UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1980_A6_7 D66.pdf [ 6.07MB ]
Metadata
JSON: 831-1.0095410.json
JSON-LD: 831-1.0095410-ld.json
RDF/XML (Pretty): 831-1.0095410-rdf.xml
RDF/JSON: 831-1.0095410-rdf.json
Turtle: 831-1.0095410-turtle.txt
N-Triples: 831-1.0095410-rdf-ntriples.txt
Original Record: 831-1.0095410-source.json
Full Text
831-1.0095410-fulltext.txt
Citation
831-1.0095410.ris

Full Text

C.I IN VITRO CULTURE OF FOUR RUBUS SPECIES B.Sc. (Agr.), Macdonald College, McGill University, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES (The Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apr i l , 1980 (c) Danielle Juli e Donnelly by Danielle Julie Donnelly i n In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P l a n t S c i e n c e The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e A p r i l 1980 6 A b s t r a c t M e r i s t e m t i p c u l t u r e f o l l o w e d b y i n v i t r o m i c r o p r o p a -g a t i o n o f c u l t u r e d p l a n t s i s a v a l u a b l e t e c h n i q u e f o r p r o -d u c i n g a n d m a s s p r o p a g a t i n g d i s e a s e - f r e e s e l e c t g e r m p l a s m . T h i s i n v e s t i g a t i o n w a s i n i t i a t e d t o d e t e r m i n e t h e n u t r i t i o n -a l a n d e n v i r o n m e n t a l r e q u i r e m e n t s f o r t h e c u l t u r e o f r e d r a s p b e r r y ( R u b u s i d a e u s L . ) . A p r o c e d u r e w a s f i r s t d e v e l o p e d u s i n g b l a c k r a s p b e r r y ( R . o c c i d e n t a l i s L . ) , l o g a n b e r r y ( R . l o g a n o b a c c u s B a i l e y ) , a n d t a y b e r r y , a l o g a n b e r r y - l i k e h y b r i d . S a t i s f a c t o r y l e v e l s o f m a j o r a n d m i n o r e l e m e n t s , v i t a m i n s , i n o s i t o l , h o r m o n e s , c a r b o h y d r a t e s , a n d p H w e r e t h e n e s t a b l i s h e d f o r r e d r a s p b e r r y i n c u b a t i o n t e m p e r a t u r e a n d p h o t o p e r i o d w e r e d e t e r m i n e d . S o m e r e d r a s p b e r r y p l a n t s w e r e g i v e n e t i o l a t i o n p r e - t r e a t m e n t p r i o r t o c u l t u r e . S o m e b l a c k r a s p b e r r y , l o g a n b e r r y , a n d r e d r a s p b e r r y p l a n t s w e r e g i v e n h e a t - t r e a t m e n t p r i o r t o c u l t u r e . E x p l a n t s i z e a f f e c t e d s u r v i v a l , b u t n o t r o o t i n g i n v i t r o . A p i c a l m e r i s t e m t i p s w e r e s u p e r i o r t o l a t e r a l m e r i s -t e m t i p s o n l y i n s o m e m e d i a . S e a s o n o f e x c i s i o n d i d n o t i n f l u e n c e s u r v i v a l i n c u l t u r e . S p r i n g - t i m e d i d a p p e a r t o i n c r e a s e v i g o u r i n c u l t u r e d p l a n t s . E t i o l a t i n g s o u r c e p l a n t s w a s n o t a d v a n t a g e o u s . T h e 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 p r i o r t o m e r i s t e m t i p c u l t u r e v a r i e d w i t h t h e R u b u s s p e c i e s . W i t h b l a c k r a s p b e r r y , s u r v i v a l a n d g r o w t h r a t e w e r e i n c r e a s e d , a n d r o o t i n g h a s t e n e d . i i i . Heat-treatment of loganberry source p l a n t s had no e f f e c t on meristem t i p s . With red r a s p b e r r y , s u r v i v a l was o n l y s l i g h t l y i n c r e a s e d ; growth r a t e was not a f f e c t e d . Three media used i n s u c c e s s i o n induced meristem t i p development, shoot p r o l i f e r a t i o n , and r o o t i n g , r e s p e c t i v e l y . The b a s a l medium used was t h a t of Murashige and Skoog (1962). E f f e c t of d i l u t i n g macroelements, even by a 1:4 d i l u t i o n was not dramatic. I n o s i t o l a t 150 mg/L s t i m u l a t e d a x i l l a r y bud p r o l i f e r a t i o n . I n o s i t o l i n the medium i n h i b i t e d r o o t -i n g . E f f e c t s of the hormones i n d o l e b u t y r i c a c i d (IBA) and 6-benzylaminopurine (BAP) were c o n s i s t a n t w i t h other r e p o r t s . Naphthalene a c e t i c a c i d (NAA) caused c a l l u s p r o l i f e r a t i o n and induced a d v e n t i t i o u s r o o t formation from l e a f p e t i o l e s , l e a f lamina, and c a l l u s e d shoot bases. Rooting was normal wi t h IBA. L e v e l s of pH and carbohydrates were c r i t i c a l , and e f f e c t s were r e l a t e d to c o n c e n t r a t i o n of the c y t o k i n i n BAP i n the medium. Incub a t i o n temperature used was 27°C. T h i s temperature may not have been optimum f o r red r a s p b e r r y micropropagation, although i t was adequate f o r meristem t i p c u l t u r e . Moderate l i g h t i n t e n s i t y , and the use of c o o l white f l u o r e s c e n t l i g h t s were s a t i s f a c t o r y . A photoperiod of 16 hours was adequate. A l l f o u r Rubus s p e c i e s t e s t e d had s i m i l a r c u l t u r e r e -quirements, and micropropagation p o t e n t i a l . T h e i r n u t r i t i o n -a l requirements are w e l l w i t h i n the range r e p o r t e d i n the l i t e r a t u r e f o r Rosaceous p l a n t s . I V . T a b l e o f C o n t e n t s Page T i t l e Page A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f F i g u r e s v i L i s t o f T a b l e s i x Acknowledgement x I n t r o d u c t i o n 1 M a t e r i a l s and Methods 10 A. B l a c k R a s p b e r r y 13 1. C u l t u r e Media 13 2. Source P l a n t P r e - t r e a t m e n t 16 3. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s 16 B. L o g a n b e r r y 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. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s 19 C. T a y b e r r y 19 1. C u l t u r e Media 19 D. Red R a s p b e r r y 20 1. C u l t u r e Media 20 2. Source P l a n t P r e - t r e a t m e n t 23 3. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s 24 V . T a b l e o f C o n t e n t s (cont'd) R e s u l t s Page 25 A. B l a c k R a s p b e r r y 25 25 36 1. C u l t u r e Media 2. Source P l a n t P r e - t r e a t m e n t 3. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s 3 6 B. L o g a n b e r r y 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 4 2 3. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s 44 C. T a y b e r r y 44 1. C u l t u r e Media 44 D. Red R a s p b e r r y 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. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s ^2 D i s c u s s i o n 55 Summary -74 L i t e r a t u r e C i t e d 77 B i o g r a p h i c a l I n f o r m a t i o n v i . L i s t o f F i g u r e s F i g u r e P a g e 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 2 6 m o n t h s o f c u l t u r e i n m e d i u m M S w i t h k i n e t i n a t 0 . 0 4 m g / L , s h o w i n g s l o w , e r r a t i c d e v e l o p -m e n t . 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 f o r m e d i n s o m e 3 4 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 w e r e t h o u g h t t o b e r u d i m e n t a r y f l o w e r s . 3 . E f f e c t o f h e a t - t r e a t i n g b l a c k r a s p b e r r y 3 7 s o u r c e p l a n t s o n d e v e l o p m e n t o f m e r i s t e m t i p s i n m e d i u m M S w i t h c y s t e i n e H C l a t 1 0 m g / L , B A P a t 0 . 1 m g / L , a n d I B A a t 1 . 0 m g / L , p H . 5 . 7 . T h e r o o t e d p l a n t l e t s a r o s e f r o m m e r i s t e m t i p s e x c i s e d f r o m h e a t - t r e a t e d s o u r c e p l a n t s ; t h e s e t o o k 4 - 5 w e e k s t o r o o t . T h e r o o t l e s s s h o o t w a s f r o m a n o n -h e a t - t r e a t e d s o u r c e p l a n t ; t h e s e t o o k f r o m 5 - 8 w e e k s t o r o o t . 4 . L o g a n b e r r y c l o n e s i l l u s t r a t i n g t h e e f f e c t o f 3 9 t w o c a r b o h y d r a t e s o u r c e s , a n d t w o l e v e l s o f B A P , o n 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 m e d i u m M S w i t h B A P a t 1 . 0 m g / L a n d I B A a t 0 . 1 m g / L . A : A f t e r 2 0 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 , t h e p l a n t l e t w a s d i v i s i b l e i n t o 1 5 c u t t i n g s . B : A f t e r 1 2 w e e k s o f c u l t u r e 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 , t h e s h o o t w a s t r a n s f e r r e d t o m e d i u m M S w i t h B A P i n -c r e a s e d t o 3 . 0 m g / L . A f t e r a n o t h e r 6 w e e k s , t h e l e a v e s w e r e s m a l l e r t h a n o n A , a n d s o m e - . .. w h a t c u r l e d , b u t t h e p l a n t l e t w a s d i v i s i b l e i n t o 2 5 c u t t i n g s . C : A f t e r 1 4 w e e k s o f c u l t u r e i n s t a r t i n g m e d i u m w i t h g l u c o s e , t h e s h o o t w a s d i v i s i b l e i n t o 6 0 c u t t i n g s . D : A f t e r 8 w e e k s o f c u l t u r e i n s t a r t i n g m e d i u m w i t h g l u c o s e , t h e s h o o t w a s t r a n s -f e r r e d t o m e d i u m w i t h B A P i n c r e a s e d t o 3 . 0 m g / L . A f t e r a n o t h e r 6 w e e k s , t h e l e a v e s w e r e s m a l l e r t h a n o n C , a n d s o m e w h a t c u r l e d , a n d t h e p l a n t l e t w a s d i v i s i b l e i n t o 2 0 c u t -t i n g s . v i i . L i s t of F i g u r e s (cont'd) F i g u r e Page 5. Tayberry 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 45 carbohydrate source, and two l e v e l s of 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 with glucose, and wit 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, the shoot was t r a n s f e r r e d to medium wit h sucrose, and BAP a t 3.0 mg/L. A f t e r another 4 weeks, the l e a v e s were l a r g e r and the r o o t s not as w e l l developed as on A. 6. E f f e c t s of carbohydrate source, sucrose (S) 53 or g lucose (G), i n o s i t o l ( I ) , or hormones (H) on the growth of meristem t i p s of red ra 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 ) times are i n d i c a t e d by an arrow. 7. E f f e c t s of i n o s i t o l c o n c e n t r a t i o n on develop- 54 ment of red 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 i n c u l t u r e . 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. I n o s i t o l i n h i b i t e d 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 i n c r e a s e d pro-l i f e r a t i o n of a x i l l a r y buds. 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 - 55 t u r e showing response of some 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. A, B, C, and D were a l l s t a r t e d i n medium wit h sucrose, 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 to medium wit h BAP i n c r e a s e d to 3.0 mg/L. A f t e r 4 more months i n c u l t u r e the p l a n t l e t s were 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 r e s p e c t i v e l y . B and C developed from buds e x c i s e d from the 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 t h a t can occur w i t h i n s e l e c t i o n s . A and D developed from buds of two other s e l e c t i o n s , and i l l u s t r a t e the v a r i a b i l i t y i n response to BAP i n the growth medium; s e l e c t i o n A grew bes t i n s t a r t i n g medium, D grew bes t i n medium wit h BAP a t 3.0 mg/L. v i i i . L i s t o f F i g u r e s ( c o n t ' d ) F i g u r e P a g e 9. R e d 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 56 o f p H o n l e a f s i z e a n d b r o w n i n g . B o t h p l a n t l e t s w e r e g r o w n i n m e d i u m M S w i t h B A P a t 1.0 m g / L , I B A a t 0.1 m g / L w i t h . g l u c o s e , f o r 7 m o n t h s . A : A t p H 6.1 B : A t p H 5.7. 10. R e d r a s p b e r r y c l o n e s s h o w i n g d i f f e r e n t 57 r e s p o n s e o f t w o 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 m e d i u m w i t h g l u c o s e . A f t e r 4 m o n t h s i n s t a r t i n g m e d i u m w i t h g l u c o s e , I B A a t 0.1 m g / L , a n d B A P a t 1.0 m g / L , a n d 3 m o n t h s i n m e d i u m w i t h B A P i n c r e a s e d t o 3.0 m g / L , A w a s 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. C l o n e s i n m e d i u m w i t h g l u c o s e h a d l e a v e s t h a t w e r e u s u a l l y l a r g e r a n d m o r e r u g o s e t h a n i n m e d i u m w i t h s u c r o s e . 11. R e d 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 60 m e d i u m M S w i t h B A P a t 1.0 m g / L a n d I B A a t 0.1 m g / L a f t e r 3 w e e k s i n r o o t i n g m e d i u m M S w i t h I B A a t 0.5 m g / L . 12. : R e d r a s p b e r r y a p i c a l ( t o p r o w ) a n d l a t e r a l 64 ( b o t t o m r o w ) p a i r s o f m e r i s t e m t i p s s h o w -i n g t h e i n f l u e n c e o f c a r b o h y d r a t e t y p e a n d c o n c e n t r a t i o n o n d e v e l o p m e n t a f t e r 2 m o n t h s i n m e d i u m M S w i t h B A P a t 1.0 m g / L a n d I B A a t 0.1 m g / L . C a r b o h y d r a t e s o u r c e s w e r e ( l e f t t o r i g h t ) : s u c r o s e a t 20, a n d 30 g / L a n d g l u c o s e a t 20, 30, a n d 40 g / L . i x . L i s t o f T a b l e s T a b l e Page 1. The e f f e c t o f the hormones BAP and GA 3, and 28 the a n t i o x i d a n t s PVP and c y s t e i n e HC1, 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 m e r i s t e m t i p s i n medium MS w i t h IBA a t 1.0 mg/L 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 , and BAP 29 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 m e r i s t e m t i p s 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 31 medium, and BAP and IBA 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 m e r i s t e m t i p s 4. The e f f e c t s o f BAP and IBA c o n c e n t r a t i o n i n 33 l i q u i d o r s o l i d medium on new l e a f and r o o t f o r m a t i o n o f b l a c k r a s p b e r r y s h o o t s 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 - 41 h y d r a t e 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 l o g a n b e r r y 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 6. Comparison o f l o g a n b e r r y c u t t i n g s r o o t e d i n 4 3 s o i l and i n v i t r o 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 - 50 t i o n on s u r v i v a l and growth o f r e d r a s p b e r r y m eristem t i p s i n n u t r i e n t medium 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 51 on s u r v i v a l and growth 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 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 61 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 f o u r media 10. E f f e c t o f h e a t - t r e a t m e n t (37°C) o f sour c e 63 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 media X . A c k n o w l e d g m e n t I w i s h t o e x p r e s s g r a t i t u d e t o t h e m e m b e r s o f m y c o m m i t t e e : F r a n c e s C . M e l l o r a n d D r . R . S t a c e - S m i t h , A g r i c u l t u r e C a n a d a , R e s e a r c h S t a t i o n , V a n c o u v e r ; D r . V . C . R u n e c k l e s a n d D r . R . J . C o p e m a n , D e p a r t m e n t o f P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , f o r t h e i r g u i d a n c e a n d s u g g e s t i o n s p e r t a i n i n g t o t h i s t h e s i s . I a m g r a t e f u l t o t h e N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a f o r t h e i r f i n a n c i a l s u p p o r t . I w o u l d l i k e t o t h a n k D r . P . T o w n s l e y a n d W , C . C h a l m e r s o f t h e F o o d S c i e n c e d e p a r t -m e n t , U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r t h e i r e n c o u r a g e -m e n t a n d s u g g e s t i o n s . I a l s o w i s h t o t h a n k M r . S . W . M a c D i a r m i d f o r h i s e x c e l l e n t p h o t o g r a p h y , D r . H . D a u b e n y f o r h i s d o n a t i o n o f r e d r a s p b e r r y a n d t a y b e r r y p l a n t s , a n d t h e o t h e r m e m b e r s o f t h e s t a f f o f A g r i c u l t u r e C a n a d a , R e s e a r c h S t a t i o n , V a n c o u v e r f o r t h e i r a s s i s t a n c e a n d e n c o u r a g e m e n t . S p e c i a l t h a n k s t o m y d e a r h u s b a n d W a y n e f o r h i s e n t h u s i a s t i c s u p p o r t . 1. I n t r o d u c t i o n 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 h e i n i t i a -t i o n o f meri s t e m c u l t u r e and were t h e f i r s t t o use t h i s t e c h n i q u e f o r r i d d i n g p l a n t s o f v i r u s e s . I n 1960 M o r e l e x c i s e d young m e r i s t e m a t i c r e g i o n s o f cymbidiums and, by c u l t u r i n g t h e s e e x p l a n t s on n u t r i e n t b r o t h , d e v e l o p e d v i r u s - f r e e c l o n e s . T h i s p r o c e d u r e a l s o r e v e a l e d an im-p o r t a n t p o t e n t i a l f o r p l a n t m u l t i p l i c a t i o n ( m i c r o p r o p a g a t i o n ) . 