Biotechnology in Crop Improvement - ACS Symposium Series (ACS

34 Biotechnology in Crop Improvement JOHN T. MARVEL

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch034

Monsanto Agricultural Products Company, St. Louis, MO 63167

Biotechnology promises to play a significant role in crop improvement and productivity in the 1990's and beyond. Early advances will probably be in the development of selective and safe microbial pesticides and the transfer of one to three gene traits to agronomic crops. While microbial pesti cides are technically fairly straightforward, genetically improving crop plants, using recombi nant techniques, will require the solution of numer ous technical problems. Of initial importance is the development of transformation vectors and re generation technology in key crop plants, particu larly legumes and cereals. Once these hurdles have been overcome, the key emphasis will shift to the discovery of genes to be transferred. This paper reviews the status of regeneration and transformation technology in the major crop plants and highlights recent progress in plant biochem istry which may serve as a source of important traits for genetic engineering.

A g r i c u l t u r a l b i o t e c h n o l o g y h a s been i n t h e p u b l i c eye a good d e a l r e c e n t l y . However, t h e b a s i c t h r u s t o f b i o t e c h n o l o g y i n a g r i c u l t u r e i s a c t u a l l y mundane. I n f a c t , g e n e t i c e n g i n e e r i n g o f p l a n t s w i l l be j u s t a n o t h e r t o o l f o r p l a n t b r e e d e r s t o u s e i n t h e i r c o n t i n u i n g e f f o r t s t o improve p l a n t p r o d u c t i v i t y . C l a s s i c a l b r e e d i n g h a s been t h e m a i n s t a y o f c r o p improvement s i n c e the r e d i s c o v e r y o f Mendelian g e n e t i c s at the beginning o f t h i s century. The improvements have been s i g n i f i c a n t , e.g., t h e d e v e l o p ment o f h y b r i d c o r n r e s u l t e d i n a s t e a d y 1-2% i n c r e a s e i n y i e l d p e r year. Other c r o p b r e e d i n g programs l e d t o t h e development o f s t r a i n s t h a t would s u s t a i n f o o d p r o d u c t i o n i n p r e v i o u s l y s t e r i l e e n v i r o n ments. C e l l b i o l o g y , i n c o n j u n c t i o n w i t h g e n e t i c e n g i n e e r i n g , prom i s e s new ways t o improve t h i s r e c o r d by enhancing y i e l d p o t e n t i a l , i m p r o v i n g p e s t t o l e r a n c e , d e c r e a s i n g s t r e s s e s due t o t h e environment and t o a g r i c u l t u r a l c h e m i c a l s , and i m p r o v i n g o v e r a l l agronomic

