Applications of In Vitro Selection Systems - American Chemical Society

Chapter 4 Applications of In Vitro Selection Systems: Whole-Plant of

Selection

Using

the

Haploid

Phase

Ceratopteris Leslie G. Hickok

Downloaded by FUDAN UNIV on January 13, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch004

Department of Botany, University of Tennessee, Knoxville, TN 37996

Plant genes that confer tolerance to the herbicides paraquat and glyphosate have been selected for by using the haploid gametophyte phase of the fern Ceratopteris. The gametophyte generation of this plant, because i t can be cultured in large numbers and is haploid, provides a convenient selection system for the induction and recovery of both dominant and recessive mutations. In contrast to plant cell culture or bacterial selection systems, this approach utilizes whole plants that are photosynthetic. Expression of the selected traits can be monitored in both haploid gametophytes and in diploid vascular sporophytes, which are produced by controlled self- or cross-fertilizations of selected gametophytes. This system provides a rapid and powerful means of identifying and characterizing a variety of single genes that may be beneficial for crop improvement through recombinant DNA techniques and for basic studies of plant molecular biology.

Crop improvement through g e n e t i c e n g i n e e r i n g i s dependent upon t h e a b i l i t y t o i d e n t i f y genes t h a t i n f l u e n c e t r a i t s o f p o t e n t i a l agronomic i n t e r e s t . In a d d i t i o n , t h e a v a i l a b i l i t y o f s i n g l e gene m u t a t i o n s can be o f major importance i n b a s i c s t u d i e s o f p l a n t m o l e c u l a r b i o l o g y . In r e c e n t y e a r s , t h e development o f i n v i t r o c u l t u r e t e c h n i q u e s has led t o the successful a p p l i c a t i o n of m i c r o b i a l mutation s e l e c t i o n p r o t o c o l s t o h i g h e r p l a n t s {1,2). As a r e s u l t , a number o f p l a n t genes o f p a r t i c u l a r i n t e r e s t have been i d e n t i f i e d through t h i s approach ( 3 , 4 ) . Other approaches a l s o o f f e r promise f o r t h e i d e n t i f i c a t i o n o f s p e c i f i c genes. Because t h e development o f recombinant DNA t e c h n o l o g y i s e f f e c t i v e l y circumventing natural b a r r i e r s t o gene exchange between s p e c i e s , v i r t u a l l y any organism may be a s o u r c e o f genes f o r use i n g e n e t i c e n g i n e e r i n g o r f o r b a s i c r e s e a r c h a p p l i c a t i o n s (5). Obvious p o s s i b i l i t i e s i n c l u d e y e a s t , a l g a l and b a c t e r i a l systems which can be r e a d i l y m a n i p u l a t e d under l a b o r a t o r y c o n d i t i o n s ( 6 ) . Less t r a d i t i o n a l approaches a l s o e x i s t . F o r i n s t a n c e , r e c e n t s t u d i e s w i t h t h e homosporous f e r n C e r a t o p t e r i s 0097-6156/87/0334-0053$06.00/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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B I O T E C H N O L O G Y IN A G R I C U L T U R A L C H E M I S T R Y

