f e c t of two c a r b o h y d r a t e s o u r c e s , and two l e v e l s of BAP, on m i c r o p r o p a g a t i o n p o t e n t i a l of m e r i s t e m t i p s s t a r t e d i n m e d i u m MS w i t h BAP at 1.0 mg/L and IBA at 0.1 mg/L. A: A f t e r 20 w e e k s i n s t a r t i n g m e d i u m w i t h s u c r o s e , the p l a n t l e t was d i v i s i b l e i n t o 15 c u t t i n g s . B: A f t e r 12 w e e k s o f c u l t u r e i n s t a r t i n g medium w i t h s u c r o s e , the shoot w a s t r a n s f e r r e d t o m e d i u m MS w i t h B A P i n c r e a s e d to 3.0 mg/L. A f t e r another 6 weeks, the l e a v e s were s m a l l e r than on A, and some.. w h a t c u r l e d , b u t the p l a n t l e t was d i v i s i b l e i n t o 25 c u t t i n g s . C: A f t e r 14 w e e k s of c u l t u r e i n s t a r t i n g medium w i t h g l u c o s e , the shoot was d i v i s i b l e i n t o 60 c u t t i n g s . D: A f t e r 8 weeks of c u l t u r e i n s t a r t i n g medium w i t h g l u c o s e , the shoot was t r a n s f e r r e d t o medium w i t h BAP i n c r e a s e d to 3.0 mg/L. A f t e r another 6 weeks, the l e a v e s were s m a l l e r than on C, and somewhat c u r l e d , and the p l a n t l e t was d i v i s i b l e i n t o 20 c u t t i n g s .  39  4.  .  vii. List  of Figures  (cont'd)  Figure  Page  5.  Tayberry clones i l l u s t r a t i n g the e f f e c t of c a r b o h y d r a t e s o u r c e , and two l e v e l s o f BAP, on m i c r o p r o p a g a t i o n p o t e n t i a l o f m e r i s t e m t i p s s t a r t e d i n medium MS w i t h g l u c o s e , and w i t h BAP a t 1.0 mg/L and IBA a t 0.1 mg/L. A: A f t e r 16 weeks i n s t a r t i n g medium. B: A f t e r 28 weeks i n s t a r t i n g medium. C: A f t e r 12 weeks i n s t a r t i n g medium, t h e s h o o t was t r a n s f e r r e d t o medium w i t h s u c r o s e , and BAP a t 3.0 mg/L. A f t e r a n o t h e r 4 weeks, t h e l e a v e s were l a r g e r and t h e r o o t s n o t a s w e l l d e v e l o p e d as on A.  45  6.  E f f e c t s o f c a r b o h y d r a t e s o u r c e , s u c r o s e (S) o r g l u c o s e ( G ) , i n o s i t o l ( I ) , o r hormones (H) on t h e g r o w t h o f m e r i s t e m t i p s o f r e d raspberry. Subculture (transfer) times a r e i n d i c a t e d by an a r r o w .  53  7.  E f f e c t s o f i n o s i t o l c o n c e n t r a t i o n on d e v e l o p ment o f r e d r a s p b e r r y m e r i s t e m t i p s i n c u l ture. A: A f t e r 8 weeks i n c u l t u r e . B: A f t e r 20 weeks i n c u l t u r e . Inositol l e v e l s were ( l e f t t o r i g h t ) : 0, 5 0 , 1 0 0 , and 150 mg/L. Inositol inhibited rooting, and i n c r e a s i n g c o n c e n t r a t i o n i n c r e a s e d p r o l i f e r a t i o n o f a x i l l a r y buds.  54  8.  Red r a s p b e r r y c l o n e s a f t e r 8 months i n c u l t u r e s h o w i n g r e s p o n s e o f some s e l e c t i o n s t o p r o l i f e r a t i o n medium. A, B, C, and D were a l l s t a r t e d i n medium w i t h s u c r o s e , BAP a t 1.0 mg/L, and IBA a t 0.1 mg/L. A f t e r 4 months i n s t a r t i n g medium, B, C, and D were t r a n s f e r r e d t o medium w i t h BAP i n c r e a s e d t o 3.0 mg/L. A f t e r 4 more months i n c u l t u r e t h e p l a n t l e t s were d i v i s i b l e i n t o 2 0 , 7, 1 0 , and 25 c u t t i n g s r e s p e c t i v e l y . B and C d e v e l o p e d f r o m buds e x c i s e d f r o m t h e same stem and i l l u s t r a t e the v a r i a b i l i t y that can occur within selections. A and D d e v e l o p e d f r o m buds o f two o t h e r s e l e c t i o n s , and i l l u s t r a t e t h e v a r i a b i l i t y i n r e s p o n s e t o BAP i n t h e g r o w t h medium; s e l e c t i o n A grew b e s t i n s t a r t i n g medium, D grew b e s t i n medium w i t h BAP a t 3.0 mg/L.  55  v i i i .  L i s t  of  F i g u r e s  (cont'd)  F i g u r e  Page  Red r a s p b e r r y c l o n e s showing the e f f e c t of pH o n l e a f s i z e and browning. Both p l a n t l e t s were grown i n m e d i u m MS w i t h BAP at 1.0 mg/L, IBA at 0.1 mg/L w i t h . g l u c o s e , f o r 7 months. A: At pH 6.1 B: At p H 5.7.  56  10.  Red r a s p b e r r y c l o n e s showing d i f f e r e n t response of two s e l e c t i o n s to p r o l i f e r a t i o n medium w i t h g l u c o s e . A f t e r 4 months i n s t a r t i n g medium w i t h g l u c o s e , IBA at 0.1 m g / L , a n d B A P a t 1.0 m g / L , a n d 3 months i n medium w i t h BAP i n c r e a s e d to 3.0 mg/L, A was d i v i s i b l e i n t o 9 c u t t i n g s ; B i n t o 35. Clones i n medium w i t h g l u c o s e had l e a v e s t h a t were u s u a l l y l a r g e r and more rugose than i n medium w i t h s u c r o s e .  57  11.  Red r a s p b e r r y c u t t i n g s from a c l o n e m e d i u m MS w i t h BAP at 1.0 mg/L and  60  9.  at 0.1 medium 12. :  mg/L a f t e r 3 weeks MS w i t h IBA at 0.5  i n IBA  i n r o o t i n g mg/L.  Red r a s p b e r r y a p i c a l (top row) and l a t e r a l (bottom row) p a i r s of m e r i s t e m t i p s showi n g the i n f l u e n c e of c a r b o h y d r a t e type and c o n c e n t r a t i o n on development a f t e r 2 months i n m e d i u m MS w i t h B A P at 1.0 mg/L and IBA at 0.1 mg/L. Carbohydrate s o u r c e s were ( l e f t to r i g h t ) : s u c r o s e at 20, and 30 g / L and g l u c o s e at 20, 3 0 , a n d 40 g/L.  64  ix. L i s t of  Tables  Table  Page  1.  The e f f e c t o f t h e hormones BAP a n d G A , a n d t h e a n t i o x i d a n t s PVP and c y s t e i n e HC1, o n s u r v i v a l of black raspberry meristem t i p s i n medium MS w i t h I B A a t 1.0 mg/L  28  2.  The e f f e c t o f e x p l a n t s o u r c e , s i z e , a n d BAP c o n c e n t r a t i o n on s u r v i v a l o f b l a c k r a s p b e r r y meristem t i p s  29  3.  E f f e c t o f BAP c o n c e n t r a t i o n i n s t a r t i n g medium, a n d BAP and I B A c o n c e n t r a t i o n i n r o o t i n g medium, o n r o o t i n g o f b l a c k r a s p b e r r y shoots d e r i v e d from meristem t i p s  31  4.  The e f f e c t s o f BAP a n d I B A c o n c e n t r a t i o n i n l i q u i d o r s o l i d medium o n new l e a f a n d r o o t formation o f black raspberry shoots  33  5.  E f f e c t o f BAP c o n c e n t r a t i o n , pH, and c a r b o h y d r a t e source on i n c i d e n c e and r o o t i n i t i a t i o n time, of loganberry shoot c u t t i n g s i n nutrient culture  41  6.  Comparison of loganberry s o i l and i n v i t r o  43  7.  E f f e c t o f c a r b o h y d r a t e source and c o n c e n t r a t i o n on s u r v i v a l and g r o w t h o f r e d r a s p b e r r y m e r i s t e m t i p s i n n u t r i e n t medium  50  8.  E f f e c t o f c a r b o h y d r a t e s o u r c e a n d pH l e v e l on s u r v i v a l a n d g r o w t h o f r e d r a s p b e r r y m e r i s tem t i p s i n n u t r i e n t c u l t u r e  51  9.  E f f e c t o f e t i o l a t i n g s o u r c e p l a n t s on s u r v i v a l of r e d r a s p b e r r y meristem t i p s i n four media  61  E f f e c t of heat-treatment (37°C) o f s o u r c e p l a n t s on s u r v i v a l o f r e d r a s p b e r r y m e r i s t e m t i p s i n two m e d i a  63  10.  3  cuttings rooted  in  X  Acknowledgment  I  w i s h  to  express  g r a t i t u d e  committee:  Frances  C.  A g r i c u l t u r e  Canada,  Research  V.  C.  P l a n t  Runeckles S c i e n c e ,  guidance am  g r a t e f u l  for P.  and  t h e i r  ment  s u g g e s t i o n s to  the  and  W,  for  for  h i s  d o n a t i o n  and  the  other  Research  h i s  p e r t a i n i n g  red of  a l s o  S p e c i a l  e n t h u s i a s t i c  w i s h  to  the  s t a f f for  thanks  support.  and of  t h e i r to  my  Dr.  Department  t h i s  of  f o r  t h e i r  t h e s i s .  C o u n c i l  of  to  Food  S c i e n c e  f o r  thank  t h e i r  thank  t a y b e r r y  S.  H.  d e p a r t -  W. Daubeny  p l a n t s ,  A g r i c u l t u r e a s s i s t a n c e dear  Dr.  encourage-  Mr.  Dr.  I  Canada  l i k e  photography,  r a s p b e r r y  Vancouver  the  Columbia  e x c e l l e n t  of  of  my  Vancouver;  to  would  of  S t a c e - S m i t h ,  Columbia,  Research I  members  R.  B r i t i s h  Chalmers  I  Dr.  Copeman,  of  B r i t i s h  members  S t a t i o n ,  encouragement. for  of  the  S t a t i o n ,  support. C.  h i s  and  J .  N a t i o n a l  s u g g e s t i o n s .  MacDiarmid  R.  U n i v e r s i t y  U n i v e r s i t y and  Dr.  f i n a n c i a l  Townsley  ment,  and  M e l l o r  to  husband  Canada, and Wayne  .  1. Introduction  M o r e l and M a r t i n  (1952) a r e c r e d i t e d w i t h  t i o n o f m e r i s t e m c u l t u r e and were t h e f i r s t technique f o r r i d d i n g plants of viruses. excised  young m e r i s t e m a t i c  regions  the i n i t i a -  t o use t h i s I n 1960 M o r e l  o f c y m b i d i u m s a n d , by  c u l t u r i n g these explants  on n u t r i e n t b r o t h ,  v i r u s - f r e e clones.  procedure also revealed  portant  This  developed  potential f o r plant multiplication  an i m -  (micropropagation).  P l a n t v i r o l o g i s t s have u t i l i z e d m e r i s t e m t i p c u l t u r e t o e l i m i n a t e v i r u s e s f r o m many u n i v e r s a l l y - i n f e c t e d p l a n t cultivars. excised  They h a v e d i s c o v e r e d  from h e a t - t r e a t e d  plants are often  that are not e a s i l y eliminated  large explants  tips  f r e e from  viruses  by m e r i s t e m c u l t u r e a l o n e  ( M e l l o r and S t a c e - S m i t h , 1977). relatively  that meristematic  Thermotherapy  enables  t o be v i r u s - f r e e , a n d t h e s e more  r e a d i l y develop i n c u l t u r e . Horticulturists, tal  and f o o d  p l a n t b r e e d e r s a n d g r o w e r s o f ornamen-  c r o p s h a v e b e e n a t t r a c t e d by t h e p o s s i b i l i t y  of r a p i d m i c r o p r o p a g a t i o n o f s e l e c t v a r i e t i e s . tages of micropropagation include superior lication, limited  short generation  time,  year-round  The a d v a n -  rate of multipproduction,  space r e q u i r e m e n t s , r a p i d e s t a b l i s h m e n t . o f  new s e -  l e c t i o n s , and v i r u s and pathogen e l i m i n a t i o n from i n f e c t e d clones.  In v i t r o propagation eliminates r e i n f e c t i o n r i s k s ,  common t o g r e e n h o u s e o r f i e l d lets are therefore  m u l t i p l i e d stocks.  e a s i l y and e x p e d i t i o u s l y  The p l a n t -  transported,  2. both r e g i o n a l l y of  and  internationally,  spreading pests or In the  important  tem  t i p culture,  The  genera,  berry;  and  shoot  tayberry.  these,  hybrids, and  cultured  (1978),  termine  in vitro  tigation, vitro  Fragaria,  by  by Walkey  of v i r u s  (1978),  Popov, 1970;  started,  by  shoot t i p  t h e o b j e c t was  affecting  to  t h e g r o w t h and  important  t h e need  infected  1971;  1979;  root-  inves-  w i t h the i n for virus  cultivars  Vertesy,  de-  red raspberry  primary motivation f o r t h i s  universally Putz,  and  elimination.  o f r e d r a s p b e r r y , was  from  crops.  species, strawberry  as w i t h p r e v i o u s s t u d i e s c o n c e r n e d  elimination  Murashige  or nut  been s u c c e s s f u l l y  factors  The  fruit  Two  of the a g r i c u l t u r a l l y  culture  straw-  a p p l e , c h e r r y , peach-almond  blackberry.  p r o j e c t was  (Rubus i d a e u s L . ) .  and  propagatable  as h a v i n g  some o f t h e  follows:  micropropagation.  s i x species are l i s t e d  f o r the purpose  When t h i s  and/or  for meris-  Rubus, r e d r a s p b e r r y , b l a c k b e r r y ,  red raspberry, are l i s t e d  Murashige  ing  t i p culture  strawberry,  almond, and  containing ten  Prunus, c h e r r y , peach-almond h y b r i d s ,  (1978) as c o m m e r c i a l l y These i n c l u d e :  genera,  s p e c i e s , have been u t i l i z e d  Rosa, r o s e ;  Of  five  s p e c i e s a r e as  Malus, apple;  almond, plum;  risk  pathogens.  f a m i l y Rosaceae,  commercially  with minimized  (Shchelkunova Carrg  et a l . ,  1979) . The  most f a v o r e d method f o r m i c r o p r o p a g a t i o n  w i t h meristem generate  t i p s which p a s s e s s meristemoids  meristemoids.  The  and  begins cells  normal o r g a n i z a t i o n of  the  that  3.  differentiated  s h o o t i s t h e r e f o r e r e t a i n e d , and t h e  risk  of aberrant c e l l s developing i n t o p l a n t s that are genetic deviants i s l i t t l e  o r n i l (Abbott, 1976).  This i s highly  i m p o r t a n t s i n c e t h e u n i f o r m i t y and p r o d u c t i v i t y o f c h a r a c t e r i s t i c s must be m a i n t a i n e d i n c u l t u r e  selected  (Murashige,  1977) . V a r i a b i l i t y i n regenerative capacity of meristem may  be a t t r i b u t e d  to i n i t i a l  size,  l a t e r a l or apical  d i f f e r e n c e s i n source p l a n t p h y s i o l o g i c a l  s t a t u s and  and t h e s e a s o n i n w h i c h e x p l a n t s a r e t a k e n The  tips origin, age,  (Murashige, 1974).  o n l y one o f t h e s e f a c t o r s t h a t h a s b e e n  investigated  f o r Rubus s p e c i e s i s s e a s o n a l e f f e c t o n r e d r a s p b e r r y e x p l a n t survival  in culture.  Shchelkunova  (1974)  excised  etiolated  m e r i s t e m t i p s f r o m s p r o u t e d r o o t c u t t i n g s , and f o u n d  the  g r e a t e s t number o f r e g e n e r a n t s c o r r e l a t e d w i t h M a r c h o r April-June excisions. t i p s a t monthly  CarrS et a l .  s u r v i v a l was  From J u l y - S e p t e m b e r m e r i s t e m t i p s  formed o n l y c a l l u s . tips  took meristem  i n t e r v a l s from v i g o r o u s l y growing  and f o u n d t h a t f r o m M a r c h - J u n e , to 50%).  (1979)  From September-Febuary  plants,  maximum  (up  survived  but  no  meristem  survived. S e v e r a l r e d r a s p b e r r y t i s s u e c u l t u r i s t s p r e f e r t o ex-  c i s e e t i o l a t e d meristem t i p s from s p r o u t i n g r o o t ( S h c h e l k u n o v a and P o p o v , 1970; and  Z a t y k o , 1978;  Anderson  1979).  S h c h e l k u n o v a , 1974;  and H a n e r , 1 9 7 9 ) .  w i t h v i g o r o u s l y growing p l a n t s  cuttings  ( P u t z , 1971;  Kiss  Others begin Carrg et  al.,  4.  Vine  (1968)  w i t h Rosaceous  and M u l l e n e t a l .  (1974, 1 9 7 5 ) , w o r k i n g  p l a n t s , reported that meristem t i p s  f r o m some h e a t - t r e a t e d  source p l a n t s , w i l l  more  s u r v i v e , and grow more q u i c k l y t h a n t h o s e f r o m treated source p l a n t s . for  The  m e n t s w i t h one  l e a f a n d d o r m a n t a x i l l a r y bud  Stace-Smith, 1976). comparable  the  facility  s i z e o f Rubus e x p l a n t s may  S h c h e l k u n o v a , 1974;  root i n culture.  ( S h c h e l k u n o v a and P o p o v ,  e a s i l y rooted shoots  cm)  M e l l o r and S t a c e - S m i t h ,  were used, y i e l d e d  (James and T h u r b o n ,  1977;  from meristem t i p s  success i n rooting  0.05-0.06 x 0.10-0.20 mm  p u t r e s c i n e , spermedine, Of v i t a l tips  and  factors,  and  Carrg. e t a l .  shoots derived to the  c h a r c o a l removed t o x i c o x i d a t i v e p r o d u c t s a n d  All  Kiss  2% a c t i v a t e d c h a r c o a l i n t h e r o o t i n g medium.  that i t contained possible rooting  where  relatively  M e l l o r and S t a c e - S m i t h , 1976).  attributed their  who  1970;  However, m i c r o p r o p a g a t i o n o r r o o t i n g t r i a l s ,  Z a t y k o , 1978;  and than  r e p o r t e d by m o s t w o r k e r s  P u t z , 1971;  l a r g e e x p l a n t s (0.5-2.0  of  (Mellor  be i m p o r t a n t i n  w i t h which d e r i v e d shoots w i l l  began w i t h m e r i s t e m t i p s  (1979)  or nodal seg-  cuttings.  