0097-6156/85/0276-0477$09.50/0 © 1985 American Chemical Society

Hedin et al.; Bioregulators for Pest Control ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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acceptability. In o r d e r t o comprehend how t h e s e new improvements w i l l o c c u r , an u n d e r s t a n d i n g o f c l a s s i c a l b r e e d i n g methods i s essential. C l a s s i c a l breeding c o n s i s t s of four d i s t i n c t a c t i v i t i e s : (1) s c r e e n i n g f o r d e s i r a b l e t r a i t s and t r a n s f e r r i n g t h o s e t r a i t s t o adapted l i n e s by s e x u a l c r o s s e s (2-4 y e a r s ) ; (2) s e l e c t i n g progeny w i t h the d e s i r e d c o m b i n a t i o n s o f t r a i t s (3-4 y e a r s ) ; (3) f i e l d e v a l u a t i o n of the s e l e c t e d v a r i e t i e s f o r y i e l d and performance under s e v e r a l e n v i r o n m e n t s (3-4 y e a r s ) ; and f i n a l l y (4) seed i n c r e a s e f o r s a l e (2-3 y e a r s ) . In a l l , the p r o c e s s r e q u i r e s 10-15 y e a r s t o p r o duce a new v a r i e t y which w i l l t y p i c a l l y have a l i f e t i m e o f o n l y 6-8 years. In a d d i t i o n , t h i s p r o c e s s i s l i m i t e d by the s e x u a l c o m p a t i b i l i t y between the l i n e s used f o r a c r o s s . T y p i c a l l y , only l i n e s from the same s p e c i e s o r v e r y c l o s e l y r e l a t e d ones can be used as a s o u r c e o f new t r a i t s . B i o t e c h n o l o g y can a d d r e s s t h e s e b o t t l e n e c k s of time and gene s o u r c e s by g e n e t i c a l l y e n g i n e e r i n g p l a n t c e l l s and t h e n r e g e n e r a t i n g them i n t o whole p l a n t s w i t h the new t r a i t s . This i s p o s s i b l e because p l a n t s , a l o n e among h i g h e r o r g a n i s m s , can be r e g e n e r a t e d i n t o whole p l a n t s from s o m a t i c c e l l s . This i s a phenomenon c a l l e d " t o t i p o t e n c y " . The r e g e n e r a t i o n c y c l e i s i l l u s t r a t e d f o r a l f a l f a i n F i g u r e 1. A c u t t i n g , o r e x p i a n t , t a k e n from the p a r e n t p l a n t , i s put onto a medium c o n t a i n i n g p l a n t hormones and n u t r i e n t s . Soon the t i s s u e b e g i n s t o p r o l i f e r a t e c e l l s i n a r a t h e r d i s o r g a n i z e d mass t o form a callus. Upon t r e a t m e n t w i t h a p p r o p r i a t e p l a n t n u t r i e n t s and h o r mones, the c a l l u s w i l l form s t r u c t u r e s w h i c h d e v e l o p i n t o s h o o t s , a p r o c e s s r e f e r r e d t o as " o r g a n o g e n e s i s " . These s h o o t s may be r e moved from the c a l l u s , r o o t e d , and grown i n t o normal f e r t i l e plants (1). T h i s p r o c e s s , o u t l i n e d i n F i g u r e 2, c o u l d be u s e f u l i n a b r e e d i n g program. As i n c l a s s i c a l b r e e d i n g , f i r s t a q u a l i t y c u l t i v a r i s chosen. E s t a b l i s h e d t i s s u e c u l t u r e techniques are then utilized. The m a t e r i a l i s p l a c e d i n t o c u l t u r e which a l l o w s s e l e c t i o n by c l a s s i c a l methods o r the i n s e r t i o n o f new genes. A f t e r the t i s s u e w i t h a new t r a i t has been p r o d u c e d , i t can be r e g e n e r a t e d i n t o a q u a l i t y c u l t i v a r c o n t a i n i n g the new d e s i r e d t r a i t . The p o t e n t i a l b e n e f i t s o f t h i s scheme a r e t w o - f o l d . First, the t i m e - l i n e s t o d e v e l o p new c u l t i v a r s may be d r a m a t i c a l l y s h o r t ened. In c e l l b i o l o g y - f a c i l i t a t e d b r e e d i n g , the i d e n t i f i c a t i o n , i s o l a t i o n and c l o n i n g o f a gene r e q u i r e s 1-3 y e a r s . To c u l t u r e a p l a n t t i s s u e , t r a n s f o r m i t and r e g e n e r a t e i t t a k e s a p p r o x i m a t e l y 6 months. F i e l d e v a l u a t i o n and seed i n c r e a s e a r e unchanged by t h i s t e c h n o l o g y , so the t o t a l time i s 6-10 y e a r s , a c o n s i d e r a b l e s a v i n g s i n time o v e r c o n v e n t i o n a l b r e e d i n g . G e n e t i c e n g i n e e r i n g a l l o w s the i n t r o d u c t i o n o f genes from any s o u r c e i n t o p l a n t s ; hence, a c r o p ' s germplasm base becomes a l l l i v i n g organisms r a t h e r t h a n j u s t c l o s e l y r e l a t e d , s e x u a l l y c o m p a t i b l e plants. T h i s means t h a t genes ( F i g u r e 3A) become a v a i l a b l e from b a c t e r i a which p r o d u c e i n s e c t i c i d a l p r o t e i n s , from b a c t e r i a o r f u n g i which p r o d u c e a n t i b i o t i c s a c t i v e a g a i n s t p l a n t pathogens, o r from s t r e s s - t o l e r a n t w i l d p l a n t s which would n o r m a l l y be s e x u a l l y incompatible. The p r o m i s e o f t h i s t e c h n o l o g y i s i n i t s a b i l i t y t o " t e a c h " p l a n t s t o produce t h e i r own i n s e c t i c i d e s , f u n g i c i d e s and growth r e g u l a t o r s .