have shown t h a t i t s f r e e l i v i n g h a p l o i d gametophyte g e n e r a t i o n can be e f f e c t i v e l y used t o s e l e c t f o r a wide v a r i e t y o f m u t a t i o n s t h a t are o f i n t e r e s t i n t h e areas o f p l a n t m o l e c u l a r b i o l o g y and c r o p improvement. Comparisons w i t h more t r a d i t i o n a l approaches and s p e c i f i c advantages and a p p l i c a t i o n s o f t h e C e r a t o p t e r i s system a r e d i s c u s s e d below. H i g h e r P l a n t Systems Ιϋ * ° s e l e c t i o n t e c h n i q u e s have obvious advantages over t h e use of whole p l a n t s o r seeds f o r t h e i n d u c t i o n and r e c o v e r y o f g e n e t i c v a r i a n t s i n h i g h e r p l a n t s . M u t a t i o n i n d u c t i o n and s e l e c t i o n a t t h e single cell level avoids the complexities associated with m u l t i c e l l u l a r systems and a l l o w s l a r g e numbers o f i n d i v i d u a l c e l l s t o be screened s i m u l t a n e o u s l y . The development and a p p l i c a t i o n o f anther c u l t u r e t e c h n i q u e s i n c e r t a i n s p e c i e s has f u r t h e r improved t h e u t i l i t y o f t h i s approach by a l l o w i n g t h e s e l e c t i o n and r e c o v e r y o f both r e c e s s i v e and dominant m u t a t i o n s from h a p l o i d c e l l c u l t u r e s . As s u c h , i t has become p o s s i b l e t o d e s i g n h i g h e r p l a n t s e l e c t i o n systems t h a t can e f f e c t i v e l y s e l e c t f o r s p e c i f i c m u t a t i o n s t h a t c o n f e r i n c r e a s e d t o l e r a n c e o r r e s i s t a n c e t o p e s t i c i d e s , t o x i n s , growth r e g u l a t o r s and o t h e r c h e m i c a l and p h y s i c a l s t r e s s e s . In s p i t e of t h e s e advantages, t h e i n d u c t i o n and s e l e c t i o n o f mutants i n c e l l o r t i s s u e c u l t u r e s o f h i g h e r p l a n t s a l s o has c e r t a i n t e c h n i c a l l i m i t a t i o n s t h a t hinder the successful general a p p l i c a t i o n o f these t e c h n i q u e s (.1,7). These d i f f i c u l t i e s i n c l u d e : 1) t h e l o s s o f r e g e n e r a t i v e c a p a c i t y i n o l d e r c e l l c u l t u r e s t h a t have been s u b j e c t e d t o s e l e c t i o n p r e s s u r e s and t h e consequent l i m i t on t h e number o f p u t a t i v e mutants t h a t can be r e g e n e r a t e d from such c u l t u r e s ; 2) problems i n t h e r e c o v e r y and maintenance o f s e l e c t e d mutants because o f g e n e t i c and chromosomal i n s t a b i l i t i e s t h a t f r e q u e n t l y e x i s t i n c e l l c u l t u r e s , even though such i n s t a b i l i t i e s may be o f d i r e c t v a l u e i n g e n e r a t i n g g e n e t i c v a r i a n t s i n t h e absence o f any mutagenic t r e a t m e n t ( 8 ) ; 3) t h e i n a b i l i t y t o a p p l y i n v i t r o approaches t o many h i g h e r p l a n t s and t h e consequent r e s t r i c t i o n o f t h e s e t e c h n i q u e s t o r e l a t i v e l y few g e n e r a ; 4) t h e presence o f c e l l aggregates i n c e l l s u s p e n s i o n c u l t u r e s , which may r e s u l t i n t h e p r o d u c t i o n o f g e n e t i c chimeras t h a t may s u b s e q u e n t l y i n t e r f e r e w i t h t h e r e c o v e r y and s t a b i l i t y o f c e r t a i n m u t a n t s ; and 5) s e l e c t i o n at t h e l e v e l o f u n d i f f e r e n t i a t e d and f r e q u e n t l y n o n - p h o t o s y n t h e t i c c e l l s , which can prevent t h e r e c o v e r y o f m u t a t i o n s t h a t a r e expressed i n whole p l a n t s or i n photosynthetic t i s s u e .


V 1

t r

C o n t i n u i n g s t u d i e s w i t h h i g h e r p l a n t systems a r e r e f i n i n g t e c h n i q u e s and approaches so t h a t many o f t h e s e l i m i t a t i o n s a r e m i n i m i z e d . F o r i n s t a n c e , r e c e n t s t u d i e s have shown t h a t t h e use o f p r o t o p l a s t c u l t u r e s can s u c c e s s f u l l y a v o i d many o f t h e problems a s s o c i a t e d w i t h c e l l c u l t u r e systems (.7,9). Because p r o t o p l a s t s o b t a i n e d from green l e a f t i s s u e c o n s t i t u t e g e n e t i c a l l y homogeneous c u l t u r e s o f s i n g l e p h o t o s y n t h e t i c c e l l s , t h e use o f such c u l t u r e s f o r i n v i t r o s e l e c t i o n s i s more l i k e l y t o y i e l d mutants t h a t a r e e x p r e s s e d i n d i f f e r e n t i a t e d t i s s u e s a t t h e whole p l a n t l e v e l . This a t t r i b u t e i s e s s e n t i a l where s e l e c t i o n f o r r e s i s t a n c e t o p h o t o s y n t h e t i c a l l y active herbicides i s of i n t e r e s t . In a d d i t i o n , because p r o t o p l a s t s are n o t m a i n t a i n e d i n c u l t u r e f o r extended p e r i o d s o f t i m e , g e n e t i c


4.

HICKOK

instability problems.