D i f f i c u l t y w i t h r o o t i n g was  1976).  reported  T h e s e a l s o r o o t e d more r a p i d l y  control  Initial  readily non-heat-  same phenomenon was  sand c u l t u r e o f r e d r a s p b e r r y shoot t i p s ,  excised  inclusion  They t h o u g h t  demonstrated  the  phenolamines,  spermine.  importance i n the regeneration of meristem  i s t h e adequacy  o f t h e c u l t u r e medium c o m p o n e n t s .  c u l t u r e media s u p p l y :  an i n o r g a n i c s a l t m i x t u r e composed  5. o f t h e m a j o r and m i n o r m i n e r a l e l e m e n t s known t o be tial  f o r p l a n t growth;  and e n e r g y ;  a source of carbohydrate f o r carbon  o r g a n i c growth f a c t o r s , u s u a l l y i n c l u d i n g  v i t a m i n s , and and  essen-  s o m e t i m e s amino a c i d s o r o t h e r  B  supplements;  phytohormones. Media d i f f e r ,  t i o n s of the major  i n t h e a b s o l u t e and r e l a t i v e c o n c e n t r a i n o r g a n i c e l e m e n t s , and i n t h e c o m p l e x i t y  of the minor element f r a c t i o n .  F o r t h e c u l t u r e o f Rubus  s p e c i e s , i n o r g a n i c s a l t m i x t u r e s t h a t have been used Morel's macroelements,  Heller's  ( S h c h e l k u n o v a and P o p o v , 1970; 1979);  M u r a s h i g e and S k o o g ' s  elements 1978;  ( P u t z , 1971;  (1953)  (1962) m a c r o e l e m e n t s  James and T h u r b o n ,  (1969) m a c r o e l e m e n t s  Zatyko, 1978).  1977;  Vertesy, and m i c r o -  Harper,  C a r r g e t a l . , 1979  ammonium n i t r a t e r e d u c e d t o 4 00 mg/L); Nitsch's  microelements  S h c h e l k u n o v a , 1974;  Broome and Zimmerman, 1978;  include  and N i t s c h  and m i c r o e l e m e n t s  When t h i s i n v e s t i g a t i o n was  with  and  (Kiss  started,  and the  g r e a t e s t s u c c e s s i n n u t r i e n t c u l t u r e o f r e d r a s p b e r r y was by P u t z  (1971);  Zimmerman ( 1 9 7 8 ) . (1962)  o f any Rubus s p e c i e s , by Broome and B o t h g r o u p s u s e d M u r a s h i g e and  Skoog's  i n o r g a n i c s a l t s , w h i c h a r e c h a r a c t e r i z e d by much  g r e a t e r amounts o f n i t r a t e ,  ammonium, c a l c i u m , and  potassium  s a l t s t h a n most o t h e r media. S u c r o s e , a t a c o n c e n t r a t i o n o f 2-4%, o f c a r b o h y d r a t e f o r c a r b o n and e n e r g y . less often.  i s the usual  source  G l u c o s e i s used  No o t h e r c a r b o h y d r a t e h a s y e t b e e n shown s u -  p e r i o r t o s u c r o s e o r g l u c o s e ( S t r e e t and S h i l l i t o ,  1977).  6. I n t h e c u l t u r e o f Rubus s p e c i e s , by P u t z  (1971),  Broome and glucose  James and  Zimmerman  a t 20-30 g/L  Shchelkunova  (1974),  Thurbon  (1978), by  sucrose  and  a t 30 g/L  (1977),  Carr§ e t a l .  S h c h e l k u n o v a and  K i s s and  Harper  Zatyko  used  (1978), (1979);  Popov  (1978),  was  (1970),  and  Vertesy  (1979) . The and  B vitamins  thiamine,  nicotinic acid,  i n o s i t o l a r e u s u a l l y i n c l u d e d , and  g l y c i n e as w e l l .  Inositol,  but  not  essential.  Rubus s p e c i e s and  (0.5), pyridoxine  thiamine  Vitamin  (mg/L):  containing  glycine  Zimmerman, 197 8 who  t o 0.4  and  mixtures  (0.5), thiamine  mg/L);  (mg/L):  (0.05),  biotin  acid  (5.0), pyridoxine  acid  (5.0)  ( K i s s and  u s e d M o r e l and  and  Skoog acid  (1965) w h i c h Thurbon,  N i t s c h and . N i t s c h glycine  (0.5), thiamine  and  t h e amount o f  (James and  Zatyko, 1978).  Muller's  (Shchelkunova  ( P u t z , 1971;  Skoog  mg/L  and  culturing  (2.0), n i c o t i n i c  and  Harper, 1978);  benefi-  M u r a s h i g e and  increased  a t 0.4  for  (1964)  (0.1)  Linsmaier  c o n s i s t s o n l y of thiamine 1977;  I t i s considered  Shchelkunova, 1974);  containing  Broome and  mg/L.  i n c l u d e t h a t of Butenko  P o p o v , 1970;  (1962),  o f t e n t h e amino a c i d  a s u g a r a l c o h o l , i s a common  s u p p l e m e n t , e m p l o y e d a t 100 cial  pyridoxine,  (1969),  (2.0),  ( 0 . 5 ) , and  nicotinic folic  Carr€ et a l .  (1979)  (1964) v i t a m i n s w h i c h i n c l u d e  a t 0.01  mg/L  nicotinic  acid, pyridoxine,  calcium  p a n t o t h e n a t e e a c h a t 1.0  the  s u p p l e m e n t s c y s t e i n e HC1  200  mg/L.  I n o s i t o l a t 100  mg/L,  a t 1.0  mg/L  was  mg/L  and  thiamine,  they  and  biotin and  included  L-glutamine  u s e d by a l l Rubus  at  7. workers  except  Shchelkunova  and  Popov  and  (197 0 ) ,  Shchelkunova  (1974) . Phytohormones of  p l a n t  are  p r o p a g a t i o n  c e n t r a t i o n s  of  p l a n t  growth  monly  used  amino and  p u r i n e  the  may  c e n t r a t i o n s a  may  used  l i q u i d  l i q u i d  medium. have  a f f e c t  The  5.0-6.0  medium.  medium A l l  been  V e r t e s y  pH  of  the  d u r i n g  5.5-5.7  (1979),  w i t h  and  agar  to  et  a l . ,  1979);  o t h e r s  to  g i v e  a  5.6-5.8  of  to  a f t e r  w e l l  as  n e c e s -  and  of  Only  Zimmerman  s t a r t e d  Rubus  (1978)  c u l t u r e s  shoots  f o r  or  determine  197 7 ) .  u s u a l l y  Rubus  to  s o l i d  s p e c i e s  a d j u s t e d  workers  (Harper, pH  may  g/L.  5-7 i s  c o n -  s o l i d i t y  as  r e p o r t e d  Some  the  v a r y i n g  e s t a b l i s h e d  a u t o c l a v i n g a d j u s t  of  Zimmerman  medium  p r e p a r a t i o n . p r i o r  Broome  at  and  (BAP);  s t a t i o n a r y  (Murashige,  and  (NAA),  6 - f u r f u r y l -  c u l t u r e  h e l d  s u r v i v a l ,  media  n u t r i e n t  be  degree  t r a n s f e r r e d  pH  pH  The  w i t h  L i q u i d  may  growth  a c i d  (GA^).  agar.  c u l t u r e  s o l i d i f i e d  a c i d  of  Broome  and  other  and  coma u x i n s ,  c y t o k i n i n s ,  s o l i d i f i e d  e x p l a n t  most the  a c e t i c  or  e x t e n t s .  are  c o n -  determine  6-benzylaminopurine  g i b b e r e l l i c  and  r e l a t i v e  Those  s p e c i e s  the  and  and  components  g i b b e r e l l i n s  naphthalene  s t r u c t u r e ,  m u l t i p l i c a t i o n  (1971),  Rubus  (K),  q u a l i t i e s  and  c u l t u r e .  (IBA);  l i q u i d ,  v a r i o u s  Putz  on  be  and  to  medium  p l a n t  k i n e t i n  support  a g i t a t e d the  a c i d  g i b b e r e l l i n ,  Media  s i t a t e  or  of  (IAA),  8-(indole-3)-butyric  i n  o r g a n i c  a b s o l u t e  c y t o k i n i n s ,  c u l t u r e  a c i d  c r i t i c a l  The  development  the  i n d o l e - 3 - a c e t i c  most  media.  a u x i n s ,  and  i n  the  p r i o r  a u t o c l a v i n g  a d j u s t  1978; to  to the  Carrg  a u t o c l a v i n g  (Broome  and  8. Zimmerman, 1 9 7 8 ) . L i g h t i n t e n s i t y , q u a l i t y , and l y i n f l u e n c e p l a n t g r o w t h and c u l t u r e i n i t i a t i o n and  photoperiod,  organized  multiplication,  1,000-3,000 l u x i s u s u a l l y a d e q u a t e .  profound-  development.  For  a l i g h t i n t e n s i t y of During the  later  stages  o f c u l t u r e 3,000-10,000 l u x p r o m o t e s s u r v i v a l and  increases  g r o w t h r a t e when t h e p l a n t l e t s a r e  soil  ( M u r a s h i g e , 1974, key  1977).  t r a n s f e r r e d to  Murashige  (1974) p o s t u l a t e d  organogenetic processes i n t i s s u e c u l t u r e are  p h o g e n i c phenomena, p r o b a b l y r e g u l a t e d This  i s substantiated  v i o l e t and  blue  by  regions  shoot growth, w h i l e  the  by  f a c t t h a t l i g h t near the  requirements of each s p e c i e s .  The  the  photo-  normal  Some, p l a n t s  more s t r i n g e n t d a y - l e n g t h r e q u i r e m e n t s t h a n o t h e r s . i n t e n s i t y u s e d i n t h e c u l t u r e o f Rubus s p e c i e s 500  lux  ( P u t z , 1 9 7 1 ) , t o 2,200-4,300 l u x  Zimmerman, 1 9 7 8 ) .  The  q u a l i t y of  (Broome  l i g h t required  f o r Rubus  species  were u s u a l l y exposed t o 16-hour p h o t o p e r i o d during  of 12-hour p h o t o p e r i o d  (Vertesy,  24-hour l i g h t e x p o s u r e  (Broome and  by  temperatures i n the  the  1 9 7 9 ) , and  Rubus both  exception  trials  with  Zimmerman, 197 8 ) .  Temperature requirements d u r i n g fulfilled  extensively.  culture, with  from  and  i n v i t r o has  g r e e n h o u s e and  been s t u d i e d  have  Light  ranges  species  i n the  not  ultra-  stimulates  l i g h t enhances r o o t i n g .  p e r i o d optimum i n c u l t u r e g e n e r a l l y r e f l e c t s photoperiod  photomor-  phytochrome.  of the energy spectrum  red  that  culture generally  20's°C.  Some s p e c i e s  r e q u i r e d i u r n a l f l u c t u a t i o n s , o r have c h i l l i n g  are may  requirements  9. (Murashige,  1977).  Rubus c u l t u r e s have b e e n i n c u b a t e d  18-29°C and have n o t been d e m o n s t r a t e d to, r e q u i r e  at  chilling,  or d i u r n a l f l u c t u a t i o n s . The the  ultimate  objective of this  s t u d y was t o d e t e r m i n e  requirements f o r meristem t i p c u l t u r e , i n v i t r o  tion, plants  and r o o t i n g o f r e d r a s p b e r r y . are not only  difficult  they are a l s o d i f f i c u l t reason, other  t o grow and t o r o o t  that r e a d i l y root  i n preliminary  experiments.  (R.  o c c i d e n t a l i s L.) was t h e f i r s t  its  readiness  to root  However, r e d r a s p b e r r y  species  and  For t h i s  from t i p c u t Black  raspberry  tested  because  from t i p c u t t i n g s suggested t h a t i t  might a l s o r o o t r e a d i l y i n c u l t u r e . loganobaccus Bailey)  i n culture,  t o grow f r o m stem c u t t i n g s .  Rubus s p e c i e s  t i n g s were u s e d  cultiva-  also roots  Loganberry  easily  (R.  f r o m stem c u t t i n g s ,  because i t i s t h e progeny o f a r e d r a s p b e r r y - t r a i l i n g  blackberry  cross,  i t was t h e n e x t s p e c i e s  a loganberry-like hybrid blackberry  (Jennings,  of red raspberry  1978),  tested.  and u p r i g h t  was t h e t h i r d  Experience gained  from i n v i t r o  was u s e d t o s o l v e  the problem o f developing  plants  i n c u l t u r e from meristem  species  c u l t u r e of these  tips.  Tayberry,  tested.  species  red raspberry  10. M a t e r i a l s and  Black raspberry c u l t i v a r Munger. source  and 139,  the  source  s e v e n new and  source  explants.  source  mature  of a l l t a y b e r r y e x p l a n t s .  s e l e c t i o n s ( 7 1 - 8 - 2 8 , 115, The  red raspberry  from dormant r o o t c u t t i n g s .  provide  One  125,  tayberry Red  rasp-  Willamette, 133,  138,  supply  Black raspberry  o f young s o u r c e  normal growing c o n d i t i o n s .  Some s o u r c e  seeds  and  plants.  exposed  p l a n t s were  O t h e r s w e r e h e a t - t r e a t e d a t 37°C, i n an  l u x ) , w i t h 16-hour  fluorescent lights  to  etiolated days  incuba(3,000  photoperiod.  I n p r e p a r a t i o n f o r d i s e c t i o n , stems were c u t s e c t i o n s and  to  Most  g r e e n h o u s e - g r o w n p l a n t s i n t h e d a r k f o r 10  t i o n chamber, under c o o l w h i t e  and  p l a n t s w e r e grown  p l a n t s w e r e g r o w n i n t h e g r e e n h o u s e and  o r more.  the  r o o t c u t t i n g s were p l a n t e d a t i n t e r v a l s  a constant  by k e e p i n g  the  p l a n t s were  p l a n t s w e r e c u l t i v a r s M e e k e r and  71-15-145).  red raspberry  p l a n t s were s e e d l i n g s o f  F i v e mature loganberry  of a l l loganberry  p l a n t was berry  source  Methods  surface s t e r i l i z e d  by  into  i m m e r s i n g i n a 10%  solu-  t i o n of household bleach  ( a c t i v e i n g r e d i e n t 5.25-6% s o d i u m  h y p o c l o r i t e ) and  f o r 15 m i n u t e s on  shaker.  shaking  a wrist-action  They w e r e t h e n g i v e n t h r e e 1 5 - m i n u t e  i n d i s t i l l e d w a t e r , and  rinse-shakes  wrapped i n m o i s t paper t o w e l s  to  await d i s e c t i o n . All  e x c i s i o n s were p e r f o r m e d under a d i s e c t i o n m i c r o -  s c o p e a t 40X  magnification.  E a c h m e r i s t e m t i p was  excised  in  t u r n from base t o stem t i p , and i t s l e n g t h , p o s i t i o n  on  t h e stem, and e x c i s i o n d a t e were r e c o r d e d .  A mounted  r a z o r b l a d e f r a g m e n t was u s e d t o p e e l b a c k t h e i m m a t u r e l e a v e s o f e a c h l e a f b u d a n d t o remove t h e m e r i s t e m t i p which  i n c l u d e d t h e m e r i s t e m a t i c dome a n d o n e p a i r o f l e a f  primordia; Meristem  total  l e n g t h l e s s t h a n 1.0 mm  (Quak,  1970).  t i p s w e r e m e a s u r e d w i t h t h e a i d o f an o c u l a r  s c a l e , t h e n q u i c k l y t r a n s f e r r e d t o c u l t u r e medium. When s o l i d medium was u s e d ,  t h e meristem  p l a n t e d on t h e c e n t e r o f t h e agar the meristem  slant.  t i p was i m -  For liquid  t i p was p o s i t i o n e d o n a f i l t e r  medium,  paper.or M i r a c l o t h  w i c k t h a t p r o t r u d e d a b o v e t h e s u r f a c e o f t h e medium. M i r a c l o t h w i c k s were p r e f e r a b l e t o f i l t e r  paper  wicks  b e c a u s e t h e y w e r e s t r o n g e r when w e t a n d w e r e more Tubes w e r e c l o s e d w i t h B a c t i - C a p a l l  polypropylene  c l o s u r e s , P a r a f i l m , o r polypropylene squares e l a s t i c bands. Glassware  absorbent.  secured  B a c t i - C a p a l l c l o s u r e s were t h e most was c l e a n e d i n a T y p h o o n g l a s s w a r e  with convenient.  washer,  u s i n g a n o r g a n i c d e t e r g e n t , a n d r i n s e d f o r two r i n s e c y c l e s in  c o l d t a p water.  New t u b e s a n d j a r s w e r e a l w a y s  washed,  a u t o c l a v e d i n d i s t i l l e d w a t e r , washed, a u t o c l a v e d i n d i s tilled  water  a g a i n , a n d washed f o r a t h i r d  mended b y Thomas a n d D a v e y The  (1975).  b a s i c medium, h e r e a f t e r d e s i g n a t e d MS, was a mod-  i f i c a t i o n o f M u r a s h i g e and Skoog's for  time,- a s r e c o m -  tobacco  protoplasts.  (1962) r e v i s e d  Usually i tcontained  t h i a m i n e HC1 (0.4 m g / L ) , a s s u g g e s t e d  medium  additional  by L i n s m a i e r and  12. Skoog  (1965).  Edamin  Media  employed  i n  hormones medium  and/or  i n d i c a t i n g  the  b a s i c  used;  which  .  at  a  of  Hormones  c o r r e c t  prepared of  and  by  p e n s i n g .  When  medium  s t i r r i n g  f o l l o w e d i n  HCl  and/or  by  a  mg/L.  (C),  sub-  The  NAA,  symbol, (N);  when  tubes  h e a t i n g ,  and  u s e d ,  Phytagar and  100  the  of  r e g u l a t o r s  a d d i n g  weighed  a n t i o x i d a n t s .  and  d i s s o l v e d  then  i n  a d j u s t e d  to  the was  pH.  F i n a l l y ,  the  medium  x  mm,  per  tube,  13  agar  added  was  then  was  volumes  growth  sometimes  medium  Bacto  and  and  p r e p a r e d ,  d e s i r e d  When  a p p r o p r i a t e  w a t e r ,  and  Each  to  u s e d , medium  3  ml  was  i n c l u d e d ,  the  medium  i t  was  was  i t  was  b e f o r e  d i s s o l v e d  d i s p e n s e d  b e -  d i s -  i n  the  d u r i n g  s t i r r i n g .  Media  were  s t e r i l i z e d  d i s p e n s i n g  i n t o  s t e r i l e  c l a v i n g  l e t t e r  a d d i t i v e  (I);  v i t a m i n s ,  f r e s h l y  the  g l a s s  adding  d i s t i l l e d  s o l v e n t s .  d i s s o l v e d  v i g o r o u s  s a l t s ,  always  s t e r i l i z i n g .  