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 from 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 c u l t i v a r s . They have d i s c o v e r e d t h a t m e r i s t e m a t i c t i p s e x c i s e d from h e a t - t r e a t e d p l a n t s a r e o f t e n f r e e from v i r u s e s t h a t a r e not e a s i l y e l i m i n a t e d by meri 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). Thermotherapy e n a b l e s r e l a t i v e l y l a r g e e x p l a n t s t o be v i r u s - f r e e , and t h e s e more r e a d i l y d e v e l o p i n c u l t u r e . H o r t i c u l t u r i s t s , p l a n t b r e e d e r s and growers o f ornamen-t a l and food c r o p s have been a t t r a c t e d by t h e p o s s i b i l i t y o f 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 . The advan-t a g e s o f m i c r o p r o p a g a t i o n i n c l u d e s u p e r i o r r a t e o f m u l t i p -l i c a t i o n , s h o r t g e n e r a t i o n t i m e , y e a r - r o u n d p r o d u c t i o n , l i m i t e d 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 se-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 c l o n e s . I n v i t r o p r o p a g a t i o n e l i m i n a t e s r e i n f e c t i o n r i s k s , common t o greenhouse o r f i e l d m u l t i p l i e d s t o c k s . The p l a n t -l e t s a r e t h e r e f o r e e a s i l y and e x p e d i t i o u s l y t r a n s p o r t e d , 2. both r e g i o n a l l y and i n t e r n a t i o n a l l y , w i t h minimized r i s k of spreading pests or pathogens. In the f a m i l y Rosaceae, f i v e genera, c o n t a i n i n g ten commercially important s p e c i e s , have been u t i l i z e d f o r meris-tem t i p c u l t u r e , shoot t i p c u l t u r e and/or micropropagation. The genera, and s p e c i e s are as f o l l o w s : F r a g a r i a , straw-berry; Malus, apple; Prunus, c h e r r y , peach-almond h y b r i d s , almond, plum; Rosa, rose; Rubus, red r a s p b e r r y , b l a c k b e r r y , t a y b e r r y . Of these, s i x s p e c i e s are l i s t e d by Murashige (1978) as commercially propagatable f r u i t or nut crops. These i n c l u d e : strawberry, apple, c h e r r y , peach-almond h y b r i d s , almond, and b l a c k b e r r y . Two s p e c i e s , strawberry and red r a s p b e r r y , are l i s t e d by Walkey (1978), and by Murashige (1978), as having been s u c c e s s f u l l y shoot t i p c u l t u r e d f o r the purpose of v i r u s e l i m i n a t i o n . When t h i s p r o j e c t was s t a r t e d , the o b j e c t was to de-termine some of the f a c t o r s a f f e c t i n g the growth and r o o t -in g i n v i t r o of the a g r i c u l t u r a l l y important red r a s p b e r r y (Rubus idaeus L . ) . The primary m o t i v a t i o n f o r t h i s i n v e s -t i g a t i o n , as w i t h p r e v i o u s s t u d i e s concerned w i t h the i n v i t r o c u l t u r e of red r a s p b e r r y , was the need f o r v i r u s e l i m i n a t i o n from u n i v e r s a l l y i n f e c t e d c u l t i v a r s (Shchelkunova and Popov, 1970; Putz, 1971; V e r t e s y , 1979; Carrg e t a l . , 1979) . The most favored method f o r micropropagation begins w i t h meristem t i p s which p a s s e s s meristemoids and c e l l s t h a t generate meristemoids. The normal o r g a n i z a t i o n of the 3. d i f f e r e n t i a t e d s h oot i s t h e r e f o r e r e t a i n e d , and t h e r i s k o f a b e r r a n t c e l l s d e v e l o p i n g i n t o p l a n t s t h a t a r e g e n e t i c d e v i a n t s i s l i t t l e o r n i l ( A b b o t t , 1976). T h i s i s h i g h l y 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 s e l e c t e d 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 (Murashige, 1977) . V a r i a b i l i t y i n r e g e n e r a t i v e c a p a c i t y o f m e r i s t e m t i p s may be a t t r i b u t e d t o i n i t i a l s i z e , l a t e r a l o r a p i c a l o r i g i n , d i f f e r e n c e s i n s o u r c e 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 age, and the season i n w h i c h e x p l a n t s a r e t a k e n (Murashige, 1974). The o n l y one o f t h e s e f a c t o r s t h a t has been i n v e s t i g a t e d 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 on r e d r a s p b e r r y e x p l a n t s u r v i v a l i n c u l t u r e . Shchelkunova (1974) e x c i s e d e t i o l a t e d m e r i s t e m t i p s from s p r o u t e d r o o t c u t t i n g s , and found t h e 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 March o r A p r i l - J u n e e x c i s i o n s . C a r r S e t a l . (1979) t o o k meristem t i p s a t monthly i n t e r v a l s from v i g o r o u s l y g rowing p l a n t s , and found t h a t from March-June, s u r v i v a l was maximum (up t o 5 0 % ) . From July-September m e r i s t e m t i p s s u r v i v e d but formed o n l y c a l l u s . From September-Febuary no m e r i s t e m t i p s s u r v i v e d . 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 m e r i s t e m t i p s from s p r o u t i n g r o o t c u t t i n g s (Shchelkunova and Popov, 1970; Shchelkunova, 1974; K i s s and Zatyko, 1978; Anderson and Haner, 1979). O t h e r s b e g i n w i t h v i g o r o u s l y growing p l a n t s ( P u t z , 1971; C a r r g e t a l . , 1979). 4. V i n e (1968) and M u l l e n e t a l . (1974, 1975), w o r k i n g w i t h Rosaceous p l a n t s , r e p o r t e d t h a t m e r i s t e m t i p s e x c i s e d from some h e a t - t r e a t e d s o u r c e p l a n t s , w i l l more r e a d i l y 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 from non-heat-t r e a t e d s o u r c e p l a n t s . The same phenomenon was r e p o r t e d f o r 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 , o r n o d a l s e g -ments w i t h one l e a f and dormant a x i l l a r y bud ( M e l l o r and S t a c e - S m i t h , 1976). These a l s o r o o t e d more r a p i d l y t h a n comparable c o n t r o l c u t t i n g s . I n i t i a l s i z e o f Rubus e x p l a n t s may be i m p o r t a n t i n the f a c i l i t y w i t h which d e r i v e d s h o o t s w i l l r o o t i n c u l t u r e . D i f f i c u l t y w i t h r o o t i n g was r e p o r t e d by most workers who began w i t h m e r i s t e m t i p s (Shchelkunova and Popov, 1970; Shchelkunova, 1974; P u t z , 1971; M e l l o r and S t a c e - S m i t h , 1976). 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 , where l a r g e e x p l a n t s (0.5-2.0 cm) were used, y i e l d e d r e l a t i v e l y e a s i l y r o o t e d s h o o t s (James and Thurbon, 1977; K i s s and Zatyko, 1978; M e l l o r and S t a c e - S m i t h , 1976). Carrg. e t a l . (1979) a t t r i b u t e d t h e i r s u c c e s s i n r o o t i n g s h o o t s d e r i v e d from m e r i s t e m t i p s 0.05-0.06 x 0.10-0.20 mm t o t h e i n c l u s i o n of 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. They thought 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 and demonstrated t h a t i t c o n t a i n e d p o s s i b l e r o o t i n g f a c t o r s , the phenolamines, p u t r e s c i n e , spermedine, and spermine. Of v i t a l i m p o r t a n c e i n t h e r e g e n e r a t i o n o f m e r i s t e m t i p s i s t h e adequacy o f the c u l t u r e medium components. A l l 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 major and minor m i n e r a l elements known t o be e s s e n -t i a l f o r p l a n t growth; a s o u r c e o f c a r b o h y d r a t e f o r c a r b o n and energy; 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 B v i t a m i n s , and sometimes amino a c i d s o r o t h e r supplements; and phytohormones. Media d i f f e r , 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 -t i o n s o f t h e major 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 t h e 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 i n c l u d e M o r e l ' s macroelements, H e l l e r ' s (1953) m i c r o e l e m e n t s (Shchelkunova and Popov, 1970; Shchelkunova, 1974; V e r t e s y , 1979); Murashige and Skoog's (1962) macroelements and m i c r o -elements ( P u t z , 1971; James and Thurbon, 1977; H a r p e r , 1978; Broome and Zimmerman, 1978; C a r r g e t a l . , 1979 w i t h ammonium n i t r a t e reduced t o 4 00 mg/L); and N i t s c h and N i t s c h ' s (1969) macroelements and m i c r o e l e m e n t s ( K i s s and Zatyko, 1978). When t h i s i n v e s t i g a t i o n was s t a r t e d , t h e 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); o f any Rubus s p e c i e s , by Broome and Zimmerman (1978). B o t h groups used Murashige and Skoog's (1962) 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 of n i t r a t e , ammonium, c a l c i u m , and p o t a s s i u m 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%, i s t h e u s u a l s o u r c e of c a r b o h y d r a t e f o r c a r b o n and energy. G l u c o s e i s used l e s s o f t e n . No o t h e r c a r b o h y d r a t e has y e t been 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 the c u l t u r e o f Rubus s p e c i e s , s u c r o s e a t 30 g/L was used by P u t z (1971), James and Thurbon (1977), Harper (1978), Broome and Zimmerman (1978), and Carr§ e t a l . (1979); g l u c o s e a t 20-30 g/L by Shchelkunova and Popov (1970), Shchelkunova (1974), K i s s and Zatyko (1978), and V e r t e s y (1979) . The B v i t a m i n s t h i a m i n e , n i c o t i n i c a c i d , p y r i d o x i n e , and 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 o f t e n t h e amino a c i d g l y c i n e as w e l l . I n o s i t o l , a sugar a l c o h o l , i s a common supplement, employed a t 100 mg/L. I t i s c o n s i d e r e d b e n e f i -c i a l but n o t e s s e n t i a l . V i t a m i n m i x t u r e s f o r c u l t u r i n g Rubus s p e c i e s i n c l u d e t h a t o f Butenko (1964) (Shchelkunova and Popov, 1970; Shchelkunova, 1974); M u r a s h i g e and Skoog (1962), c o n t a i n i n g (mg/L): g l y c i n e ( 2 . 0 ) , n i c o t i n i c a c i d ( 0 . 5 ) , p y r i d o x i n e ( 0 . 5 ) , t h i a m i n e (0.1) ( P u t z , 1971; and Broome and Zimmerman, 197 8 who i n c r e a s e d t h e amount o f t h i a m i n e t o 0.4 mg/L); L i n s m a i e r and Skoog (1965) w h i c h c o n s i s t s o n l y o f t h i a m i n e a t 0.4 mg/L (James and Thurbon, 1977; and Harper, 1978); and N i t s c h and . N i t s c h (1969), c o n t a i n i n g (mg/L): b i o t i n ( 0 .05), g l y c i n e ( 2 . 0 ) , n i c o t i n i c a c i d ( 5 . 0 ) , p y r i d o x i n e ( 0 . 5 ) , t h i a m i n e ( 0 . 5 ) , and f o l i c a c i d (5.0) ( K i s s and Z a t y k o , 1978). C a r r € e t a l . (1979) used M o r e l and M u l l e r ' s (1964) v i t a m i n s w h i c h i n c l u d e b i o t i n a t 0.01 mg/L and n i c o t i n i c a c i d , p y r i d o x i n e , t h i a m i n e , and c a l c i u m p a n t o t h e n a t e each a t 1.0 mg/L, and t h e y i n c l u d e d t h e supplements c y s t e i n e HC1 a t 1.0 mg/L and L - g l u t a m i n e a t 200 mg/L. I n o s i t o l a t 100 mg/L was used by a l l Rubus 7. w o r k e r s e x c e p t S h c h e l k u n o v a a n d P o p o v (197 0 ) , a n d S h c h e l k u n o v a (1974) . P h y t o h o r m o n e s a r e t h e m o s t c r i t i c a l o r g a n i c c o m p o n e n t s o f p l a n t p r o p a g a t i o n m e d i a . T h e a b s o l u t e a n d r e l a t i v e c o n -c e n t r a t i o n s o f a u x i n s , c y t o k i n i n s , a n d g i b b e r e l l i n s d e t e r m i n e p l a n t g r o w t h a n d d e v e l o p m e n t i n c u l t u r e . T h o s e m o s t c o m -m o n l y u s e d i n t h e c u l t u r e o f R u b u s s p e c i e s a r e t h e a u x i n s , i n d o l e - 3 - a c e t i c a c i d ( I A A ) , n a p h t h a l e n e a c e t i c a c i d ( N A A ) , a n d 8 - ( i n d o l e - 3 ) - b u t y r i c a c i d ( I B A ) ; t h e c y t o k i n i n s , 6 - f u r f u r y l -a m i n o p u r i n e o r k i n e t i n ( K ) , a n d 6 - b e n z y l a m i n o p u r i n e ( B A P ) ; a n d t h e g i b b e r e l l i n , g i b b e r e l l i c a c i d ( G A ^ ) . M e d i a m a y b e l i q u i d , o r s o l i d i f i e d w i t h v a r y i n g c o n -c e n t r a t i o n s a n d q u a l i t i e s o f a g a r . L i q u i d c u l t u r e m a y n e c e s -s i t a t e a s u p p o r t s t r u c t u r e , a n d m a y b e h e l d s t a t i o n a r y o r a g i t a t e d t o v a r i o u s e x t e n t s . T h e d e g r e e o f s o l i d i t y o f t h e m e d i u m m a y a f f e c t e x p l a n t s u r v i v a l , a s w e l l a s d e t e r m i n e p l a n t m u l t i p l i c a t i o n a n d g r o w t h ( M u r a s h i g e , 197 7 ) . O n l y P u t z (1971), V e r t e s y (1979), a n d B r o o m e a n d Z i m m e r m a n (1978) u s e d l i q u i d m e d i u m . B r o o m e a n d Z i m m e r m a n s t a r t e d c u l t u r e s o n l i q u i d m e d i u m a n d t r a n s f e r r e d e s t a b l i s h e d s h o o t s t o s o l i d m e d i u m . A l l o t h e r c u l t u r e m e d i a r e p o r t e d f o r R u b u s s p e c i e s h a v e b e e n s o l i d i f i e d w i t h a g a r a t 5-7 g / L . T h e p H o f t h e n u t r i e n t m e d i u m i s u s u a l l y a d j u s t e d t o 5.0-6.0 d u r i n g p r e p a r a t i o n . S o m e R u b u s w o r k e r s a d j u s t t h e p H t o 5.5-5.7 p r i o r t o a u t o c l a v i n g ( H a r p e r , 1978; C a r r g e t a l . , 1979); o t h e r s a d j u s t t h e p H p r i o r t o a u t o c l a v i n g t o g i v e a p H o f 5.6-5.8 a f t e r a u t o c l a v i n g ( B r o o m e a n d 8. Zimmerman, 1978). L i g h t i n t e n s i t y , q u a l i t y , and p h o t o p e r i o d , p r o f o u n d -l y i n f l u e n c e p l a n t growth and o r g a n i z e d development. For c u l t u r e i n i t i a t i o n and m u l t i p l i c a t i o n , a l i g h t i n t e n s i t y o f 1,000-3,000 l u x i s u s u a l l y adequate. D u r i n g t h e l a t e r s t a g e s of c u l t u r e 3,000-10,000 l u x promotes s u r v i v a l and i n c r e a s e s growth r a t e when the p l a n t l e t s a r e t r a n s f e r r e d t o s o i l ( Murashige, 1974, 1977). M u rashige (1974) p o s t u l a t e d t h a t key o r g a n o g e n e t i c p r o c e s s e s i n t i s s u e c u l t u r e a r e photomor-phogenic phenomena, p r o b a b l y r e g u l a t e d by phytochrome. T h i s i s s u b s t a n t i a t e d by t h e f a c t t h a t l i g h t near t h e u l t r a -v i o l e t and b l u e r e g i o n s o f t h e energy spectrum s t i m u l a t e s shoot growth, w h i l e r e d l i g h t enhances r o o t i n g . The p hoto-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 t h e normal p h o t o p e r i o d r e q u i r e m e n t s of each s p e c i e s . Some, p l a n t s have 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 . L i g h t i n t e n s i t y used i n t h e c u l t u r e o f Rubus s p e c i e s ranges from 500 l u x ( P u t z , 1971), t o 2,200-4,300 l u x (Broome and Zimmerman, 1978). The q u a l i t y o f l i g h t r e q u i r e d f o r Rubus s p e c i e s i n v i t r o has not been s t u d i e d e x t e n s i v e l y . Rubus s p e c i e s were u s u a l l y exposed t o 16-hour p h o t o p e r i o d b o t h i n t h e greenhouse and d u r i n g c u l t u r e , w i t h t h e e x c e p t i o n o f 12-hour p h o t o p e r i o d ( V e r t e s y , 1979), and t r i a l s w i t h 24-hour l i g h t exposure (Broome and Zimmerman, 197 8 ) . Temperature r e q u i r e m e n t s d u r i n g c u l t u r e g e n e r a l l y a r e f u l f i l l e d by t e m p e r a t u r e s i n t h e 20's°C. Some s p e c i e s may 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 r e q u i r e m e n t s 9. (Murashige, 1977). Rubus c u l t u r e s have been incubated a t 18-29°C and have not been demonstrated to, r e q u i r e c h i l l i n g , or d i u r n a l f l u c t u a t i o n s . The u l t i m a t e o b j e c t i v e of t h i s study was to determine the requirements f o r meristem t i p c u l t u r e , i n v i t r o c u l t i v a -t i o n , and r o o t i n g of red r a s p b e r r y . However, red r a s p b e r r y p l a n t s are not onl y d i f f i c u l t t o grow and to r o o t i n c u l t u r e , they are a l s o d i f f i c u l t to grow from stem c u t t i n g s . For t h i s reason, other Rubus s p e c i e s t h a t r e a d i l y r o o t from t i p c u t -t i n g s were used i n p r e l i m i n a r y experiments. Black r a s p b e r r y (R. o c c i d e n t a l i s L.) was the f i r s t s p e c i e s t e s t e d because i t s r e a d i n e s s to r o o t 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 . Loganberry (R. loganobaccus B a i l e y ) a l s o r o o t s e a s i l y from stem c u t t i n g s , and because i t i s the progeny of a red r a s p b e r r y - t r a i l i n g b l a c k b e r r y c r o s s , i t was the next s p e c i e s t e s t e d . Tayberry, a l o g a n b e r r y - l i k e h y b r i d of red r a s p b e r r y and u p r i g h t b l a c k b e r r y (Jennings, 1978), was the t h i r d s p e c i e s t e s t e d . Experience gained from i n v i t r o c u l t u r e of these s p e c i e s was used t o s o l v e the problem of de v e l o p i n g red r a s p b e r r y p l a n t s i n c u l t u r e from meristem t i p s . 10. M a t e r i a l s and Methods B l a c k r a s p b e r r y s o u r c e p l a n t s were s e e d l i n g s o f t h e c u l t i v a r Munger. F i v e mature l o g a n b e r r y p l a n t s were the s o u r c e o f a l l l o g a n b e r r y e x p l a n t s . One mature t a y b e r r y p l a n t was t h e s o u r c e o f a l l t a y b e r r y e x p l a n t s . Red r a s p -b e r r y s o u r c e p l a n t s were c u l t i v a r s Meeker and W i l l a m e t t e , and seven new s e l e c t i o n s (71-8-28, 115, 125, 133, 138, and 139, and 71-15-145). The r e d r a s p b e r r y p l a n t s were grown from dormant r o o t c u t t i n g s . B l a c k r a s p b e r r y seeds and r e d r a s p b e r r y 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 t o p r o v i d e a c o n s t a n t s u p p l y o f young s o u r c e p l a n t s . Most so u r c e p l a n t s were grown i n t h e greenhouse and exposed t o normal growing c o n d i t i o n s . Some s o u r c e p l a n t s were e t i o l a t e d by k e e p i n g greenhouse-grown p l a n t s i n t h e d a r k f o r 10 days o r more. Oth e r s were h e a t - t r e a t e d a t 37°C, i n an i n c u b a -t i o n chamber, 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 (3,000 l u x ) , w i t h 16-hour p h o t o p e r i o d . 