55

Applications of In Vitro Selection Systems and l o s s of

regenerative

c a p a c i t y are not

significant


A l t e r n a t i v e Approaches M i c r o b i a l systems p r o v i d e more r a p i d and e f f i c i e n t s e l e c t i o n systems than h i g h e r p l a n t approaches and may t h e r e f o r e be a u s e f u l s o u r c e f o r c e r t a i n types of a g r o n o m i c a l l y i m p o r t a n t genes. F o r i n s t a n c e , s t u d i e s w i t h t h e b a c t e r i a A e r o b a c t e r aeroqenes (10) and S a l m o n e l l a typhimurium (11) have r e s u l t e d i n t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f mutant s t r a i n s t h a t possess i n c r e a s e d t o l e r a n c e t o t h e h e r b i c i d e g l y p h o s a t e (N - p h o s p h o n o m e t h y l g l y c i n e ) . The r e s p o n s i b l e gene i n typhimurium has been i d e n t i f i e d and c l o n e d . Long-range g o a l s of t h i s r e s e a r c h i n c l u d e t h e i n c o r p o r a t i o n and e x p r e s s i o n o f t h i s gene i n t o h i g h e r p l a n t s i n o r d e r t o a c h i e v e g e n e t i c a l l y engineered h e r b i c i d e r e s i s t a n c e i n s e l e c t e d c r o p s ( 1 2 ) . These approaches o f f e r e x c i t i n g o p p o r t u n i t i e s i n the a r e a o f p l a n t g e n e t i c e n g i n e e r i n g . They a l s o p r o v i d e b a s i c i n f o r m a t i o n about p o s s i b l e r e s i s t a n c e mechanisms t h a t may be s e l e c t e d f o r i n h i g h e r p l a n t s y s t e m s . However, because s e l e c t i o n i s c a r r i e d out i n a b a c t e r i a l s y s t e m , t h e e x p r e s s i o n and p o t e n t i a l agronomic v a l u e o f the gene i n a h i g h e r p l a n t cannot be i m m e d i a t e l y a s s e s s e d . T h i s c o u l d be of c o n s i d e r a b l e p r a c t i c a l c o n c e r n . For i n s t a n c e , c e r t a i n m u t a t i o n s (see b e l o w ) , w h i l e c o n f e r r i n g t o l e r a n c e t o a p a r t i c u l a r s t r e s s , may s i g n i f i c a n t l y reduce a p l a n t ' s o v e r a l l v i g o r and f i t n e s s . Such a s i t u a t i o n c o u l d l e a d t o an u n a c c e p t a b l e " t r a d e o f f " between t o l e r a n c e and p r o d u c t i v i t y , w h i c h would o n l y be e v i d e n t a f t e r a s u b s t a n t i a l commitment of time and r e s o u r c e s was made i n o r d e r t o i n c o r p o r a t e and a c h i e v e e x p r e s s i o n of t h e gene i n a h i g h e r p l a n t . In a d d i t i o n , m i c r o b i a l s e l e c t i o n systems cannot be u t i l i z e d f o r h e r b i c i d e s t h a t are p r i m a r i l y a c t i v e i n p h o t o s y n t h e t i c t i s s u e s . A l t e r n a t i v e approaches t o t h e use o f h i g h e r p l a n t c e l l o r b a c t e r i a l c u l t u r e s i n i n v i t r o s e l e c t i o n s are p o s s i b l e t h r o u g h t h e use o f organisms w i t h d i f f e r e n t l i f e c y c l e c h a r a c t e r i s t i c s , such as t h e homosporous f e r n C e r a t o p t e r i s . S t u d i e s w i t h t h i s p l a n t have shown t h a t because i t possesses a f r e e l i v i n g h a p l o i d phase, f e a t u r e s o f both i n v i t r o and whole p l a n t s e l e c t i o n can be combined t o d e s i g n an e f f i c i e n t means o f s e l e c t i n g f o r a wide v a r i e t y o f m u t a t i o n s . In a d d i t i o n , p a r t i c u l a r f e a t u r e s of both t h e gametophyte and s p o r o p h y t e g e n e r a t i o n s p r o v i d e an e x c e p t i o n a l l y u s e f u l system f o r p h y s i o l o g i c a l , b i o c h e m i c a l and g e n e t i c s t u d i e s of s e l e c t e d m u t a t i o n s . The l i f e c y c l e o f C e r a t o p t e r i s c o n s i s t s o f two independent a u t o t r o p h i c p h a s e s , a d i p l o i d sporophyte and a h a p l o i d gametophyte. The d i p l o i d v a s c u l a r sporophyte phase can be r e a d i l y c u l t u r e d i n t h e greenhouse and c o n s i s t s of p l a n t s w i t h a s h o r t u p r i g h t stem, a s s o c i a t e d r o o t s and u p r i g h t l e a v e s r a n g i n g i n l e n g t h from 0 . 2 t o 0 . 5 m. The margins o f the l e a v e s r e g u l a r l y produce s p o r o p h y t i c buds which can be d e t a c h e d , s u r f a c e s t e r i l i z e d and c u l t u r e d under l a b o r a t o r y o r greenhouse c o n d i t i o n s t o produce a d d i t i o n a l p l a n t s . Mature s p o r o p h y t e s c o n t i n u o u s l y produce l a r g e numbers of s i n g l e c e l l e d h a p l o i d spores as d i r e c t p r o d u c t s o f m e i o t i c d i v i s i o n s . Spores can be c o l l e c t e d i n e s s e n t i a l l y u n l i m i t e d numbers and s t o r e d d r y f o r s e v e r a l y e a r s . To e s t a b l i s h gametophyte c u l t u r e s , spores are s u r f a c e s t e r i l i z e d and sown on a g a r - s o l i d i f i e d o r l i q u i d medium c o n t a i n i n g a p p r o p r i a t e macro- and m i c r o n u t r i e n t s ( 1 3 ) . Because t h e gametophyte g e n e r a t i o n i s a u t o t r o p h i c and c o n s i s t s of i n t a c t p l a n t s , no carbon