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Figure 1. Alfalfa regeneration (clockwise). A cutting i s t a k e n from a p e t i o l e ( e x p i a n t ) , p l a c e d on medium t o induce t h e f o r m a t i o n o f a c a l l u s , t h e n t r a n s f e r r e d t o an a l t e r e d medium c a u s i n g shoots t o form.

QUALITY CULTIVAR

\ TISSUE EXPLANT

CALLUS INITIATION

SELECTION

/

* \

SUSPENSION CULTURE

SX

CALLUS I

PROTOPLASTS s

TISSUE WITH NEW TRAIT

GENETIC ENGINEER

I REGENERATION •

QUALITY CULTIVAR WITH NEW TRAIT

F i g u r e 2. T i s s u e c u l t u r e crop improvement. Sequence shows t h e i n t e g r a t i o n o f c e l l b i o l o g y t e c h n i q u e s i n t o crop improvement. H u r d l e s t o u s i n g t h e scheme i n c l u d e c a l l u s i n i t i a t i o n , p r o t o plast preparation, selection in culture, and plant regeneration.


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A

Insecticidal Bacterium

Fungicidal Streptomyces

Stress Tolerant Wild Plant

Novel Soybean Cultivar

Β

Plant Cell

F i g u r e 3. Examples o f d e s i r a b l e genes t o be i n s e r t e d i n t o crop plants. A. U n r e l a t e d organisms may have genes b e n e f i c i a l t o crop p l a n t s . B. Suggested e x t e n s i o n o f a b i o c h e m i c a l pathway in plants. B a c t e r i a produces d e s i r e d m o l e c u l e Ε b y enzymatic s t e p s E i t o E ; p l a n t pathway s t o p s a t i n t e r m e d i a t e C. I n t r o d u c t i o n o f b a c t e r i a l genes f o r s t e p s E3 and E causes p l a n t c e l l t o produce E . 4

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Even more s o p h i s t i c a t e d improvements may be p o s s i b l e ( F i g u r e 3 B ) . F o r example, i f a m i c r o b e produces a m o l e c u l e Ε w h i c h i s n e m a t o c i d a l , and t h e p l a n t has t h e b i o s y n t h e t i c machinery to make a key i n t e r m e d i a t e o f t h i s m o l e c u l e , C then perhaps genes, c o d i n g f o r t h e enzymes n e c e s s a r y t o complete t h e b i o s y n t h e t i c p a t h way, c o u l d be moved i n t o t h e p l a n t , c a u s i n g t h e p l a n t t o produce i t s own nematocide. The r e s u l t i s l i t e r a l l y c h e m i c a l s y n t h e s i s i n living tissues. P l a n t g e n e t i c e n g i n e e r i n g c o u l d be c o m p e t i t i v e with the chemical p e s t i c i d e business.