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 i n t o s e c t i o n s and s u r f a c e s t e r i l i z e d by immersing i n a 10% s o l u -t i o n o f h o u s e h o l d b l e a c h ( a c t i v e i n g r e d i e n t 5.25-6% sodium h y p o c l o r i t e ) and s h a k i n g f o r 15 minutes on a w r i s t - a c t i o n s h a k e r . They were th e n g i v e n t h r e e 15-minute r i n s e - s h a k e s i n d i s t i l l e d w a t e r , and wrapped i n m o i s t paper t o w e l s t o a w a i t d i s e c t i o n . A l l e x c i s i o n s were performed under a d i s e c t i o n m i c r o -scope a t 40X m a g n i f i c a t i o n . Each m e r i s t e m t i p was e x c i s e d i n 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 fragment was used t o p e e l back t h e immature l e a v e s o f each l e a f bud and t o remove the me r i s t e m t i p whic h i n c l u d e d the m e r i s t e m a t i c dome and one p a i r o f l e a f p r i m o r d i a ; t o t a l l e n g t h l e s s than 1.0 mm (Quak, 1970). M e r i s t e m t i p s were measured 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 used, t h e meris t e m t i p was im-p l a n t e d on the c e n t e r o f the agar s l a n t . F o r l i q u i d medium, the m e r i s t e m t i p was p o s i t i o n e d on a f i l t e r p a p e r . o r 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 above 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 w i c k s because t h e y were s t r o n g e r when wet and were more a b s o r b e n t . Tubes were c l o s e d w i t h B a c t i - C a p a l l p o l y p r o p y l e n e c l o s u r e s , P a r a f i l m , o r p o l y p r o p y l e n e squares s e c u r e d w i t h e l a s t i c bands. B a c t i - C a p a l l c l o s u r e s were t h e most c o n v e n i e n t . G l a s s w a r e was c l e a n e d i n a Typhoon g l a s s w a r e washer, u s i n g an o r g a n i c d e t e r g e n t , and r i n s e d f o r two r i n s e c y c l e s i n c o l d t a p water. New tubes and j a r s were always 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 -t i l l e d water a g a i n , and washed f o r a t h i r d time,- as recom-mended by Thomas and Davey (1975). The 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 Murashige and Skoog's (1962) r e v i s e d medium f o r t o b a c c o p r o t o p l a s t s . U s u a l l y i t c o n t a i n e d a d d i t i o n a l t h i a m i n e HC1 (0.4 mg/L), as suggested by L i n s m a i e r and 1 2 . S k o o g ( 1 9 6 5 ) . E d a m i n ( c a s e i n h y d r o l y s a t e ) w a s o m i t t e d . M e d i a e m p l o y e d i n t h i s i n v e s t i g a t i o n w e r e c o m p o s e d o f M S , h o r m o n e s a n d / o r o t h e r a d d i t i v e s . E a c h f r e q u e n t l y - u s e d m e d i u m f o r m u l a t i o n h a s b e e n g i v e n a s e q u e n c e o f l e t t e r s y m b o l s i n d i c a t i n g t h e b a s i c m e d i u m , t h e a d d i t i v e a n d / o r h o r m o n e s u s e d ; e a c h h o r m o n e s y m b o l i s f o l l o w e d b y a s u b -s c r i p t w h i c h i n d i c a t e s t h e a m o u n t u s e d , i n m g / L . T h e h o r m o n e s m o s t o f t e n u s e d a n d t h e i r r e p r e s e n t a t i v e s y m b o l , a r e : B A P , ( B ) ; k i n e t i n , ( K ) ; I B A , ( I ) ; N A A , ( N ) ; a n d G A ^ , ( G ) . T h e a n t i o x i d a n t c y s t e i n e H C l ( C ) , w h e n u s e d , w a s a l w a y s a t 1 0 m g / L . M e d i a w e r e p r e p a r e d b y a d d i n g a p p r o p r i a t e v o l u m e s o f s t o c k s o l u t i o n s o f s a l t s , v i t a m i n s , a n d g r o w t h r e g u l a t o r s t o a l a r g e v o l u m e o f d i s t i l l e d w a t e r , t h e n a d d i n g w e i g h e d a m o u n t s o f s u g a r , i n o s i t o l , a n d s o m e t i m e s a n t i o x i d a n t s . H o r m o n e s w e r e a l w a y s f r e s h l y p r e p a r e d , a n d d i s s o l v e d i n a p p r o p r i a t e s o l v e n t s . E a c h m e d i u m w a s t h e n a d j u s t e d t o t h e c o r r e c t v o l u m e a n d t h e d e s i r e d p H . F i n a l l y , t h e m e d i u m w a s d i s p e n s e d i n t o g l a s s t u b e s 1 0 0 x 1 3 m m , 3 m l p e r t u b e , b e -f o r e s t e r i l i z i n g . W h e n B a c t o a g a r w a s i n c l u d e d , i t w a s d i s s o l v e d b y h e a t i n g , a n d a d d e d t o t h e m e d i u m b e f o r e d i s -p e n s i n g . W h e n P h y t a g a r w a s u s e d , i t w a s d i s s o l v e d i n t h e m e d i u m b y s t i r r i n g a n d t h e m e d i u m w a s d i s p e n s e d d u r i n g v i g o r o u s s t i r r i n g . M e d i a w e r e s t e r i l i z e d b y m i l l i p o r e f i l t r a t i o n b e f o r e d i s p e n s i n g i n t o s t e r i l e t u b e s o r , m o r e c o m m o n l y , b y a u t o -c l a v i n g a f t e r d i s p e n s i n g , a t 5 . 5 . k g p r e s s u r e f o r 1 2 m i n u t e s . When IBA was used 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 volume w i t h s t e r i l e d i s t i l l e d water and a s e p t i c a l l y d i s p e n s e d i n t o i n d i v i d u a l t u b e s subsequent t o a u t o c l a v i n g . Racks o f s t e r i l i z e d media were p l a c e d i n t o p l a s t i c bags, a l l o w e d t o c o o l t o room t e m p e r a t u r e , t h e n r e f r i g e r a t e d (4°C). Unused medium was d i s c a r d e d a f t e r 6 weeks s t o r a g e . C u l t u r e s were i n c u b a t e d i n growth chambers 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 (3,000 l u x ) , w i t h 16-hour p h o t o p e r i o d . Shoots o r p l a n t l e t s t h a t outgrew t h e i r t e s t tube c o n t a i n e r s were t r a n s f e r r e d t o 100-ml j a r s , con-t a i n i n g 9-12 ml o f medium. Shoots o r p l a n t l e t s were sup-p o r t e d a g a i n s t t h e s i d e s o f t h e j a r s w i t h s t r i p s o f M i r a c l o t h o r chromatography paper. When p l a n t l e t s were moved t o s o i l , h i g h r e l a t i v e h u m i d i t y was m a i n t a i n e d f o r 10 days t o 2 weeks by c o v e r i n g them w i t h a b e a k e r , o r e n c l o s i n g 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 were t r a n s f e r r e d t o s o i l , t h e i r bases were d u s t e d w i t h r o o t i n g hormone powder, c o n t a i n i n g 0.8% IBA, b e f o r e p l a n t i n g . B l a c k R a s p b e r r y C u l t u r e Media To d e v e l o p a medium t h a t would s u p p o r t t h e growth o f b l a c k r a s p b e r r y m e r i s t e m t i p s , s e v e r a l media were t e s t e d w i t h d i f f e r e n t hormones and hormone c o n c e n t r a t i o n s . I n some t e s t s , a n t i o x i d a n t s were used i n c l u d i n g p o l y v i n y l p y r r o l i d o n e (PVP) and c y s t e i n e H C l , b o t h i n the media and f o r r i n s i n g t o o l s d u r i n g e x c i s i o n . The media most f r e ^ q u e n t l y used were MS w i t h the hormones BAP a t 0.1 mg/L and IBA a t 1.0 mg/L, (MSB Q ^1^ Q) ; MS w i t h c y s t e i n e HCl a t 10 mg/L, (MSC); or b o t h , (MSCB Q Q ) . V a r i a t i o n s i n 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 C, and t h e r o t a t i o n of some c u l t u r e s a t 1 rpm i n s t e a d o f h o l d i n g them s t a t i o n a r y . P l a n t s a t two s t a g e s o f m a t u r i t y were e v a l u a t e d as s o u r c e s o f m e r i s t e m t i p s : s e e d l i n g s 3-6 months o l d , w h i c h were v i g o r o u s , and u s u a l l y l e s s than 50 cm t a l l ; and mature, p r o s t r a t e p l a n t s , w i t h a d v e n t i t i o u s r o o t s a t t h e i r t i p s . 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 t h e a d v e n t i t i o u s r o o t zone o f t h e o l d e r p l a n t s . From t h e s e s o u r c e p l a n t s , m e r i s t e m t i p s , and l a r g e r t i p s (1.0-2.0 and 2.1-3.0 mm), were e x c i s e d i n t o each of two media, MSCB^ ^ 1 ^ ^ and MSCB^ g l ^ Q . To d e t e r m i n e t h e optimum l e v e l o f t h e hormone IBA, m e r i s t e m t i p s -0.25 mm were e x c i s e d i n t o media w i t h BAP a t 0.1 mg/L and s i x l e v e l s o f IBA, from 0.0-1.0 mg/L. I n r o o t i n g e x p e r i m e n t s , s h o o t s d e v e l o p e d i n media M S C B Q ^ 1 ^ Q o r M S C B ^ ^ 1 ^ Q were s u b c u l t u r e d t o t h e same two media, o r t o media w i t h MSC and a range o f IBA c o n c e n t r a -t i o n s from 0.0-1.0 mg/L. To d e t e r m i n e whether d a r k n e s s a f f e c t e d the i n c i d e n c e o r q u a l i t y o f r o o t i n g , t h e base of some c u l t u r e tubes were c o v e r e d w i t h f o i l wrap t o keep shoot bases i n t h e d a r k . M i c r o p r o p a g a t i o n 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 would s t i m u l a t e shoot p r o l i f e r a t i o n i n c u l t u r e . The p r o l i f e r a t i o n media, o f w h i c h t h e r e were 18 c o m b i n a t i o n s , c o n t a i n e d MSC w i t h BAP a t 1.0, 3.0, and 5.0 mg/L and IBA a t 0.0, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/L. The media were l i q u i d , o r s o l i d i f i e d w i t h P h y t a g a r o r Bacto agar a t 7 g/L. L e a f y s h o o t s from medium MSCB^ ^ 1 ^ ^ were c u t i n t o p i e c e s and 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 media. A l l p l a n t s were h a r v e s t e d a f t e r about 6 weeks i n t h e p r o l i f e r a t i o n media and t h e i r l e a v e s and r o o t s examined and counted. 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 m i c r o p r o p a g a t i o n , t h e f o l l o w i n g t r i a l was made. Two 8-week o l d r o o t e d p l a n t l e t s from meristem t i p s , g rowing i n medium MSCB.^ q , each w i t h 10-20 l e a v e s and 12-15 r o o t s , were 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 e n s u i n g p l a n t l e t s were 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 t i m e s . 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 and t h e i r r o o t s and l e a v e s were examined and counted. An e s t i m a t e was made o f t h e number o f m i c r o p r o p a g a t e d p l a n t l e t s t h a t c o u l d be produced 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 from r o o t s t h a t had been s e v e r e d from s h o o t s , t h e s h o o t s o f seven p l a n t l e t s were c u t o f f below t h e l o w e s t l e a f , and t h e s h o o t s o f two 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 l e a f . The s e v e r e d shoot and the r o o t p o r t i o n were each r e t u r n e d t o medium M S C B Q ^ 1 ^ Q . A f t e r a month, the 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 ; t h e t o p p o r t i o n s f o r r e g e n e r a t i o n o f r o o t s . To e v a l u a t e growth from e x c i s e d n o d a l segments, 15 s i n g l e l e a v e s , each a t t a c h e d t o a s m a l l (^1.0 cm) s e c -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 p l a n t -l e t , and r e t u r n e d t o medium MSCB ,1 . A f t e r 10 d a y s , the segments were examined f o r growth o f stems, l e a v e s , and r o o t s . The t y p e o f m i c r o p r o p a g a t i o n t h a t o c c u r s i n media. M S C B Q ^ 1 ^ Q o r M S C B ^ ^ I ^ was compared t o m i c r o p r o p a g a -t i o n i n v o l v i n g a x i l l a r y bud p r o l i f e r a t i o n , when p l a n t l e t s from e i t h e r o f t h e s e media were s u b c u l t u r e d t o a medium w i t h BAP i n c r e a s e d t o 3.0 mg/L, and IBA d e c r e a s e d t o 0.1 mg/L ( M S C B 3 > 0 I 0 > 1 ) . Source P l a n t P r e - t r e a t m e n t To e x p l o r e t h e e f f e c t o f heat t r e a t m e n t on s u r v i v a l , growth and r o o t i n g o f b l a c k r a s p b e r r y i n c u l t u r e , f i v e b l a c k r a s p b e r r y s o u r c e p l a n t s were h e a t - t r e a t e d , and ex-p l a n t s t a k e n a t 1-week i n t e r v a l s f o r 5 weeks. Each week, 10 m e r i s t e m t i p s -0.35 mm were e x c i s e d i n t o each o f media MSCB Q 1 I 1 and MSCB.^ . B o t h media were a d j u s t e d t o pH 6.1 p r i o r t o a u t o c l a v i n g . A f t e r 8 weeks o f h e a t - t r e a t m e n t , 31 m e r i s t e m t i p s -0.5 mm l o n g were p l a c e d i n t o medium ^ W i . o ' p H 5 - 7 -C o l d S t o r a g e of I n V i t r o P l a n t l e t s S i x r o o t e d p l a n t l e t s i n v i t r o were h e l d a t 10°C i n the 17. d a r k f o r 3 months, th 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. Those t h a t produced new l e a v e s were p l a n t e d i n s o i l a f t e r 2 weeks. The p o s s i b i l i t y o f u s i n g t h i s p r o c e d u r e t o p r e s e r v e b l a c k r a s p b e r r y germ-plasm was e v a l u a t e d . L o g a n b e r r y C u l t u r e Media A few l o g a n b e r r y m e r i s t e m t i p s -0.35 mm l o n g were p l a c e d i n t o media MSBg ^ 1 ^ ^ o r MSB Q ^ 1 ^ g w i t h a 1:2 d i l u t i o n o f macroelements. S u r v i v i n g s h o o t s were s u b c u l -t u r e d t o e i t h e r o f two media; MSB^ gig 1 o r MSB^ gig pH 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 s o u r c e and pH was i n -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 0.05-0.50 mm l o n g i n t o medium MSB^ gig ^ w i t h e i t h e r 30 g/L s u c r o s e (pH 5.7, 6.1) o r 25 g/L g l u c o s e (pH 5.7). T i p s t h a t s u r v i v e d t o the s m a l l shoot s t a g e were s u b c u l t u r e d t o b o t t l e s w i t h t h e same medium, o r w i t h BAP c o n c e n t r a t i o n i n c r e a s e d t o 3.0 mg/L (MSB^ gig ^ ) . Subsequent c l o n e s were used i n 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 e x p e r i m e n t s . To e s t i m a t e the 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 m i c r o p r o p a g a t i o n , t h e f o l l o w i n g t r i a l was made. Two m e r i s -tem t i p s i n medium MSB^ gig ^, pH 6.1, and two i n g l u c o s e (25 g/L) medium, were t r a n s f e r r e d t o medium MSB^ gig 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 former 1 8 . p a i r o f m e r i s t e m t i p s h a d d e v e l o p e d i n t o c l o n e s a n d w e r e d i v i d e d t h e f i r s t , t i m e ; t h e l a t t e r t w o c l o n e s w e r e d i v i d e d a f t e r 6 w e e k s ; s u b s e q u e n t d i v i s i o n s w e r e a t t h r e e 4 - w e e k i n t e r v a l s . I n r o o t i n g e x p e r i m e n t s , 2 4 l o g a n b e r r y c l o n e s , 1 4 f r o m s t a r t i n g m e d i u m M S B ^ gig ^ w i t h s u c r o s e , 1 f r o m t h i s m e d i u m w i t h g l u c o s e , 7 h a v i n g b e e n i n m e d i u m M S B ^ Q I Q w i t h s u c r o s e , a n d 2 i n t h i s m e d i u m w i t h g l u c o s e f o r t h e p r e c e e d i n g 6 w e e k s , w e r e d i v i d e d a s e p t i c a l l y i n t o p i e c e s h a v i n g t h r e e o r m o r e l e a v e s e a c h . F r o m e a c h o f t h e f o u r s o u r c e m e d i a , t h r e e c u t t i n g s , o n e p e r t u b e , w e r e p l a c e d i n t o e a c h r o o t i n g m e d i u m . R o o t i n g m e d i a w e r e c o m p o s e d o f M S w i t h I B A c o n c e n t r a t i o n f r o m 0 . 0 - 1 . 0 m g / L , a n d w i t h s u c r o s e a t 3 0 g / L o r g l u c o s e a t 2 5 g / L , p H 5 . 7 o r 6 . 1 . T h e e f f i c i e n c y o f r o o t i n g l o g a n b e r r y c u t t i n g s i n v i t r o , i n m e d i u m M S I g ^ , p H 5 . 7 o r 6 . 1 , w a s 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 r o o t i n g p o w d e r i n s o i l i n t h e g r e e n h o u s e . T w o l o g a n b e r r y c l o n e s , i n m e d i u m M S B ^ gig ^ w e r e s u b d i v i d e d i n t o c u t t i n g s . T w e n t y c u t t i n g s f r o m e a c h c l o n e w e r e u s e d i n e a c h t r e a t m e n t . R o o t i n i t i a t i o n t i m e w a s n o t e d f o r i n v i t r o p l a n t l e t s , a s w a s t h e t i m e r e q u i r e d f o r r o o t d e v e l o p -m e n t a n d e l o n g a t i o n p r i o r t o p l a n t i n g . C u t t i n g s i n t h e g r e e n h o u s e w e r e e x a m i n e d p e r i o d i c a l l y f o r p r o d u c t i o n o f r o o t s a n d n e w l e a v e s . R o o t i n g i n v i t r o w a s c o m p a r e d t o r o o t i n g o f c u t t i n g s i n t h e g r e e n h o u s e . A f t e r 2 a n d 4 w e e k s i n v i t r o , r o o t e d c u t t i n g s w e r e p l a n t e d i n s o i l . O n e m o n t h a f t e r p l a n t i n g t h e l a s t g r o u p , f i v e p l a n t l e t s f r o m e a c h o f t h e t h r e e groups were removed from s o i l ; average w e i g h t , h e i g h t , number o f l e a v e s , and number o f main r o o t s were compared f o r each group. Source P l a n t P r e - t r e a t m e n t To i n v e s t i g a t e t h e p o s s i b i l i t y o f s p e e d i n g up growth and r o o t i n g o f l o g a n b e r r y c u l t u r e s , f i v e l o g a n b e r r y s o u r c e p l a n t s were grown a t 37°C f o r 1-8 weeks and e x p l a n t s t a k e n weekly. Each week t e n m e r i s t e m t i p s were e x c i s e d i n t o each of two media; MS w i t h o u t i n o s i t o l o r hormones, and MSB^ Q I Q Both media were a d j u s t e d t o pH 6.1. Those p l a c e d i n medium w i t h o u t i n o s i t o l o r hormones were moved t o t h e o t h e r medium a t t h e t i m e o f t h e f i r s t s u b c u l t u r e (28 d a y s ) . The e f f e c t 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 h e s u r v i v a l and growth o f t i p s c u l t u r e d i n t h e s e two media was e v a l u a t e d . C o l d S t o r a g e o f I n V i t r o P l a n t l e t s Twenty-seven r o o t e d l o g a n b e r r y p l a n t l e t s i n v i t r o were k e p t a t 10°C i n t h e d a r k , f o r 2 months, t h e n p l a n t e d d i r e c t l y i n t o s o i l i n t h e greenhouse. The e f f e c t i v e n e s s o f c o l d s t o r a g e f o r t h i s p e r i o d was e v a l u a t e d as a means of p r e s e r -v i n g l o g a n b e r r y germplasm. T a y b e r r y 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 were e x c i s e d i n t o medium 2 0 . M S B ^ g i g ^ , p H 6 . 