56


s o u r c e o r growth r e g u l a t o r s are needed i n t h e medium. G e r m i n a t i o n of spores o c c u r s w i t h i n 5 days under c o n t i n u o u s l i g h t at 25 C o r w i t h i n 3 days at 30 C. Sexual development i n m u l t i s p o r e c u l t u r e s i s moderated by a p h e r o m o n e - l i k e system i n v o l v i n g the growth r e g u l a t o r , antheridiogen (14). A l l gametophytes i n a c u l t u r e have t h e c a p a c i t y t o d e v e l o p i n t o b i s e x u a l p l a n t s t h a t can s e l f f e r t i l i z e when water i s applied to t h e i r surface to l i b e r a t e m o t i l e spermatozoids. These gametophytes, w h i c h a r e e s s e n t i a l l y two d i m e n s i o n a l and h e a r t - s h a p e d , reach s e x u a l m a t u r i t y w i t h i n 10-14 days from s o w i n g , at w h i c h time t h e y are c a . 1.0 mm? . A l t h o u g h gametophyte development proceeds n o r m a l l y i n t h e absence o f exogenously s u p p l i e d growth r e g u l a t o r s , t h e a d d i t i o n o f growth r e g u l a t o r s t o t h e medium can d r a m a t i c a l l y a l t e r development ( 1 5 ) . F o r i n s t a n c e , t h e a d d i t i o n of a b s c i s i c a c i d t o t h e medium a l t e r s s e x u a l d i f f e r e n t i a t i o n and s i g n i f i c a n t l y r e t a r d s gametophyte growth ( 1 6 ) . These responses have been used as t h e b a s i s t o s e l e c t f o r a b s c i s i c a c i d - r e s i s t a n t mutants i n C e r a t o p t e r i s ( 1 7 , 1 8 ) . Because gametophytes are haploid and bisexual, self f e r t i l i z a t i o n r e s u l t s i n t h e p r o d u c t i o n o f c o m p l e t e l y homozygous sporophytes. In a d d i t i o n , gametophytes can be c r o s s e d r e a d i l y t o produce defined h y b r i d combinations i n F l sporophytes (18). Homozygous or h y b r i d sporophytes can be c u l t u r e d and v e g e t a t i v e l y c l o n e d t o p r o v i d e an u n l i m i t e d s u p p l y o f g e n e t i c a l l y d e f i n e d d i p l o i d v e g e t a t i v e m a t e r i a l and h a p l o i d s p o r e s . The e n t i r e l i f e c y c l e (spore t o s p o r e ) can be completed i n l e s s than 120 days under l a b o r a t o r y and greenhouse c o n d i t i o n s . These f e a t u r e s of t h e C e r a t o p t e r i s l i f e c y c l e f a c i l i t a t e g e n e t i c s t u d i e s and p r o v i d e t h e means of d e s i g n i n g an e f f i c i e n t and r a p i d s e l e c t i o n system f o r t h e i n d u c t i o n and r e c o v e r y of s p e c i f i c m u t a t i o n s . H a p l o i d F e r n S e l e c t i o n System A s e l e c t i o n system u s i n g C e r a t o p t e r i s and c a p i t a l i z i n g on many o f t h e a t t r i b u t e s o f the l i f e c y c l e d i s c u s s e d above has been developed f o r t h e purpose of r e c o v e r i n g m u t a t i o n s a f f e c t i n g c e r t a i n developmental processes or c o n f e r r i n g t o l e r a n c e t o s p e c i f i c s e l e c t i o n agents (17,18). F i g u r e 1 i l l u s t r a t e s t h e d e s i g n of t h e s c r e e n . Singlec e l l e d h a p l o i d spores a r e mutagenized by exposure t o X - i r r a d i a t i o n and sown a x e n i c a l l y on a g a r - s o l i d i f i e d m i n e r a l n u t r i e n t medium c o n t a i n i n g the s e l e c t i o n agent. Because o f t h e i r s m a l l s i z e , l a r g e numbers of spores ( c a . 12,500) can be sown i n a s i n g l e 100 mm p e t r i d i s h , a l l o w i n g as many as 1.5 χ 1 0 i n d i v i d u a l s t o be screened i n an a r e a of c a . 1.0 m . W i l d - t y p e s p o r e s sown on medium c o n t a i n i n g an a p p r o p r i a t e c o n c e n t r a t i o n o f a s e l e c t i o n agent ( e . g . a h e r b i c i d e o r growth r e g u l a t o r ) e i t h e r f a i l t o germinate or produce s m a l l abnormal gametophytes t h a t f r e q u e n t l y d i e w i t h i n a s h o r t p e r i o d o f t i m e . Any gametophytes c o n t a i n i n g m u t a t i o n s t h a t c o n f e r r e s i s t a n c e o r t o l e r a n c e t o t h e s e l e c t i o n agent d e v e l o p n o r m a l l y and are e v i d e n t as m a c r o s c o p i c green p l a n t s ( c a . 1-2 mm diam.) u s u a l l y w i t h i n 21 days from s o w i n g . These p u t a t i v e mutants can be s c o r e d v i s u a l l y and then t r a n s f e r r e d t o i n d i v i d u a l c u l t u r e d i s h e s and s e l f f e r t i l i z e d . The r e s u l t i n g sporophytes are homozygous f o r the s e l e c t e d m u t a t i o n and can be c u l t u r e d t o produce u n l i m i t e d q u a n t i t i e s of g e n e t i c a l l y u n i f o r m M2 spores. To i l l u s t r a t e t h e u t i l i t y o f t h i s s e l e c t i o n system and i t s a p p l i c a b i l i t y t o s t u d i e s o r i e n t e d toward t h e g e n e t i c improvement o f 6