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However, enough must be u n d e r s t o o d about p l a n t metabolism t o support the u s e f u l m a n i p u l a t i o n o f t h e p l a n t s ' b i o s y n t h e t i c appara tus t o respond b e t t e r t o s t r e s s e s o r d i s e a s e . G e n e t i c m a n i p u l a t i o n of complex pathways, such as t h e s h i k i m a t e pathway ( F i g u r e 4 ) , w i l l be a l a r g e t a s k r e q u i r i n g c o n s i d e r a b l y more b i o c h e m i c a l knowledge about p l a n t s t h a n i s c u r r e n t l y a v a i l a b l e . Having c o n s i d e r e d t h e k i n d s o f advances t h a t t i s s u e c u l t u r e and g e n e t i c e n g i n e e r i n g c a n make i n c r o p improvements, i t i s n e c e s s a r y t o e x p l o r e t h e t e c h n i c a l l i m i t a t i o n s t o making t h o s e k i n d s o f changes ( F i g u r e 2 ) . F o r t h e major c r o p s one must f i r s t have t h e a b i l i t y t o c u l t u r e and r e g e n e r a t e p l a n t s from v a r i o u s e x p l a n t s . U n f o r t u n a t e l y , n o t a l l c r o p s respond t o c u r r e n t t i s s u e c u l t u r e t e c h n i q u e s and r e g e n e r a t e i n v i t r o . I t i s a l s o n e c e s s a r y t o have t h e t e c h n o l o g y t o e i t h e r s e l e c t new c e l l l i n e s i n c u l t u r e o r t o g e n e t i c a l l y e n g i n e e r new t r a i t s i n t o t h o s e t i s s u e s t h a t a r e i n c u l t u r e , and then r e g e n e r a t e p l a n t s t h a t w i l l e x p r e s s t h e new t r a i t . F i n a l l y , t h e s e new c u l t i v a r s must be e x t e n s i v e l y e v a l u a t e d i n t h e f i e l d t o a s s u r e t h a t t h e d e s i r e d t r a i t has been i n s e r t e d and i s e x p r e s s e d a t t h e p r o p e r time and i n t h e p r o p e r p l a n t t i s s u e . Much p r o g r e s s has been made i n t h e r e g e n e r a t i o n o f p l a n t s and i n u n d e r s t a n d i n g t h e r e g e n e r a t i o n p r o c e s s . T h i s i n c l u d e s t h e de velopment o f s e l e c t i o n t e c h n o l o g y w i t h p a r t i c u l a r emphasis on r e s i s t a n c e and t h e development o f g e n e t i c e n g i n e e r i n g t e c h n o l o g y u s i n g t h e A g r o b a c t e r i u m t u m e f a c i e n s v e c t o r system. The p r o c e s s o f p l a n t r e g e n e r a t i o n b e g i n s w i t h t h e s e l e c t i o n of t h e p r o p e r e x p i a n t w h i c h , when p l a c e d i n t h e a p p r o p r i a t e c u l t u r e media, w i l l form a c a l l u s . I n t h e case o f a l f a l f a , s o m a t i c embryos w i l l form on t h e c a l l u s s u r f a c e ( F i g u r e 5A) a f t e r t h e c a l l i have been exposed t o t h e a p p r o p r i a t e r a t i o o f c y t o k i n i n s and a u x i n s . E v e n t u a l l y t h e s e embryos w i l l p r e c o c i o u s l y germinate and form shoots. The d e v e l o p i n g embryos c a n be e x c i s e d and p l a c e d onto a r o o t i n g media t o d e v e l o p a r o o t system ( F i g u r e 5B). A f i n a l trans f e r o f t h e r o o t e d p l a n t s t o g r a v e l t u b s and g r a d u a l exposure t o greenhouse c o n d i t i o n s r e s u l t i n t h e development o f normal p l a n t s ( F i g u r e 5C). I n time t h e y f l o w e r and p r o d u c e s e e d s . When t h e s e seeds a r e p l a n t e d , they p r o d u c e normal f e r t i l e p l a n t s . Some v a r i a b i l i t y i n phenotype has been o b s e r v e d i n p l a n t s a r i s i n g from t h e t i s s u e c u l t u r e p r o c e s s . These s o m a c l o n a l v a r i a t i o n s may be p r e s e n t i n 2-20% o f t h e o p o u l a t i o n , and may depend upon t h e s t r e s s e s e n countered during the t i s s u e c u l t u r e process. T h i s k i n d o f media m a n i p u l a t i o n i s i n d i s p e n s a b l e i n t i s s u e c u l t u r e f o r m a x i m i z i n g t h e f r e q u e n c y o f r e g e n e r a t i o n . When t h e r e g e n e r a t i o n system f o r a new s p e c i e s i s i n t h e e a r l y s t a g e s o f development, e x p e r i m e n t a l m o d i f i c a t i o n s a r e n e c e s s a r y i n o r d e r t o