by  by  i n o s i t o l ,  i n t o  IBA,  MS,  mg/L.  sugar,  volume  of  r e p r e s e n t a t i v e  c y s t e i n e  of  were  d i s p e n s e d  (K);  i s  u s e d ,  t h e i r  of  f r e q u e n t l y - u s e d  the  symbol  o m i t t e d .  composed  sequence  medium,  and  volume  a p p r o p r i a t e  f o r e  10  s o l u t i o n s  amounts  used  a  amount  a n t i o x i d a n t  were  l a r g e  the  was  were  Each  g i v e n  hormone  k i n e t i n ,  The  Media  to  o f t e n  (B);  always  s t o c k  each  i n d i c a t e s  most  BAP, (G)  was  a d d i t i v e s . been  hormones  GA^,  other  h y d r o l y s a t e )  i n v e s t i g a t i o n  has  hormones  a r e :  t h i s  f o r m u l a t i o n  symbols  s c r i p t  ( c a s e i n  a f t e r  by  tubes  d i s p e n s i n g ,  at  m i l l i p o r e o r ,  more  5 . 5 . k g  f i l t r a t i o n commonly,  p r e s s u r e  f o r  b e f o r e  by  a u t o -  12  m i n u t e s .  When I B A was u s e d i t was d i s s o l v e d i n a s m a l l amount o f 70%  e t h a n o l , made t o v o l u m e w i t h  and  a s e p t i c a l l y dispensed i n t o i n d i v i d u a l tubes  to autoclaving.  6 weeks  (4°C).  U n u s e d medium was d i s c a r d e d  into  then  after  storage. i n g r o w t h c h a m b e r s a t 27°C,  under c o o l w h i t e f l u o r e s c e n t l i g h t s hour p h o t o p e r i o d .  their  w e r e t r a n s f e r r e d t o 100-ml j a r s ,  9-12 m l o f medium. against  (3,000 l u x ) , w i t h 1 6 -  Shoots o r p l a n t l e t s t h a t outgrew  t e s t tube containers  ported  subsequent  t o c o o l t o room t e m p e r a t u r e ,  C u l t u r e s were i n c u b a t e d  taining  water  Racks o f s t e r i l i z e d media were p l a c e d  p l a s t i c bags, allowed refrigerated  sterile distilled  con-  Shoots o r p l a n t l e t s were sup-  the sides of the j a r s with  strips of Miracloth  or chromatography paper. When p l a n t l e t s w e r e moved t o s o i l , was  high  relative  m a i n t a i n e d f o r 10 d a y s t o 2 weeks b y c o v e r i n g  a beaker, o r enclosing  humidity  them  with  p l a n t and p o t i n a p l a s t i c bag.  When u n r o o t e d s h o o t s w e r e t r a n s f e r r e d t o s o i l ,  their  bases  w e r e d u s t e d w i t h r o o t i n g hormone p o w d e r , c o n t a i n i n g 0.8% IBA,  Black  before  Raspberry  Culture To black  planting.  Media d e v e l o p a medium t h a t w o u l d s u p p o r t t h e g r o w t h o f  raspberry  meristem t i p s ,  s e v e r a l media were  w i t h d i f f e r e n t hormones a n d hormone c o n c e n t r a t i o n s .  tested In  some t e s t s , a n t i o x i d a n t s w e r e u s e d i n c l u d i n g p o l y v i n y l  pyrrolidone  (PVP)  and  c y s t e i n e HCl, both i n the media  for r i n s i n g tools during  excision.  The  media most f r e ^  quently  u s e d w e r e MS  w i t h t h e hormones BAP  and  a t 1.0  (MSB  IBA  a t 10 mg/L,  mg/L,  (MSC);  ^1^  Q  or both,  ) ;  MS  Q  (MSCB  and  a t 0.1  mg/L  with cysteine  Q  Q  ) .  HCl  Variations in  c u l t u r a l c o n d i t i o n s i n c l u d e d i n c u b a t i o n a t 20 C o r a t 27 and  t h e r o t a t i o n o f some c u l t u r e s a t 1 rpm  i n s t e a d of  C,  holding  them s t a t i o n a r y . P l a n t s a t two sources  stages  of m a t u r i t y were e v a l u a t e d  of meristem t i p s :  were v i g o r o u s ,  and  s e e d l i n g s 3-6  as  months o l d , w h i c h  u s u a l l y l e s s t h a n 50 cm  tall;  and  prostrate plants, with adventitious roots at their  mature,  tips.  M e r i s t e m t i p s were e x c i s e d o n l y from the a d v e n t i t i o u s zone o f t h e o l d e r p l a n t s . tips,  and  larger tips  i n t o e a c h o f two  From t h e s e  (1.0-2.0 and  source  p l a n t s , meristem  2.1-3.0 mm),  m e d i a , MSCB^ ^ 1 ^ ^ and  were e x c i s e d  MSCB^ g l ^ Q .  To d e t e r m i n e t h e o p t i m u m l e v e l o f t h e hormone m e r i s t e m t i p s -0.25 a t 0.1  mg/L  and  mm  In r o o t i n g experiments, MSCBQ  ^1^  Q  or  MSCB^  ^1^  shoots  t i o n s f r o m 0.0-1.0 mg/L.  of  f r o m 0.0-1.0  To  and  mg/L.  to the  a r a n g e o f IBA  determine whether  shoot bases i n the  same  darkness base  w i t h f o i l wrap t o keep  dark.  Micropropagation  two  concentra-  i n c i d e n c e or q u a l i t y of r o o t i n g , the  some c u l t u r e t u b e s w e r e c o v e r e d  BAP  developed i n media  were s u b c u l t u r e d  Q  media, o r t o media w i t h MSC  a f f e c t e d the  IBA,  were e x c i s e d i n t o media w i t h  s i x l e v e l s o f IBA,  root  t r i a l s were aimed a t d e v e l o p i n g  a  medium t h a t w o u l d s t i m u l a t e s h o o t p r o l i f e r a t i o n  i n culture.  The p r o l i f e r a t i o n m e d i a , o f w h i c h t h e r e w e r e 18  combinations,  c o n t a i n e d MSC  and IBA  at  0.0, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/L.  liquid, Leafy and  w i t h BAP a t 1.0, 3.0, a n d 5.0 mg/L  or s o l i d i f i e d  shoots  o r B a c t o a g a r a t 7 g/L.  f r o m medium MSCB^ ^ 1 ^ ^ w e r e c u t i n t o  pieces  s u b c u l t u r e d t o one o f t h e p r o l i f e r a t i o n m e d i a . A l l  p l a n t s were h a r v e s t e d m e d i a and t h e i r  a f t e r a b o u t 6 weeks i n t h e p r o l i f e r a t i o n  l e a v e s and r o o t s examined and c o u n t e d .  To e s t i m a t e  t h e p o t e n t i a l r a t e o f p l a n t i n c r e a s e by  micropropagation, old  w i t h Phytagar  The m e d i a w e r e  t h e f o l l o w i n g t r i a l was made.  rooted p l a n t l e t s from meristem t i p s ,  MSCB.^  q  Two  8-week  g r o w i n g i n medium  , e a c h w i t h 10-20 l e a v e s a n d 12-15 r o o t s , w e r e  d i v i d e d and s u b c u l t u r e d i n t h e same medium.  The  ensuing  p l a n t l e t s w e r e r e s u b d i v i d e d a t 3-week i n t e r v a l s > t h r e e times. and  A l l c u t t i n g s were t h e n h a r v e s t e d  l e a v e s w e r e e x a m i n e d and c o u n t e d .  made o f t h e number o f m i c r o p r o p a g a t e d  and t h e i r  roots  An e s t i m a t e  was  plantlets that  could  be p r o d u c e d i n 1 y e a r . To d e t e r m i n e t h e e x t e n t o f r e g r o w t h had  been severed  from shoots,  were c u t o f f below t h e l o w e s t  the shoots leaf,  from r o o t s o f seven  plantlets  and t h e s h o o t s  o t h e r p l a n t l e t s were c u t o f f j u s t above t h e l o w e s t The s e v e r e d to  medium  s h o o t and t h e r o o t p o r t i o n w e r e e a c h  MSCBQ  ^1^ Q.  that  o f two leaf.  returned  A f t e r a month, t h e r o o t s were  examined f o r r e g e n e r a t i o n o f stems and l e a v e s ; portions f o r regeneration of roots.  the top  To 15  evaluate  growth from e x c i s e d nodal  s i n g l e l e a v e s , each attached  to a small  segments,  (^1.0  cm)  sec-  t i o n o f stem, were c u t from a s i n g l e b l a c k r a s p b e r r y let,  and  returned  t o medium MSCB  ,1  .  A f t e r 10  the segments were examined f o r growth o f stems, and  days,  leaves,  roots. The  type of micropropagation  ^1^  M S C B Q  or  Q  M S C B ^  ^I^  was  t i o n i n v o l v i n g a x i l l a r y bud  that occurs  w i t h BAP mg/L  i n c r e a s e d t o 3.0  ( M S C B  3  >  0  I  Source P l a n t To  p r o l i f e r a t i o n , when p l a n t l e t s  >  1  p l a n t s taken  1  6.1  I  1  the e f f e c t of heat treatment  source  0.1  on  in culture, five  a t 1-week i n t e r v a l s f o r 5 w e e k s .  and  mm  MSCB.^  31 m e r i s t e m t i p s -0.5 p H  5  mm  ex-  E a c h week,  were e x c i s e d i n t o each of media .  B o t h media were a d j u s t e d A f t e r 8 weeks o f  l o n g were p l a c e d  to  heat-treatment,  i n t o medium  -7-  Cold Storage of In V i t r o Six rooted  survival,  p l a n t s w e r e h e a t - t r e a t e d , and  p r i o r to autoclaving.  ^ W i . o '  IBA d e c r e a s e d t o  ) .  10 m e r i s t e m t i p s -0.35 Q  and  r o o t i n g of black r a s p b e r r y  black raspberry  MSCB  mg/L,  t o a medium  Pre-treatment  explore  g r o w t h and  0  i n media.  compared t o m i c r o p r o p a g a -  from e i t h e r o f these media were s u b c u l t u r e d  pH  plant-  Plantlets  p l a n t l e t s i n v i t r o w e r e h e l d a t 10°C  in  the  17. d a r k f o r 3 m o n t h s , t h e n k e p t a t room t e m p e r a t u r e f o r 1 day  p r i o r t o i n c u b a t i o n a t 27°C.  l e a v e s were p l a n t e d  in soil  after  Those t h a t p r o d u c e d 2 weeks.  of using t h i s procedure t o preserve p l a s m was  The  new  possibility  black raspberry  germ-  evaluated.  Loganberry C u l t u r e Media A few l o g a n b e r r y placed  m e r i s t e m t i p s -0.35 mm  i n t o m e d i a MSBg ^ 1 ^ ^ o r M S B  d i l u t i o n of macroelements.  ^1^ g with a  S u r v i v i n g shoots  t u r e d t o e i t h e r o f two m e d i a ; pH  Q  MSB^  gig  1  were  were 1:2 subcul-  o r MSB^ g i g  6.1. The i n f l u e n c e o f c a r b o h y d r a t e  source  v e s t i g a t e d by e x c i s i n g 102 m e r i s t e m t i p s i n t o medium MSB^ 6.1)  o r 25 g/L g l u c o s e  (pH 5 . 7 ) .  (MSB^ g i g ^ ) .  micropropagation,  long  (pH 5.7,  to b o t t l e s with the increased  t o 3.0  Subsequent c l o n e s were used i n m i c r o -  or rooting  To e s t i m a t e  0.05-0.50 mm  Tips that survived to the  same medium, o r w i t h BAP c o n c e n t r a t i o n  propagation  and pH was i n -  g i g ^ w i t h e i t h e r 30 g/L s u c r o s e  s m a l l shoot stage were s u b c u l t u r e d  mg/L  long  experiments.  t h e p o t e n t i a l r a t e o f p l a n t i n c r e a s e by t h e f o l l o w i n g t r i a l was made.  tem t i p s i n medium MSB^  Two  meris-  g i g ^, pH 6.1, and two i n g l u c o s e  (25 g/L) medium, w e r e t r a n s f e r r e d t o medium MSB^ g i g pH 5.7.  A f t e r 6 and 12 weeks r e s p e c t i v e l y , t h e f o r m e r  18. p a i r  of  m e r i s t e m  d i v i d e d  the  a f t e r  weeks;  6  t i p s  had  f i r s t , t i m e ;  developed the  subsequent  l a t t e r  i n t o two  d i v i s i o n s  c l o n e s c l o n e s  were  at  and  were  were  d i v i d e d  t h r e e  4-week  i n t e r v a l s . In  r o o t i n g  s t a r t i n g w i t h and  medium  g l u c o s e , 2  were  i n  R o o t i n g from at  25  The i n  w i t h  were mg/L, 5.7  MSIg w i t h  v i t r o  ment  and  5.7  a f t e r  i n  p l a n t i n g  l e a v e s . i n  l a s t  medium  was  s u c r o s e , 6  or  weeks,  more  t h r e e  r o o t i n g  medium.  c o n c e n t r a t i o n g/L  or  g l u c o s e  the to  i n  MSB^ from  time  the  i n  each time  group,  c l o n e was  f o r  p l a n t e d  s u b d i v i d e d were  noted f o r  used f o r  r o o t i n  d e v e l o p the  p r o d u c t i o n  was  of  compared  A f t e r i n  v i t r o ,  r o o t i n g  were  C u t t i n g s  v i t r o  f i v e  ^  to  i n  greenhouse.  r e q u i r e d  greenhouse. were  the  gig  p l a n t i n g .  R o o t i n g  c u t t i n g s  compared  p e r i o d i c a l l y  c u t t i n g s  the  t h r e e  each  30  w i t h  m e d i a ,  IBA  at  s o i l  i n i t i a t i o n  examined  r o o t e d  w i t h  medium  p r e c e e d i n g  source i n t o  from  t h i s  QIQ  h a v i n g  was  i n  c u t t i n g s  Root  c u t t i n g s  the  l o g a n b e r r y  6.1,  powder  p r i o r  of  v i t r o ,  or  e l o n g a t i o n  new  p l a c e d  r o o t i n g  as  and  r o o t i n g  w i t h  of  r o o t i n g  were  four  MS  MSB^  f o r  p i e c e s  the  were  p l a n t l e t s ,  greenhouse r o o t s  i n t o  1  14  6.1.  Twenty  i n  medium  s u c r o s e  pH  treatment.  from  and  c u t t i n g s .  each  s u c r o s e ,  of  c l o n e s ,  i n  c l o n e s ,  composed  or  ^,  i n  l o g a n b e r r y  g l u c o s e  of  t u b e ,  l o g a n b e r r y  i n t o  been  each  e f f i c i e n c y  c u t t i n g s  i n  per  pH  medium  Two  From  media  g/L,  having  24  w i t h  ^  a s e p t i c a l l y  one  0 . 0 - 1 . 0  gig  medium  each.  c u t t i n g s ,  MSB^  7  t h i s  d i v i d e d  l e a v e s  e x p e r i m e n t s ,  s o i l .  p l a n t l e t s  2  and  4  One from  to weeks month  each  o f t h e t h r e e g r o u p s w e r e removed f r o m s o i l ; h e i g h t , number o f l e a v e s , and  average  weight,  number o f m a i n r o o t s w e r e  compared f o r each group.  Source P l a n t To and  Pre-treatment  i n v e s t i g a t e the p o s s i b i l i t y of speeding  r o o t i n g of loganberry  cultures, five  p l a n t s w e r e g r o w n a t 37°C f o r 1-8 weekly. o f two  growth  loganberry  w e e k s and  source  explants  taken  E a c h week t e n m e r i s t e m t i p s w e r e e x c i s e d i n t o media;  MS  without  without  QI  6.1.  Those p l a c e d  Q  i n medium  i n o s i t o l o r hormones w e r e moved t o t h e o t h e r medium  the time of the f i r s t  of heat-treatment  subculture  of source  Cold Storage  of In V i t r o  Twenty-seven r o o t e d k e p t a t 10°C into s o i l  The  two  m e d i a was  effect and  evaluated.  Plantlets loganberry  i n the dark,  plantlets in vitro  f o r 2 months, then p l a n t e d  i n the greenhouse.  f o r t h i s p e r i o d was  ving loganberry  (28 d a y s ) .  p l a n t s on t h e s u r v i v a l  growth of t i p s c u l t u r e d i n these  storage  each  i n o s i t o l o r h o r m o n e s , and MSB^  B o t h m e d i a w e r e a d j u s t e d t o pH  at  up  The  were  directly  e f f e c t i v e n e s s of c o l d  evaluated  a s a means o f  preser-  germplasm.  Tayberry C u l t u r e Media F i v e t a y b e r r y m e r i s t e m t i p s w e r e e x c i s e d i n t o medium  20. MSB^  gig  pH  ^,  6.1,  and  medium,  except  s u c r o s e  and  pH  a d j u s t e d  the  were  was  t r a n s f e r r e d  Shoots  were  g l u c o s e , To  to  5.7  and  e s t i m a t e  s t a r t e d  MSB  12  medium  weeks,  s u c r o s e .  F u r t h e r  an  e s t i m a t e  was  be  produced  i n  Rooting to  r o o t i n g  medium i n  MSB^  s o i l .  1  a f t e r  r a t e  w i t h  medium  were  made of  c u l t u r e . w i t h  01  pH  i n c r e a s e  0.15  to  shoots  s u c r o s e ,  p l a n t  same  g/L)  of  l  Q  w i t h  g l u c o s e ,  number  (25  months  t i p  the  t a y b e r r y  MSB^  of  meristem  the  2  Q I Q  i n t o  g l u c o s e  medium  MSB^  s u b d i v i s i o n s of  by  S u r v i v i n g  j a r s  ^  e x c i s e d  mm  was MSB^  at  5.7. by  l o n g , s u b d i v i d e d Q I Q ^  w i t h  i n t e r v a l s  c u t t i n g s  t h a t  ,  was  and c o u l d  year. v i t r o  i n  medium  F i f t e e n  were  c u t t i n g s  dusted of  r o o t e d  MSI^  w i t h  c u t t i n g s  i n  pH  from  r o o t i n g from  the  f o r  r e d  5.7 a  compared  p l a n t l e t  powder  i n  and  p l a n t e d  m i c r o p r o p a g a t i o n  v i t r o .  Raspberry  C u l t u r e  Media  P r e l i m i n a r y t i p  gl^  1  b a l a n c e  were  5.7.  t r a n s f e r r e d  s o i l .  Q I Q  The  s i n g l e  made  i n  i n  experiment  Red  and  r e p l a c e d  p o t e n t i a l  a  were  between  medium  the  t i p s  to  100-ml  m i c r o p r o p a g a t i o n ,  a f t e r  was  d i s t r i b u t e d  pH  i n  10  c u l t u r e  at  t h r e e  To  e s t a b l i s h  i n i t i a t e  t r i a l s  i n c l u d e d  d i l u t i o n s ,  the  the  w i t h  m a c r o n u t r i e n t s and  BAP  c o r r e c t  growth  media  of  and  hormone  red  IBA  at  f u l l  each  at  r a s p b e r r y s t r e n g t h , s e v e r a l  c o n c e n t r a t i o n  r a s p b e r r y  meristem  meristem  i n  and l e v e l s .  