1 , a n d 1 0 t i p s w e r e e x c i s e d i n t o t h e s a m e m e d i u m , e x c e p t s u c r o s e w a s r e p l a c e d b y g l u c o s e ( 2 5 g / L ) a n d t h e p H w a s a d j u s t e d t o 5 . 7 . S u r v i v i n g t a y b e r r y s h o o t s w e r e t r a n s f e r r e d t o 1 0 0 - m l j a r s a f t e r 2 m o n t h s o f c u l t u r e . S h o o t s w e r e d i s t r i b u t e d b e t w e e n m e d i u m M S B ^ Q l 0 1 w i t h g l u c o s e , p H 5 . 7 a n d m e d i u m M S B ^ Q I Q w i t h s u c r o s e , p H 5 . 7 . T o 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 b y m i c r o p r o p a g a t i o n , a s i n g l e m e r i s t e m t i p 0 . 1 5 m m l o n g , s t a r t e d i n m e d i u m M S B 1 g l ^ ^ w i t h g l u c o s e , w a s s u b d i v i d e d a f t e r 1 2 w e e k s , a n d t r a n s f e r r e d t o m e d i u m M S B ^ Q I Q ^ w i t h s u c r o s e . F u r t h e r s u b d i v i s i o n s w e r e m a d e a t i n t e r v a l s a n d a n e s t i m a t e w a s m a d e o f t h e n u m b e r o f c u t t i n g s t h a t c o u l d b e p r o d u c e d i n 1 y e a r . R o o t i n g i n v i t r o i n m e d i u m M S I ^ , p H 5 . 7 w a s c o m p a r e d t o r o o t i n g i n s o i l . F i f t e e n c u t t i n g s f r o m a p l a n t l e t i n m e d i u m M S B ^ Q I Q w e r e d u s t e d w i t h r o o t i n g p o w d e r a n d p l a n t e d i n s o i l . T h e b a l a n c e o f c u t t i n g s f r o m t h e m i c r o p r o p a g a t i o n e x p e r i m e n t w e r e r o o t e d i n v i t r o . R e d R a s p b e r r y C u l t u r e M e d i a P r e l i m i n a r y t r i a l s w i t h m e d i a f o r r e d r a s p b e r r y m e r i s t e m t i p c u l t u r e i n c l u d e d m a c r o n u t r i e n t s a t f u l l s t r e n g t h , a n d a t t h r e e d i l u t i o n s , a n d B A P a n d I B A e a c h a t s e v e r a l l e v e l s . T o e s t a b l i s h t h e c o r r e c t h o r m o n e c o n c e n t r a t i o n i n w h i c h t o i n i t i a t e t h e 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 , 6 : 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 each o f 35 media w i t h BAP a t 0.1, 0.3, 0.5, 0.7, 1.0, 3.0, and 5.0 mg/L and IBA a t 0.1, 0.3, 0.5, 0.7, and 1.0 mg/L. A l l media were a d j u s t e d t o pH 5.7. A f t e r 6 weeks i n c u l t u r e , a l l s u r v i v i n g t i p s were h a r v e s t e d , and c o l o r and growth were e v a l u a t e d . To e v a l u a t e s e v e r a l s t a r t i n g media, more th a n 8 0 mer i s t e m t i p s , t h e m a j o r i t y -0.25 mm l o n g , were e x c i s e d i n t o each o f media MSB 1 Q I Q ^ and MSB^ 0 I Q ^ . Three t i m e s t h i s number o f t i p s were i n i t i a t e d i n medium MS w i t h no hormones, w i t h i n o s i t o l a t 100 mg/L, o r w i t h o u t i n o s i t o l ; a t t h e t i m e o f .the f i r s t s u b c u l t u r e t h e s e were d i s t r i b u t e d i n t o media w i t h hormones and i n o s i t o l (MSB^ ^ I Q ^ and MSB^ Q I Q ^ ) . E f f e c t s on s u r v i v a l and growth were r e c o r d e d . To d e t e r m i n e t h e optimum 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 l o n g were e x c i s e d i n t o each o f two media, MSB  ^ QIQ 1 and MSB^ I 2 , a d j u s t e d t o pH 5.5, 5.7, 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 . T i p s were examined a f t e r 30 and 60 days, and t h e i r appearance and growth were compared. T h i s e xperiment was performed t w i c e , and t h e r e s u l t s from t h e two t r i a l s were averaged. To examine 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 concen-t r a t i o n o f sugar i n t h e medium, s u c r o s e a t 20 o r 30 g/L, o r g l u c o s e a t 20, 30, o r 40 g/L were used i n medium MSB^ Q I Ten meristem t i p s -0.375 mm l o n g were e x c i s e d i n t o each of the f i v e media. T i p s were measured a t 30-day i n t e r v a l s . S u r v i v a l and growth r a t e were compared f o r mer i s t e m t i p s 2 2 . i n e a c h s u g a r c o n c e n t r a t i o n . T h i s e x p e r i m e n t w a s r e p e a t e d a n d t h e r e s u l t s f r o m b o t h t r i a l s w e r e a v e r a g e d . F o l l o w i n g t h e c a r b o h y d r a t e t r i a l s , t w o p H l e v e l s w e r e r e - t e s t e d b y e x c i s i n g 1 4 0 m e r i s t e m t i p s i n t o m e d i u m M S B 1 Q I q 1 c o n t a i n i n g g l u c o s e a t 2 5 g / L , a n d a d j u s t e d t o p H 5 . 7 o r p H 6 . 1 . T o d e t e r m i n e t h e r e q u i r e m e n t s f o r i n o s i t o l i n m e r i s t e m t i p c u l t u r e s o f r e d r a s p b e r r y , t i p s w e r e e x c i s e d i n t o m e d i u m M S w i t h n o i n o s i t o l w i t h s u c r o s e a t 3 0 g / L , o r g l u c o s e a t 2 5 g / L . T h e p H w a s a d j u s t e d t o 5 . 7 . A f t e r 1 m o n t h o f c u l t u r e , s u r v i v i n g s h o o t s w e r e t r a n s f e r r e d t o e i t h e r m e d i u m M S w i t h o u t h o r m o n e s , o r m e d i u m M S B ^ g I Q ^ , w i t h i n o s i t o l a t 0, 5 0 , 1 0 0 , o r 1 5 0 m g / L . A l l t i p s w e r e m e a s u r e d a t 1 0 - d a y i n t e r v a l s i n o r d e r t o f a c i l i t a t e d i r e c t c o m p a r i s o n o f i n o s i t o l e f f e c t s . T h i s e x p e r i m e n t w a s r e p e a t e d t w i c e u s i n g g l u c o s e , a n d t h r e e t i m e s u s i n g s u c r o s e a s t h e c a r b o -h y d r a t e s o u r c e i n t h e m e d i u m . T h e r e s u l t s w e r e c o m b i n e d f o r t i p s i n e a c h c a r b o h y d r a t e s o u r c e . T o i n d u c e p r o l i f e r a t i o n o f a x i l l a r y b u d s , h e a l t h y , l e a f y s h o o t s i n m e d i u m M S B ^ ^ 1 ^ ^ c o n t a i n i n g s u c r o s e a t 3 0 g / L o r g l u c o s e a t 2 5 g / L , p H 5.7 o r 6 . 1 w e r e t r a n s f e r r e d t o m i c r o p r o p a g a t i o n m e d i a w i t h i n c r e a s e d B A P ( M S B ^ Q I Q ^) w i t h s u c r o s e a t 3 0 g / L o r g l u c o s e a t 2 5 g / L , p H 5.7 o r 6 . 1 . T o d e t e r m i n e t h e i n f l u e n c e o f h o r m o n e l e v e l s o n r o o t i n g , t w o s h o o t s 8 - 1 6 w e e k s o l d , u s u a l l y 1 . 0 - 2 . 0 c m l o n g w i t h s e v e r a l l e a v e s , i n s t a r t i n g m e d i u m M S B ^ ^ 1 ^ ^ , a n d t h r e e c u t t i n g s , e a c h w i t h o n e o r m o r e l e a v e s , f r o m f i v e c l o n e s t h a t 23. had been i n medium MSB^ g l ^ ^  f o r t h e p r e c e e d i n g f o u r 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 IBA o r NAA, each a t 0.0, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/L. There were 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 media. Time f o r r o o t i n g 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 whether 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 from medium MSB^ ^ I Q ^  i n d u c e d r o o t s t o form more q u i c k l y , o r whether l e v e l o f BAP i n t h e shoot growth medium was more i m p o r t a n t , a second r o o t i n g e x p e r i m e n t was done. The e f f e c t o f IBA l e v e l s from 0.0-1.0 mg/L on r o o t i n g o f 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 . Three c l o n e s from t h e two s o u r c e media MSB^ QIQ 1 and MSB 1 0 I Q 1 were each d i v i d e d i n t o segments w i t h t h r e e o r more l e a v e s . Four c u t t i n g s from each o f t h e two s o u r c e media were t r a n s f e r r e d t o each r o o t i n g medium. Appearance o f f i r s t r o o t s and r o o t q u a l i t y were e v a l u a t e d i n each r o o t i n g medium f o r each o f t h e two s o u r c e s o f c u t t i n g s . The e f f i c i e n c y o f 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 i n v i t r o was compared t o r o o t i n g c u t t i n g s w i t h r o o t i n g powder i n s o i l i n t h e greenhouse. T h i r t y - f i v e c u t t i n g s each, from media KSB^ QIQ ^ o r MSB^ Q I 0 1 w e r e P l a c e d i n t o r o o t i n g medium MSI_ j . . Twenty c u t t i n g s o f comparable s i z e were d u s t e d w i t h r o o t i n g hormone and p l a n t e d i n t o s o i l . Source P l a n t P r e - t r e a t m e n t The e f f e c t o f e t i o l a t i o n o f sou 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 m e r i s t e m 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 days. S u r v i v a l o f t h e s e t i p s was compared 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 . Average growth a f t e r 30 and 60 days i n c u l t u r e was r e c o r d e d f o r e t i o l a t e d 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. The 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 meris t e m t i p s u r v i v a l and growth 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 weeks. Each week, 10 t i p s were 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 hormones, and 10 t i p s i n t o medium MSB^ Q I 0 ^ , pH 6.1. A t the time o f t h e f i r s t s u b c u l t u r e , t i p s from medium MS w i t h -o u t hormones and i n o s i t o l were t r a n s f e r r e d t o medium w i t h b o t h (MSB-j^  Q I Q ^ ) . Shoots were measured a t 30-day i n t e r v a l s M e r i s t e m t i p s u r v i v a l i n two media was compared u s i n g t i p s from two s o u r c e s . T i p s o u r c e s i n c l u d e d p o t t e d p l a n t -l e t s d e r i v e d from 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 from r o o t c u t t i n g s . T i p s were p l a c e d i n t o medium MSB 1 Q I 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. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s To e v a l u a t e c o l d s t o r a g e as 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 germplasm, 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 were k e p t a t 10°C i n t h e dark f o r 3 months, th e n p l a n t e d d i r e c t l y i n t o s o i l i n t h e greenhouse. 2 5 . R e s u l t s B l a c k R a s p b e r r y C u l t u r e Media M e r i s t e m t i p s e x c i s e d i n t o media MS ( t h i a m i n e a t 0 . 1 mg/L) w i t h g l u c o s e a t 4 0 g/L, agar a t 8 g/L, BAP a t 0 . 1 o r 1. 0 mg/L, and IBA a t 1 . 0 mg/L and 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, IAA a t 0 . 5 mg/L, and GA 3 a t 0 . 1 mg/L, i n c u b a t e d a t 2 0°C o r a t 27°C d i d not s u r v i v e . 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 time i n c u l t u r e , and many c u l t u r e s became c o n t a m i n a t e d . The most 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 i n l i q u i d medium, a t 2 7 ° C . L i q u i d medium caused t i p s t o s w e l l , and some 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 s u p p o r t s d i s p e r s e d t o 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 , and appeared t o i n h i b i t growth. On medium M S K Q Q^, 1 4 / 5 2 m e r i s t e m t i p s produced r o o t e d p l a n t l e t s b u t t h e r a t e o f growth was e x t r e m e l y slow and development was e r r a t i c . A f t e r 5 months o f c u l t u r e , some t i p s had not de v e l o p e d a t a l l , w h i l e some were w e l l r o o t e d and ready f o r p l a n t i n g ( F i g . 1 ) . When meristem t i p c u l t u r e s i n medium MSB^ Q I ^ QGQ ^  were r o t a t e d a t 1 rpm, t i p s became lo d g e d on 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 con-t a m i n a t e d . Due t o the 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 F i g u r e 1. Meristem t i p s of b l a c k r a s p b e r r y a f t e r 5 months of c u l t u r e i n medium MS wit h k i n e t i n a t 0.04 mg/L, showing slow, e r r a t i c development. 27. s t a g e o f c u l t u r e . W h i l e none o f the s t a t i o n a r y c o n t r o l t i p s 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 not a problem. A n t i o x i d a n t s , such as PVP (0.5-2%) i n c o r p o r a t e d i n t o t h e above medium, and PVP o r c y s t e i n e H C l used d u r i n g ex-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 HCl 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 promoted s u r v i v a l and growth o f meris t e m 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 d e v e l o p i n g p l a n t l e t s . When t h e concen-t r a t i o n o f BAP i n t h e medium was reduced 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 i n c r e a s e d (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 s o u r c e m a t e r i a l ; s u r v i v a l o f meri s t e m t i p s was h i g h e r (Table 2 ) . M e r i s t e m t i p s from o l d e r s o u r c e p l a n t s sometimes formed c a l l u s . W i t h b o t h young and o l d s o u r c e p l a n t s , d e c r e a s i n g t h e c o n c e n t r a t i o n o f BAP from 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 s i z e , i n c r e a s e d the number o f t i p s t h a t s u r v i v e d (Table 2 ) . I n d o l e b u t y r i c a c i d was b e s t added subsequent t o a u t o -c l a v i n g . A u t o c l a v i n g IBA s i g n i f i c a n t l y a l t e r e d i t s e f f e c t s (0.05 l e v e l ) . When IBA a t 1.0 mg/L was added a f t e r a u t o -c l a v i n g medium MSCBg ^, l e a f development was good, and 10/30 s h o o t s r o o t e d . R e s u l t s were n ot enhanced by f i l t e r -s t e r i l i z a t i o n ; 7/30 sh o o t s r o o t e d , b u t shoot growth was not as e x t e n s i v e as t h a t i n a u t o c l a v e d medium, w i t h IBA added a f t e r a u t o c l a v i n g . When IBA was a u t o c l a v e d w i t h t h e medium, sh o o t s were v e r y s m a l l and o n l y 3/30 s h o o t s r o o t e d . 28. T a b l e 1. The e f f e c t o f t h e hormones BAP and GA^, and th e a n t i o x i d a n t s PVP and c y s t e i n e H C l , 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 m e r i s t e m t i p s i n medium MS w i t h IBA a t 1.0 mg/L Hormones A n t i o x i d a n t s No. o f t i p s BAP GA^ PVP C y s t e i n e HCl e x c i s e d s u r v i v e d (mg/L) (mg/L) (mg/L) (mg/L) 1.0 0.1 - . - 58 0 1.0 0.1 5-20x10 - 50 0 1.0 0.1 - 10 56 9 1.0 - - 24 0 1.0 - - 10 256 56 0.1 - - 10 156 54 2 9 . T a b l e 2 . T h e 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 B A P c o n c e n t r a t i o n o n s u r v i v a l o f b l a c k r a s p b e r r y m e r i s t e m t i p s B A P * T i p s o u r c e * * N o . o f e x p l a n t s s u r v i v e d / n o . e x c i s e d ( m g / L ) t i p s i z e ( m m ) ^ 1 . 0 1 . 1 - 2 . 0 2 . 1 - 3 . 0 1 . 0 s t a g e 1 5 6 / 2 5 6 3 9 / 7 2 9 / 1 2 1 . 0 s t a g e 2 5 / 1 3 8 2 / 1 4 0 . 1 s t a g e 1 5 4 / 1 5 6 4 3 / 6 7 2 1 / 3 0 0 . 1 s t a g e 2 1 / 2 7 M e d i u m w a s M S w i t h c y s t e i n e H C l , I B A a t 1 . 0 m g / L , a n d B A P a t 0 . 1 o r 1 . 0 m g / L . S t a g e 1 t i p s w e r e f r o m y o u n g , s u c c u l e n t p l a n t s 1 5 - 5 0 c m t a l l . S t a g e 2 t i p s w e r e f r o m t h e a d v e n t i t i o u s r o o t z o n e o f o l d e r p l a n t s t h a t h a d d e v e l o p e d a p r o s t r a t e h a b i t . A f t e r t h i s t r i a l , w h e n I B A w a s u s e d , i t w a s a l w a y s a d d e d a f t e r t h e m e d i u m w a s a u t o c l a v e d . W h e n l e v e l s o f t h e h o r m o n e I B A w e r e v a r i e d f r o m 0 . 0 -1 . 0 m g / L , t h e n u m b e r o f e x p l a n t s t h a t s u r v i v e d a n d r o o t e d i n e a c h m e d i u m , a n d t h a t w e r e e v e n t u a l l y t r a n s f e r r e d t o s o i l , w e r e t h e f o l l o w i n g ( I B A , m g / L ) : ( 0 . 0 ) , 8 / 4 3 ; ( 0 . 1 ) , 6 / 4 4 ; ( 0 . 3 ) , 5 / 4 4 ; ( 0 . 5 ) , 3 / 4 4 ; ( 0 . 7 ) , 5 / 4 1 ; a n d ( 1 . 0 ) , 9 / 4 3 . W h i l e r o o t i n g a m o n g e x p l a n t s i n t h e s e m e d i a w e r e n o t s i g n i f i c a n t l y d i f f e r e n t , r a t e o f g r o w t h w a s i n c r e a s e d s l i g h t l y , a n d r o o t i n g i n i t i a t e d m o r e q u i c k l y , a s t h e l e v e l s o f I B A w e r e i n c r e a s e d . A l m o s t a l l ( 9 4 % ) o f t h e s h o o t s r o o t e d w h e n s u b c u l t u r e d i n t h e b e s t s t a r t i n g m e d i u m , M S C B Q ^ 1 ^ Q a n d a l l s h o o t s r o o t e d w h e n t r a n s f e r r e d f r o m m e d i u m M S C B ^ ^ 1 ^ ^ t o m e d i u m M S C B Q ^ 1 ^ 0 ( T a b l e 3 ) . R o o t i n g o f s h o o t s f r o m m e d i u m w i t h B A P a t 1 . 0 m g / L i n c r e a s e d i f B A P w a s r e d u c e d t o 0 . 1 m g / L o r w a s o m i t t e d , a n d I B A l e v e l s v a r i e d b e t w e e n 0 . 0 a n d 1 . 0 m g / L ( T a b l e 3 ) . R o o t i n g w a s f a s t e s t w h e n t h e r o o t i n g m e d i u m c o n t a i n e d t h e h i g h e s t l e v e l o f I B A t e s t e d , 1 . 0 m g / L . C o v e r i n g t h e b a s e o f t h e t e s t t u b e w i t h f o i l w r a p t o k e e p s h o o t b a s e s i n t h e d a r k , d i d n o t h a v e a n y d i s c e r n a b l e e f f e c t o n i n c i d e n c e o r t i m e o f r o o t i n g , c o m p a r e d t o s h o o t s i n t u b e s f u l l y e x p o s e d t o t h e l i g h t . S o m e s h o o t s i n m e d i u m M S C B N , 1 , n t h a t h a d b e e n i n c u l t u r e f o r c l o s e t o 4 m o n t h s w i t h o u t f o r m i n g r o o t s , w e r e r o o t e d w i t h i n a v e r y f e w d a y s b y s u b c u l t u r e t o n e w m e d i u m . T h e s t i m u l a t o r y e f f e c t o f s u b c u l t u r e w a s o f t e n o b s e r v e d . 