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F i g u r e 4. Shikimate-derived metabolism i n p l a n t s . A compli c a t e d b i o s y n t h e t i c pathway i s a p o s s i b l e g e n e t i c engineering target.



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Figure 5. Alfalfa embryogenesis. A. An a l f a l f a somatic embryo, E , about t o g e r m i n a t e , which i s surrounded by c a l l u s C. B. Plantlets rooting. C. Regenerated p l a n t s i n g r a v e l i n t h e greenhouse.


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achieve optimal r e s u l t s . F i g u r e 6 shows t h e e f f e c t o f m a n i p u l a t i n g t h e amino a c i d c o m p o s i t i o n o f t h e media on t h e f r e q u e n c y o f r e g e n e r a t i o n ( 2 ) . I t i s e v i d e n t t h a t when e i t h e r Shenk and H i l d e b r a n d t s o r B l a y d e s b a s a l media a r e u s e d , the f r e q u e n c y o f embryo f o r m a t i o n i s low. However, i f e i t h e r media i s supplemented w i t h a l a n i n e o r p r o l i n e , t h e f r e q u e n c y o f embryogenesis i s g r e a t l y enhanced.


1

R e s e a r c h o f a more fundamental n a t u r e i s a l s o n e c e s s a r y i n o r d e r t o u n d e r s t a n d and e f f e c t i v e l y m a n i p u l a t e t h e r e g e n e r a t i o n process. T h i s i s p a r t i c u l a r l y t r u e i n c r o p s such as soybean and c e r e a l s w h i c h a r e r e c a l c i t r a n t t o r e g e n e r a t i o n . F o r example, h i s t o l o g i c a l and h i s t o c h e m i c a l s t u d i e s can be conducted d u r i n g r e g e n e r a t i o n i n o r d e r t o u n d e r s t a n d t h e growth and development p r o c e s s . The f o r m a t i o n o f a l f a l f a s o m a t i c embryos ( F i g u r e 5A) i s w e l l s u i t e d f o r such a b a s i c i n v e s t i g a t i o n . Very e a r l y i n r e g e n e r a t i o n , a l f a l f a c a l l u s (as v i s u a l i z e d i n a c r o s s - s e c t i o n s t a i n e d w i t h s a f r a n i n and f a s t g r e e n i n F i g u r e 7A) has a l r e a d y d i f f e r e n t i a t e d i n t o d i s t i n c t tissues. The d a r k l y s t a i n i n g p u r p l e t i s s u e h i g h l i g h t s t h e embryo i n an e a r l y s t a g e o f f o r m a t i o n . The l i g h t e r b l u e s t a i n i n g c e l l s below t h e embryo have been named the " p r o r e g e n e r a t i v e mass and appear t o f u n c t i o n as c e l l u l a r p r o g e n i t o r s t o t h e embryo ( 3 ) . From t h i s p r o r e g e n e r a t i v e mass, t h e i n c i p i e n t embryo d e v e l o p s from a single c e l l . T h i s i s an i n t e r e s t i n g and s i g n i f i c a n t f i n d i n g b e c a u s e i t c l a r i f i e s an e a r l y o r g a n i z a t i o n a l event w h i c h o c c u r s i n r e g e n e r a tion. I n F i g u r e 7B t h e p r o r e g e n e r a t i v e mass remains as the embryo grows l a r g e r ; i n f a c t , when t h e embryo i s a p p r o x i m a t e l y a t t h e s t a g e o f t h e whole embryo shown i n F i g u r e 5A, t h e p r o r e g e n e r a t i v e mass remains a t t a c h e d t o t h e embryo ( F i g u r e 7C). 11