which  t i p s ,  6:  to  m e r i s t e m t i p s -0.25 mm  l o n g were p l a c e d  i n t o e a c h o f 35  m e d i a w i t h BAP a t 0.1, 0.3, 0.5, 0.7, 1.0, 3.0, and mg/L  a n d I B A a t 0.1, 0.3, 0.5, 0.7, a n d 1.0 mg/L.  m e d i a w e r e a d j u s t e d t o pH 5.7. all  and c o l o r a n d g r o w t h  evaluated. To e v a l u a t e  meristem t i p s ,  s e v e r a l s t a r t i n g m e d i a , more t h a n 8 0  t h e m a j o r i t y -0.25 mm  i n t o each o f media MSB  l o n g , were e x c i s e d  Q I Q ^ and MSB^  1  t h i s number o f t i p s w e r e i n i t i a t e d  a t t h e t i m e o f .the f i r s t  I  Q  ^.  Three times  or without  s u b c u l t u r e t h e s e were  i n t o m e d i a w i t h hormones a n d i n o s i t o l QIQ ^).  0  i n medium MS w i t h no  h o r m o n e s , w i t h i n o s i t o l a t 100 mg/L,  MSB^  A l l  A f t e r 6 weeks i n c u l t u r e ,  s u r v i v i n g t i p s were h a r v e s t e d ,  were  5.0  inositol; distributed  (MSB^ ^ I Q ^ and  E f f e c t s on s u r v i v a l and g r o w t h w e r e  recorded.  To d e t e r m i n e t h e o p t i m u m pH o f t h e medium, e i g h t m e r i s t e m t i p s -0.25 mm m e d i a , MSB^  I  Q Q  1  l o n g w e r e e x c i s e d i n t o e a c h o f two  and MSB^  I  2  , adjusted  5.9, 6.1, o r 6.3 p r i o r t o a u t o c l a v i n g . after  t o pH 5.5,  5.7,  T i p s were examined  30 and 60 d a y s , and t h e i r a p p e a r a n c e and g r o w t h w e r e  compared.  T h i s e x p e r i m e n t was p e r f o r m e d t w i c e , a n d t h e  r e s u l t s f r o m t h e two t r i a l s w e r e a v e r a g e d . To e x a m i n e t h e e f f e c t s o f v a r y i n g t h e t y p e and c o n c e n t r a t i o n o f s u g a r i n t h e medium, s u c r o s e or glucose  a t 20 o r 30 g/L,  a t 20, 30, o r 40 g/L w e r e u s e d i n medium MSB^  Ten m e r i s t e m t i p s -0.375 mm of the f i v e media.  l o n g were e x c i s e d i n t o  T i p s w e r e m e a s u r e d a t 30-day  QI  each  intervals.  S u r v i v a l and g r o w t h r a t e w e r e c o m p a r e d f o r m e r i s t e m  tips  22. i n  each  and  sugar  the  c o n c e n t r a t i o n .  r e s u l t s  from  F o l l o w i n g  the  were  r e - t e s t e d  by  MSB  1  Q I  pH  5.7  pH  w i t h  25  g/L.  e x c i s i n g  determine  no  of  red  pH  at  0,  50,  at  10-day  i n t e r v a l s  of  i n o s i t o l  hormones,  i n  To  induce  i n  i n  s e v e r a l c u t t i n g s ,  at  i n t o  and  medium  a d j u s t e d  media  the  each  i n w i t h  or  to  The  of  or  t i p s  month  of medium  measured comparison  repeated  r e s u l t s  at  i n o s i t o l  d i r e c t  s u c r o s e  medium  e i t h e r  were  was  a x i l l a r y  ^ 1 ^ ^ pH  i n f l u e n c e  one  ^,  Q  i n t o  as  t w i c e  the  were  c a r b o -  combined  s o u r c e .  u s u a l l y medium more  or  6.1  i n c r e a s e d  of  buds,  c o n t a i n i n g  5.7  g l u c o s e  s t a r t i n g  1  meristem  g l u c o s e  w i t h  u s i n g  w i t h  o l d ,  A f t e r  f a c i l i t a t e  times  g/L,  or  MSB^  experiment  MSB^  25  8-16 weeks  l e a v e s ,  to  g/L,  to  gI  i n  e x c i s e d  t r a n s f e r r e d  A l l  medium.  g/L  30  determine  shoots  T h i s  medium  g l u c o s e  at  o r d e r  p r o l i f e r a t i o n  m i c r o p r o p a g a t i o n  two  repeated  l e v e l s  i n o s i t o l  30  5.7.  were  carbohydrate  to  To  i n  the  g/L  s u c r o s e  to  medium  t h r e e  30  w i t h  or  and  each  shoots  pH  t i p s  were  at  150 mg/L.  e f f e c t s .  source  t i p s  or  100, or  g l u c o s e ,  l e a f y  a d j u s t e d  f o r  t i p s  s u c r o s e  shoots  w i t h o u t  for  two  g/L,  25  requirements  w i t h  s u r v i v i n g  hydrate  at  was  averaged.  t r i a l s ,  r a s p b e r r y ,  was  MS  u s i n g  were  140 m e r i s t e m  g l u c o s e  the  i n o s i t o l  The  c u l t u r e ,  experiment  6.1.  c u l t u r e s  MS  t r i a l s  carbohydrate  c o n t a i n i n g  1  or  To t i p  q  both  T h i s  at  25  h e a l t h y ,  sucrose  were  BAP g/L,  t r a n s f e r r e d  (MSB^ pH  l e v e l s  1.0-2.0  cm  l e a v e s ,  long  ^ 1 ^ ^, from  QIQ or  5.7  hormone  MSB^  at  and  f i v e  on  ^) 6.1.  r o o t i n g ,  w i t h t h r e e c l o n e s  t h a t  23. had b e e n i n medium MSB^  g l ^ ^ f o r the preceeding four  weeks, were t r a n s f e r r e d t o r o o t i n g media w i t h e a c h a t 0.0, 0.1, 0.3, 0.5, 0.7, a n d 1.0 mg/L. 11 hormone c o m b i n a t i o n s i n t h e r o o t i n g m e d i a . rooting  I B A o r NAA, There  were  Time f o r  t o b e g i n , and r o o t q u a l i t y were compared f o r b o t h  s h o o t s and c u t t i n g s . To d e t e r m i n e w h e t h e r  d i v i s i o n o r a c t u a l c u t t i n g up  o f c l o n e s f r o m medium MSB^ more q u i c k l y , o r w h e t h e r  ^ I Q^ induced roots  t o form  l e v e l o f BAP i n t h e s h o o t  growth  medium was more i m p o r t a n t , a s e c o n d r o o t i n g e x p e r i m e n t was done. of  The e f f e c t o f I B A l e v e l s f r o m 0.0-1.0 mg/L o n r o o t i n g  l a r g e r c u t t i n g s was a l s o t e s t e d .  two  s o u r c e m e d i a MSB^ I Q  i n t o segments w i t h  Q  and MSB  1  1  Three c l o n e s from t h e I  0  were each  Q1  t h r e e o r more l e a v e s .  Four  divided  cuttings  f r o m e a c h o f t h e two s o u r c e m e d i a w e r e t r a n s f e r r e d t o e a c h r o o t i n g medium.  Appearance  of f i r s t  roots  and r o o t  quality  w e r e e v a l u a t e d i n e a c h r o o t i n g medium f o r e a c h o f t h e two sources of c u t t i n g s . The  e f f i c i e n c y of rooting red raspberry cuttings i n  v i t r o was c o m p a r e d t o r o o t i n g c u t t i n g s w i t h in  soil  i n t h e greenhouse.  m e d i a KSB^ I Q  Q  ^ o r MSB^  rooting  powder  T h i r t y - f i v e c u t t i n g s each,  Q 0 1 I  w  e  r  e  P  l  a  MSI_  j . . Twenty c u t t i n g s o f comparable  with  r o o t i n g hormone a n d p l a n t e d  into  c  e  d  into rooting  from medium  s i z e were d u s t e d soil.  Source P l a n t Pre-treatment The  e f f e c t o f e t i o l a t i o n o f s o u r c e p l a n t s on s u r v i v a l  o f m e r i s t e m t i p s i n c u l t u r e was d e t e r m i n e d by e x c i s i n g  meristem  t i p s from p l a n t s t h a t had been grown i n d a r k n e s s  f o r a t l e a s t 10 d a y s .  S u r v i v a l o f t h e s e t i p s was c o m p a r e d  t o s u r v i v a l o f t i p s from greenhouse-grown p l a n t s . growth  after  etiolated The meristem  Average  30 a n d 60 d a y s i n c u l t u r e was r e c o r d e d f o r  and c o n t r o l t i p s  i n each o f s e v e r a l media.  e f f e c t s o f h e a t - t r e a t m e n t o f s o u r c e p l a n t s on t i p s u r v i v a l and g r o w t h  r a t e were e v a l u a t e d by  e x p o s i n g s o u r c e p l a n t s t o 37°C f o r 1-8 w e e k s .  E a c h week,  10 t i p s w e r e e x c i s e d i n t o medium MS w i t h o u t i n o s i t o l o r h o r m o n e s , and 10 t i p s i n t o medium MSB^ the time of the f i r s t  subculture, tips  Q  I  0  ^, pH 6.1.  At  f r o m medium MS  with-  o u t hormones and i n o s i t o l w e r e t r a n s f e r r e d t o medium w i t h both  (MSB-j^ Q I Q ^) . Meristem  S h o o t s w e r e m e a s u r e d a t 30-day  t i p survival  t i p s f r o m two s o u r c e s .  intervals  i n two m e d i a was c o m p a r e d u s i n g  T i p sources included potted p l a n t -  l e t s d e r i v e d f r o m c u l t u r e , and c o n t r o l p l a n t s r a i s e d root cuttings.  T i p s w e r e p l a c e d i n t o medium M S B  1  Q I  from Q  1  w i t h s u c r o s e a t 30 g/L o r g l u c o s e a t 25 g / L , pH 5.7.  Cold Storage of In V i t r o To e v a l u a t e c o l d  Plantlets  s t o r a g e a s a means o f p r e s e r v i n g r e d  r a s p b e r r y g e r m p l a s m , 12 r o o t e d r e d r a s p b e r r y p l a n t l e t s i n v i t r o w e r e k e p t a t 10°C i n t h e d a r k f o r 3 m o n t h s , t h e n planted d i r e c t l y  into soil  i n t h e greenhouse.  25.  Results  Black  Raspberry  Culture  Media  M e r i s t e m t i p s e x c i s e d i n t o m e d i a MS  (thiamine at 0.1  mg/L) w i t h g l u c o s e a t 4 0 g / L , a g a r a t 8 g / L , BAP a t 0 . 1 o r 1 . 0 mg/L, a n d I B A a t 1 . 0 mg/L a n d MS  ( t h i a m i n e a t 0 . 1 mg/L)  w i t h BAP a t 0 . 1 mg/L, o r K a t 0 . 0 4 mg/L, I A A a t 0 . 5 mg/L, and  GA  3  a t 0 . 1 mg/L,  survive.  incubated  a t 2 0°C o r a t 2 7 ° C d i d n o t  T i p s t u r n e d brown o r b l a c k a f t e r a s h o r t t i m e i n  c u l t u r e , a n d many c u l t u r e s became c o n t a m i n a t e d .  The m o s t  p r o m i s i n g c u l t u r e s i n i t i a l l y were t i p s  medium,  at 27°C.  i n liquid  L i q u i d medium c a u s e d t i p s t o s w e l l ,  e l o n g a t i o n was o b s e r v e d .  F i l t e r paper  a n d some  supports dispersed  to a l i m i t e d e x t e n t a p u r p l i s h pigment from t h e t i p s .  On  s o l i d medium t h i s m a t e r i a l was n o t d i s p e r s e d , a n d a p p e a r e d to i n h i b i t  growth.  On medium  MSKQ  14/52  Q^,  meristem  t i p s produced  rooted  p l a n t l e t s b u t t h e r a t e o f g r o w t h was e x t r e m e l y s l o w and d e v e l o p m e n t was e r r a t i c .  A f t e r 5 months o f c u l t u r e ,  t i p s had n o t d e v e l o p e d a t a l l , and r e a d y f o r p l a n t i n g When m e r i s t e m  (Fig.  some  w h i l e some w e r e w e l l r o o t e d 1 ) .  t i p c u l t u r e s i n medium MSB^ Q I ^ Q G Q ^  w e r e r o t a t e d a t 1 rpm, t i p s became l o d g e d o n t h e s i d e s o f t h e t e s t t u b e s and d r i e d o u t , and a l l 3 5 t i p s became c o n taminated.  Due t o t h e d i f f i c u l t y o f m a i n t a i n i n g t i p s i n  s o l u t i o n , r o t a t i n g c u l t u r e s were i n a p p r o p r i a t e a t t h i s  Figure  1.  M e r i s t e m t i p s o f b l a c k r a s p b e r r y a f t e r 5 months o f c u l t u r e i n medium MS w i t h k i n e t i n a t 0.04 mg/L, showing s l o w , e r r a t i c d e v e l o p m e n t .  27.  stage tips not  of culture.  W h i l e none o f t h e s t a t i o n a r y c o n t r o l  s u r v i v e d i n t h i s medium  (Table  1) c o n t a m i n a t i o n  was  a problem. Antioxidants,  s u c h a s PVP (0.5-2%) i n c o r p o r a t e d  into  t h e a b o v e medium, a n d PVP o r c y s t e i n e H C l u s e d d u r i n g e x c i s i o n , were n o t u s e f u l ;  however, c y s t e i n e H C l i n c o r p o r a t e d  i n t o t h e medium a t 10 mg/L p r o m o t e d s u r v i v a l a n d g r o w t h o f meristem t i p s  (Table  1).  G i b b e r e l l i c a c i d a t 0.1 mg/L  caused c h l o r o s i s o f developing  plantlets.  When t h e c o n c e n -  t r a t i o n o f BAP i n t h e medium was r e d u c e d t o 0.1 mg/L, t h e number o f s u r v i v i n g m e r i s t e m t i p s  increased  (Table 1 ) .  Young, s u c c u l e n t p l a n t s were p r e f e r a b l e t o o l d e r p l a n t s f o r source  material;  was  2).  higher  (Table  s u r v i v a l o f meristem  Meristem t i p s from o l d e r  p l a n t s sometimes formed c a l l u s . source  plants, decreasing  tips source  W i t h b o t h young and o l d  t h e c o n c e n t r a t i o n o f BAP f r o m  1.0 t o 0.1 mg/L, o r i n c r e a s i n g e x p l a n t number o f t i p s t h a t s u r v i v e d  size,  increased the  (Table 2 ) .  I n d o l e b u t y r i c a c i d was b e s t a d d e d s u b s e q u e n t t o a u t o claving. (0.05  Autoclaving  level).  IBA s i g n i f i c a n t l y  altered i t s effects  When I B A a t 1.0 mg/L was a d d e d a f t e r  auto-  c l a v i n g medium MSCBg ^, l e a f d e v e l o p m e n t was g o o d , a n d 10/30  shoots  rooted.  sterilization; not  R e s u l t s were n o t enhanced by f i l t e r -  7/30 s h o o t s  r o o t e d , b u t s h o o t g r o w t h was  as e x t e n s i v e as t h a t i n autoclaved  added a f t e r a u t o c l a v i n g . medium, s h o o t s  were v e r y  medium, w i t h I B A  When I B A was a u t o c l a v e d  with the  s m a l l a n d o n l y 3/30 s h o o t s  rooted.  28. T a b l e 1.  The e f f e c t o f t h e hormones BAP a n d GA^, a n d t h e a n t i o x i d a n t s PVP a n d c y s t e i n e H C l , on s u r v i v a l of black raspberry meristem t i p s i n medium MS w i t h IBA a t 1.0 mg/L  Hormones BAP (mg/L)  1.0 1.0 1.0 1.0 1.0 0.1  GA^ (mg/L)  0.1 0.1 0.1 -  Antioxidants PVP  Cysteine  (mg/L)  (mg/L)  . 5-20x10 -  10 10 10  No. o f t i p s HCl  excised  58 50 56 24 256 156  survived  0 0 9 0 56 54  29. T a b l e  2.  BAP*  The e f f e c t of e x p l a n t s o u r c e , s i z e , c o n c e n t r a t i o n on s u r v i v a l of b l a c k meristem t i p s  Tip  s o u r c e * *  No.  of  e x p l a n t s  (mg/L)  s u r v i v e d / n o .  t i p ^1.0  s i z e  1 . 1 - 2 . 0  1.0 1.0  stage stage  1 2  56/256 5/138  39/72 2/14  0.1 0.1  stage stage  1 2  54/156 1/27  43/67  Medium BAP at  was 0.1  MS or  w i t h 1.0  c y s t e i n e mg/L.  and BAP r a s p b e r r y  H C l ,  IBA  at  e x c i s e d  (mm) 2 . 1 - 3 . 0  9/12  21/30  1.0  mg/L,  and  Stage 1 t i p s were from young, s u c c u l e n t p l a n t s 15-50 cm t a l l . Stage 2 t i p s were from the a d v e n t i t i o u s r o o t zone of o l d e r p l a n t s t h a t had developed a p r o s t r a t e h a b i t .  A f t e r  t h i s  a f t e r  the  medium  was  When  l e v e l s  of  mg/L,  1.0 i n  each  s o i l ,  the  were  were  A l m o s t i n  and  u s e d ,  hormone  of t h a t  were (IBA,  ( 0 . 5 ) ,  among  r a t e  i n i t i a t e d  a l l  ( 9 4 % ) of  the  t r a n s f e r r e d  from  (Table  R o o t i n g  mg/L  or  1.0  mg/L  medium  i n  tubes  M S C B  N  o m i t t e d ,  and  (Table  by  3 ) . the  the  bases  f u l l y n  i n  t h a t  s u b c u l t u r e was  had  new  o f t e n  i f  and  0 . 0 r o o t e d  t h e s e  ( 0 . 1 ) ,  and  ( 1 . 0 ) ,  media was  q u i c k l y ,  to  8 / 4 3 ;  5 / 4 1 ;  growth  were  i n c r e a s e d  as  the  l e v e l s  l e v e l  of  the or  the  t e s t  d a r k , time to  been  d i d  of  the i n  were  not  l i g h t . c u l t u r e  r o o t e d  medium. o b s e r v e d .  The  w i t h  f o i l  any  Some  w i t h i n  to  a  to  v e r y  s t i m u l a t o r y  and  r o o t i n g mg/L.  1.0 wrap  to  d i s c e r n a b l e  s h o o t s  c l o s e  0 . 1  0.0  the  compared  f o r  medium  medium  t e s t e d ,  have  r o o t i n g ,  to  between  when  IBA  tube  s h o o t s  r e d u c e d  v a r i e d  of  a l l  from  was  f a s t e s t  s u b c u l t u r e d  ^ 1 ^ ^  s h o o t s  l e v e l s  when  and  Q  M S C B ^  BAP  h i g h e s t  r o o t s ,  to  IBA  of  was  exposed  f o r m i n g  s u b c u l t u r e  ( 0 . 0 ) ,  ^ 1 ^  Q  R o o t i n g  base  i n c i d e n c e  , 1 ,  w i t h o u t  3 ) .  was  on  from  t r a n s f e r r e d  r o o t e d  medium  i n c r e a s e d  shoot  of  more  M S C B  1 . 0 mg/L  c o n t a i n e d  e f f e c t  medium,  at  C o v e r i n g keep  s u r v i v e d  i n  s h o o t s  when  BAP  added  i n c r e a s e d .  r o o t e d  w i t h  always  v a r i e d  ( 0 . 7 ) ,  e x p l a n t s  s t a r t i n g  0  t h a t  3 / 4 4 ;  d i f f e r e n t ,  r o o t i n g  were  mg/L):  b e s t  ^ 1 ^  was  e v e n t u a l l y  the  MSCBQ  i t  IBA  e x p l a n t s  f o l l o w i n g  s i g n i f i c a n t l y  IBA  the  r o o t i n g  and  I B A was  a u t o c l a v e d .  5 / 4 4 ;  W h i l e  s l i g h t l y , of  the  ( 0 . 3 ) ,  9/4 3.  when  number  medium,  6/44;  not  t r i a l ,  to i n  s h o o t s medium  4  months  few  days  e f f e c t  of  31. T a b l e 3.  