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 IBA 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 m e r i s t e m t i p s S t a r t i n g medium* R o o t i n g medium** No. r o o t e d / n o . s h o o t s BAP BAP IBA (mg/L) (mg/L) (mg/L) 1.0 1.0 1.0 9/22 41 1.0 0.1 1.0 3/3 100 1.0 - - 8/10 80 1.0 - 0.1 - 0.7 9/9 100 1.0 — 1.0 24/34 71 53/78 68 0.1 1.0 1.0 13/22 59 0.1 0.1 1.0 47/50 94 0.1 - - 1/3 33 0.1 - 0.1 - 0.7 8/12 67 0.1 — 1.0 1/3 33 70/90 78 *: S t a r t i n g medium was MSC w i t h IBA a t 1.0 mg/L and BAP a t 0.1 o r 1.0 mg/L. **: 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 and IBA a t 0.0-1.0 mg/L. 32. M i c r o p r o p a g a t i o n t r i a l s d emonstrated t h a t r a i s i n g t h e l e v e l o f BAP i n t h e medium gave r i s e t o l e a f i e r s h o o t s and m i n i m i z e d t h e tendency t o r o o t . The b e s t p r o l i f e r a t i o n t o o k p l a c e i n l i q u i d media MSCB 1 QIQ ^ Q (Table 4 ) . 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 s o l i d medium. M i c r o b i o l o g i c a l B a c t o agar i n h i b i t e d r o o t i n g ( T able 4 ) . When 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 m i c r o -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 o b s e r v a t i o n s were made. Two 8-week o l d b l a c k r a s p b e r r y p l a n t l e t s y i e l d e d f i v e c u t t i n g s each. A t 3-week i n t e r v a l s , two f u r t h e r sub-d i v i s i o n s y i e l d e d 25, th e n 50 c u t t i n g s . A f t e r 3 weeks o f c u l t u r e , t h e 50 c u t t i n g s had 4-35 l e a v e s and 1-20 r o o t s each. These p l a n t l e t s were d i v i s i b l e i n t o 3-10 p i e c e s each. The most c o n s e r v a t i v e e s t i m a t e o f t h e number o f c u t t i n g s o b t a i n a b l e i n 1 y e a r i n v o l v e s d i v i s i o n i n t o t h r e e c u t t i n g s per p l a n t l e t e v e r y t h i r d week from t h e e i g t h week onwards. T h i s y i e l d s 1.4 3 x 10 r o o t e d c u t t i n g s by t h e end o f 1 y e a r . These can be p l a n t e d o u t i m m e d i a t e l y w i t h n e g l i g i b l e l o s s e s . I t seems l i k e l y t h a t p r o p a g a t i o n o f t h i s s o r t c o u l d be c a r r i e d o u t i n d e f i n i t e l y , y i e l d i n g v a s t numbers o f r o o t e d c u t t i n g s . W h i l e e x t e n s i v e p r o l i f e r a t i o n i n medium MSCBg ^ 1 ^ ( was not i n v e s t i g a t e d , c u t t i n g s had no apparent d i f f i c u l t y r o o t i n g o r growing i n t h i s medium. An o d d i t y t h a t appeared i n some o l d e r 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 m i n i a t u r e w h i t e s t r u c t u r e s ( F i g . 2 ) . These were tho u g h t t o be r u d i m e n t a r y f l o w e r s . 33. T a b l e 4. The e f f e c t s o f BAP and IBA 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 on new l e a f and r o o t f o r m a t i o n o f b l a c k r a s p b e r r y s h o o t s BAP IBA Medium* No. o f c u t t i n g s No. o f c u t t i n g s w i t h (mg/L) (mg/L) new l e a v e s r o o t s 1.0 0.0-0.7 L 22 17 10 1.0 0.0-0.7 P 13 8 4 1.0 1.0 L 20 18 7 1.0 1.0 P 20 12 8 1.0 1.0 B 20 20 0 3.0 0.0-1.0 L 19 12 6 3.0 0.0-1.0 P 14 4 1 5.0 0.0-1.0 L 15 15 2 5.0 0.0-1.0 P 2 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 Bacto agar ( B ) . F i g u r e 2 . M i n i a t u r e white s t r u c t u r e s formed i n some bl a c k r a s p b e r r y c u l t u r e s which were thought to be rudimentary f l o w e r s . 35. D e c a p i t a t i o n e x p e r i m e n t s i l l u s t r a t e d t h e r e g e n e r a t i o n p o t e n t i a l of s e v e r e d r o o t s and s h o o t s . When t h e stem was s e v e r e d below th e p o i n t o f attachment t o t h e r o o t s , r o o t s d i d n ot produce new l e a v e s w i t h i n t h e n e x t month. When one o r more l e a v e s were r e t a i n e d , new s h o o t ( s ) grew from th e s e v e r e d stem. I f t h e stem was s e v e r e d above th e p o i n t o f attachment t o t h e r o o t s , a bud u s u a l l y formed on t h e s e v e r e d s t a l k and new l e a v e s emerged. I n a l l c a s e s , s e v e r e d s h o o t s q u i c k l y r o o t e d and c o n t i n u e d t o grow. A l l 15 n o d a l segments c u t from a b l a c k r a s p b e r r y p l a n t l e t and r e t u r n e d t o medium M S C B Q ^ 1 ^ Q , when examined a f t e r 10 d a y s , had produced new l e a v e s from t h e i r a x i l l a r y bud. Four of t h e 15 n o d a l s e c t i o n s had produced s u b s t a n t i a l r o o t s by t h i s t i m e ; a l l had r o o t e d 2 weeks l a t e r . T h i s t r i a l demonstrated t h a t i t i s p o s s i b l e t o improve t h e 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 r e d u c i n g t h e s i z e o f t h e b l a c k r a s p b e r r y c u t t i n g s , u t i l i z i n g n o d a l segments i n s t e a d o f t h e u s u a l 3-5 l e a f c u t t i n g s . Shoots and p l a n t l e t s from medium MSC w i t h IBA a t 1.0 mg/L and BAP a t e i t h e r 0.1 o r 1.0 mg/L d e v e l o p e d much a x i l l a r y growth when t r a n s f e r r e d t o medium MSB^ QI-^ Q and they c o u l d be s u b s e q u e n t l y d i v i d e d i n t o many c u t t i n g s . The r o o t s o f 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 a l t h o u g h t o p growth was s u b s t a n t i a l . C u t t i n g s from t h e s e p l a n t l e t s were 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. F o r t h i s r e a s o n , t h e t y p e o f p r o l i f e r a t i o n a c h i e v e d by r e p e a t e d d i v i -s i o n o f p l a n t l e t s i n media MSCB^ ^ 1 ^ ^ o r MSCB.^ ^ 1 ^ Q was 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 by t r a n s f e r t o medium MSB^ Q I 1 s i n c e t h i s i n v o l v e s more th a n one medium, and d i s c o u r a g e s r o o t i n g . Source P l a n t P r e - t r e a t m e n t 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 p l a n t s f o r 1-5 weeks, on growth i n media MSCB Q ^1^ Q o r MSB-j^ I. Q ^, b o t h a d j u s t e d t o pH 6.1, were 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 were o n l y 5/33 i n t h e former medium and 3/23 i n t h e l a t t e r o v e r t h e e n t i r e 5-week p e r i o d . T h i s was a t t r i b u t e d t o t h e h i g h pH o f t h e media. When me r i s t e m t i p s from s o u r c e p l a n t s exposed t o 8 weeks o f h e a t - t r e a t m e n t were e x c i s e d i n t o medium MSCB Q ^1^ , a d j u s t e d t o pH 5.7, t h e y had a h i g h e r s u r v i v a l r a t e (17/28) th a n t i p s from n o n - h e a t - t r e a t e d c o n t r o l p l a n t s (54/156). Shoots from h e a t - t r e a t e d s o u r c e 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 ; none o f t h o s e from c o n t r o l p l a n t s d i d ( F i g . 3 ) . Shoots from c o n t r o l p l a n t s r o o t e d i n 5-8 weeks o f c u l t u r e . C o l d S t o r a g e o f I n V i t r o P l a n t l e t s Four o f t h e s i x p l a n t l e t s s u b j e c t e d t o c o l d s t o r a g e grew new s h o o t s 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 tempera-t u r e . 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 . The f o u r p l a n t l e t s w i t h new growth were p l a n t e d i n s o i l and s u r v i v e d . They c o u l d p r o b a b l y have been p l a c e d d i r e c t l y from t h e c o l d i n t o s o i l . C o l d s t o r a g e a t t h i s t e m p e r a t u r e e l i m i n a t e d shoot growth, but some new r o o t s were formed i n v i t r o . T h i s 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 germplasm. 37. F i g u r e 3. E f f e c t o f h e a t - t r e a t i n g b l a c k r a s p b e r r y s o u r c e p l a n t s o n d e v e l o p m e n t o f m e r i s t e m t i p s i n m e d i u m M S w i t h c y s t e i n e HC1 a t 10 m g / L , B A P a t 0.1 m g / L , a n d I B A a t 1.0 m g / L , p H 5.7. T h e r o o t e d p l a n t -l e t s a r o s e f r o m m e r i s t e m t i p s e x c i s e d f r o m h e a t -t r e a t e d s o u r c e p l a n t s ; t h e s e t o o k 4-5 w e e k s t o r o o t . T h e r o o t l e s s s h o o t w a s f r o m a n o n - h e a t -t r e a t e d s o u r c e p l a n t ; t h e s e t o o k f r o m 5-8 w e e k s t o r o o t . L o g a n b e r r y C u l t u r e 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 showed t h a t s u r v i v a l was moderate ( 8 / 1 5 ) i n medium M S B Q ^ 1 ^ Q , and low ( 1 / 8 ) i n medium M S B ^ ^ 1 ^ Q w i t h a t w o - f o l d d i l u t i o n o f macroelements, and t h e s e s m a l l r e s u l t a n t s h o o t s were d e c l i n i n g a f t e r a 2 month p e r i o d . When t h e s e s h o o t s 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 r e c o v e r e d q u i c k l y , f o r m i n g t a l l ( 5 - 8 cm), ab u n d a n t l y r o o t e d p l a n t l e t s w i t h i n 2 months. Of 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 and were d i s c a r d e d . S u r v i v a l t o t h e s m a l l shoot s t a g e was 1 5 / 3 2 i n medium MSB^ Q I Q ^ w i t h g l u c o s e , pH 5 . 7 . I n medium MSB^ Q I Q 1 w i t h s u c r o s e , 1 9 / 4 2 s u r v i v e d when t h e medium was a d j u s t e d t o pH 6 . 1 and 1 2 / 1 3 s u r v i v e d when t h e pH was a d j u s t e d t o 5 . 7 . When two meri s t e m t i p s i n medium MSB-^  Q I Q ^, pH 6 . 1 , were t r a n s f e r r e d t o medium MSB^ Q I Q pH 5 . 7 , a f t e r 6 weeks and 1 2 weeks r e s p e c t i v e l y , t h e y had formed l a r g e c l o n e s . These were th 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 each. Four weeks l a t e r t h e y were r e s u b d i v i d e d i n t o 5 0 and 7 6 c u t t i n g s , a f t e r a n o ther 4 weeks, i n t o 1 1 2 and 2 0 5 c u t t i n g s . T i p s from medium c o n t a i n i n g g l u c o s e gave a com-p a r a b l e number o f c u t t i n g s a t t h i s s t a g e ( F i g s . 4 A - D ) . The number o f c u t t i n g s o b t a i n e d by c o n t i n u e d s u b d i v i s i o n a t 4-week i n t e r v a l s would be comparable t o 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 . F i g u r e 4 . L o g a n b e r r y c l o n e s i l l u s t r a t i n g t h e e f f e c t o f t w o c a r b o h y d r a t e s o u r c e s , a n d t w o l e v e l s o f B A P , o n 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 m e d i u m M S w i t h B A P a t 1 . 0 m g / L a n d I B A a t 0 . 1 m g / L . A : A f t e r 2 0 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 , t h e p l a n t l e t w a s d i v i s i b l e i n t o 1 5 c u t t i n g s . B : A f t e r 1 2 w e e k s o f c u l t u r e 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 , t h e s h o o t w a s t r a n s f e r r e d t o m e d i u m M S w i t h B A P i n -c r e a s e d t o 3 . 0 m g / L . A f t e r a n o t h e r 6 w e e k s , t h e l e a v e s w e r e s m a l l e r t h a n o n A , a n d s o m e w h a t c u r l e d , b u t t h e p l a n t l e t w a s d i -v i s i b l e i n t o 2 5 c u t t i n g s . C : A f t e r 1 4 w e e k s o f c u l t u r e i n s t a r t i n g m e d i u m w i t h g l u c o s e , t h e s h o o t w a s d i v i s i b l e i n t o 6 0 c u t t i n g s . D : A f t e r 8 w e e k s o f c u l t u r e i n s t a r t i n g m e d i u m w i t h g l u c o s e , t h e s h o o t w a s t r a n s f e r r e d t o m e d i u m w i t h B A P i n c r e a s e d t o 3 . 0 m g / L . A f t e r a n o t h e r 6 w e e k s , t h e l e a v e s w e r e s m a l l e r t h a n o n C , a n d s o m e w h a t c u r l e d , a n d t h e p l a n t l e t w a s d i v i s i b l e i n t o 2 0 c u t t i n g s . 3 9 . 4 0 . R o o t i n g o f l o g a n b e r r y s h o o t c u t t i n g s , a f t e r t r a n s f e r f r o m s t a r t i n g m e d i a w i t h B A P a t t w o l e v e l s t o r o o t i n g m e d i a w i t h o u t B A P , w a s i n f l u e n c e d b y t h e B A P l e v e l i n t h e s t a r t i n g m e d i u m , b y p H , a n d t o s o m e e x t e n t b y c a r b o h y d r a t e s o u r c e i n b o t h m e d i a ( T a b l e 5 ) . T h e c o n c e n t r a t i o n o f I B A i n t h e r o o t i n g m e d i u m h a d n o s i g n i f i c a n t e f f e c t o n t i m e o f r o o t i n i t i a t i o n o r p e r c e n t a g e r o o t i n g , b u t n u m b e r o f r o o t s p e r c u t t i n g w a s h i g h e s t a n d r o o t e l o n g a t i o n w a s b e s t w i t h I B A a t 0 . 7 m g / L . W i t h B A P a t 1 . 0 m g / L , r o o t i n g w a s f a s t e r a n d p e r c e n t a g e r o o t i n g w a s g r e a t e r t h a n w i t h B A P a t 3 . 0 m g / L . W i t h B A P a t 1 . 0 m g / L , p e r c e n t a g e r o o t i n g w a s n o t a f f e c t e d b y p H . W i t h B A P a t 3 . 0 m g / L , p e r c e n t a g e r o o t i n g w a s h i g h e r w h e n t h e p H w a s a d j u s t e d t o 6 . 1 . R e s u l t s w e r e i n c o n c l u s i v e f o r t e s t s o f c a r b o h y d r a t e s o u r c e b u t , f o r t h e t w o g r o u p s w i t h 1 0 0 % r o o t i n g , t h e s h o o t g r o w t h m e d i u m c o n t a i n e d s u c r o s e , a n d t h e r o o t i n g m e d i u m , g l u c o s e . R o o t i n g o f l o g a n b e r r y s h o o t c u t t i n g s , a f t e r t r a n s f e r f r o m s t a r t i n g m e d i u m M S B ^ Q I Q p H 5 . 7 t o r o o t i n g m e d i u m M S I n _ w i t h t w o p H l e v e l s , w a s i n f l u e n c e d b y t h e p H o f t h e r o o t i n g m e d i u m . R o o t i n g w a s 8 7 % , a n d t o o k f r o m 1 - 3 w e e k s . T h e r e w a s n o d i f f e r e n c e i n r o o t i n g b e t w e e n t h e t w o c l o n e s . T h e l o w e r p H w a s s l i g h t l y s u p e r i o r f o r t h e r o o t i n g m e d i u m . A t p H 5 . 7 , 3 6 / 3 9 c u t t i n g s r o o t e d ( 9 2 % ) ; a t p H 6 . 1 , 3 2 / 3 9 ( 8 2 % ) r o o t e d . S h o o t c u t t i n g s t r a n s f e r r e d d i r e c t l y t o s o i l r o o t e d w i t h i n t h e s a m e p e r i o d a s t h o s e r o o t e d i n v i t r o b u t r o o t e d c u t t i n g s , p l a n t e d a f t e r 2 w e e k s i n r o o t i n g m e d i u m , w e r e a l i t t l e l a r g e r t h a n t h o s e r o o t e d i n s o i l . T h e g r o u p 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 c a r b o h y d r a t e 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 l o g a n b e r r y s h oot 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 Shoot medium* R o o t i n g medium** No. o f c u t t i n g s % Average r o o t i n g BAP Sugar* pH Sugar pH r o o t e d / t r a n s f e r r e d t i m e (mg/L) * (days) 3.0 S 6.1 S 6.1 14/17 82 30 3.0 S 6.1 G 6.1 18/18 100 24 3.0 S 5.7 S 5.7 11/18 61 21 3.0 S 5.7 G 5.7 11/18 61 35 3.0 G 5.7 G 5.7 14/18 78 14 1.0 S 6.1 S 6.1 12/13 92 18 1.0 S 6.1 G 6.1 14/18 78 25 1.0 S 5.7 S 5.7 14/18 78 24 1.0 S 5.7 G 5.7 17/17 100 15 1.0 G 5.7 G 5.7 13/16 81 16 *: Shoot growth media were MS w i t h IBA a t 0.1 mg/L and BAP a t 1.0 or 0.1 mg/L. **: R o o t i n g media were MS w i t h IBA up t o 1.0 mg/L. *.: S: s u c r o s e a t 30 g/L; G: g l u c o s e a t 25 g/L. o f 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 were as l a r g e as e i t h e r o f t h e o t h e r two groups. A month a f t e r 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 , f i v e p l a n t s from each group were weighed, measured, and t h e i r r o o t s and l e a v e s were counted (Table 6 ) . On p l a n t l e t s r o o t e d i n s o i l r o o t s were s m a l l e r i n d i a m e t e r , and had a g r e a t e r number o f b r a n c h i n g r o o t s than t h o s e on p l a n t l e t s r o o t e d i n c u l t u r e . P l a n t l e t s t r a n s f e r r e d t o s o i l a f t e r 2 weeks i n r o o t i n g medium, were s l i g h t l y t a l l e r and more r o b u s t t h a n p l a n t l e t s i n t h e o t h e r two gr o u p s , and weighed al m o s t h a l f as much a g a i n . I t appears t h a t r o o t i n g i n v i t r o may be advantageous but t h a t p l a n t l e t s r o o t e d i n c u l t u r e s h o u l d be p l a n t e d i n s o i l w i t h i n a s h o r t t i m e o f r o o t i n g . I f r o o t e d p l a n t l e t s are a l l o w e d t o remain i n c u l t u r e f o r t o o l o n g , t h e i r ad-vantage i s l e s s e n e d . Source P l a n t P r e - t r e a t m e n t 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 not improved when so u r c e p l a n t s were exposed t o 1-8 weeks o f h e a t - t r e a t m e n t . S u r v i v a l o f c o n t r o l t i p s s t a r t e d i n medium MS w i t h o u t hormones o r i n o s i t o l , and s u b c u l t u r e d t o medium MSB^ ^ 1 ^ ^, was 24/45 (56%) compared t o h e a t - t r e a t e d m e r i s t e m t i p s , 25/58 ( 4 3 % ) . I n medium MSB^ 0 I Q ^ , pH 6.1, s u r v i v a l o f mer i s t e m t i p s from c o n t r o l p l a n t s was 19/42 ( 4 5 % ) ; from h e a t - t r e a t e d p l a n t s , i t was 23/67 ( 3 4 % ) . These d i f f e r e n c e s were not s i g n i f i c a n t a t t h e 0.05 l e v e l . T a b l e 6. Comparison of l o g a n b e r r y c u t t i n g s r o o t e d i n s o i l and i n v i t r o 43. Group* Average w e i g h t Average no. o f Average l e n g t h o f of p l a n t l e t main r o o t s l e a v e s r o o t s l e a v e s (g) (mm) (mm) 1 0.692 2 1.017 3 0.693 10 10 8 12 7 13 12.26 8.60 12.54 10.44 9.16 9.44 *: Group 1 was r o o t e d i n s o i l ; group 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 weeks; 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 were examined 1 month a f t e r p l a n t -i n g group 3. F i v e p l a n t l e t s from each group were examined and t h e r e s u l t s averaged. 