I t i s s p e c u l a t e d t h a t t h i s mass s u b s t i t u t e s f o r t h e s u s p e n s o r , an o r g a n t h a t n o r m a l l y a i d s i n f e e d i n g d e v e l o p i n g embryos i n p l a n t a . R e c a l c i t r a n t soybean and c e r e a l t i s s u e c u l t u r e systems a r e c u r r e n t l y being i n v e s t i g a t e d f o r evidence that these kinds of proregenerative c e l l masses a r e formed ( 4 ) . Another approach to s t u d y i n g the developmental process i n r e g e n e r a t i o n i s t o o b s e r v e t h e h i s t o c h e m i c a l changes i n c e l l s . F i g u r e 8A i l l u s t r a t e s a l f a l f a c e l l s w h i c h have been s t a i n e d w i t h a n i l i n e b l u e - b l a c k f o r t o t a l p r o t e i n b e f o r e i n d u c t i o n o f the r e generation process. These normal c a l l u s c e l l s a r e e l o n g a t e d and not densely s t a i n i n g . F i g u r e 8B shows a c e l l mass w h i c h has been induced to regenerate. The c e l l s a r e v e r y compact and t i g h t l y a s s o c i a t e d , and t h e c y t o p l a s m i s d a r k l y s t a i n i n g w i t h a n i l i n e b l u e , i n d i c a t i n g a h i g h c o n c e n t r a t i o n o f p r o t e i n s w h i c h may be n e c e s s a r y f o r the r e g e n e r a t i o n process. Studies of t h i s type w i l l a i d i n the design of b i o c h e m i c a l experiments designed to b e t t e r understand the molecular basis f o r regeneration i n p l a n t s . The development o f s e l e c t i o n t e c h n o l o g y i s n e c e s s a r y i n o r d e r t o d e r i v e c e l l l i n e s w i t h s p e c i f i c t r a i t s , such as h e r b i c i d e r e s i s t a n c e , from t i s s u e c u l t u r e . F i g u r e 9 d e p i c t s the somatic c e l l s e l e c t i o n process using c e l l c u l t u r e techniques. A t t h e bottom l e f t i s a f l a s k c o n t a i n i n g a suspension of a l f a l f a c e l l s . These c e l l s have been c u l t u r e d f o r 4-8 weeks and t h e n s i e v e d t o y i e l d v e r y s m a l l c e l l clumps which w i l l be used f o r s e l e c t i o n i n v i t r o . I n t h e example shown h e r e , s e l e c t i o n was made f o r h e r b i c i d e r e sistance. In the upper l e f t - h a n d c o r n e r , g l y p h o s a t e , t h e a c t i v e i n g r e d i e n t i n Roundup, i s added t o t h e growth media. A f t e r s e v e r a l



MARVEL


F i g u r e 6. E f f e c t on a l f a l f a r e g e n e r a t i o n o f amino a c i d a d d i t i o n t o media. The t o p row shows p e t r i p l a t e s o f Shenk and H i l d e b r a n d t medium w i t h no amino a c i d a d d i t i o n , SHO; w i t h t h e a d d i t i o n o f L - a l a n i n e , SHA; o r w i t h L - p r o l i n e , SHP. The bottom row shows B l a d y e s medium, w i t h no amino a c i d a d d i t i o n , BI2Y; w i t h the a d d i t i o n o f L - a l a n i n e , BIA; o r w i t h L - p r o l i n e , BIP. These amino acid additions enhance the frequency of embryogenesis.




486

Figure 7. Histology of alfalfa somatic embryogenesis. A. C r o s s - s e c t i o n o f c a l l u s a f t e r i n d u c t i o n o f embryogenesis. Lightly staining cells are the p r o r e g e n e r a t i v e mass (PRM) which g i v e s r i s e t o t h e d a r k l y s t a i n i n g c e l l s , t h e embryo ( Ε ) , surrounded by v e r y l i g h t l y s t a i n i n g n o n - r e g e n e r a t i n g c a l l u s (C) B. A s o m a t i c embryo a t a l a t e r s t a g e o f development. C. Somatic embryo b e g i n n i n g t o g e r m i n a t e , comparable t o the whole embryo shown i n F i g u r e 5A.