2


HIC ΚΟΚ

Applications of In Vitro Selection Systems

Selection medium Irradiate 2 - 4 weeks


Sow

Resistant gametophyte

Haploid spores

Isolate

• Confirmation of resistance in gametophytes and /or sporophytes • Crosses between mutant and wild type gametophytes • Biochemical /physiological studies of gametophytes and/or sporophytes

Mature sporophyte

Homozygous sporophyte

F i g u r e 1. The C e r a t o p t e r i s h a p l o i d s e l e c t i o n s y s t e m . M e a s u r e m e n t s o f g a m e t o p h y t e a r e a s w e r e made w i t h B l o q u a n t i m a g e a n a l y s i s s y s t e m a t 18 d a y s f o l l o w i n g s o w i n g . Data are r e p r e s e n t e d as t h e p e r c e n t o f t h e s i z e o f each s t r a i n on t h e p l a i n medium c o n t r o l .


58


c r o p s , two examples of s u c c e s s f u l s e l e c t i o n s f o r h e r b i c i d e are d i s s c u s s e d below.


S e l e c t i o n f o r Herbicide Tolerance

tolerance

in Ceratopteris

Paraquat T o l e r a n c e . Mutants of C e r a t o p t e r i s t o l e r a n t t o t h e nonselective h e r b i c i d e paraquat (l,r-dimethyl-4,4'-bipyridinium d i c h l o r i d e ) were o b t a i n e d from i n v i t r o s e l e c t i o n s i n t h e gametophyte g e n e r a t i o n u s i n g 0 . 5 yM paraquat as t h e s e l e c t i v e agent ( 1 9 ) . One hundred and t h i r t y - e i g h t p u t a t i v e mutants were r e c o v e r e d from t h e s e l e c t i o n , a t a f r e q u e n c y o f c a . 2 x 1 0 - 5 . To d a t e , 19 o f the s e l e c t i o n s have been t e s t e d f o r t o l e r a n c e i n t h e next (M2) g e n e r a t i o n and 17 were c o n f i r m e d as s t a b l e m u t a n t s . Two o f the s e l e c t i o n s were c h a r a c t e r i z e d e x t e n s i v e l y by comparative s t u d i e s w i t h t h e w i l d - t y p e . T o l e r a n c e was assayed i n both the gametophyte and s p o r o p h y t e generations by m o n i t o r i n g c h l o r o p h y l l r e t e n t i o n i n response to exposure t o p a r a q u a t . In a d d i t i o n , t o l e r a n c e was assayed i n t h e gametophyte g e n e r a t i o n by a s s e s s i n g t h e e f f e c t o f paraquat on g r o w t h . T o l e r a n c e t o paraquat was e v i d e n t i n both gametophytes and sporophytes and t h e two mutants e x h i b i t e d i n c r e a s e d t o l e r a n c e t o paraquat o f 1 0 t o 5 0 - f o l d over t h e w i l d - t y p e . A l t h o u g h t h e two mutants were g e n e r a l l y s i m i l a r i n t h e i r l e v e l of t o l e r a n c e , i t was p o s s i b l e t o d i s t i n g u i s h between them on t h e b a s i s of s l i g h t d i f f e r e n c e s i n c h l o r o p h y l l l o s s i n both gametophytes and sporophytes and by comparison of growth i n h i b i t i o n i n response t o i n c r e a s i n g c o n