44. C o l d S t o r a g e o f I n V i t r o P l a n t l e t s Of t h e 27 l o g a n b e r r y p l a n t l e t s p l a c e d i n t o c o l d s t o r a g e i n v i t r o , 23 s u r v i v e d p l a n t i n g d i r e c t l y i n t o s o i l . When th e n o n - s u r v i v o r s were d i s c a r d e d a f t e r 2 weeks from t h e t i m e o f p l a n t i n g , a l l appeared t o be i n f e c t e d w i t h powdery mildew; some had 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 and lower l e a v e s . Mildew was a l s o p r e s e n t on t h e dead 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 . A f t e r a l l i n f e c t e d l e a v e s were removed from th e r e m a i n i n g p l a n t s t h e y resumed v i g o r o u s growth. C o l d s t o r a g e i s a p o t e n t i a l p r o c e d u r e f o r p r e s e r v i n g l o g a n b e r r y germplasm i n v i t r o , s i n c e s u r v i v a l o f p l a n t l e t s i s v e r y h i g h . T a y b e r r y C u l t u r e Media None o f t h e f i v e t a y b e r r y m e r i s t e m t i p s s t a r t e d i n medium MSB^ ^ 1 ^ ^, pH 6.1, s u r v i v e d . Of t h e t e n t i p s s t a r t e d i n t h e same medium w i t h g l u c o s e , pH 5.7, s i x s m a l l t i p s 0.05-0.15 mm l o n g , d i e d , but f o u r t i p s 0.15-0.25 mm l o n g , 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 stems, and many l e a v e s . A f t e r 2 months on t h i s medium, two l a r g e shoots were 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 MSB^ Q I Q w i t h s u c r o s e , pH 5.7. Two s h o o t s were k e p t i n t h e s t a r t i n g medium. Two months l a t e r , p l a n t s i n s t a r t i n g medium had s m a l l , w e l l expanded l e a v e s , many s h o r t , compact s h o o t s , and v e r y l o n g r o o t s ( F i g . 5A). A f t e r 28 weeks i n s t a r t i n g medium ( F i g . 5B) a t a y b e r r y c l o n e had a v a s t number 45. A. B. F i g u r e 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 -bohydrate source, and two l e v e l s of 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 wit h g l u c o s e , and wit h BAP at 1.0 mg/L and IBA at 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, the shoot was t r a n s f e r r e d to medium with sucrose, and BAP at 3.0 mg/L. A f t e r another 4 weeks, the leaves were l a r g e r and the r o o t s not as w e l l developed as on A. o f r o o t s . I n medium w i t h BAP a t 3.0 mg/L, l e a v e s were l a r g e r , s h o o t s t a l l e r , and i n t e r n o d e s l o n g e r , b u t o n l y one shoot r o o t e d , and t h e r o o t s were few and v e r y s h o r t ( F i g . 5C) . I n m i c r o p r o p a g a t i o n t e s t s i n medium w i t h e l e v a t e d 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 l o n g d e v e l o p e d i n t o a shoot d i v i s i b l e i n t o 15 c u t t i n g s w i t h i n 12 weeks o f 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 BAP l e v e l , t h e s e c u t t i n g s were s u b d i v i d e d i n t o 4 0 c u t t i n g s , and 4 weeks a f t e r t h i s , 160 c u t t i n g s were made. Based on t h i s l i m i t e d sample, t h e a n n u a l m i c r o p r o p a g a t i o n r a t e o f t a y b e r r y would be s i m i l a r t o t h a t o f b l a c k r a s p b e r r y or l o g a n b e r r y . 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 , a l l r o o t e d and p r o -duced new l e a v e s w i t h i n 2 weeks. I n c u l t u r e , c u t t i n g s r o o t e d i n medium M S I Q w i t h i n t h e same p e r i o d and were t r a n s p l a n t e d t o s o i l w i t h o u t d i f f i c u l t y . Red R a s p b e r r y C u l t u r e M e d ia 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 media 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 c u l t u r e . None o f 59 mer i s t e m t i p s s u r v i v e d i n medium MSC w i t h BAP a t 0.1 mg/L and IBA a t 0.1-0.7 mg/L, and o n l y 2/36 s u r v i v e d and r o o t e d w i t h IBA a t 1.0 mg/L. Most t u r n e d brown. When 50 meristems each were p l a c e d i n t o medium m s b Q -^ 1 ^  Q w i t h m a c r o n u t r i e n t s a t f u l l s t r e n g t h , d i l u t e d 3:4, 2:4, and 1:4, the 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 the s m a l l shoot s t a g e was 8 , 8 , 3, and 1 r e s p e c t i v e l y . S i n c e browning was t h e major symptom s t i l l , major n u t r i e n t t o x i c i t y was u n l i k e l y t o be i n v o l v e d . F u r t h e r t e s t s showed t h a t low t o t a l hormone c o n c e n t r a t i o n s promoted me r i s t e m t i p s u r v i v a l . W i t h BAP a t 0.1 mg/L, IBA l e v e l s c o u l d be i n c r e a s e d t o 0.5 mg/L; w i t h IBA l e v e l a t 0.1 mg/L, BAP c o n c e n t r a t i o n c o u l d be i n c r e a s e d t o 3.0 mg/L w i t h o u t d e c r e a s i n g s u r v i v a l . M e r i s t e m t i p growth was s u p e r i o r a t t h e l o w e s t hormone l e v e l t e s t e d (MSB^ ^ 1 ^ ^) b u t c h l o r o p h y l l f o r m a t i o n , r e -f l e c t e d i n the i n t e n s i t y o f g r e e n c o l o u r , improved as c y t o k i n i n was i n c r e a s e d from 0.1 t o 5.0 mg/L. From t h i s t r i a l two media were s e l e c t e d f o r f u r t h e r s t u d y , MSB^ Q I Q ^ and MSB_ I . ,. I t was d e c i d e d t h a t t h r e e o t h e r media s h o u l d be i n v e s t i g a t e d as w e l l , MSB^ ^ 1 ^ ^, and MS w i t h o u t hormones, w i t h i n o s i t o l a t 100 mg/L, o r w i t h o u t i n o s i t o l . The h i g h e s t r a t e o f s u r v i v a l t o t h e s m a l l shoot s t a g e (65/86) was among meri s t e m t i p s e x c i s e d i n t o medium MS w i t h n e i t h e r i n o s i t o l nor hormones, and t r a n s f e r r e d t o medium MSB-j^ 0 I Q ^ a t t h e f i r s t s u b c u l t u r e (28 days) . S u r v i v a l r a t e was a l s o h i g h (60/84) f o r t i p s i n i t i a t e d i n medium w i t h o u t hormones, b u t c o n t a i n i n g i n o s i t o l a t 100 mg/L, and t r a n s f e r r e d t o medium MSB 1 Q I q ^ a t t h e f i r s t s u b c u l t u r e . When t i p s were s t a r t e d i n medium w i t h o u t hormones, and s u b c u l t u r e d t o medium w i t h BAP a t 3.0 mg/L, s u r v i v a l was r educed t o 24/86 and growth was p o o r e r than 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 reduced (33/86) when meristem t i p s were 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 ^) , but growth r a t e o f t h e s u r v i v o r s was b e t t e r t h a n t h a t o f t i p s s t a r t e d i n medium w i t h o u t hormones, whi c h grew v e r y l i t t l e u n t i l s u b c u l t u r e d t o medium w i t h hormones. I f meristem t i p s were a l l o w e d t o remai n i n medium w i t h o u t hormones o r i n o s i t o l t h e y d i e d w i t h i n 5 weeks. I n medium w i t h i n o s i t o l , b u t no hormones, t h e y grew so s l o w l y t h a t t hey were e v e n t u a l l y d i s c a r d e d . Of mer i s t e m t i p s s t a r t e d i n medium w i t h BAP a t 3.0 mg/L (MSB^ Q I Q ^ ) , o n l y 18/81 s u r v i v e d t o t h e s m a l l shoot s t a g e . T e s t s o f media MSB^ ^ 1 ^ ^ and MSB^ Q I Q ^, each a t f i v e pH l e v e l s , showed t h a t 5.7 was t h e most e f f e c t i v e pH f o r s u p p o r t i n g growth o f r e d r a s p b e r r y t i p s . E f f e c t s o f pH were s i m i l a r i n b o t h media. The average growth r a t e and s u r v i v a l d e c r e a s e d w i t h i n c r e a s e o r d e c r e a s e o f pH v a l u e s from t h i s p o i n t . The g r e a t e s t average growth was i n medium MSB^ ^ I Q ^ , pH 5.7. E x p l a n t s i n c r e a s e d i n l e n g t h 1 0 - f o l d i n 30 days; 3 0 - f o l d o r more i n 60 da y s , a t w h i c h t i m e t h e y 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 . A t a l l o t h e r pH l e v e l s , t i p s were g e n e r a l l y p a l e r and n o t as r o b u s t i n appearance as t h o s e a t pH 5.7. A t pH 5.9, the average growth was 7 - f o l d i n 30 days and 1 6 - f o l d i n 60 days. A t pH 6.1, growth was 5 - f o l d i n 30 days and 1 0 - f o l d i n 60 days. A t pH 5.5 or 6.3 s u r v i v a l and growth iwere s e v e r e l y reduced. A d j u s t i n g 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 -c l a v i n g . T h i s v a l u e d r i f t s down, s l o w l y i n r e f r i g e r a t e d tubes, more q u i c k l y i n tubes i n the i n c u b a t i o n chamber (pH 5.1 a f t e r 1 week), most q u i c k l y i n tubes where p l a n t s are a c t i v e l y growing (pH 4.5-4.7 a f t e r 4 weeks). Carbon source and c o n c e n t r a t i o n i n the medium d r a -m a t i c a l l y a f f e c t e d meristem t i p s u r v i v a l and average growth r a t e (Table 7). T i p s i n medium MSB^ ^1^ ^ w i t h glucose at 20 g/L grew the f a s t e s t and had the g r e a t e s t s u r v i v a l r a t e . Leaves were fewer and l a r g e r than i n media wi t h sucrose but they tended to t u r n brown a t t h e i r edges. I t was decided t h a t glucose at 25 g/L i n the s t a r t i n g medium might make use of the h i g h e r growth r a t e enjoyed by meristem t i p s i n glucose, and p o s s i b l y a v o i d the browning e f f e c t s of medium wit h 20 g/L g l u c o s e . In t e s t s of two carbohydrate sources, each a t two pH l e v e l s , growth of red r a s p b e r r y meristem t i p s to the s m a l l shoot stage was s l i g h t l y f a s t e r , and s u r v i v a l g r e a t e s t (70%) i n the medium wit h glucose, a t pH 6.1 (Table 8). At pH 5.7, s u r v i v a l was o n l y 35%, although growth a f t e r 30 and 60 days was not s i g n i f i c a n t l y d i f f e r e n t from t h a t i n the glucose medium a t the higher pH. In the medium w i t h sucrose, s u r v i v a l was b e t t e r at pH 6 . 1 than a t pH 5.7. Growth was not s i g n i f i c a n t l y a f f e c t e d by pH l e v e l ; r e s u l t s may t h e r e f o r e be i n f l u e n c e d by i n h e r e n t d i f f e r e n c e s i n the two s e l e c t i o n s used i n the pH and i n the carbohydrate t r i a l s . In the presence of i n o s i t o l , w i t h sucrose as the carbohydrate source, a few meristem t i p s s u r v i v e d and grew i n t o l a r g e shoots i n the absence of any growth 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 and growth 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* C a r b o h y d r a t e Shoot s u r v i v a l * * Average i n c r e a s e i n shoot l e n g t h t y p e amount p r o p o r t i o n % (g/L) (mm) 30 days 60 days s u c r o s e 20 6/18 s u c r o s e .30 6/15 g l u c o s e 20 9/14 g l u c o s e 30 5/16 g l u c o s e 40 3/14 33 1.05 4.75 40 0.86 2.38 64 6.33 11.49 31 3.75 5.19 21 1.15 3.96 *: N u t r i e n t medium was MS w i t h BAP a t 1.0 mg/L and IBA a t 0.1 mg/L, pH 6.1. **: 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 shoot 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 were combined. 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 growth 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 c u l t u r e * C a r b o h y d r a t e pH Shoot s u r v i v a l * * Average i n c r e a s e i n shoot l e n g t h t y p e amount p r o p o r t i o n % (g/L) (mm) 3 0 days 6 0 days g l u c o s e g l u c o s e 25 25 s u c r o s e 30 s u c r o s e 30 6.1 5.7 6.1 5.7 48/69 24/68 8/23 19/83 70 35 35 23 4.05 3. 04 1, 1. 35 76 7, 7. 2, 3, 33 28 43 67 *: N u t r i e n t medium was MS w i t h BAP a t 1.0 mg/L and IBA 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 the s m a l l s h oot s t a g e / no. e x c i s e d . 52. ( F i g . 6 ) . W i t h g l u c o s e as the c a r b o h y d r a t e s o u r c e , t i p s 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 o r w i t h o u t i n o s i t o l . I n medium c o n t a i n i n g s u c r o s e and hormones, s u r v i v a l was n o t a f f e c t e d by i n o s i t o l but b o t h growth r a t e and morphology were a f f e c t e d . As t h e i n o s i t o l l e v e l was i n c r e a s e d from 0 t o 150 mg/L, growth was s t i m u -l a t e d and a x i l l a r y b r a n c h i n g i n c r e a s e d ( F i g s . 7A, B ) . I n media c o n t a i n i n g g l u c o s e , average growth r a t e o f sh o o t s was f a s t e r t h a n i n any media w i t h s u c r o s e r e g a r d l e s s o f i n o s i t o l l e v e l . N e i t h e r s u r v i v a l nor 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 the f a s t e s t growth o c c u r e d w i t h t i p s i n t h e h i g h e s t i n o s i t o l concen-t r a t i o n t e s t e d (150 mg/L). M i c r o p r o p a g a t i o n e x p e r i m e n t s w i t h r e d r a s p b e r r y s h o o t s i n d i c a t e d v a r i a b i l i t y between s e l e c t i o n s , and even among sho o t s d e r i v e d from t h e same p l a n t s . I n medium c o n t a i n i n g s u c r o s e , 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 as good a s , o r b e t t e r t h a n , i n medium w i t h e l e v a t e d BAP (MSB 3 0 I Q ^) (compare F i g . 8A t o F i g s . 8B and 8C). Other 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 e l e v a t e d BAP l e v e l ( F i g . 8D). 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 an 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 c o n c e n t r a t i o n (compare F i g s . 9B, 10A); 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 medium w i t h e l e v a t e d BAP (compare F i g s . 10A and B ) . S u r v i v a l o f s h o o t s o r c u t t i n g s t r a n s f e r r e d t o p r o l i f e r a t i o n media 53. Key Days after excision F i g u r e 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 growth o f me 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 arrow. 54. Figure 7. Eff e c t s of i n o s i t o l concentration on development of red raspberry meristem t i p s in culture. A: After 8 weeks i n culture. B: After 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. I n o s i t o l i n h i b i t e d rooting, and increasing concentration 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 showing response 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 20, 7, 10, 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 from buds e x c i s e d from t h e same stem 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 t h a t can o c c u r 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 from 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 response t o BAP i n t h e growth 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. F i g u r e 9. Red r a s p b e r r y clones showing the e f f e c t of pH on l e a f s i z e and browning. Both p l a n t l e t s were grown i n medium MS wit h BAP a t 1.0 mg/L, IBA at 0.1 mg/L with glucose, f o r 7 months. A: At pH 6 . 1 . B: At pH 5 . 7 . A . B . 5 7 . F i g u r e 1 0 . R e d r a s p b e r r y c l o n e s s h o w i n g d i f f e r e n t r e s p o n s e o f t w o 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 m e d i u m w i t h g l u c o s e . A f t e r 4 m o n t h s i n s t a r t i n g m e d i u m w i t h g l u c o s e , I B A a t 0 . 1 m g / L a n d B A P a t 1 . 0 m g / L , a n d 3 m o n t h s i n m e d i u m w i t h B A P i n c r e a s e d t o 3 . 0 m g / L , A w a s 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 3 5 . C l o n e s i n m e d i u m w i t h g l u c o s e h a d l e a v e s t h a t w e r e u s u a l l y l a r g e r a n d m o r e r u g o s e t h a n i n m e d i u m w i t h s u c r o s e . 58. was always s u p e r i o r i n media 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. W h i l e growth was f a s t a t pH 6.1, e s p e c i a l l y i n media 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 by p e r i p h e r a l , t h e n t o t a l , l e a f browning (compare F i g s . 9A and B ) . Shoots 8-16 weeks o l d and 1.0-2.0 cm l o n g , grown 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 IBA o r NAA a t 0.0-1.0 mg/L, took an average o f 23 days t o r o o t , w i t h 12/23 sh o o t s r o o t e d i n IBA, 17/26 i n NAA. When c u t t i n g s w i t h one o r more l e a v e s , from p l a n t l e t s grown i n medium MSB^ Q I Q ^, 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 i t h e r IBA o r NAA a t 0.0-1.0 mg/L, r o o t i n g took an average o f 15 days, and 17/35 c u t t i n g s r o o t e d i n IBA, 15/30 i n NAA. Roots t h a t formed i n t h e p r e s e n c e o f NAA were d i f f e r e n t from th o s e formed i n media w i t h IBA, o r w i t h o u t a u x i n s . W i t h NAA, r o o t s a r o s e from l e a f b l a d e s , p e t i o l e s , o r shoot bases 'in c o n t a c t w i t h t h e medium, and were u s u a l l y accompanied by c a l l u s f o r m a t i o n on 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 shoot bases was sometimes v e r y e x t e n s i v e . 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 ( c o l o u r , r o o t h a i r f o r m a t i o n , e l o n g a t i o n ) e x c e p t p o i n t o f o r i g i n . T h i s 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 problem s i n c e i m p e r f e c t v a s -c u l a r c o n n e c t i o n s would l e a d t o poor i n i t i a l growth i n t h e greenhouse, o r i n h i b i t s u r v i v a l . The use o f NAA was d i s -c o n t i n u e d . D i f f e r e n t c a r b o h y d r a t e s o u r c e s i n t h e medium d i d n o t i n f l u e n c e t h e s e r e s u l t s . D i v i s i o n o f c l o n e s from s t a r t i n g medium M S B . ^ ^ I Q ^, and medium M S B ^ ^ I Q ^ i n t o l a r g e r c u t t i n g s w i t h two o r more l e a v e s each, i n c r e a s e d r o o t i n g o f c u t t i n g s 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 I B A a t 0 . 1 - 1 . 0 mg/L. From medium w i t h B A P a t 3 . 0 mg/L, 1 9 / 2 2 c u t t i n g s r o o t e d w i t h i n 2 - 3 weeks o f t r a n s f e r t o r o o t i n g media. From medium w i t h B A P a t 1 . 0 mg/L, 2 2 / 2 2 c u t t i n g s r o o t e d w i t h i n 1 - 2 weeks of t r a n s f e r t o r o o t i n g media. The l o n g e s t and b e s t - f o r m e d r o o t s o c c u r e d a t t h e l o w e s t l e v e l s o f I B A , o r no I B A i n t h e r o o t i n g medium ( F i g . 