MARVEL

Figure 8. Histochemical studies of a l f a l f a callus cells. A. Normal c a l l u s c e l l s s t a i n e d w i t h a n i l i n e b l u e b l a c k f o r t o t a l p r o t e i n before induction of regeneration. B. Callus cells stained with a n i l i n e blue black after induction of regeneration; very darkly s t a i n i n g material i s p r o t e i n .


488



1

days exposure t o g l y p h o s a t e , t h e c e l l s a r e p l a t e d onto s o l i d media as shown i n t h e c e n t e r p i c t u r e . From t h e s e p l a t e d c e l l s o n l y a few w i l l develop i n t o c a l l i . Once t h e s e c a l l i a r e formed, t h e hormone l e v e l s c a n be ma n i p u l a t e d t o i n d u c e shoot f o r m a t i o n . T h i s i s f o l l o w e d by t h e r o o t i n g , h a r d e n i n g and t r a n s f e r - t o - g r e e n h o u s e p r o c e s s e s . D u r i n g t h i s sequence t h e s e l e c t e d l i n e s c a n be s c r e e n e d a t t h e c e l l u l a r l e v e l , at t h e r e g e n e r a t e d p l a n t l e t l e v e l , a t t h e whole p l a n t l e v e l i n t h e greenhouse, and f i n a l l y i n t h e f i e l d w h i c h i s t h e " a c i d t e s t " . F i g u r e 10 shows, i n c o n s i d e r a b l y more d e t a i l , t h e sequence o f m u t a g e n e s i s and s e l e c t i o n w h i c h has been a c t u a l l y used t o d e v e l o p herbicide resistant lines. C a l l i were i n i t i a t e d from a h e a l t h y a l falfa plant. A f t e r 4-8 weeks, t h e s e c a l l i were b r o k e n up i n t o s u s p e n s i o n s , and e i t h e r t r e a t e d w i t h a m u t a g e n i z i n g agent o r s c r e e n e d s i m p l y by s e l e c t i o n f o r spontaneous m u t a t i o n s . After either pro c e d u r e , t h e s e l e c t e d l i n e s , i . e . , t h e l i n e s which s u r v i v e d exposure to t h e h e r b i c i d e , were t h e n r e g e n e r a t e d , and t h e p l a n t s were e v a l u a t e d i n a number o f schemes. I n a d d i t i o n , p l a n t s s e l e c t e d a t t h e c e l l u l a r l e v e l f o r r e s i s t a n c e were r e c y c l e d t h r o u g h t h e e n t i r e s y s tem o f mutagenesis and s e l e c t i o n t o enhance t h e d e s i r e d r e s i s t a n c e trait. A number o f c e l l l i n e s i d e n t i f i e d i n c e l l c u l t u r e were r e s i s t a n t t o 10 m i l l i m o l a r g l y p h o s a t e . These l i n e s were r e g e n e r a t e d and t h e p l a n t l e t s were p l a c e d on media c o n t a i n i n g 10 o r 100 m i l l i molar g l y p h o s a t e . F o r comparison, r e g e n e r a t e d b u t n o t s e l e c t e d c o n t r o l p l a n t l e t s were p l a c e d on s i m i l a r media. Some o f t h e s e l e c t e d l i n e s grew and d e v e l o p e d on t h e g l y p h o s a t e - c o n t a i n i n g media. In c o n t r a s t , u n s e l e c t e d c o n t r o l p l a n t l e t s f a i l e d t o s u r v i v e . Sur v i v o r s were r o o t e d and t r a n s f e r r e d t o t h e greenhouse where they were s p r a y e d w i t h Roundup a t r a t e s e q u i v a l e n t t o 2 o r 4 pounds p e r a c r e . S u r v i v o r s o f t h i s t e s t were s u b s e q u e n t l y e v a l u a t e d f o r t h e i r r e s i s t a n c e t o Roundup i n t h e f i e l d . The d a t a f o r t h i s f i e l d experiment a r e summarized i n T a b l e 1. Data a r e p r e s e n t e d h e r e f o r 13 l i n e s w h i c h were d e r i v e d from c u l t u r e and f i e l d - e v a l u a t e d f o r r e s i s t a n c e t o Roundup h e r b i c i d e . Each o f t h e s e l i n e s was s i g n i f i c a n t l y more t o l e r a n t t o Roundup than t h e r e g e n e r a t e d n o n - s e l e c t e d c o n t r o l B74. However, t h e l e v e l o f r e s i s t a n c e i n t h e s e 13 c e l l l i n e s was n o t c o m m e r c i a l l y s i g n i f i c a n t . Neverthe l e s s , t h i s does i n d i c a t e t h a t r e s i s t a n t p l a n t s c a n be d e r i v e d by s e l e c t i o n s at the c e l l u l a r l e v e l . E l e v e n - t h o u s a n d c e l l l i n e s were mutagenized and s c r e e n e d t o i d e n t i f y t h e s e 13 l i n e s w h i c h were r e s i s t a n t a t t h e whole p l a n t l e v e l i n the f i e l d . T h i s i s a frequency o f approximately one-tenth of one p e r c e n t ; u n d o u b t e d l y t h i s f r e q u e n c y c a n be improved by genetic engineering.