E f f e c t o f BAP c o n c e n t r a t i o n i n s t a r t i n g medium, and BAP and I B A c o n c e n t r a t i o n i n r o o t i n g medium, on r o o t i n g o f b l a c k r a s p b e r r y s h o o t s d e r i v e d from meristem t i p s  S t a r t i n g medium* BAP (mg/L)  1.0 1.0 1.0 1.0 1.0  0.1 0.1 0.1 0.1 0.1  *: **:  Rooting BAP (mg/L)  medium** No. r o o t e d / n o . IBA (mg/L)  1.0 0.1  1.0 1.0  —  0.1 - 0.7 1.0  -  -  1.0 0.1  1.0 1.0  —  0.1 - 0.7 1.0  -  shoots  -  9/22 3/3 8/10 9/9 24/34  41 100 80 100 71  53/78  68  13/22 47/50 1/3 8/12 1/3  59 94 33 67 33  70/90  78  S t a r t i n g medium was MSC w i t h I B A a t 1.0 mg/L a t 0.1 o r 1.0 mg/L.  a n d BAP  R o o t i n g medium was MSC w i t h BAP a t 0.0, 0.1, o r 1.0 mg/L a n d I B A a t 0.0-1.0 mg/L.  32. Micropropagation t r i a l s  demonstrated that r a i s i n g the  l e v e l o f BAP i n t h e medium g a v e r i s e t o l e a f i e r minimized the tendency t o r o o t . took place  i n l i q u i d media MSCB  s h o o t s and  The b e s t p r o l i f e r a t i o n 1  Q  I  ^  Q  (Table 4 ) .  Q  These media a l s o promoted r o o t growth.  Growth o f c u t t i n g s  i n l i q u i d medium u s u a l l y s u r p a s s e d t h a t  i n solid  M i c r o b i o l o g i c a l Bacto agar i n h i b i t e d r o o t i n g When t h e p o t e n t i a l r a t e o f p l a n t  w e r e made.  Two 8-week o l d b l a c k  f i v e c u t t i n g s each.  (Table 4 ) .  increase  p r o p a g a t i o n was i n v e s t i g a t e d t h e f o l l o w i n g raspberry  by m i c r o -  observations plantlets yielded  A t 3-week i n t e r v a l s , two f u r t h e r  d i v i s i o n s y i e l d e d 2 5 , t h e n 50 c u t t i n g s .  After  c u l t u r e , t h e 50 c u t t i n g s h a d 4-35 l e a v e s  a n d 1-20  each. The  These p l a n t l e t s were d i v i s i b l e  most c o n s e r v a t i v e  obtainable per  sub-  3 weeks o f roots  i n t o 3-10 p i e c e s  each.  e s t i m a t e o f t h e number o f c u t t i n g s  i n 1 year involves d i v i s i o n i n t o three  cuttings  p l a n t l e t e v e r y t h i r d week f r o m t h e e i g t h week o n w a r d s .  This  y i e l d s 1.4 3 x 10  T h e s e c a n be p l a n t e d I t seems l i k e l y  rooted  cuttings.  c u t t i n g s b y t h e end o f 1 y e a r .  out immediately with  negligible losses.  that propagation of t h i s sort could  carried out i n d e f i n i t e l y , While extensive  y i e l d i n g vast  numbers o f  r o o t i n g o r g r o w i n g i n t h i s medium. i n some o l d e r miniature  rooted  An o d d i t y  difficulty that  appeared  c u l t u r e s , i n t h e m i c r o p r o p a g a t i o n t r i a l s , were  white structures  rudimentary  be  p r o l i f e r a t i o n i n medium MSCBg ^ 1 ^  was n o t i n v e s t i g a t e d , c u t t i n g s h a d no a p p a r e n t  be  medium.  flowers.  (Fig. 2).  These were t h o u g h t t o  (  33. T a b l e 4.  BAP  IBA  (mg/L)  1.0 1.0 1.0 1.0 1.0 3.0 3.0 5.0 5.0  *:  The e f f e c t s o f BAP a n d I B A c o n c e n t r a t i o n i n l i q u i d o r s o l i d medium o n new l e a f a n d r o o t formation of black raspberry shoots  Medium*  No. o f c u t t i n g s  (mg/L)  0.0-0.7 0.0-0.7 1.0 1.0 1.0 0.0-1.0 0.0-1.0 0.0-1.0 0.0-1.0  No. o f c u t t i n g s w i t h new l e a v e s  L P L P B L P L P  22 13 20 20 20 19 14 15 2  17 8 18 12 20 12 4 15 2  roots  10 4 7 8 0 6 1 2 0  Medium was composed o f MS; l i q u i d (L) o r s o l i d i f i e d w i t h P h y t a g a r (P) o r B a c t o a g a r ( B ) .  Figure  2.  M i n i a t u r e w h i t e s t r u c t u r e s formed i n some b l a c k r a s p b e r r y c u l t u r e s w h i c h were t h o u g h t t o be rudimentary flowers.  35. D e c a p i t a t i o n experiments p o t e n t i a l of severed severed  illustrated  r o o t s and  shoots.  n o t p r o d u c e new  one  o r more l e a v e s w e r e r e t a i n e d , new  the severed  stem.  I f t h e s t e m was  s t a l k and  new  severed  had  p r o d u c e d new  F o u r o f t h e 15 n o d a l  r o o t s by t h i s t i m e ;  grew f r o m  raspberry  ^ 1 ^ Q , when e x a m i n e d  leaves from t h e i r produced  axillary substantial  r o o t e d 2 weeks l a t e r .  demonstrated t h a t i t i s p o s s i b l e to improve  e f f i c i e n c y of b l a c k r a s p b e r r y m i c r o p r o p a g a t i o n  by  reducing  BAP  cuttings.  p l a n t l e t s f r o m medium MSC  at either  o r 1.0  w i t h IBA  at  1.0  developed  much  axillary  g r o w t h when t r a n s f e r r e d t o medium MSB^  QI-^ Q and  they  could  subsequently  0.1  nodal  mg/L  be  and  leaf  This the  the s i z e of the b l a c k raspberry c u t t i n g s , u t i l i z i n g  S h o o t s and  severed  t o grow.  M S C B Q  s e g m e n t s i n s t e a d o f t h e u s u a l 3-5  the  In a l l cases,  s e c t i o n s had  a l l had  When  u s u a l l y f o r m e d on  continued  r e t u r n e d t o medium  a f t e r 10 d a y s ,  mg/L  was  above t h e p o i n t  15 n o d a l s e g m e n t s c u t f r o m a b l a c k  p l a n t l e t and  trial  shoot(s)  l e a v e s emerged.  s h o o t s q u i c k l y r o o t e d and  bud.  When t h e s t e m  l e a v e s w i t h i n t h e n e x t month.  o f a t t a c h m e n t t o t h e r o o t s , a bud  All  regeneration  below the p o i n t of attachment to the r o o t s , r o o t s  did  severed  the  d i v i d e d i n t o many c u t t i n g s .  The  p l a n t l e t s d e t e r i o r a t e d i n medium w i t h h i g h BAP g r o w t h was  substantial.  although  top  C u t t i n g s from these p l a n t l e t s  u n l i k e l y t o r o o t i n medium w i t h e l e v a t e d BAP. reason,  roots of  the type of p r o l i f e r a t i o n achieved  For  were  this  by r e p e a t e d  s i o n o f p l a n t l e t s i n m e d i a MSCB^ ^ 1 ^ ^ o r MSCB.^ ^ 1 ^ Q  diviwas  s u p e r i o r t o a x i l l a r y bud p r o l i f e r a t i o n a c h i e v e d t o medium MSB^  Q  I  s i n c e t h i s i n v o l v e s more t h a n  1  medium, and d i s c o u r a g e s  Source P l a n t  by t r a n s f e r one  rooting.  Pre-treatment  The e f f e c t s o f h e a t - t r e a t i n g s o u r c e w e e k s , on g r o w t h i n m e d i a M S C B  Q  ^1^  Q  p l a n t s f o r 1-5  o r MSB-j^  I. ^,  both  Q  a d j u s t e d t o pH 6.1, w e r e i n c o n c l u s i v e s i n c e s u r v i v a l and r o o t i n g w e r e o n l y 5/33 i n t h e f o r m e r medium and 3/23 i n t h e l a t t e r over  t h e e n t i r e 5-week p e r i o d .  t o t h e h i g h pH o f t h e m e d i a .  T h i s was  When m e r i s t e m t i p s f r o m  p l a n t s e x p o s e d t o 8 weeks o f h e a t - t r e a t m e n t i n t o medium M S C B higher  Q  ^1^  survival rate  control plants  (17/28) t h a n  (54/156).  were e x c i s e d  t i p s from  non-heat-treated  Shoots from h e a t - t r e a t e d  from c o n t r o l p l a n t s d i d ( F i g . 3 ) .  source  none o f  those  Shoots from c o n t r o l p l a n t s  i n 5-8 weeks o f c u l t u r e .  Cold Storage  of In V i t r o  Plantlets  Four of the s i x p l a n t l e t s subjected g r e w new s h o o t s ture.  source  , a d j u s t e d t o pH 5.7, t h e y h a d a  p l a n t s r o o t e d w i t h i n 4-5 weeks o f c u l t u r e ;  rooted  attributed  to cold  and r o o t s when r e t u r n e d t o i n c u b a t i o n t e m p e r a -  Two p l a n t l e t s d i d n o t s u r v i v e c o l d s t o r a g e .  p l a n t l e t s w i t h new g r o w t h w e r e p l a n t e d i n s o i l They c o u l d p r o b a b l y into s o i l .  storage  have been p l a c e d d i r e c t l y  Cold storage  The  four  and s u r v i v e d . from t h e c o l d  a t t h i s temperature e l i m i n a t e d shoot  g r o w t h , b u t some new r o o t s w e r e f o r m e d i n v i t r o .  This i s a  p o t e n t i a l method f o r s t o r a g e o f b l a c k r a s p b e r r y g e r m p l a s m .  37.  F i g u r e  3.  E f f e c t  of  h e a t - t r e a t i n g  p l a n t s on developmen M S w i t h c y s t e i n e HC1 and IBA at 1.0 mg/L, l e t s arose from m e r i t r e a t e d source p l a n t r o o t . The r o o t l e s s t r e a t e d source p l a n t to r o o t .  t  b l a c k  r a s p b e r r y  of meristem t i p s at 10 m g / L , BAP at p H 5.7. The r o o t e s t e m t i p s e x c i s e d f s ; these took 4-5 shoot was from a n ; these took from  source i n medium 0.1 mg/L, d p l a n t rom h e a t weeks to o n - h e a t 5-8 weeks  Loganberry Culture  Media  T r i a l s w i t h a s m a l l number o f e x p l a n t s s u r v i v a l was m o d e r a t e low  (1/8)  i n medium  (8/15)  MSBQ  showed t h a t  i n medium M S B ^ ^ 1 ^ Q w i t h a t w o - f o l d  o f macroelements, and t h e s e declining after  Q , and  ^ 1 ^  dilution  s m a l l r e s u l t a n t shoots were  a 2 month p e r i o d .  When t h e s e  shoots were  t r a n s f e r r e d t o medium M S B ^ g i g ^ o r medium M S B ^ Q I Q pH 6 . 1 , t h e y  recovered  abundantly rooted Of  q u i c k l y , forming  p l a n t l e t s w i t h i n 2 months.  S u r v i v a l t o t h e small shoot stage  i n medium MSB^ Q I Q ^ w i t h g l u c o s e , MSB^ Q I Q  1  with sucrose,  was a d j u s t e d adjusted  19/42  pH 5 . 7 .  to 5 . 7 . i n medium MSB-^ Q I Q ^, pH 6 . 1 , pH 5 . 7 , a f t e r 6  weeks a n d 1 2 weeks r e s p e c t i v e l y , t h e y clones.  had formed  large  These were t h e n d i v i d e d i n t o 1 2 and 2 0 c u t t i n g s F o u r weeks l a t e r t h e y were r e s u b d i v i d e d  7 6cuttings, after  cuttings.  The  I n medium  s u r v i v e d when t h e pH was  w e r e t r a n s f e r r e d t o medium MSB^ Q I Q  parable  was 1 5 / 3 2  s u r v i v e d when t h e medium  t o pH 6 . 1 a n d 1 2 / 1 3  When two m e r i s t e m t i p s  and  ( 5 - 8 cm),  1 0 2 c u l t u r e s , 1 5 became c o n t a m i n a t e d a n d w e r e  discarded.  each.  tall  /  into 5 0  a n o t h e r 4 weeks, i n t o 1 1 2 and 2 0 5  T i p s f r o m medium c o n t a i n i n g g l u c o s e  number o f c u t t i n g s a t t h i s  number o f c u t t i n g s o b t a i n e d  stage  g a v e a com-  (Figs. 4A-D).  by c o n t i n u e d  subdivision  a t 4 - w e e k i n t e r v a l s w o u l d be c o m p a r a b l e t o b l a c k micropropagation.  raspberry  F i g u r e  4.  Loganberry c l o n e s i l l u s t r a t i n g the e f f e c t of two c a r b o h y d r a t e s o u r c e s , and two l e v e l s of BAP, on m i c r o p r o p a g a t i o n p o t e n t i a l of m e r i s t e m t i p s s t a r t e d i n m e d i u m MS w i t h BAP at 1.0 mg/L and IBA at 0.1 mg/L. A: A f t e r 20 w e e k s i n s t a r t i n g m e d i u m w i t h s u c r o s e , the p l a n t l e t was d i v i s i b l e i n t o 15 c u t t i n g s . B: A f t e r 12 w e e k s o f c u l t u r e i n s t a r t i n g medium w i t h s u c r o s e , the shoot w a s t r a n s f e r r e d t o m e d i u m MS w i t h BAP i n c r e a s e d to 3.0 mg/L. A f t e r another 6 weeks, the l e a v e s were s m a l l e r than on A, and somewhat c u r l e d , but the p l a n t l e t was d i v i s i b l e i n t o 25 c u t t i n g s . C: A f t e r 14 weeks of c u l t u r e i n s t a r t i n g medium w i t h g l u c o s e , the shoot was d i v i s i b l e i n t o 60 c u t t i n g s . D: A f t e r 8 weeks of c u l t u r e i n s t a r t i n g medium w i t h g l u c o s e , the shoot was t r a n s f e r r e d t o medium w i t h BAP i n c r e a s e d to 3.0 mg/L. A f t e r another 6 weeks, the l e a v e s were s m a l l e r than on C, and somewhat c u r l e d , and the p l a n t l e t was d i v i s i b l e i n t o 20 c u t t i n g s .  39.  40.  R o o t i n g from  s t a r t i n g  w i t h o u t  BAP,  medium,  by  b o t h  was  or  was  BAP  pH.  when f o r w i t h and  1 . 0 mg/L,  pH  was  of  r o o t  w i t h  3 . 0 mg/L, to  the  medium,  of  two  pH  medium.  MSB^  R o o t i n g  shoot  was  was  r o o t e d  l i t t l e  a  i n  r o o t i n g  c u t t i n g s p e r i o d  p l a n t e d  l a r g e r  the  medium  and  mg/L.  3.0  a f f e c t e d was  h i g h e r  i n c o n c l u s i v e two  groups  c o n t a i n e d  than  5 . 7 to  8 7 % , and  c u t t i n g s  were  f o r  i n f l u e n c e d  5 . 7 , 3 6 / 3 9  c u t t i n g s ,  IBA  r o o t i n g  s u p e r i o r  r o o t e d  w i t h  pH  s l i g h t l y  same  per  a f t e r  was  the  were  r o o t  r o o t s  c u t t i n g s ,  pH  w i t h i n  of  r o o t i n g  R e s u l t s  i n  the  f a s t e r  not  lower  r o o t e d  was  was  growth  QIQ  l e v e l s ,  Shoot  r o o t i n g  b u t ,  s o u r c e  b e s t  r o o t i n g  d i f f e r e n c e  r o o t e d .  of  at  no  (82%)  time  s t a r t i n g  i n  BAP  was  pH  on  media  s u c r o s e ,  g l u c o s e .  l o g a n b e r r y  medium  IBA  w i t h  6 . 1 .  shoot  the  of  was  p e r c e n t a g e  s o u r c e  r o o t i n g i n  number  e l o n g a t i o n  than  t r a n s f e r  c a r b o h y d r a t e  but  p e r c e n t a g e  to  l e v e l  e f f e c t  1.0 mg/L,  a d j u s t e d  r o o t i n g  by  r o o t i n g ,  c a r b o h y d r a t e  _  There  and  at  BAP  s i g n i f i c a n t  p e r c e n t a g e  BAP  the  a f t e r  l e v e l s  c o n c e n t r a t i o n  g r e a t e r  M S I  r o o t i n g  The  was  s t a r t i n g  n  no  two  e x t e n t  r o o t i n g  from  At  had  by  at  R o o t i n g  The  5 ) .  1 0 0 %r o o t i n g , the  some  c u t t i n g s ,  at  BAP  W i t h  t e s t s  BAP  With  at  the  to  h i g h e s t  0.7 mg/L.  p e r c e n t a g e  w i t h  shoot  i n f l u e n c e d  and  medium  c u t t i n g  by  media  (Table  i n i t i a t i o n  W i t h  l o g a n b e r r y  pH,  media  r o o t i n g  at  of  by  took  t r a n s f e r  the  from  pH  the  weeks.  1 - 3  the  two  c l o n e s .  f o r  r o o t i n g  medium.  the a t  ( 9 2 % ) ;  pH  6 . 1 ,  d i r e c t l y  as  those  r o o t e d  2  weeks  i n  r o o t i n g  r o o t e d  i n  s o i l .  t h o s e  of  between  t r a n s f e r r e d  a f t e r  medium  i n  3 2 / 3 9  to  v i t r o  s o i l but  medium, The  group  41. T a b l e 5.  E f f e c t o f BAP c o n c e n t r a t i o n , pH, and carbohydrate s o u r c e on i n c i d e n c e and r o o t i n i t i a t i o n t i m e , o f loganberry shoot c u t t i n g s i n n u t r i e n t c u l t u r e  S h o o t medium* BAP  Rooting  S u g a r * pH  medium** No.  Sugar  pH  of c u t t i n g s  %  rooted/transferred  (mg/L)  *  Average rooting time (days)  3.0 3.0 3.0 3.0 3.0  S S S S G  6.1 6.1 5.7 5.7 5.7  S G S G G  6.1 6.1 5.7 5.7 5.7  14/17 18/18 11/18 11/18 14/18  82 100 61 61 78  30 24 21 35 14  1.0 1.0 1.0 1.0 1.0  S S S S G  6.1 6.1 5.7 5.7 5.7  S G S G G  6.1 6.1 5.7 5.7 5.7  12/13 14/18 14/18 17/17 13/16  92 78 78 100 81  18 25 24 15 16  *: **: *.:  S h o o t g r o w t h m e d i a w e r e MS w i t h IBA a t 0.1 mg/L a t 1.0 o r 0.1 mg/L. Rooting S:  m e d i a w e r e MS w i t h IBA up t o 1.0  sucrose  a t 30 g/L;  G:  glucose  a t 25  mg/L. g/L.  and  BAP  of c u t t i n g s p l a n t e d  a f t e r 4 weeks i n r o o t i n g medium w e r e a s  l a r g e as e i t h e r o f t h e o t h e r  two g r o u p s .  p l a n t i n g the t h i r d group o f p l a n t l e t s ,  A month a f t e r  five plants  from  e a c h g r o u p w e r e w e i g h e d , m e a s u r e d , and t h e i r r o o t s l e a v e s were counted r o o t s were s m a l l e r branching  (Table  6).  r o o t s t h a n t h o s e on p l a n t l e t s r o o t e d  the other  again. but  and more r o b u s t  number o f  i n culture.  than p l a n t l e t s  two g r o u p s , and w e i g h e d a l m o s t h a l f as much  I t a p p e a r s t h a t r o o t i n g i n v i t r o may be a d v a n t a g e o u s  that p l a n t l e t s rooted  i n c u l t u r e should  s o i l w i t h i n a short time of r o o t i n g . are  in soil  a f t e r 2 weeks i n r o o t i n g  medium, w e r e s l i g h t l y t a l l e r in  On p l a n t l e t s r o o t e d  i n d i a m e t e r , and h a d a g r e a t e r  P l a n t l e t s transferred to s o i l  and  allowed  be p l a n t e d i n  I f rooted p l a n t l e t s  t o remain i n c u l t u r e f o r too l o n g , t h e i r ad-  vantage i s lessened.  