c e n t r a t i o n s o f p a r a q u a t . Subsequent s t u d i e s (19,20) have shown t h a t t h e two mutants are i n h e r i t e d as s i n g l e gene n u c l e a r m u t a t i o n s which are r e c e s s i v e i n heterozygous diploid sporophytes. In addition, tests for complementation i n t h e s p o r o p h y t e g e n e r a t i o n have shown t h a t t h e two mutants do not complement each o t h e r and a r e , t h e r e f o r e , f u n c t i o n a l l y allelic. E x t e n s i v e t e s t s f o r recombinants i n which c a . 1.25 χ 10° gametophytes were m o n i t o r e d have not d e t e c t e d any r e c o m b i n a t i o n between t h e two m u t a t i o n s . S t u d i e s are c o n t i n u i n g t o f u r t h e r d e f i n e t h e g e n e t i c b a s i s of t h e m u t a n t s . F o r i n s t a n c e , spore p o p u l a t i o n s d e r i v e d from one o f the mutant s t r a i n s have been mutagenized and s u b j e c t e d t o s e l e c t i o n c o n d i t i o n s of 2 . 0 y M , which i s f o u r times t h e c o n c e n t r a t i o n used i n t h e i n i t i a l s e l e c t i o n p r o t o c o l . A t o t a l of s i x h i g h l y t o l e r a n t p u t a t i v e mutants have been r e c o v e r e d from t h i s s e l e c t i o n and are b e i n g s t u d i e d f u r t h e r ( 2 0 ) . The r e c o v e r y and c h a r a c t e r i z a t i o n s of t h e two paraquat t o l e r a n t mutants i l l u s t r a t e some of t h e advantages of the Ceratopteris s e l e c t i o n s y s t e m . Because a l a r g e number of spores can be s c r e e n e d , a l a r g e number o f p u t a t i v e mutants can be r e c o v e r e d . Thus, i t i s p o s s i b l e t o be h i g h l y s e l e c t i v e i n c h o o s i n g w h i c h i n d i v i d u a l s t o characterize further. F o r example, i n t h e paraquat s t u d i e s , i n d i v i d u a l s w i t h m o r p h o l o g i c a l a b n o r m a l i t i e s or reduced v i g o r i n t h e sporophyte g e n e r a t i o n were not i n v e s t i g a t e d i n i t i a l l y . The two i n d i v i d u a l s t h a t have been f u l l y c h a r a c t e r i z e d (see above) were chosen because of t h e i r s l i g h t l y d i f f e r e n t responses t o paraquat i n i n i t i a l growth t e s t s and t h e p o s s i b i l i t y t h a t t h e y r e p r e s e n t e d different mutations. Because gametophytes are h a p l o i d , i t was p o s s i b l e t o i s o l a t e r e c e s s i v e m u t a n t s . I t i s i n t e r e s t i n g t o note t h a t a l t h o u g h c o n s i d e r a b l e s u c c e s s was r e a l i z e d by u s i n g a b a s i c d i p l o i d (n=x=39) s t o c k of C e r a t o p t e r i s , attempts t o s e l e c t paraquat t o l e r a n t mutants from an e s t a b l i s h e d p o l y p o i d ( a m p h i d i p l o i d ) s t r a i n w i t h n=2x=78 were