MARVEL

Biotechnology in Crop improvement

Figure 9. Somatic c e l l s e l e c t i o n f o r h e r b i c i d e resistance. Bottom l e f t , a f l a s k o f a l f a l f a c e l l s i n s u s p e n s i o n . Top l e f t , addition of herbicide t o the c e l l s . Center, c e l l s plated onto s o l i d medium c o n t a i n i n g herbicide; a resistant callus growing on h e r b i c i d e - c o n t a i n i n g medium. Top r i g h t , r e s i s t a n t p l a n t l e t s regenerating. Bottom r i g h t , t o l e r a n t p l a n t s s e l e c t e d from t i s s u e c u l t u r e growing i n t h e f i e l d a f t e r b e i n g s p r a y e d with the h e r b i c i d e .

Mutagenesis

Initiate suspension cultures

Spontaneous mutations

Selection

Modified cell lines Morphogensis and embryogenesis Plant with new characteristics

F i g u r e 10. G e n e r a l i z e d scheme f o r c e l l u l a r spontaneous o r i n d u c e d m u t a g e n e s i s .

selection

involving


490


Table

1.

S u p e r i o r A l f a l f a C l o n e s from the G l y p h o s a t e F i e l d Based on S u r v i v a l 21 Days P o s t - A p p l i c a t i o n .

Test

P e r c e n t S u r v i v a l and One-Tailed Ρ V a l u e

6

Clone

M u t a g e n e s i s and Selection Conditions

ID

B74

Lbs/Acre

4

Lbs/Acre 0%

5%

None

tested)

30%

.0288

57%

.0012

30%

.0288

31%

.0386

57%

.0004

29%

.0586

70%

.0000

63%

.0001

27%

.0425

44%

.0014

38%

.0139

63%

.0001

B62-3-13

47%

.0031

B62-3-26

42%

.0076

B89-4

31%

.0490

64s-8 65S-1

EMS-AMP

(not

2

1

65s-2 65S-8


2

1

141s-3 141S-6

1

80s-3

PF-GLY

ls-1

3

5FU-GLY

4

NQO-GLY

5

ls-2 B62-3-10

1

^"Also s u p e r i o r t o t h e 20% 2 l b / a c r e treatment.

^Ethylmethanesulfonate

s u r v i v a l l e v e l of

RA3- 24

(p

Biotechnology in Crop Improvement - ACS Symposium Series (ACS

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