Source P l a n t  Pre-treatment  L o g a n b e r r y m e r i s t e m t i p s u r v i v a l was n o t i m p r o v e d when s o u r c e p l a n t s w e r e e x p o s e d t o 1-8 weeks o f h e a t - t r e a t m e n t . Survival of control tips hormones o r i n o s i t o l , was 24/45 25/58  s t a r t e d i n medium MS  and s u b c u l t u r e d  t o medium MSB^  (56%) c o m p a r e d t o h e a t - t r e a t e d  (43%).  I n medium MSB^  0  I  Q  meristem  p l a n t s , i t was 23/67  w e r e n o t s i g n i f i c a n t a t t h e 0.05  ^ 1 ^ ^,  tips,  ^, pH 6.1, s u r v i v a l o f  m e r i s t e m t i p s f r o m c o n t r o l p l a n t s was 19/42 heat-treated  without  (34%). level.  (45%);  from  These d i f f e r e n c e s  43. T a b l e 6.  Group*  Comparison of loganberry and i n v i t r o  Average weight of p l a n t l e t  cuttings rooted  A v e r a g e no. o f main r o o t s  leaves  (g)  in soil  Average l e n g t h of roots  leaves  (mm)  (mm)  1  0.692  10  10  12.26  8.60  2  1.017  8  12  12.54  10.44  3  0.693  7  13  9.16  9.44  *:  G r o u p 1 was r o o t e d i n s o i l ; g r o u p 2 was r o o t e d i n v i t r o and t r a n s f e r r e d t o s o i l a f t e r 2 w e e k s ; group 3 was r o o t e d i n v i t r o and t r a n s f e r r e d t o s o i l a f t e r 4 weeks. P l a n t l e t s w e r e e x a m i n e d 1 month a f t e r p l a n t i n g g r o u p 3. F i v e p l a n t l e t s f r o m e a c h g r o u p w e r e e x a m i n e d and t h e r e s u l t s a v e r a g e d .  44.  Cold Storage of In V i t r o Of  the  in vitro,  27  23  Plantlets  loganberry  p l a n t l e t s placed  survived planting d i r e c t l y  the non-survivors  were d i s c a r d e d  after  t i m e o f p l a n t i n g , a l l a p p e a r e d t o be mildew; and  some had  lower  leaves.  When  2 weeks f r o m  the  infected with  M i l d e w was  Cold  also present  storage  for preserving loganberry of p l a n t l e t s  powdery  green t i p s w i t h h e a v i l y i n f e c t e d stem  l e a v e s w e r e removed f r o m t h e r e m a i n i n g growth.  storage  into s o i l .  l e a v e s o f some o f t h e s u r v i v i n g p l a n t s .  vigorous  into cold  i s very  on  the  dead  After a l l infected p l a n t s they  resumed  i s a p o t e n t i a l procedure  germplasm i n v i t r o ,  since  survival  high.  Tayberry Culture  Media  None o f t h e medium MSB^  f i v e tayberry meristem t i p s  ^ 1 ^ ^, pH  6.1,  survived.  i n t h e same medium w i t h g l u c o s e ,  pH  Of 5.7,  the ten t i p s s t a r t e d s i x small  tips  0.05-0.15 mm  l o n g , d i e d , but  s u r v i v e d and  grew q u i c k l y , w i t h v i g o r o u s l y b r a n c h i n g  and  many l e a v e s .  shoots MSB^ the  0.15-0.25 mm  long,  A f t e r 2 m o n t h s on t h i s medium, two  stems, large  w e r e t r a n s f e r r e d t o b o t t l e s c o n t a i n i n g medium  QIQ  with sucrose,  s t a r t i n g medium.  medium had shoots,  four t i p s  started in  and  Two  pH  5.7.  Two  shoots  were k e p t i n  months l a t e r , p l a n t s i n  starting  s m a l l , w e l l e x p a n d e d l e a v e s , many s h o r t , c o m p a c t very  s t a r t i n g medium  long roots ( F i g . 5B)  ( F i g . 5A).  A f t e r 28 weeks i n  a t a y b e r r y c l o n e had  a v a s t number  45. A.  Figure  B.  5.  Tayberry clones i l l u s t r a t i n g the e f f e c t of carb o h y d r a t e s o u r c e , and two l e v e l s o f BAP, on micropropagation p o t e n t i a l of meristem t i p s s t a r t e d i n medium MS w i t h g l u c o s e , and w i t h BAP a t 1.0 mg/L and IBA a t 0.1 mg/L. A: After 16 weeks i n s t a r t i n g medium. B: A f t e r 28 weeks i n s t a r t i n g medium. C: A f t e r 12 weeks i n s t a r t i n g medium, t h e s h o o t was t r a n s f e r r e d t o medium w i t h s u c r o s e , and BAP a t 3.0 mg/L. A f t e r another 4 weeks, t h e l e a v e s were l a r g e r and t h e r o o t s n o t as w e l l d e v e l o p e d as on A.  of r o o t s . larger,  I n medium w i t h BAP a t 3.0 mg/L,  shoots  taller,  one s h o o t r o o t e d , (Fig.  and i n t e r n o d e s  leaves  were  longer, but only  and t h e r o o t s were few and v e r y  short  5C) . In micropropagation  t e s t s i n medium w i t h  BAP c o n c e n t r a t i o n , a m e r i s t e m t i p 0.15 mm i n t o a shoot d i v i s i b l e  elevated  long  developed  i n t o 15 c u t t i n g s w i t h i n 12 weeks  of c u l t u r e .  A f t e r 5 weeks i n medium w i t h e l e v a t e d  level,  c u t t i n g s were s u b d i v i d e d  and  these  4 weeks a f t e r t h i s ,  on t h i s l i m i t e d  into 40 cuttings,  160 c u t t i n g s w e r e made.  Based  sample, t h e annual m i c r o p r o p a g a t i o n  o f t a y b e r r y w o u l d be s i m i l a r t o t h a t o f b l a c k or  BAP  rate  raspberry  loganberry. Of 15 c u t t i n g s p l a c e d ; j i n t o s o i l ,  d u c e d new l e a v e s w i t h i n 2 w e e k s . rooted  i n medium  MSIQ  a l l rooted  and p r o -  In culture, cuttings  w i t h i n t h e same p e r i o d and w e r e  transplanted to s o i l without  difficulty.  Red R a s p b e r r y Culture Media Red r a s p b e r r y m e r i s t e m t i p s d i d p o o r l y i n t h e m e d i a t h a t were s u i t a b l e f o r b l a c k r a s p b e r r y 59 m e r i s t e m t i p s  culture.  None o f  s u r v i v e d i n medium MSC w i t h BAP a t 0.1  mg/L a n d I B A a t 0.1-0.7 mg/L, a n d o n l y 2/36 s u r v i v e d a n d r o o t e d w i t h I B A a t 1.0 mg/L.  Most turned  50 m e r i s t e m s e a c h w e r e p l a c e d  i n t o medium  brown. m  s  b  Q  When  -^1 ^ Q w i t h  macronutrients 1:4,  at f u l l  8,8,  3, and  W i t h BAP  involved.  a t 0.1  mg/L;  mg/L,  w i t h IBA  c o u l d be  Further  IBA  i n c r e a s e d t o 3.0  level tested flected  (MSB^  i n the  c y t o k i n i n was trial  two  MSB_  s h o u l d be  browning  l e v e l s c o u l d be  increased  was low  mg/L  mg/L,  BAP  without  but  increased  to  concentration  decreasing lowest  survival.  hormone  chlorophyll formation,  i n t e n s i t y of green c o l o u r , improved f r o m 0.1  t o 5.0  mg/L.  re-  as  From  this QIQ  m e d i a w e r e s e l e c t e d f o r f u r t h e r s t u d y , MSB^ I . ,.  I t was  decided  that three other ^ 1 ^ ^,  i n v e s t i g a t e d as w e l l , MSB^  hormones, w i t h i n o s i t o l The  Since  t e s t s showed t h a t  s u p e r i o r at the  ^ 1 ^ ^)  small  promoted meristem t i p s u r v i v a l .  l e v e l a t 0.1  M e r i s t e m t i p g r o w t h was  a t 100  h i g h e s t r a t e of  (65/86) was  medium MSB-j^  mg/L,  0  I  Q  ^ at the  S u r v i v a l r a t e was medium w i t h o u t  s u r v i v a l to the  h o r m o n e s , and first  also high  without  inositol.  small shoot  subculture  QI  q  ^ at the  When t i p s w e r e s t a r t e d i n medium w i t h o u t t o medium w i t h BAP  r e d u c e d t o 24/86 and  g r o w t h was  a t 3.0 poorer  MS  (28 d a y s ) . initiated  containing i n o s i t o l 1  stage  t r a n s f e r r e d to  (60/84) f o r t i p s  hormones, b u t  t r a n s f e r r e d t o medium MSB  subcultured  and MS  or without  ^  media  among m e r i s t e m t i p s e x c i s e d i n t o medium  w i t h n e i t h e r i n o s i t o l nor  and  and  major n u t r i e n t t o x i c i t y  t o t a l hormone c o n c e n t r a t i o n s  and  2:4,  1 respectively.  t h e m a j o r symptom s t i l l ,  u n l i k e l y t o be  0.5  3:4,  t h e number o f m e r i s t e m t i p s s u r v i v i n g t o t h e  s h o o t s t a g e was was  strength, diluted  a t 100  first  hormones,  mg/L,  in mg/L,  subculture. and  survival  was  t h a n i n medium w i t h  48. BAP a t 1.0 mg/L.  S u r v i v a l was a l s o r e d u c e d  (33/86) when  m e r i s t e m t i p s w e r e s t a r t e d i n medium c o n t a i n i n g  hormones  (MSB^ Q I Q ^) , b u t g r o w t h r a t e o f t h e s u r v i v o r s was than that of t i p s w h i c h grew v e r y hormones.  s t a r t e d i n medium w i t h o u t h o r m o n e s ,  little until  subcultured  t o medium  I f m e r i s t e m t i p s were a l l o w e d  medium w i t h o u t hormones o r i n o s i t o l weeks.  I n medium w i t h  they died w i t h i n 5  i n o s i t o l , b u t no h o r m o n e s , t h e y g r e w  tips  s t a r t e d i n medium w i t h BAP a t 3.0 mg/L  only  18/81 s u r v i v e d  to the small  shoot  s u r v i v a l decreased with from t h i s p o i n t .  10-fold  Q I Q ^, e a c h a t  growth o f r e d raspberry  o f pH w e r e s i m i l a r i n b o t h m e d i a .  i n medium MSB^  (MSB^ Q I Q ^ ) ,  showed t h a t 5.7 was t h e m o s t e f f e c t i v e  pH f o r s u p p o r t i n g  values  Of m e r i s t e m  stage.  T e s t s o f m e d i a MSB^ ^ 1 ^ ^ a n d MSB^  and  i n 30 d a y s ;  Effects  The a v e r a g e g r o w t h  increase  The g r e a t e s t  ^ I Q ^, pH 5.7.  tips.  pH l e v e l s ,  rate  o r d e c r e a s e o f pH a v e r a g e g r o w t h was  Explants  increased  i n length  3 0 - f o l d o r more i n 60 d a y s , a t w h i c h  time they were t r a n s f e r r e d t o l a r g e r c o n t a i n e r s . other  with  t o remain i n  so s l o w l y t h a t t h e y w e r e e v e n t u a l l y d i s c a r d e d .  f i v e pH l e v e l s ,  better  At a l l  t i p s were g e n e r a l l y p a l e r and n o t as  i n a p p e a r a n c e a s t h o s e a t pH 5.7.  robust  A t pH 5.9, t h e a v e r a g e  g r o w t h was 7 - f o l d i n 30 d a y s a n d 1 6 - f o l d  i n 60 d a y s .  pH 6.1, g r o w t h was 5 - f o l d i n 30 d a y s a n d 1 0 - f o l d  At  i n 60 d a y s .  A t pH 5.5 o r 6.3 s u r v i v a l a n d g r o w t h iwere s e v e r e l y  reduced.  Adjusting  t h e pH t o 5.7 r e s u l t s i n a pH o f 5.3 a f t e r a u t o -  claving.  This value  d r i f t s down, s l o w l y  i n refrigerated  t u b e s , more q u i c k l y (pH  5.1  after  are a c t i v e l y  i n tubes  rate at  growing  (pH  (Table 7).  rate.  sucrose but I t was  were f e w e r  they tended  browning  tips  and  had  levels,  than  20 g/L  carbohydrate  i n t h e medium w i t h g l u c o s e , a t pH  slightly  s u r v i v a l was not  faster,  o n l y 35%,  and  and  in  t h e g l u c o s e medium a t t h e h i g h e r pH. s u r v i v a l was  G r o w t h was  not  t h e r e f o r e be  influenced  two  s e l e c t i o n s used In the presence  into  large  source,  shoots  affected by  i n t h e pH of  the  6.1  at  tips  two  to  (Table 8). after  from  inositol,  that  I n t h e medium w i t h than  by pH  a t pH  5.7.  level;  results  inherent differences and  the  survival greatest  different  6.1  a t pH  significantly  may  carbohydrate  significantly  better  enjoyed  although growth  30  sucrose,  starting  s o u r c e s , each  (70%)  60 d a y s was  edges.  avoid  growth of r e d r a s p b e r r y meristem s t a g e was  with  glucose.  shoot  5.7,  survival  o f the h i g h e r growth r a t e possibly  growth  glucose  i n media  i n the  small  A t pH  ^ with  t o t u r n brown a t t h e i r  i n g l u c o s e , and  o f two  average  the g r e a t e s t  larger  e f f e c t s o f medium w i t h  In t e s t s pH  and  and  ^1^  g l u c o s e a t 25 g/L  medium m i g h t make u s e by m e r i s t e m  t i p survival  T i p s i n medium MSB^  decided that  4 weeks).  c o n c e n t r a t i o n i n t h e medium d r a -  grew t h e f a s t e s t  Leaves  i n t u b e s where p l a n t s  4.5-4.7 a f t e r  a f f e c t e d meristem  20 g/L  i n c u b a t i o n chamber  1 w e e k ) , most q u i c k l y  C a r b o n s o u r c e and matically  i n the  i n the  i n the carbohydrate with  a few m e r i s t e m  s u c r o s e as  tips  i n t h e a b s e n c e o f any  the  s u r v i v e d and growth  trials.  grew  hormones  50. T a b l e 7.  E f f e c t o f c a r b o h y d r a t e s o u r c e and c o n c e n t r a t i o n on s u r v i v a l a n d g r o w t h o f r e d r a s p b e r r y m e r i s t e m t i p s i n n u t r i e n t medium*  Carbohydrate type  amount  Shoot s u r v i v a l * * proportion  Average i n c r e a s e i n shoot l e n g t h  %  (g/L)  (mm) 30 d a y s  60 d a y s  sucrose sucrose  20 .30  6/18 6/15  33 40  1.05 0.86  4.75 2.38  glucose glucose glucose  20 30 40  9/14 5/16 3/14  64 31 21  6.33 3.75 1.15  11.49 5.19 3.96  *: **:  N u t r i e n t medium was MS w i t h BAP a t 1.0 mg/L a t 0.1 mg/L, pH 6.1.  and IBA  No. o f t i p s t h a t s u r v i v e d t o t h e s m a l l s h o o t s t a g e / no. e x c i s e d . R e s u l t s o f two e x p e r i m e n t s w e r e c o m b i n e d .  51. T a b l e 8.  E f f e c t o f c a r b o h y d r a t e s o u r c e and pH l e v e l on s u r v i v a l and g r o w t h o f r e d r a s p b e r r y m e r i s t e m tips i n nutrient culture*  Carbohydrate type  pH  amount  Shoot s u r v i v a l * * proportion  Average increase i n shoot length  %  (g/L)  (mm) 3 0 days  6 0 days  glucose glucose  25 25  6.1 5.7  48/69 24/68  70 35  4.05 3. 04  7, 33 7. 28  sucrose sucrose  30 30  6.1 5.7  8/23 19/83  35 23  1, 35 1. 76  2, 43 3, 67  *: **:  N u t r i e n t medium was MS w i t h BAP a t 1.0 mg/L a t 0.1 mg/L. No. o f t i p s t h a t s u r v i v e d t o t h e s m a l l s h o o t no. e x c i s e d .  and I B A stage/  52. (Fig.  6).  With glucose  as t h e c a r b o h y d r a t e  source,  tips  were u n a b l e t o s u r v i v e i n t h e absence o f hormones, w i t h or without  inositol.  I n medium c o n t a i n i n g s u c r o s e  and  h o r m o n e s , s u r v i v a l was n o t a f f e c t e d b y i n o s i t o l b u t b o t h g r o w t h r a t e and m o r p h o l o g y w e r e a f f e c t e d . l e v e l was i n c r e a s e d  As t h e i n o s i t o l  f r o m 0 t o 150 mg/L, g r o w t h was  l a t e d and a x i l l a r y b r a n c h i n g  increased  (Figs.  In media c o n t a i n i n g g l u c o s e ,  average growth r a t e of  was f a s t e r t h a n i n a n y m e d i a w i t h s u c r o s e inositol  level.  Neither  stimu-  7A, B ) . shoots  regardless of  s u r v i v a l n o r growth r a t e were  d r a m a t i c a l l y a f f e c t e d by t h e c o n c e n t r a t i o n o f i n o s i t o l . S u r v i v a l was b e s t w i t h i n o s i t o l a t 100 mg/L b u t t h e f a s t e s t growth occured  with tips  tration tested  i n the highest  inositol  concen-  (150 mg/L).  Micropropagation  experiments with r e d raspberry  shoots  i n d i c a t e d v a r i a b i l i t y b e t w e e n s e l e c t i o n s , a n d e v e n among shoots  d e r i v e d f r o m t h e same p l a n t s .  sucrose,  I n medium c o n t a i n i n g  p r o l i f e r a t i o n o f some s e l e c t i o n s i n s t a r t i n g medium  MSB^ Q I Q ^ was a s g o o d a s , o r b e t t e r t h a n , e l e v a t e d BAP 8C).  (MSB  3  0  I  Q  ^)  i n medium w i t h  (compare F i g . 8A t o F i g s . 8B a n d  O t h e r s e l e c t i o n s d i d b e t t e r i n medium w i t h  BAP l e v e l  ( F i g . 8D).  elevated  I n medium c o n t a i n i n g g l u c o s e ,  some  s e l e c t i o n s d i d b e t t e r i n s t a r t i n g medium MSB^ Q I Q ^ t h a n when t r a n s f e r r e d t o medium w i t h e l e v a t e d BAP (compare F i g s . 9B, 1 0 A ) ; medium w i t h e l e v a t e d BAP of  shoots  concentration  o t h e r s grew v e r y p r o l i f i c a l l y i n (compare F i g s . 10A a n d B ) .  or cuttings transferred to p r o l i f e r a t i o n  Survival  media  53.  Key  Days  F i g u r e 6.  after  excision  E f f e c t s o f c a r b o h y d r a t e s o u r c e , s u c r o s e (S) o r g l u c o s e ( G ) , i n o s i t o l ( I ) , o r h o r m o n e s (H) on t h e g r o w t h o f m e r i s t e m t i p s o f r e d r a s p b e r r y . S u b c u l t u r e ( t r a n s f e r ) t i m e s a r e i n d i c a t e d by an a r r o w .  