4.

HIC κοκ


59

u n s u c c e s s f u l , even though repeated attempts were made ( 2 0 ) . The f a i l u r e t o r e c o v e r mutants i n t h e s e attempts may have been r e l a t e d t o the i n a b i l i t y t o e f f e c t i v e l y s e l e c t f o r r e c e s s i v e mutants i n a gametophyte phase t h a t was not t r u l y h a p l o i d f o r t h e l o c u s i n q u e s t i o n . S i m i l a r d i f f i c u l t i e s may a r i s e i n o t h e r n o n - h a p l o i d systems such as d i p l o i d c e l l o r p r o t o p l a s t c u l t u r e s and h a p l o i d c u l t u r e s t h a t a r e d e r i v e d from a m p h i d i p l o i d s , such as N i c o t i a n a tabacum. It is also i m p o r t a n t t o note t h a t t o l e r a n t sporophytes were s u c c e s s f u l l y r e c o v e r e d by s e l e c t i n g f o r t o l e r a n c e i n t h e gametophyte g e n e r a t i o n . P a r a q u a t ' s p r i m a r y mode of h e r b i c i d a l a c t i v i t y i s a s s o c i a t e d w i t h f r e e r a d i c a l p r o d u c t i o n t h a t i s d r i v e n by e l e c t r o n s d e r i v e d from photosystem I ( 2 1 ) . T h u s , i f t h e t o l e r a n c e mechanism i s not s p e c i f i c t o e i t h e r sporophytes o r gametophytes, one would expect both phases t o respond i n a s i m i l a r manner, s i n c e both are p h o t o s y n t h e t i c . This s i t u a t i o n c o n t r a s t s w i t h t h e apparent d i s p a r i t y between paraquat tolerance in selected calli and i n s u b s e q u e n t l y regenerated sporophytes i n s e l e c t i o n s from tobacco and tomato c u l t u r e s ( 2 2 , 2 3 ) . In both of t h e s e s t u d i e s , t o l e r a n c e was g e n e r a l l y not e v i d e n t i n p l a n t s r e g e n e r a t e d from s e l e c t e d c a l l i , a l t h o u g h c a l l i d e r i v e d s u b s e q u e n t l y from t h e r e g e n e r a t e d p l a n t s were a g a i n t o l e r a n t t o m o d e r a t e l y h i g h l e v e l s of p a r a q u a t . I t i s apparent t h a t t h e s e l e c t e d c a l l i , which were n o n p h o t o s y n t h e t i c and s e l e c t e d i n t h e dark i n t h e tomato s t u d y , d i d not possess m u t a t i o n s t h a t were e f f e c t i v e a g a i n s t p a r a q u a t ' s p r i m a r y mode of h e r b i c i d a l a c t i v i t y i n p h o t o s y n t h e t i c t i s s u e . A p o s s i b l e e x p l a n a t i o n of t h i s b e h a v i o r i s t h a t the c a l l u s or c e l l c u l t u r e s were s u b j e c t e d t o h i g h c o n c e n t r a t i o n s of paraquat w h i c h , because the t i s s u e was m i n i m a l l y or not at a l l p h o t o s y n t h e t i c , were o n l y m o d e r a t e l y t o x i c . Paraquat has been r e p o r t e d t o e x h i b i t moderate t o x i c i t y t o c u l t u r e s grown i n the dark o r t h a t are n o n p h o t o s y n t h e t i c (24). As a consequence of t h i s , when sporophytes were r e g e n e r a t e d from t h i s c a l l u s and t e s t e d f o r t o l e r a n c e , t h e i r p h o t o s y n t h e t i c a c t i v i t y may have i n c r e a s e d t h e i r s u s c e p t i b i l i t y t o such an e x t e n t t h a t any t o l e r a n c e s e l e c t e d f o r i n t h e c e l l c u l t u r e s would be e f f e c t i v e l y masked by t h e i n c r e a s e d t o x i c i t y of paraquat i n green t i s s u e . As s u c h , i t would be expected t h a t o n l y m u t a t i o n s c o n f e r r i n g h i g h l e v e l s of t o l e r a n c e would be d e t e c t e d by such a s e l e c t i o n system and/or t h a t t o l e r a n c e expressed i n both c a l l u s and regenerated sporophytes would i n v o l v e a l t e r e d uptake o r t r a n s p o r t o f p a r a q u a t . In a d d i t i o n , because t h e s e s e l e c t i o n s were c a r r i e d out i n d i p l o i d s y s t e m s , one would expect the recovery o f o n l y dominant o r semidominant m u t a t i o n s , which i s a p p a r e n t l y t h e case i n t h e tomato example ( 2 3 ) . Because t h e mechanism o f a c t i o n o f paraquat i n qreen t i s s u e i s r e a s o n a b l y w e l l known and c e r t a i n p r o t e c t i v e enzymes (e.g.superoxide dismutase (SOD), p e r o x i d a s e , c a t a l a s e ) are p o s s i b l y a s s o c i a t e d w i t h t o l e r a n c e t o f r e e r a d i c a l s generated by t h e h e r b i c i d e ' s a c t i v i t y ( 2 5 2 7 ) , s t u d i e s are b e i n g pursued t o determine i f any d i f f e r e n c e s i n t h e a c t i v i t y of these enzymes are e v i d e n t between t h e w i l d - t y p e and mutant s t r a i n s of C e r a t o p t e r i s . I n i t i a l s t u d i e s have shown no d i f f e r e n c e s i n e i t h e r c o n s t i t u t i v e l e v e l s of SOD, c a t a l a s e or p e r o x i d a s e , o r i n t h e uptake o f l a b e l e d paraquat by whole gametophytes ( 2 8 ) . This c o n t r a s t s w i t h s t u d i e s of Harper and Harvey (29) i n which e l e v a t e d l e v e l s of SOD, c a t a l a s e and p e r o x i d a s e were d e t e c t e d i n paraquat t o l e r a n t b i o t y p e s of Loiiurn perenne. However, r e c e n t s t u d i e s by