1 1 ) . R o o t i n g medium M S I Q 5 was b e s t ; i t combined the f a s t e s t r o o t i n g w i t h s a t i s f a c t o r y r o o t f o r m a t i o n and t o p growth. Of t h e c u t t i n g s p l a c e d i n s o i l , a l l 2 0 r o o t e d and formed new growth w i t h i n 2. weeks. Of t h e c u t t i n g s t r a n s -f e r r e d from media M S B ^ Q I 1 or M S B 3 0 I Q ^, o n l y 3 5 / 7 0 r o o t e d b u t , 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 u n r o o t e d c u t t i n g s had produced dozens of new l e a v e s and stems, and each was d i v i s i b l e i n t o 2 0 - 8 0 c u t t i n g s w h i c h r o o t e d i n s o i l w i t h o u t d i f f i c u l t y . Source P l a n t P r e - t r e a t m e n t D i f f e r e n c e s i n s u r v i v a l and growth r a t e between c o n t r o l and e t i o l a t e d m e r i s t e m t i p s were n o t s i g n i f i c a n t a t t h e 0 . 0 5 l e v e l . The e t i o l a t e d m e r i s t e m t i p s took about 1 0 days t o t u r n g r een and t h e r e a f t e r were v i s u a l l y i n d i s t i n g u i s h a b l e from c o n t r o l t i p s (Table 9 ) . H e a t - t r e a t m e n t e x p e r i m e n t s i n d i c a t e d t h a t growing F i g u r e 11. Red r a s p b e r r y c u t t i n g s from a cl o n e i n medium MS wit h BAP at 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 with IBA at 0.5 mg/L. T a b l e 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 o n 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 f o u r m e d i a M e d i u m * S h o o t s u r v i v a l * * p r o p o r t i o n d a r k l i g h t ( c o n t r o l ) 1 1 2 / 3 0 2 1 / 3 0 2 1 0 / 3 0 8 / 3 0 3 2 0 / 3 0 1 3 / 3 0 4 5 / 3 0 7 / 3 0 * : M e d i u m 1 w a s M S w i t h n o i n o s i t o l , n o h o r m o n e s ; t i p s w e r e s u b c u l t u r e d t o m e d i u m 3 a t 1 m o n t h . M e d i u m 2 w a s M S w i t h n o i n o s i t o l , n o h o r m o n e s ; t i p s w e r e s u b c u l t u r e d t o m e d i u m 4 a t 1 m o n t h . M e d i u m 3 w a s M S w i t h B A P a t 1 . 0 m g / L , I B A a t 0 . 1 m g / L M e d i u m 4 w a s M S w i t h B A P a t 3 . 0 m g / L , I B A a t 0 . 1 m g / L * * : N o . 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 / n o . e x c i s e d . 62. s o u r c e p l a n t s f o r up t o 8 weeks a t 37°C had l i t t l e e f f e c t 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 ( T able 1 0 ) . Comparison o f s u r v i v a l and growth r a t e o f mer i s t e m t i p s e x c i s e d from p o t t e d p l a n t l e t s d e r i v e d from c u l t u r e , and from c o n t r o l p l a n t s r a i s e d from r o o t c u t t i n g s , showed no d i f -f e r e n c e s between s o u r c e p l a n t s i n e i t h e r o f two media. How-e v e r , s u r v i v a l i n medium MSB^ ^ 1 ^ 1 w i t h g l u c o s e a t 2 5 g/L was a g a i n b e t t e r ( c o n t r o l : 1 2 / 1 7 ; c u l t u r e d p l a n t s : 15/16) th a n i n t h e same medium w i t h s u c r o s e a t 30 g/L ( c o n t r o l : 1/27; c u l t u r e d p l a n t s : 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 bud on t h e stem had no ap p a r e n t e f f e c t i n most e x p e r i m e n t s . However, 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 , and c o n c e n t r a t i o n s were v a r i e d , from 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 appeared t o grow and 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 th 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 , from t h e same s o u r c e p l a n t s ( F i g . 1 2). C o l d S t o r a g e o f I n V i t r o P l a n t l e t s A f t e r 3 months o f c o l d s t o r a g e , a l l b u t 4 o f t h e 12 c o l d s t o r e d p l a n t l e t s appeated green and s t u r d y . Two weeks a f t e r t r a n s f e r t o s o i l , s i x had produced new l e a v e s , s i x were s h r i v e l e d and showed e v i d e n c e o f mildew i n f e c t i o n . C o l d s t o r a g e a t 10°C i s a p o t e n t i a l method o f p r e s e r v i n g r e d r a s p b e r r y germplasm, f o r up t o 3 months. 63. T a b l e 10. E f f e c t o f h e a t - t r e a t m e n t (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 media* H e a t - t r e a t m e n t No. e x p l a n t s s u r v i v e d / n o . e x c i s e d (weeks) Medium 1 (%) Medium 2 (%) 0 180/237 (76) 36/86 1 8/9 6/7 2 5/5 4/8 3 4/4 3/5 4 5/5 0/3 5 3/5 2/4 6 5/6 2/3 7 2/3 0/3 8 8/10 2/10 40/47 (85) 19/43 (42) *: Medium 1 was MS w i t h no hormones, 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, sh o o t s were 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, IBA a t 0.1 mg/L, pH 6.1. Figure 12. Red raspberry a p i c a l (top row) and l a t e r a l (bottom row) pairs of meristem t i p s showing the influence of carbohydrate type and con-centration on development after 2 months i n medium MS with BAP at 1.0 mg/L and IBA at 0.1 mg/L. Carbohydrate sources were ( l e f t to r i g h t ) : sucrose at 20, and 30 g/L and glucose at 20, 30, and 40 g/L. 6 5 . D i s c u s s i o n The p r e s e n t i n v e s t i g a t i o n o f f a c t o r s t h a t a f f e c t t h e growth and r o o t i n g i n v i t r o o f f o u r Rubus s p e c i e s r e v e a l e d 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 t o t h r e e c u l t i v a r s o f b l a c k b e r r y d e s c r i b e d by Harper (1978) and Broome and Zimmerman (1978). A comprehensive r e v i e w o f t h e l i t e r a -t u r e on t h e t i s s u e c u l t u r e o f Rosaceous p l a n t s i n d i c a t e s many s i m i l a r i t i e s between t h e s e f i v e Rubus s p e c i e s , and members o f f o u r o t h e r genera i n t h i s f a m i l y t h a t have been c u l t u r e d . Many f a c e t s o f c u l t u r e have never been t h o r o u g h l y e x p l o r e d nor e x p l a i n e d f o r Rosaceous p l a n t s . S i n c e t h e Rubus s p e c i e s u t i l i z e d i n t h i s i n v e s t i g a t i o n would appear t o r e a c t t o c u l t u r e i n a manner t y p i c a l o f Rosaceous p l a n t s , an e n l a r g e d u n d e r s t a n d i n g o f t h e t i s s u e c u l t u r e o f t h i s i m p o r t a n t f a m i l y may be p o s s i b l e . C o n v e r s e l y , some anoma-l i e s o b s e r v e d i n t h e c u l t u r e o f t h e s e Rubus s p e c i e s a r e e x p l a i n e d i n t h e l i t e r a t u r e on Rosaceae. Many i n t e r d e p e n d a n t f a c t o r s may a f f e c t r e g e n e r a t i o n o f m e r i s t e m t i p s o f Rubus s p e c i e s : i n i t i a l s i z e , l a t e r a l o r a p i c a l o r i g i n , d i f f e r e n c e s i n s o u r c e 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 age, season o f e x c i s i o n , and t h e adequacy o f t h e c u l t u r e medium and i n c u b a t i o n c o n d i t i o n s . T h i s s t u d y showed t h a t i n c r e a s i n g t h e s i z e o f m e r i s t e m t i p s i n c r e a s e d s u r v i v a l , but d i d not i n f l u e n c e r o o t i n g o f the s u r v i v o r s . A p i c a l m e r i s t e m t i p s were n o t c o n s i s t e n t l y s u p e r i o r t o a x i l l a r y o r l a t e r a l m e r i s t e m t i p s , as assumed by many w o r k e r s . N e i t h e r were t h e y i n f e r i o r as found by Harper (197 8) w i t h b l a c k b e r r y and t a y b e r r y . A p i c a l m e r i s t e m t i p s a r e o f t e n s l i g h t l y l a r g e r t h a n l a t e r a l m e r i s t e m t i p s , w h i c h s h o u l d g i v e them an advantage i n r e g e n e r a t i n g s h o o t s , b u t d i f -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 i n growth r a t e among t i p s o f e q u a l s i z e . However, a p i c a l 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 o f t e n outgrew t h o s e from 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 c a r b o h y d r a t e s o u r c e , a t l e v e l s o f 20 and 25 g/L. E f f e c t o f s o u r c e p l a n t age was 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 . M e r i s t e m t i p s from mature p l a n t s had l e s s r e g e n e r a t i v e p o t e n t i a l t h a n t h o s e from young s e e d l i n g s . I n c o n t r a s t t o t h e r e s u l t s o f C a r r e e t a l . (1979), r e d r a s p b e r r y m e r i s t e m t i p s e x c i s e d from J u l y - F e b u a r y d i d not g e n e r a t e c a l l u s , and s u r v i v a l was not i m p a i r e d . The use o f c o n t i n u o u s c r o p s o f young p l a n t s p r o b a b l y e x p l a i n s the l a c k o f s e a s o n a l e f f e c t . I t may be t h a t hormonal c o m p o s i t i o n o f t h e s o u r c e p l a n t s i s governed more by age t h a n by season. However, d u r i n g t h e s p r i n g , ( F e b u a r y - A p r i l ) p l a n t s i n c u l t u r e appeared t o 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 were e x c i s e d . E t i o l a t i n g r e d r a s p b e r r y s o u r c e p l a n t s b e f o r e e x c i s i n g m eristem t i p s appears t o have no advantage, and i t may de-l a y growth a f t e r e x c i s i o n . E t i o l a t e d m e r i s t e m t i p s t o o k a p p r o x i m a t e l y 10 days t o t u r n green i n v i t r o , and t o become v i s u a l l y i n d i s t i n g u i s h a b l e from t i p s s p r o u t e d i n t h e l i g h t . They were 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 terms of s u r v i v a l , 67. and growth r a t e was not s i g n i f i c a n t l y d i f f e r e n t a f t e r t h e i n i t i a l l a g p e r i o d . The e f f e c t s o f h e a t - t r e a t m e n t of s o u r c e p l a n t s p r i o r t o m e r i s t e m t i p c u l t u r e v a r i e d w i t h t h e Rubus s p e c i e s . H e a t - t r e a t m e n t o f b l a c k r a s p b e r r y i n c r e a s e d s u r v i v a l and growth r a t e , and promoted f a s t e r r o o t i n g . W i t h r e d r a s p -b e r r y , s u r v i v a l was o n l y s l i g h t l y i n c r e a s e d ; growth r a t e was not a f f e c t e d . H e a t - t r e a t m e n t of l o g a n b e r r y s o u r c e p l a n t s had no e f f e c t . R e p o r t s on o t h e r Rosaceous p l a n t s a l s o show marked d i f f e r e n c e s . Popov (1976) found h e a t -t r e a t m e n t o f sour c h e r r y had no e f f e c t on c u l t u r e s ; P o s n e t t e and Jha (1960) found a d e c r e a s e i n t h e number o f s t r a w b e r r y c u t t i n g s t h a t became e s t a b l i s h e d ; V i n e (1968) found t h a t c u t t i n g s from h e a t - t r e a t e d s t r a w b e r r y grew more q u i c k l y ; M e l l o r and S t a c e - S m i t h (1976) found t h a t h e a t - t r e a t m e n t of r e d r a s p b e r r y s o u r c e p l a n t s i n c r e a s e d t h e s u r v i v a l , growth and r o o t i n g o f e x c i s e d shoot t i p s . Most r e v e a l i n g i s t h e work o f M u l l e n e t a l . (1974, 1975) w o r k i n g w i t h s t r a w b e r r y , who found t h a t h e a t - t r e a t m e n t of t h r e e s t r a w b e r r y s e l e c t i o n s i n c r e a s e d t h e s u r v i v a l and r o o t i n g o f m e r i s t e m t i p s , b u t had no e f f e c t on a f o u r t h . Sward and H a l l a m (1976) des-c r i b e d a number of 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 i n m e r i s t e m t i p s o f h e a t - t r e a t e d p o t a t o p l a n t s . They demonstrated t h a t thermotherapy c o n t r i b u t e s t o t h e d i s o r g a n i z a t i o n o f growth p r o c e s s e s i n e x c i s e d m eristem t i p s . I t i s d i f f i c u l t t o r e c o n c i l e t h i s e v i d e n c e w i t h improvements i n v i t r o o r s t i m u -l a t i o n o f 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 and 6 8 . 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 remains t o be e l u c i d a t e d . The e f f e c t i s apparent i n th e m e r i s t e m d u r i n g c u l t u r e . Of g r e a t e s t i m p o r t a n c e i n meristem t i p r e g e n e r a t i o n , shoot growth, 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 o f c u l t u r e media. Of t h e s e , c a r b o h y d r a t e c o n c e n t r a t i o n and pH had t h e g r e a t e s t e f f e c t on growth; phytohormone con-c e n t r a t i o n and pH had t h e g r e a t e s t e f f e c t on s u r v i v a l and morphogenesis. R a r e l y have d i f f e r e n t c a r b o h y d r a t e s o u r c e s , c o n c e n t r a t i o n s , o r pH l e v e l s been e v a l u a t e d i n t h e c u l t u r e o f 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 t h a t pH l e v e l and 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 have 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 growth. I d e a l l y , c o r r e c t o s m o l a r i t y s h o u l d be e s t a b l i s h e d f o r any c u l t i v a r i n v i t r o a f t e r , o r p r e f e r a b l y i n c o n j u n c t i o n w i t h , phytohormone and pH a d j u s t m e n t s . L e v e l o f pH and o s m o l a r i t y were found t o be i n t e r d e p e n d e n t f a c t o r s w i t h r e d r a s p b e r r i e s , c o m p l i c a t e d f u r t h e r by c y t o k i n i n (BAP) l e v e l s i n t h e medium. E f f e c t s o f d i l u t i n g m a c r o n u t r i e n t s , even by a 1:4 d i l u t i o n , were not d r a m a t i c by comparison. D i f f e r i n g l e v e l s o f i n o s i t o l i n t h e medium have 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 ; a l m o s t i n v a r i a b l y 100 mg/L a r e added t o c u l t u r e media. W h i l e i n o s i t o l s t i m u l a t e d shoot growth and, a t 150 mg/L, promoted 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 . Red r a s p b e r r y s h o o t s i n t h e shoot growth medium r o o t e d more o f t e n when i n o s i t o l was o m i t t e d t h a n when t h e medium con-69. t a i n e d i n o s i t o l a t 50, 100, o r 150 mg/L. The c o n c e n t r a t i o n s of B v i t a m i n s , and g l y c i n e , i n MS medium appear t o be adequate. The a n t i o x i d a n t c y s t e i n e HC1 seemed t o be n e c e s s a r y i n t h e c u l t u r e medium f o r b l a c k r a s p b e r r y , e x p e c i a l l y when the t i p s were newly e x c i s e d , b u t not f o r t h e o t h e r t h r e e Rubus s p e c i e s ; p r o b a b l y b l a c k r a s p b e r r y c o n t a i n s more p h e n o l i c s u b s t a n c e s t h a t t h e o t h e r t h r e e s p e c i e s . D i f f e r e n c e s i n t h e c o n c e n t r a t i o n o f phytohormones employed 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 i n c l u d e d t h e range 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 Rosaceous p l a n t s . E f f e c t s o f IBA and BAP a r e c o n s i s t e n t w i t h t h o s e i n o t h e r r e p o r t s . 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 sh o o t s seems t o be due t o t h e enhancement of a x i l l a r y b r a n c h i n g 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 i n t e r e s t i s t h e l a c k o f t o l e r a n c e o f b l a c k r a s p b e r r y c u l t u r e s t o GA^ w h i c h , a t 0.1 mg/L, caused c h l o r o s i s . Of p a r t i c u l a r i m p o r t a r e t h e r e s p o n s e s of l o g a n b e r r y and r e d r a s p b e r r y c u t t i n g s t o NAA i n the r o o t i n g medium. Naphthalene a c e t i c a c i d i s commonly used as a r o o t i n g hormone f o r Rosaceous p l a n t s , b u t i n t h e s e 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 , and i n d u c e d r o o t f o r m a t i o n from l e a f p e t i o l e s , l e a f l a m i n a and c a l l u s e d shoot bases i n c o n t a c t w i t h t h e medium. W i t h IBA, however, t h e r e was r a r e l y any c a l l u s and r o o t s were produced n o r m a l l y , from t h e base o f t h e s h o o t . W h i l e few w o r kers used 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 groups have found i t s t i m u l a t o r y f o r i n i t i a t i n g c u l t u r e s (Broome and Zimmerman, 1978). T h i s 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 d u r i n g a l l s t a g e s o f p l a n t growth. The d i s a d v a n t a g e o f l i q u i d medium i s t h e need f o r a s u p p o r t s t r u c t u r e . I t s advantages a r e improved s u r v i v a l and r a t e o f growth. I n l i q u i d medium t h e r o o t s o f b l a c k r a s p b e r r y p l a n t l e t s were s h o r t e r and more numerous, and had many more r o o t h a i r s , t h a n r o o t s i n s o l i d medium. The i n c r e a s e d number o f r o o t h a i r s , and consequent i n c r e a s e d a b s o r b t i v e c a p a c i t y , may account f o r t h e more v i g o r o u s growth o f l a r g e s h o o t s i n l i q u i d medium. Murashige and S k o o g 1 s (1962) medium f o r m u l a t i o n i s p o o r l y b u f f e r e d , and t h e pH l e v e l tends t o d e c r e a s e w i t h t i m e , d e c r e a s i n g the s t o r a g e l i f e o f l i q u i d medium. 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 h i g h e r pH. The pH l e v e l o f t h e medium a f f e c t e d s u r v i v a l and growth r a t e 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 , o f some s e l e c t i o n s , and a f f e c t e d shoot p r o l i f e r a t i o n , and r o o t i n g o f c u t t i n g s . S u r v i v a l o f b l a c k r a s p b e r r y m e r i s t e m t i p s was much lower a t pH 6.1 tha n a t 5.7. T h i s might e x p l a i n why f i l t e r - s t e r i l i z e d medium seemed d i f f e r e n t i n i t s e f f e c t s from a u t o c l a v e d medium. The d i f f e r e n c e s may be due t o pH s i n c e f i l t e r -s t e r i l i z i n g does not a l t e r t h e pH, and a u t o c l a v i n g d e p r e s s e s i t . L i g h t i n g , i n i t s photomorphogenic c a p a c i t y , i s p r o b a b l y much more i m p o r t a n t i n development o f a d v e n t i t i o u s growth from c a l l i t h a n i t i s i n development from m e r i s t e m t i p s . 