54.  F i g u r e 7.  E f f e c t s o f i n o s i t o l c o n c e n t r a t i o n on development of r e d r a s p b e r r y meristem t i p s i n c u l t u r e . A: A f t e r 8 weeks i n c u l t u r e . B: A f t e r 20 weeks in culture. I n o s i t o l l e v e l s were ( l e f t to r i g h t ) : 0, 50, 100, and 150 mg/L. Inositol inhibited r o o t i n g , and i n c r e a s i n g c o n c e n t r a t i o n increased p r o l i f e r a t i o n of a x i l l a r y buds.  F i g u r e 8.  Red r a s p b e r r y c l o n e s a f t e r 8 months i n c u l t u r e s h o w i n g r e s p o n s e o f some s e l e c t i o n s t o p r o l i f e r a t i o n medium. A, B, C, a n d D w e r e a l l s t a r t e d i n medium w i t h s u c r o s e , BAP a t 1.0 mg/L, and I B A a t 0.1 mg/L. A f t e r 4 months i n s t a r t i n g medium, B, C, a n d D w e r e t r a n s f e r r e d t o medium w i t h BAP i n c r e a s e d t o 3.0 mg/L. After 4 more m o n t h s i n c u l t u r e t h e p l a n t l e t s w e r e d i v i s i b l e i n t o 20, 7, 10, and 25 c u t t i n g s respectively. B and C d e v e l o p e d from buds e x c i s e d f r o m t h e same s t e m and i l l u s t r a t e t h e v a r i a b i l i t y that can occur w i t h i n s e l e c t i o n s . A and D d e v e l o p e d f r o m b u d s o f two o t h e r s e l e c t i o n s , and i l l u s t r a t e t h e v a r i a b i l i t y i n r e s p o n s e t o BAP i n t h e g r o w t h medium; selection A g r e w b e s t i n s t a r t i n g medium, D g r e w b e s t i n medium w i t h BAP a t 3.0 mg/L.  55.  Figure  9.  Red r a s p b e r r y c l o n e s s h o w i n g t h e e f f e c t o f pH on l e a f s i z e and b r o w n i n g . Both p l a n t l e t s were grown i n medium MS w i t h BAP a t 1.0 mg/L, IBA a t 0.1 mg/L w i t h g l u c o s e , f o r 7 months. A: A t pH 6 . 1 . B: A t pH 5 . 7 .  57. A.  F i g u r e  B.  10.  Red r a s p b e r r y c l o n e s showing d i f f e r e n t response of two s e l e c t i o n s to p r o l i f e r a t i o n medium w i t h g l u c o s e . A f t e r 4 months i n s t a r t i n g medium w i t h g l u c o s e , IBA at 0.1 mg/L a n d BAP at 1.0 mg/L, and 3 months i n medium w i t h BAP i n c r e a s e d to 3.0 mg/L, A was d i v i s i b l e i n t o 9 c u t t i n g s ; B i n t o 35. Clones i n medium w i t h g l u c o s e had l e a v e s t h a t were u s u a l l y l a r g e r and more rugose than i n medium w i t h s u c r o s e .  58. was a l w a y s s u p e r i o r i n m e d i a a d j u s t e d t o pH 5.7, r a t h e r t h a n pH 6.1.  While  g r o w t h was f a s t a t pH 6.1, e s p e c i a l l y  i n m e d i a w i t h g l u c o s e , i t was o f t e n f o l l o w e d b y p e r i p h e r a l , then t o t a l ,  l e a f browning  (compare F i g s .  9A a n d B ) .  S h o o t s 8-16 weeks o l d a n d 1.0-2.0 cm l o n g , g r o w n i n medium MSB^  Q I Q ^ and t r a n s f e r r e d t o r o o t i n g media w i t h  e i t h e r I B A o r NAA a t 0.0-1.0 mg/L, d a y s t o r o o t , w i t h 12/23 s h o o t s NAA.  r o o t e d i n I B A , 17/26 i n  When c u t t i n g s w i t h one o r more l e a v e s , f r o m  g r o w n i n medium MSB^ media w i t h e i t h e r  15/30 i n NAA.  I B A o r NAA a t 0.0-1.0 mg/L, r o o t i n g a n d 17/35 c u t t i n g s r o o t e d i n  Roots t h a t formed i n t h e presence  NAA w e r e d i f f e r e n t f r o m t h o s e or without auxins.  plantlets  Q I Q ^, w e r e t r a n s f e r r e d t o r o o t i n g  t o o k an a v e r a g e o f 15 d a y s , IBA,  t o o k a n a v e r a g e o f 23  of  formed i n media w i t h IBA,  W i t h NAA, r o o t s a r o s e  from l e a f  blades,  p e t i o l e s , o r s h o o t b a s e s ' i n c o n t a c t w i t h t h e medium, and w e r e u s u a l l y a c c o m p a n i e d by c a l l u s f o r m a t i o n o n t h e l e a f b l a d e , a l o n g t h e l e a f p e t i o l e o r a t t h e base o f t h e shoot. C a l l u s f o r m a t i o n on t h e s h o o t extensive.  b a s e s was s o m e t i m e s v e r y  Roots t h a t formed from l e a f b l a d e s o r l e a f  p e t i o l e s appeared normal i n a l l r e s p e c t s formation, elongation) except  (colour, root  point of origin.  This ad-  v e n t i t i o u s r o o t i n g c o u l d be a p r o b l e m s i n c e i m p e r f e c t cular connections  would l e a d t o poor i n i t i a l  greenhouse, o r i n h i b i t continued.  survival.  D i f f e r e n t carbohydrate  d i d not i n f l u e n c e these  results.  vas-  growth i n the  The u s e o f NAA was sources  hair  dis-  i n t h e medium  f r o m s t a r t i n g medium M S B . ^  D i v i s i o n of clones and  medium M S B ^  more l e a v e s  ^IQ  ^ into larger cuttings with  each, i n c r e a s e d I B A  w i t h B A P a t 3 . 0 mg/L,  1 9 / 2 2 cuttings rooted  at  mg/L.  0 . 1 - 1 . 0  weeks o f t r a n s f e r t o r o o t i n g m e d i a . BAP  at  1.0  mg/L,  2 2 / 2 2  the  occured at the  r o o t i n g medium  best; root  (Fig.  Of  the  f o r m e d new  and  top  M S B ^  Q  I  s t e m s , and  e a c h was  i n s o i l without  I B A in  M S I Q  was  5  or  a l l 2 0 rooted  Of  M S B  3  the 0  I  Q  ^,  and  cuttings only  p r o d u c e d d o z e n s o f new  divisible  into  trans-  3 5 / 7 0  2 0 - 8 0  leaves  cuttings  which  difficulty.  i n s u r v i v a l and  growth r a t e between c o n t r o l  e t i o l a t e d m e r i s t e m t i p s were not level.  best-formed  Pre-treatment  Differences  0.05  and  w i t h i n a month of t r a n s f e r , b o t h p l a n t l e t s  and  and  weeks  1-2  R o o t i n g medium  in soil,  1  u n r o o t e d c u t t i n g s had  Source P l a n t  with  growth.  and  rooted  within  longest  g r o w t h w i t h i n 2. w e e k s .  but,  within 2 - 3  fastest rooting with satisfactory  cuttings placed  f e r r e d from media rooted  1 1 ) .  or  F r o m medium  l o w e s t l e v e l s o f I B A , o r no  i t combined the formation  The  ^,  transferred  From medium  cuttings rooted  of t r a n s f e r to r o o t i n g media. roots  two  r o o t i n g of c u t t i n g s  to r o o t i n g media w i t h  ^IQ  The  s i g n i f i c a n t at  the  e t i o l a t e d meristem t i p s took about 1 0 days  t o t u r n g r e e n and  t h e r e a f t e r were v i s u a l l y  from c o n t r o l t i p s  (Table 9 ) .  indistinguishable  Heat-treatment experiments i n d i c a t e d that  growing  Figure  11.  Red r a s p b e r r y c u t t i n g s f r o m a c l o n e i n medium MS w i t h BAP a t 1.0 mg/L and IBA a t 0.1 mg/L a f t e r 3 weeks i n r o o t i n g medium MS w i t h IBA a t 0.5 mg/L.  Table  9.  E f f e c t of r e d  of e t i o l a t i n g source p l a n t s on r a s p b e r r y m e r i s t e m t i p s i n four  Medium*  Shoot  s u r v i v a l media  s u r v i v a l * *  p r o p o r t i o n dark  * :  * * :  l i g h t  (control)  1  12/30  21/30  2  10/30  8/30  3  20/30  13/30  4  5/30  7/30  Mediu were Mediu were Mediu Mediu  m 1 w a s MS w i t h n o i n s u b c u l t u r e d to medium m 2 w a s MS w i t h n o i n s u b c u l t u r e d to medium m 3 w a s MS w i t h BAP a m 4 w a s MS w i t h B A P a  No.  of  no.  e x c i s e d .  t i p s  t h a t  s u r v i v e d  o s 3 o s 4 t t to  i t o l , no at 1 mo i t o l , no at 1 mo 1.0 mg/L, 3.0 mg/L, the  hormones; nth. hormones; nth. IBA at 0.1 IBA at 0.1  s m a l l  shoot  t i p s t i p s mg/L mg/L  s t a g e /  62. source plants  f o r up t o 8 weeks a t 37°C h a d l i t t l e  on s u r v i v a l o f r e d r a s p b e r r y m e r i s t e m t i p s  effect  (Table 10).  C o m p a r i s o n o f s u r v i v a l and g r o w t h r a t e o f m e r i s t e m t i p s excised  from p o t t e d p l a n t l e t s d e r i v e d  control plants  raised  from r o o t  f e r e n c e s between source p l a n t s  cuttings,  (control:12/17;  t h a n i n t h e same medium w i t h 1/27;  cultured  plants:  with  1  showed no d i f How-  g l u c o s e a t 2 5 g/L  cultured  plants:  s u c r o s e a t 30 g/L  15/16)  (control:  5/27).  The o r i g i n a l p o s i t i o n o f t h e e x c i s e d had  and f r o m  i n e i t h e r o f two m e d i a .  e v e r , s u r v i v a l i n medium MSB^ ^ 1 ^ was a g a i n b e t t e r  from c u l t u r e ,  bud on t h e stem  no a p p a r e n t e f f e c t i n m o s t e x p e r i m e n t s .  H o w e v e r , when  c a r b o h y d r a t e s o u r c e s , g l u c o s e and s u c r o s e , a n d c o n c e n t r a t i o n s were v a r i e d ,  f r o m 20-40 g / L , t h e a p i c a l m e r i s t e m t i p s a p p e a r e d  t o grow a n d d e v e l o p f a s t e r i n g l u c o s e medium, 20 g/L t h a n l a t e r a l m e r i s t e m t i p s o f t h e same s i z e , f r o m t h e same s o u r c e plants  (Fig. 12).  Cold Storage of In V i t r o After cold  stored  3 months o f c o l d  s i x were s h r i v e l e d  red  s t o r a g e , a l l b u t 4 o f t h e 12  p l a n t l e t s a p p e a t e d g r e e n and s t u r d y .  weeks a f t e r t r a n s f e r  Cold  Plantlets  to soil,  s i x h a d p r o d u c e d new  Two leaves,  a n d showed e v i d e n c e o f m i l d e w i n f e c t i o n .  s t o r a g e a t 10°C i s a p o t e n t i a l m e t h o d o f r a s p b e r r y g e r m p l a s m , f o r up t o 3 m o n t h s .  preserving  63. T a b l e 10.  E f f e c t of heat-treatment (37°C) o f s o u r c e p l a n t s on s u r v i v a l o f r e d r a s p b e r r y m e r i s t e m t i p s i n two m e d i a *  Heat-treatment (weeks)  0 1 2 3 4 5 6 7 8  *:  No. e x p l a n t s  survived/no.  excised  Medium 1  (%)  Medium 2  180/237 8/9 5/5 4/4 5/5 3/5 5/6 2/3 8/10  (76)  36/86 6/7 4/8 3/5 0/3 2/4 2/3 0/3 2/10  40/47  (85)  19/43  (%)  (42)  Medium 1 was MS w i t h no h o r m o n e s , no i n o s i t o l , pH 6.1. A f t e r 1 month i n t h i s medium, s h o o t s w e r e s u b c u l t u r e d t o medium 2. Medium 2 was MS w i t h BAP a t 1.0 mg/L, I B A a t 0.1 mg/L, pH 6.1.  F i g u r e 12.  Red r a s p b e r r y a p i c a l (top row) and l a t e r a l (bottom row) p a i r s o f meristem t i p s showing the i n f l u e n c e of carbohydrate type and conc e n t r a t i o n on development a f t e r 2 months i n medium MS with BAP a t 1.0 mg/L and IBA a t 0.1 mg/L. Carbohydrate sources were ( l e f t to r i g h t ) : sucrose a t 20, and 30 g/L and glucose a t 20, 30, and 40 g/L.  65.  Discussion  The  p r e s e n t i n v e s t i g a t i o n of f a c t o r s t h a t a f f e c t  g r o w t h and  r o o t i n g i n v i t r o of  f o u r Rubus s p e c i e s  t h a t t h e s e behave i n c u l t u r e s i m i l a r l y of blackberry  described  Zimmerman ( 1 9 7 8 ) . t u r e on  the  by H a r p e r  to three  (1978) and  the  revealed cultivars  Broome  A comprehensive review of the  t i s s u e c u l t u r e of Rosaceous p l a n t s  and  litera-  indicates  many s i m i l a r i t i e s b e t w e e n t h e s e f i v e Rubus s p e c i e s ,  and  members o f  four other genera i n t h i s  cultured.  Many f a c e t s o f c u l t u r e h a v e n e v e r b e e n t h o r o u g h l y  explored  nor  Rubus s p e c i e s to react an  explained utilized  f o r Rosaceous p l a n t s . in this  enlarged understanding of the be  i n the  literature  on  t h e s e Rubus s p e c i e s  s t a t u s and  age,  that increasing but  d i d not  the  incubation  size,  i n source p l a n t the  conditions.  i n f l u e n c e r o o t i n g of the  lateral  physiological  adequacy of This  study  increased  survivors.  consistently superior  l a t e r a l m e r i s t e m t i p s , as  are  regeneration  initial  s i z e of meristem t i p s  m e r i s t e m t i p s were not or  affect  s e a s o n o f e x c i s i o n , and  c u l t u r e medium and  this  Rosaceae.  o f m e r i s t e m t i p s o f Rubus s p e c i e s : differences  appear  C o n v e r s e l y , some anoma-  Many i n t e r d e p e n d a n t f a c t o r s may  apical origin,  the  of Rosaceous p l a n t s ,  t i s s u e c u l t u r e of  possible.  l i e s observed i n the c u l t u r e of explained  Since  i n v e s t i g a t i o n would  t o c u l t u r e i n a manner t y p i c a l  i m p o r t a n t f a m i l y may  or  f a m i l y t h a t have been  to  the  showed survival,  Apical axillary  assumed by many w o r k e r s .  N e i t h e r w e r e t h e y i n f e r i o r a s f o u n d by H a r p e r b l a c k b e r r y and slightly  tayberry.  A p i c a l meristem  l a r g e r than l a t e r a l meristem  (197 8)  with  t i p s are o f t e n  t i p s , which  should  g i v e them an a d v a n t a g e i n r e g e n e r a t i n g s h o o t s , b u t  dif-  f e r e n c e s i n i n t e r n a l hormone c o m p o s i t i o n among t i p s  from  a p i c a l t o b a s a l p o s i t i o n were not u s u a l l y r e f l e c t e d  in  g r o w t h r a t e among t i p s o f e q u a l s i z e . meristem  However,  t i p s of red r a s p b e r r y o f t e n outgrew those  l a t e r a l p o s i t i o n s i n media w i t h g l u c o s e as a s o u r c e , a t l e v e l s o f 20 and age was  25 g/L.  E f f e c t of source p l a n t tips  than  In c o n t r a s t to the r e s u l t s  Carre e t a l . (1979), red r a s p b e r r y meristem from J u l y - F e b u a r y d i d not generate The  Meristem  less regenerative potential  t h o s e from young s e e d l i n g s .  not impaired.  from  carbohydrate  marked i n b l a c k r a s p b e r r y c u l t u r e s .  f r o m m a t u r e p l a n t s had  was  apical  tips excised  c a l l u s , and  use o f c o n t i n u o u s  survival  c r o p s o f young  p l a n t s probably e x p l a i n s the l a c k of seasonal e f f e c t . may  be  of  It  t h a t hormonal c o m p o s i t i o n of the source p l a n t s i s  g o v e r n e d more by age spring,  t h a n by  season.  However, d u r i n g  (Febuary-April) p l a n t s i n c u l t u r e appeared to  the be  more v i g o r o u s , r e g a r d l e s s o f when t h e y w e r e e x c i s e d . E t i o l a t i n g red raspberry source p l a n t s before meristem  t i p s a p p e a r s t o h a v e no a d v a n t a g e , and  l a y growth a f t e r e x c i s i o n . approximately visually  E t i o l a t e d meristem  10 d a y s t o t u r n g r e e n  i n d i s t i n g u i s h a b l e from  tips  in vitro,  excising  i t may tips  and  took  t o become  sprouted i n the  They w e r e e q u i v a l e n t t o c o n t r o l t i p s i n t e r m s o f  de-  light.  survival,  67. and  g r o w t h r a t e was  initial  lag  The  not  significantly different after  period.  e f f e c t s of heat-treatment of  to meristem t i p c u l t u r e v a r i e d with Heat-treatment of black g r o w t h r a t e , and berry, was  not  affected. no  raspberry  only  slightly  effect.  R e p o r t s on  treatment of sour cherry Jha  increased  had  other  and  Stace-Smith  of red r a s p b e r r y  increased  e t a l . (1974, 1975)  no  the  s u r v i v a l and  e f f e c t on  cultures;  strawberry that  the  survival,  potato  plants.  working with strawberry  Hallam  the  strawberry, selections but  (1976) d e s i n meristem  They d e m o n s t r a t e d  to the d i s o r g a n i z a t i o n of  r e c o n c i l e t h i s evidence with  growth  Most r e v e a l i n g i s  Sward and  processes i n excised meristem t i p s .  l a t i o n of  Posnette  heat-treatment  c r i b e d a number o f u l t r a s t r u c t u r a l a b n o r m a l i t i e s  thermotherapy c o n t r i b u t e s  heat-  r o o t i n g of meristem t i p s ,  a fourth.  t i p s of h e a t - t r e a t e d  plants  (1968) f o u n d  (1976) f o u n d t h a t  shoot t i p s .  rate  g r e w more q u i c k l y ;  found t h a t heat-treatment of three  increased had  strawberry  source plants  r o o t i n g of excised  work o f M u l l e n who  (1976) f o u n d  Vine  rasp-  source  Rosaceous  e f f e c t on  and  growth  (1960) f o u n d a d e c r e a s e i n t h e number o f  c u t t i n g s from h e a t - t r e a t e d  and  With red  loganberry  Popov no  survival  increased;  c u t t i n g s t h a t became e s t a b l i s h e d ;  Mellor  t h e Rubus s p e c i e s .  Heat-treatment of  a l s o show m a r k e d d i f f e r e n c e s .  and  source plants p r i o r  promoted f a s t e r r o o t i n g .  s u r v i v a l was  p l a n t s had  the  growth  It is difficult  improvements i n v i t r o or  s u r v i v a l , growth or r o o t i n g .  Some s p e c i e s  that  to stimuand  68.  c u l t i v a r s a r e e v i d e n t l y a f f e c t e d by a t h e r m a l mechanism t h a t r e m a i n s t o be e l u c i d a t e d . the meristem during Of  greatest  The e f f e c t i s a p p a r e n t i n  culture.  importance i n meristem t i p  regeneration,  shoot g r o w t h , p r o l i f e r a t i o n and r o o t i n g a r e t h e components of c u l t u r e media.  Of t h e s e ,  pH h a d t h e g r e a t e s t  carbohydrate concentration  e f f e c t on g r o w t h ;  c e n t r a t i o n a n d pH h a d t h e g r e a t e s t  phytohormone con-  e f f e c t on s u r v i v a l a n d  morphogenesis.  Rarely  concentrations,  o r pH l e v e l s b e e n e v a l u a t e d  have d i f f e r e n t c a r b o h y d r a t e  sources,  i n the culture  of Rosaceous p l a n t s y e t t h i s i n v e s t i g a t i o n i n d i c a t e s pH l e v e l a n d c a r b o h y d r a t e s o u r c e a n d c o n c e n t r a t i o n a d r a m a t i c i n f l u e n c e on r a t e o f g r o w t h . osmolarity  should  pH a d j u s t m e n t s . be  i n conjunction  i n vitro  w i t h , phytohormone and were found t o  interdependent factors with red raspberries,  complicated  (BAP) l e v e l s i n t h e medium.  of d i l u t i n g macronutrients, not  Ideally, correct  L e v e l o f pH a n d o s m o l a r i t y  f u r t h e r by c y t o k i n i n  that  have  be e s t a b l i s h e d f o r any c u l t i v a r  after, or preferably  and  Effects  e v e n b y a 1:4 d i l u t i o n ,  were  d r a m a t i c by c o m p a r i s o n . Differing  levels of i n o s i t o l  i n t h e medium h a v e n o t  been e x p l o i t e d i n t h e c u l t u r e o f Rosaceous p l a n t s ; invariably inositol  100 mg/L  stimulated  a r e added t o c u l t u r e media.  raspberry  While  s h o o t g r o w t h a n d , a t 150 mg/L,  a x i l l a r y , bud p r o l i f e r a t i o n ,  i t may i n h i b i t r o o t i n g .  s h o o t s i n t h e s h o o t g r o w t h medium r o o t e d  o f t e n when i n o s i t o l was o m i t t e d  almost  promoted Red more  t h a n when t h e medium c o n -  69. tained  inositol  The  a t 50,  concentrations  medium a p p e a r t o be HC1  not  mg/L.  adequate.  The  glycine, in  f o r the other  MS  antioxidant cysteine  i n t h e c u l t u r e medium f o r b l a c k  e x p e c i a l l y when t h e t i p s w e r e n e w l y  raspberry three  o r 150  o f B v i t a m i n s , and  seemed t o be n e c e s s a r y  raspberry, but  100,  t h r e e Rubus s p e c i e s ;  c o n t a i n s more p h e n o l i c  excised,  probably  black  substances that the  other  species. D i f f e r e n c e s i n the c o n c e n t r a t i o n of  phytohormones  e m p l o y e d i n t h e c u l t u r e o f t h e f o u r Rubus s p e c i e s  included  t h e r a n g e u t i l i z e d by o t h e r s w o r k i n g w i t h R o s a c e o u s p l a n t s . E f f e c t s o f IBA reports.  and  BAP  are c o n s i s t e n t w i t h those  i n other  P r o l i f e r a t i o n o f i n v i t r o - d e r i v e d Rubus  seems t o be due  t o t h e enhancement o f a x i l l a r y  shoots  branching  i n r e s p o n s e t o c y t o k i n i n i n t h e medium.  Of  the l a c k of t o l e r a n c e of black raspberry  cultures to  w h i c h , a t 0.1  mg/L,  caused c h l o r o s i s .  are the responses of loganberry t o NAA  i n t h e r o o t i n g medium.  and  Of  interest i s  particular  red raspberry  GA^ import  cuttings  Naphthalene a c e t i c a c i d i s  commonly u s e d as a r o o t i n g hormone f o r R o s a c e o u s p l a n t s , but  i n these  induced callused IBA,  t e s t s i t caused c a l l u s p r o l i f e r a t i o n ,  root formation  from l e a f p e t i o l e s ,  l e a f lamina  s h o o t b a s e s i n c o n t a c t w i t h t h e medium.  h o w e v e r , t h e r e was  produced normally,  r a r e l y any  c a l l u s and  from the base of the  and and  With  r o o t s were  shoot.  W h i l e few w o r k e r s u s e d l i q u i d medium f o r a l l s t a g e s o f c u l t u r e , some g r o u p s h a v e f o u n d i t s t i m u l a t o r y f o r initiating  c u l t u r e s (Broome and  Zimmerman, 1 9 7 8 ) .  This  i n v e s t i g a t i o n showed t h a t l i q u i d medium was s u p e r i o r t o s o l i d during of  a l l stages of p l a n t growth.  The d i s a d v a n t a g e  l i q u i d medium i s t h e n e e d f o r a s u p p o r t s t r u c t u r e .  a d v a n t a g e s a r e improved s u r v i v a l and r a t e o f g r o w t h . l i q u i d medium t h e r o o t s o f b l a c k shorter  i n s o l i d medium.  The i n c r e a s e d  and c o n s e q u e n t i n c r e a s e d  a c c o u n t f o r t h e more v i g o r o u s liquid  p l a n t l e t s were  absorbtive  number o f r o o t capacity,  may  growth o f l a r g e shoots i n  medium.  M u r a s h i g e and S k o o g s 1  poorly time,  In  and more n u m e r o u s , a n d h a d many more r o o t h a i r s ,  than roots hairs,  raspberry  Its  buffered, decreasing  (1962) medium f o r m u l a t i o n i s  a n d t h e pH l e v e l  tends t o decrease  the storage  o f l i q u i d medium.  life  with Sub-  c u l t u r e o f i n v i t r o p l a n t s t o f r e s h medium i s s t i m u l a t o r y , p a r t l y because o f t h e r e t u r n t o a higher  pH.  The pH  level  o f t h e medium a f f e c t e d s u r v i v a l a n d g r o w t h r a t e o f r e d raspberry  meristem t i p s ,  shoot p r o l i f e r a t i o n , of black  raspberry  t h a n a t 5.7.  o f some s e l e c t i o n s , a n d a f f e c t e d  and r o o t i n g o f c u t t i n g s .  m e r i s t e m t i p s was much l o w e r a t pH  T h i s m i g h t e x p l a i n why  6.1  filter-sterilized  medium seemed d i f f e r e n t i n i t s e f f e c t s f r o m medium.  Survival  autoclaved  The d i f f e r e n c e s may be due t o pH s i n c e  filter-  s t e r i l i z i n g d o e s n o t a l t e r t h e pH, and a u t o c l a v i n g  depresses  it. Lighting,  i n i t s photomorphogenic c a p a c i t y ,  i s probably  much more i m p o r t a n t i n d e v e l o p m e n t o f a d v e n t i t i o u s  growth  from c a l l i  tips.  than i t i s i n development from meristem  71. Moderate l i g h t  i n t e n s i t y and t h e use o f c o o l w h i t e  c e n t l i g h t s a p p e a r e d t o be adequate d u r i n g t h i s  fluores-  investigation.  When some b l a c k r a s p b e r r y c u l t u r e s w e r e i n a d v e r t e n t l y e x p o s e d to  continuous  l i g h t f o r s e v e r a l weeks, they  s u f f e r e d no  apparent i l l e f f e c t s . An  i n c u b a t i o n t e m p e r a t u r e o f 27°<Z was shown t o be s u -  p e r i o r f o r s u r v i v a l of meristem t i p s of black  raspberry  and was t h e t e m p e r a t u r e u s e d f o r a l l f u r t h e r s t u d i e s . may n o t h a v e b e e n o p t i m u m f o r r e d r a s p b e r r y although  R o s a t i e t a l . (1980) d e s c r i b e d  d i s c o l o r a t i o n and abnormal l e a f b l a d e 26°C i n c u l t u r e s o f J a p a n e s e p l u m  cv. C a l i t a ) . trials  proliferation,  i t d i d n o t seem t o i n h i b i t g r o w t h o f t h e o t h e r  t h r e e Rubus s p e c i e s .  at  expansion  (Prunus s a l i c i n a  S i m i l a r symptoms o c c u r r e d  during  a greater temperature s e n s i t i v i t y i n o l d e r important  tissue  occurring Lundl.  proliferation  o f r e d r a s p b e r r y , but never i n s m a l l shoots,  An  It  suggesting  tissues.  consideration f o r the production of large  numbers o f c u l t u r e d p l a n t s i s t h e s p a c e r e q u i r e m e n t s . bator or c o l d storage  space requirements  Incu-  are roughly  900  2 cuttings/m  (90 100-ml j a r s w i t h 10 c u t t i n g s / j a r ) .  p l a n t l e t s c a n be s e t o u t i n 0.33 x 0.66 m f l a t s , lets/flat, in  so greenhouse space r e q u i r e m e n t s  an i n c u b a t o r o r c o l d chamber.  Rooted  100 p l a n t -  are twice  that  A f t e r 4-6 w e e k s i n f l a t s ,  p l a n t s m u s t be t r a n s p l a n t e d , and c o u l d be s e t o u t i n t h e field. In  B r i t i s h Columbia approximately  1,215 h a a r e c u r -  r e n t l y d e v o t e d t o r e d r a s p b e r r y , w i t h 243 h a s e t o u t i n  72. 1978 and a n o t h e r  486 h a i n 1979.  t o c o n t i n u e i n 1980  (B.C.M.A., 1 9 7 9 ) .  c r o p i n 1979 was t h e l a r g e s t rative  Expansion  (> $12,000.000)  The r e d r a s p b e r r y  (6,820,000 kg) and m o s t  to date.  grown h a v e b e e n d e t e r m i n e d  i s projected  The c u l t i v a r s  luc-  currently  l a r g e l y by a v a i l a b i l i t y when t h e  p l a n t i n g s were s e t o u t . L o g a n b e r r i e s h a v e f o r many y e a r s b e e n a d e c l i n i n g i n d u s t r y i n B.C.,  s u f f e r i n g from a c o s t - p r i c e squeeze.  t h e p a s t 10 y e a r s p l a n t i n g s h a v e d e c l i n e d f r o m o v e r to approximately  3 ha.  Y i e l d s p e r h e c t a r e have  t i a l l y d e c l i n e d o v e r t h e same t i m e p e r i o d B.C.M.A., 1 9 7 9 ) .  During 28 ha  substan-  (B.C.D.A., 1 9 7 2 ,  I n 1979 l o g a n b e r r i e s s o l d f o r $ 0 . 3 4 / k g  ( p i c k e d ) , up s u b s t a n t i a l l y f r o m  1977 and 1978.  Probably  t h e a v a i l a b i l i t y o f l a r g e numbers o f d i s e a s e - f r e e p l a n t s through micropropagation would r e v e r s e the d e c l i n i n g i n g s , and h e l p t o g e n e r a t e  plant-  i n t e r e s t i n y i e l d problems,  and  i n loganberry production. Black raspberry p l a n t s are not c u l t i v a t e d  i n B.C.,  p r i m a r i l y b e c a u s e o f v i r u s d i s e a s e s and l a c k o f c o l d tolerance. rights; in  Tayberry  plants are s t i l l  under breeders'  they are c u r r e n t l y being t e s t e d f o r production  B.C.. Meristem  t i p c u l t u r e and m i c r o p r o p a g a t i o n  o f Rubus  species demonstrates  t h a t enormous numbers o f r o o t e d  l e t s c a n be p r o d u c e d  annually.  This procedure  a l t e r n a t i v e to present propagation techniques;  plant-  i s a viable i t will  a i d i n d i s e a s e e l i m i n a t i o n and a l l o w r a p i d p r o d u c t i o n o f  73.  planting material. t a b l i s h a new record  time.  Micropropagation procedures could  industry,  s u c h as t a y b e r r y  plantings,  in  es-  74. Summary  Black  Raspberry Meristem t i p c u l t u r e , micropropagation,  of b l a c k raspberry out  and  c v . Munger w e r e s u c c e s s f u l l y c a r r i e d  i n m e d i a MSCB„ , , _ I , „, pH 5.7. 0.1 o r 1.0 1.0  of b l a c k r a s p b e r r y  Heat-treatment  source p l a n t s improved s u r v i v a l ,  accelerated rooting  plantlets i n vitro  and r o o t i n g  growth,  ( F i g . 3) o f c u l t u r e d t i p s .  survived  Munger  3 months o f c o l d s t o r a g e  at  10°C. Loganberry M e r i s t e m t i p s u r v i v a l was b e s t pH 5.7;  micropropagation,  MSB^  -^;  QIQ  i n medium MSB^  i n t h e same medium o r i n medium  a n d r o o t i n g , i n medium  t h e c y t o k i n i n (BAP) c o n c e n t r a t i o n ,  M S I Q  ^.  Carbohydrates,  a n d pH l e v e l a r e i n t e r -  dependant f a c t o r s a f f e c t i n g growth and r o o t i n g o f cuttings not  (Table  5).  loganberry  Heat-treatment of source p l a n t s d i d  a f f e c t s u r v i v a l of loganberry  berry p l a n t l e t s i n v i t r o but  ^ 1 ^ ^,  survived  meristem t i p s .  Logan-  2 months o f c o l d  storage,  p o w d e r y m i l d e w i n f e c t i o n was a p r o b l e m when p l a n t l e t s  were t r a n s f e r r e d t o s o i l .  Tayberry Meristem t i p s a t 25 g/L, pH 5.7;  s u r v i v e d i n medium MSB^ micropropagation  medium o r i n medium MSB, „ I  N  ,;  ^1^ ^ with  took place  glucose  i n this  and r o o t i n g i n medium  75.  MSI  Red  , along w i t h s u b s t a n t i a l t o p growth.  Raspberry Meristem  t i p s u r v i v a l was b e s t among r e d r a s p b e r r y  t i p s e x c i s e d i n t o medium MS w i t h o u t h o r m o n e s , w i t h o r w i t h out i n o s i t o l ,  t h e n s u b c u l t u r e d t o medium MSB^ Q I Q  v i v a l was d e c r e a s e d , when m e r i s t e m  b u t growth  t i p s were s t a r t e d  Sur-  rate of survivors  improved,  i n medium w i t h h o r m o n e s .  Medium pH o f 5 . 7 was b e s t when medium h a d s u c r o s e a s t h e carbohydrate were d r a m a t i c  source.  Carbon s o u r c e and c o n c e n t r a t i o n e f f e c t s  (Table 7 ) .  Glucose promoted f a s t e r  of c u l t u r e d t i p s than sucrose;  a t 2 5 g/L g r o w t h  s a t i s f a c t o r y , pH 6 . 1 was b e t t e r t h a n 5 . 7 . and pH l e v e l w e r e i n t e r d e p e n d a n t f a c t o r s  growth r a t e was  Carbohydrate (Table 8 ) .  source  The  s u c c e s s o f m i c r o p r o p a g a t i o n i n m e d i a MSB^ Q o r 3 O ^ O 1 v a r i e d b e t w e e n r e d r a s p b e r r y s e l e c t i o n s , and among f r o m t h e same p l a n t s .  Inositol  inhibited rooting  but, as i n o s i t o l c o n c e n t r a t i o n i n c r e a s e d from a x i l l a r y branching increased.  Naphthalene  tips (Fig.7 )  0-150  mg/L,  acetic acid i s  n o t recommended f o r r o o t i n g r e d r a s p b e r r y c u t t i n g s ; is  f a r superior.  B e s t r e s u l t s w e r e i n medium  a l s o promoted c o n s i d e r a b l e t o p growth Etiolating  s o u r c e p l a n t s was n o t a d v a n t a g e o u s  (Table 1 0 ) .  (Table  t i p s w e r e s u p e r i o r i n some m e d i a  9).  t i p survival  E x p l a n t s from p r e v i o u s l y  p l a n t s d i d no b e t t e r t h a n c o n t r o l m e r i s t e m meristem  J-, w h i c h  M S I Q  (Fig. 1 1 ) .  H e a t - t r e a t i n g source p l a n t s i n c r e a s e d meristem only s l i g h t l y  IBA  tips.  cultured  Apical  (Fig. 1 2 ) .  76.  Excision  d a t e was n o t i m p o r t a n t .  in vitro  s u r v i v e d 3 months o f c o l d  Red r a s p b e r r y  plantlets  s t o r a g e , b u t powdery  m i l d e w was a p r o b l e m when p l a n t l e t s w e r e t r a n s f e r r e d soil.  I t i s recommended  to planting  that a fungicide  cold-stored plantlets  in soil.  to  be u s e d j u s t  prior  77. 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Recherches s u r l a n u t r i t i o n m i n e r a l e d e s t i s s u e vegetaux c u l t i v e s i n v i t r o . These P a r i s e t Ann. S c i . N a t . (Bot. B i o l . V e g . ) , P a r i s 1 4 : 1 - 2 2 3 . James,  D. J . and Thurbon, I. J . 1978. Growth i n Rubus h y b r i d b e r r i e s a n d s t r a w b e r r y . E a s t Res. S t n . Annu. Rep. f o r 1977. pp.177-178.  J e n n i n g s , I n s t , K i s s ,  D.  L.  1978.  Tayberry.  f o r  1977.  p . 5 5 .  Rep.  S c o t t .  v i t r o o f M a i l i n g  H o r t .  R e s .  F. and Zatyko, J . 1978. V e g e t a t i v e p r o p a g a t i o n Rubus s p e c i e s i n v i t r o . ( i n Hungarian, E n g l i s h s t r a c t ) . B o t . Kozlem. 6 5 : 6 5 - 6 8 .  of A b -  78. Literature Cited  (cont'd)  L i n s m a i e r , E. and S k o o g , F. 1965. O r g a n i c g r o w t h f a c t o r requirements of tobacco t i s s u e c u l t u r e . Physiol. P l a n t . 18:100-127. M e l l o r , F. C. a n d S t a c e - S m i t h , R. 1976. 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