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 f l u o r e s -c e n t l i g h t s appeared t o be adequate d u r i n g t h i s i n v e s t i g a t i o n . When some b l a c k r a s p b e r r y c u l t u r e s were i n a d v e r t e n t l y exposed t o c o n t i n u o u s l i g h t f o r s e v e r a l weeks, t h e y s u f f e r e d no ap p a r e n t 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 o f meristem t i p s o f b l a c k r a s p b e r r y and was t h e te m p e r a t u r e used f o r a l l f u r t h e r s t u d i e s . I t may not have been optimum f o r r e d r a s p b e r r y p r o l i f e r a t i o n , a l t h o u g h i t d i d not seem t o i n h i b i t growth o f t h e o t h e r t h r e e Rubus s p e c i e s . R o s a t i e t a l . (1980) d e s c r i b e d t i s s u e d i s c o l o r a t i o n and abnormal l e a f b l a d e e x p a n s i o n o c c u r r i n g a t 26°C i n c u l t u r e s o f Japanese plum (Prunus s a l i c i n a L u n d l . cv. C a l i t a ) . S i m i l a r symptoms o c c u r r e d d u r i n g p r o l i f e r a t i o n t r i a l s o f r e d r a s p b e r r y , but never i n s m a l l s h o o t s , s u g g e s t i n g a g r e a t e r t e m p e r a t u r e s e n s i t i v i t y i n o l d e r t i s s u e s . An i m p o r t a n t c o n s i d e r a t i o n f o r t h e p r o d u c t i o n o f l a r g e numbers of c u l t u r e d p l a n t s i s t h e space r e q u i r e m e n t s . I n c u -b a t o r o r c o l d s t o r a g e space r e q u i r e m e n t s a r e r o u g h l y 900 2 c u t t i n g s / m (90 100-ml j a r s w i t h 10 c u t t i n g s / j a r ) . Rooted p l a n t l e t s can be s e t o u t i n 0.33 x 0.66 m f l a t s , 100 p l a n t -l e t s / f l a t , so greenhouse space r e q u i r e m e n t s a r e t w i c e t h a t i n an i n c u b a t o r o r c o l d chamber. A f t e r 4-6 weeks i n f l a t s , p l a n t s must 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 f i e l d . I n B r i t i s h Columbia a p p r o x i m a t e l y 1,215 ha 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 ha s e t out i n 72. 1978 and a n o t h e r 486 ha i n 1979. E x p a n s i o n i s p r o j e c t e d t o c o n t i n u e i n 1980 (B.C.M.A., 1979). The r e d r a s p b e r r y c r o p i n 1979 was t h e l a r g e s t (6,820,000 kg) and most l u c -r a t i v e (> $12,000.000) t o d a t e . The c u l t i v a r s c u r r e n t l y grown have been d e t e r m i n e d 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 have f o r many y e a r s been 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. D u r i n g t h e p a s t 10 y e a r s p l a n t i n g s have d e c l i n e d from over 28 ha t o a p p r o x i m a t e l y 3 ha. Y i e l d s per h e c t a r e have s u b s t a n -t i a l l y d e c l i n e d over t h e same time p e r i o d (B.C.D.A., 1972, B.C.M.A., 1979). I n 1979 l o g a n b e r r i e s s o l d f o r $0.34/kg ( p i c k e d ) , up s u b s t a n t i a l l y from 1977 and 1978. P r o b a b l y 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 t h r o u g h m i c r o p r o p a g a t i o n would r e v e r s e t h e d e c l i n i n g p l a n t -i n g s , and h e l p t o g e n e r a t e i n t e r e s t i n y i e l d p roblems, and i n l o g a n b e r r y p r o d u c t i o n . B l a c k r a s p b e r r y p l a n t s a r e not c u l t i v a t e d i n B.C., p r i m a r i l y because 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 t o l e r a n c e . T a y b e r r y p l a n t s a r e s t i l l under b r e e d e r s ' r i g h t s ; t h e y a r e c u r r e n t l y b e i n g t e s t e d f o r p r o d u c t i o n i n B.C.. M e r i s t e m 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 of Rubus s p e c i e s demonstrates t h a t enormous numbers o f r o o t e d p l a n t -l e t s can be produced a n n u a l l y . T h i s p r o c e d u r e i s a v i a b l e a l t e r n a t i v e t o p r e s e n t p r o p a g a t i o n t e c h n i q u e s ; i t w i l l 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 7 3 . p l a n t i n g m a t e r i a l . M i c r o p r o p a g a t i o n p r o c e d u r e s c o u l d e s -t a b l i s h a new i n d u s t r y , such as t a y b e r r y p l a n t i n g s , i n r e c o r d t i m e . 74. Summary B l a c k R a s p b e r r y M e r i s t e m t i p c u l t u r e , m i c r o p r o p a g a t i o n , and r o o t i n g o f b l a c k r a s p b e r r y cv. Munger were s u c c e s s f u l l y c a r r i e d out i n media MSCB„ , , _ I , „, pH 5.7. H e a t - t r e a t m e n t 0.1 o r 1.0 1.0 of b l a c k r a s p b e r r y s o u r c e p l a n t s improved s u r v i v a l , growth, and a c c e l e r a t e d r o o t i n g ( F i g . 3) o f c u l t u r e d t i p s . Munger p l a n t l e t s i n v i t r o s u r v i v e d 3 months o f c o l d s t o r a g e a t 10°C. Lo 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 b e s t i n medium MSB^ ^ 1 ^ ^, pH 5.7; m i c r o p r o p a g a t i o n , i n t h e same medium o r i n medium MSB^ Q I Q -^ ; and r o o t i n g , i n medium M S I Q ^. C a r b o h y d r a t e s , the c y t o k i n i n (BAP) c o n c e n t r a t i o n , and 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 l o g a n b e r r y c u t t i n g s (Table 5 ) . 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 d i d not a f f e c t s u r v i v a l o f l o g a n b e r r y m e r i s t e m t i p s . Logan-b e r r y p l a n t l e t s i n v i t r o s u r v i v e d 2 months o f c o l d s t o r a g e , b u t powdery mildew i n f e c t i o n was a problem 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 . T a y b e r r y M e r i s t e m t i p s s u r v i v e d i n medium MSB^ ^ 1 ^ ^ w i t h g l u c o s e a t 25 g/L, pH 5.7; m i c r o p r o p a g a t i o n took p l a c e i n t h i s medium o r i n medium MSB, „ I N ,; and r o o t i n g i n medium MSI , a l o n g w i t h s u b s t a n t i a l t o p growth. 7 5 . Red R a s p 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 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 hormones, w i t h o r w i t h -o u t 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 Sur-v i v a l was d e c r e a s e d , b u t growth r a t e o f s u r v i v o r s improved, when meri s t e m t i p s were s t a r t e d i n medium w i t h hormones. Medium pH o f 5 . 7 was b e s t when medium had s u c r o s e as t h e c a r b o h y d r a t e s o u r c e . Carbon so 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 were d r a m a t i c (Table 7 ) . G l u c o s e promoted f a s t e r growth of c u l t u r e d t i p s t h a n s u c r o s e ; a t 2 5 g/L growth r a t e was 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 . 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 were i n t e r d e p e n d a n t f a c t o r s (Table 8 ) . 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 media MSB^ Q or 3 O ^ O 1 v a r i e d between r e d r a s p b e r r y s e l e c t i o n s , and among t i p s from t h e same p l a n t s . I n o s i t o l i n h i b i t e d r o o t i n g ( F i g . 7 ) b u t , 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 0 - 1 5 0 mg/L, a x i l l a r y b r a n c h i n g i n c r e a s e d . Naphthalene a c e t i c a c i d i s no 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 ; IBA i s f a r s u p e r i o r . B e s t r e s u l t s were i n medium M S I Q J-, w h i c h a l s o promoted c o n s i d e r a b l e t o p growth ( F i g . 1 1 ) . E t i o l a t i n g s o u r c e p l a n t s was n o t advantageous (Table 9 ) . H e a t - t r e a t i n g s o u r c e p l a n t s i n c r e a s e d m e r i s t e m t i p s u r v i v a l o n l y s l i g h t l y (Table 1 0 ) . E x p l a n t s from p r e v i o u s l y c u l t u r e d 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 t i p s . A p i c a l m e r i s t e m t i p s were s u p e r i o r i n some media ( F i g . 1 2 ) . 76. E x c i s i o n d a t e was n o t i m p o r t a n t . Red 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 s u r v i v e d 3 months o f c o l d s t o r a g e , b u t powdery mildew was a problem 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 . I t i s recommended t h a t a f u n g i c i d e be used j u s t p r i o r t o p l a n t i n g c o l d - s t o r e d p l a n t l e t s i n s o i l . 7 7 . L i t e r a t u r e C i t e d A b b o t t , A . J . 1 9 7 7 . P r o p a g a t i n g t e m p e r a t e w o o d y s p e c i e s i n t i s s u e c u l t u r e . S c i . H o r t i c . 2 8 : 1 5 5 - 1 6 2 . A n d e r s o n , W . C . a n d H a n e r , G . 1 9 7 9 . T i s s u e c u l t u r e p r o -p a g a t i o n o f s m a l l f r u i t s p l a n t i n g . P r o c . W e s t . W a s h . H o r t i c . A s s n . f o r 1 9 7 9 . 6 9 : 1 6 2 - 1 6 3 . B r o o m e , 0 . C . a n d Z i m m e r m a n , R . H . 1 9 7 8 . I n v i t r o p r o -p a g a t i o n o f b l a c k b e r r y . H o r t S c i e n c e 1 3 : 1 5 1 - 1 5 3 . B u t e n k o , R . G . 1 9 6 4 . C u l t i v a t i o n o f i s o l a t e d t i s s u e s a n d t h e p h y s i o l o g y o f p l a n t m o r p h o g e n e s i s . ( i n R u s s i a n ) , N a u k a . 2 7 2 p p . C a r r e , M . , M a r t i n - T a n g u y , J . , M u s s i l o n , P . , a n d M a r t i n , C . 1 9 7 9 . L a c u l t u r e d e m e r i s t e m e s e t l a m u l t i p l i c a t i o n v e g e t a t i v e i n v i t r o a u s e r v i c e d e l a p e p i n i e r e . P a g e s 7 - 6 5 i n E . B i a r , B . L a n t i n , P . M u s s i l o n , M . S c h i r m e r , a n d M . d e G o u r g u f f e d s . B u l l . P e t i t s F r u i t s N o . 1 4 . N u m e r o s p e c i a l c u l t u r e d e m e r i s t e m e s . L ' A s s o c i a t i o n N a t i o n a l e d e s P r o d u c t e u r s d e P e t i t s F r u i t s , l ' I n s t i t u t N a t i o n a l d e l a R e c h e r c h e A g r o n o m i q u e , l ' I n s t i t u t N a t i o n a l d e V u l g a r i s a t i o n p o u r l e s F r u i t s , L e g u m e s e t C h a m p i g n o n s . C a r t e r , A . C . a n d P e t e r s , W . S . 1 9 7 2 . H o r t i c u l t u r a l N e w s -l e t t e r , B r i t i s h C o l u m b i a D e p t . A g r . , 5 2 , N o . 1 0 . C a r t e r , A . C . a n d P e t e r s , W . S . 1 9 7 9 . H o r t i c u l t u r a l N e w s -l e t t e r , B r i t i s h C o l u m b i a M i n . A g r . , 5 9 , N o . 1 0 . H a r p e r , P . C . 1 9 7 8 . T i s s u e c u l t u r e p r o p a g a t i o n o f b l a c k -b e r r y a n d t a y b e r r y . H o r t i c . R e s . 1 8 : 1 4 1 - 1 4 3 . H e l l e r , R . 1 9 5 3 . R e c h e r c h e s 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 v e g e t a u x c u l t i v e s i n v i t r o . T h e s e P a r i s e t A n n . S c i . N a t . ( B o t . B i o l . V e g . ) , P a r i s 1 4 : 1 - 2 2 3 . J a m e s , D . J . a n d T h u r b o n , I . J . 1 9 7 8 . G r o w t h i n v i t r o o f R u b u s 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 M a i l i n g R e s . S t n . A n n u . R e p . f o r 1 9 7 7 . p p . 1 7 7 - 1 7 8 . J e n n i n g s , D . L . 1 9 7 8 . T a y b e r r y . R e p . S c o t t . H o r t . R e s . I n s t , f o r 1 9 7 7 . p . 5 5 . K i s s , F . a n d Z a t y k o , J . 1 9 7 8 . V e g e t a t i v e p r o p a g a t i o n o f R u b u s s p e c i e s i n v i t r o . ( i n H u n g a r i a n , E n g l i s h A b -s t r a c t ) . B o t . K o z l e m . 6 5 : 6 5 - 6 8 . 78. L i t e r a t u r e C i t e d (cont'd) L i n s m a i e r , E. and Skoog, F. 1965. O r g a n i c growth f a c t o r r e q u i r e m e n t s of t o b a c c o t i s s u e c u l t u r e . P h y s i o l . P l a n t . 18:100-127. M e l l o r , F. C. and S t a c e - S m i t h , R. 1976. I n f l u e n c e o f h e a t t h e r a p y on r o o t i n g o f , and e l i m i n a t i o n o f r a s p b e r r y bushy dwarf v i r u s from, shoot c u t t i n g s o f r e d r a s p -b e r r y . A c t a H o r t i c . (The Hague) 66:63-70. M e l l o r , F. C. and S t a c e - S m i t h , R. 1977. V i r u s - f r e e p o t a -t o e s by t i s s u e c u l t u r e . Pages 616-646 i n J . R e i n e r t and Y. P. S. B a j a j , eds. A p p l i e d and fundamental a s p e c t s o f p l a n t c e l l , t i s s u e and o r g a n c u l t u r e . S p r i n g e r - V e r l a g , B e r l i n . M o r e l , G. M. 1960. P r o d u c i n g v i r u s - f r e e cymbidiums. Am. O r c h i d Soc. B u l l . 29:495-497. M o r e l , G. and M a r t i n , C. 1952; G u e r i s o n de d a h l i a s a t t i e n t s d'une m a l a d i e a v i r u s . Compt. Rend. 235:1324-1325. M o r e l , G. and M u l l e r , J . F. 1964. La c u l t u r e i n v i t r o du mer i s t e m a p i c a l de l a pomme de t e r r e . Compt. Rend. 258:5250-5252. M u l l e n , R. H., S m i t h , S. H., F r a z i e r , N. W., S c h l e g e l , D. E. and M c C a l l , S. R. 1974. M e r i s t e m c u l t u r e f r e e s s t r a w b e r r i e s o f m i l d y e l l o w edge, p a l l i d o s i s , and m o t t l e d i s e a s e s . P h y t o p a t h o l o g y 64:1425-1429. M u l l e n , R. H., F r a z i e r , N. W. and S c h l e g e l , D. E. 1975. M e r i s t e m c u l t u r e o f F r a g a r i a c h i l o e n s i s i n f e c t e d w i t h s t r a w b e r r y p a l l i d o s i s . P l a n t D i s . Rep. 59:268. M u r a s h i g e , T. 1974. P l a n t p r o p a g a t i o n t h r o u g h t i s s u e c u l -t u r e s . Annu. Rev. P l a n t P h y s i o l . 25:135-166. M u r a s h i g e , T. 1977. C l o n a l c r o p s t h r o u g h t i s s u e c u l t u r e . Pages 392-403 i n W. B a r z , E. R e i n e r t , and M. H. Zenk eds. P l a n t t i s s u e c u l t u r e and i t s b i o - t e c h n o l o g i c a l a p p l i c a t i o n . S p r i n g e r - V e r l a g , B e r l i n . M u r a s h i g e , T. 1978. The impact o f p l a n t t i s s u e c u l t u r e on a g r i c u l t u r e . Pages 15-26 i n T..A. Thorpe ed. F r o n t i e r s i n p l a n t t i s s u e c u l t u r e . I n t e r n a t i o n a l A s s o c i a t i o n f o r P l a n t T i s s u e C u l t u r e . C a l c a r y , A l b e r t a . M u r a s h i g e , T. and Skoog, F. 1962. A r e v i s e d medium f o r r a p i d growth ;and b i o a s s a y s w i t h t o b a c c o t i s s u e c u l -t u r e s . P h y s i o l . P l a n t . 15:473-497. 79. L i t e r a t u r e C i t e d (cont'd) N i t s c h , J . P. and N i t s c h , C. 1969. H a p l o i d p l a n t s from p o l l e n g r a i n s . S c i e n c e 163:85-87. Popov, Y. G., V y s o t s k i i , V. A. and T r u s h e c h k i n , V. G. 1976. C u l t i v a t i o n o f i s o l a t e d stem a p i c e s o f sour c h e r r y . Sov. P l a n t P h y s i o l . ( E n g l i s h T r a n s l . F i z i o l . Rast.) 23:513-518 P o s n e t t e , A. F. and J h a , A. 1960. The use o f c u t t i n g s and heat t r e a t m e n t t o o b t a i n v i r u s - f r e e s t r a w b e r r y p l a n t s . E a s t M a i l i n g Res. S t n . Annu. Rep. f o r 1959. p.98. P u t z , C. 1971. O b t e n t i o n de f r a m b o i s i e r s ( v a r . ' B o i s B l a n c ' ) sans v i r u s p a r l a t e c h n i q u e des c u l t u r e s de meristems. Ann. P h y t o p a t h o l . 3:493-501. Quak, F. 1970. Review o f heat t r e a t m e n t and me r i s t e m t i p c u l t u r e as methods t o o b t a i n v i r u s f r e e p l a n t s . P r o c . 18th I n t e r n a t . H o r t i c . Cong. 3:12-25. R o s a t i , P., M a r i n o , G. and S w i e r c z e w s k i , C. 1980. I n v i t r o p r o p a g a t i o n o f Japanese plum (Prunus s a l i c i n a L i n d l . cv. C a l i t a ) . J . Amer. Soc. H o r t . S c i . 105:126-129. Shchelkunova, L. E. 1974. S e a s o n a l p e r i o d i c i t y o f p l a n t r e g e n e r a t i o n from m e r i s t e m t i p s o f r a s p b e r r y . Sov. P l a n t P h y s i o l . ( E n g l i s h T r a n s l . F i z i o l . Rast.) 21: 54-58. Shchelkunova, S. E. and Popov, Y. G. 197 0. P r o d u c t i o n of v i r u s - f r e e r a s p b e r r y p l a n t s from c u l t u r e s o f i s o l a t e d apexes. Sov. P l a n t P h y s i o l . ( E n g l i s h T r a n s l . F i z i o l . R ast.) 17:618-622. S t r e e t , H. E. and S h i l l i t o , R. D. 1977. N u t r i e n t media f o r p l a n t o r g a n , t i s s u e , and c e l l c u l t u r e . Pages 305-359 i n M. R e c h c i g l , J r . ed. C R C handbook s e r i e s i n n u t r i t i o n and f o o d . S e c t i o n G: D i e t s , c u l t u r e media, and f o o d supplements. V o l . IV: C u l t u r e media f o r c e l l s organs and embryos. C R C P r e s s I n c . Sward, R. J . and H a l l a m , N. D. 1976. Changes i n f i n e s t r u c -t u r e o f t h e p o t a t o m e r i s t e m f o l l o w i n g h e a t t r e a t m e n t f o r v i r u s e r a d i c a t i o n . A u s t . J . Bot. 24:597-605. Thomas, E. and Davey, M. R. 1975. From s i n g l e c e l l s t o p l a n t s . Wykeham S c i e n c e S e r i e s . Wykeham P u b l i c a t i o n s (London) L t d . 171pp. L i t e r a t u r e C i t e d (cont'd) V e r t e s y , J . 1979. E x p e r i m e n t s f o r the p r o d u c t i o n o f v i r u s f r e e r a s p b e r r y p r o p a g a t i o n m a t e r i a l by m e r i s t e m c u l -t u r e . 2nd I n t e r n a t . Symp. on S m a l l F r u i t V i r u s D i s e a s e s . Budapest, Hungary. ( i n p r e s s ) . V i n e , S. J . 1968. Improved c u l t u r e o f a p i c a l t i s s u e s f o r p r o d u c t i o n of v i r u s - f r e e s t r a w b e r r i e s . J . H o r t i c . S c i . 43:293-297. Walkey, D. G. A. 197 8. I n v i t r o methods f o r v i r u s e l i m i n a t i o n . Pages 245-254 i n T. A. Thorpe ed. F r o n t i e r s i n p l a n t t i s s u e c u l t u r e . The I n t e r n a t i o n a l A s s o c i a -t i o n f o r P l a n t T i s s u e C u l t u r e . C a l g a r y , A l b e r t a . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095410/manifest

Comment

Related Items