60



F u e r s t et a l . (30) have a t t r i b u t e d t o l e r a n c e i n b i o t y p e s of Conyza t o e x c l u s i o n of paraquat from i t s p r i m a r y s i t e of a c t i o n i n the chloroplast and not t o any documented d i f f e r e n c e s in enzyme a c t i v i t i e s . With t h e e x c e p t i o n o f the C e r a t o p t e r i s examples, no w e l l documented s i n g l e gene m u t a t i o n s c o n f e r r i n g paraquat t o l e r a n c e i n whole p l a n t s have been r e p o r t e d . The C e r a t o p t e r i s mutants p r o v i d e a w e l l d e f i n e d system w i t h which t o pursue f u r t h e r s t u d i e s of t h e i r g e n e t i c b a s i s and o f the mechanism o f o f t h e i r t o l e r a n c e t o p a r a q u a t . Glyphosate Tolerance. S t u d i e s have a l s o been c a r r i e d out w i t h t h e broad spectrum h e r b i c i d e g l y p h o s a t e (N-phosphonomethylglycine). S e l e c t i o n s f o r t o l e r a n c e t o g l y p h o s a t e were conducted u s i n g a c o n c e n t r a t i o n o f 0 . 1 mM g l y p h o s a t e as t h e s e l e c t i o n a g e n t . Spores were mutagenized and sown on t h e s e l e c t i v e medium and a t o t a l of c a . 450 putative glyphosate-tolerant mutants were recovered at a frequency of c a . 1 x 1 0 - 4 . P r e l i m i n a r y s t u d i e s of t h e s e s e l e c t i o n s have been c a r r i e d out and a r e p r e s e n t e d h e r e . A t o t a l of 131 p u t a t i v e mutants have been t e s t e d i n t h e M2 gametophyte g e n e r a t i o n and o f these 128 were t o l e r a n t t o g l y p h o s a t e . Maintenance o f t o l e r a n c e through a complete s e x u a l c y c l e i m p l i e s a genetic basis to the c o n d i t i o n . Two o f these mutants are f u r t h e r c h a r a c t e r i z e d here by comparison w i t h t h e w i l d - t y p e response o f gametophytes t o g l y p h o s a t e - i n d u c e d growth i n h i b i t i o n . F i g u r e s 2a and 2b i l l u s t r a t e dose-response d a t a f o r t h e two mutants (G363 and G492) and t h e w i l d - t y p e . Both mutants e x h i b i t s i g n i f i c a n t l e v e l s of t o l e r a n c e t o g l y p h o s a t e when compared w i t h t h e w i l d t y p e . F i g u r e 2a r e p r e s e n t s the d a t a as a p e r c e n t of the growth o f each s t r a i n on p l a i n medium. Both mutants and t h e w i l d - t y p e are c l e a r l y d i s t i n g u i s h e d . F o r i n s t a n c e , at 60% growth i n h i b i t i o n , s t r a i n s G363 and G492 e x h i b i t 5and 1 5 - f o l d increases in tolerance over the wild-type, respectively. F i g u r e 2b i l l u s t r a t e s dose-response d a t a as a b s o l u t e g r o w t h , e x p r e s s e d i n mm . A g a i n , d i f f e r e n c e s are c l e a r l y e v i d e n t between t h e w i l d type and t h e two mutant s t r a i n s . I t i s important t o note t h a t a l t h o u g h growth of t h e two mutant s t r a i n s i s s u p e r i o r t o t h e w i l d - t y p e at g l y p h o s a t e c o n c e n t r a t i o n s g r e a t e r than 0.03 mM, growth of t h e mutants at lower c o n c e n t r a t i o n s and on p l a i n medium i s l e s s than t h e w i l d - t y p e . T h u s , i t appears t h a t the m u t a t i o n s c o n f e r r i n g glyphosate t o l e r a n c e s i g n i f i c a n t l y a l t e r the growth p o t e n t i a l of the s e l e c t e d s t r a i n s on low c o n c e n t r a t i o n s of o r i n t h e absence of g l y p h o s a t e . G l y p h o s a t e t o l e r a n t mutants have a l s o been o b t a i n e d i n t h e b a c t e r i a S a l m o n e l l a typhimurium Q 2 ) and A e r o b a c t e r aerogenes ( 1 0 ) . In c e l l c u l t u r e s of c a r r o t , s t a b l e g l y p h o s a t e - t o l e r a n t l i n e s have been o b t a i n e d by l o n g term c u l t u r e on medium c o n t a i n i n g p r o g r e s s i v e l y h i g h e r c o n c e n t r a t i o n s of t h e h e r b i c i d e ( 3 1 ) . G l y p h o s a t e has been shown t o be a p o t e n t i n h i b i t o r of t h e s h i k i m a t e pathway enzyme 5 e n o l p y r u v y l s h i k i m a t e - 3 - p h o s p h a t e s y n t h a s e (EPSP; EC 2 . 4 . 1 . 1 9 ) i n S . typhimurium (11) and A . aerogenes and t h e h i g h e r p l a n t C o r y d a l i s sempervirens ( 3 2 ) . C e l l c u l t u r e s of C . sempervirens and A . aerogenes t h a t were exposed t o i n c r e a s i n g c o n c e n t r a t i o n s of g l y p h o s a t e o v e r t i m e , became adapted t o g l y p h o s a t e at c o n c e n t r a t i o n s up t o 10 mM ( 3 2 ) . These adapted c e l l s e x h i b i t e d a 10 t o 3 0 - f o l d i n c r e a s e i n EPSP activity. S i m i l a r r e s u l t s were r e p o r t e d i n the s t a b l e c a r r o t c e l l l i n e s (31). In t h e c a r r o t l i n e s , i t was noted t h a t growth of t h e g l y p h o s a t e - t o l e r a n t c e l l s i n medium l a c k i n g g l y p h o s a t e was much s l o w e r 2



HIC ΚΟΚ


F i g u r e 2 ( A ) . E f f e c t s o f g l y p h o s a t e on growth i n w i l d type ( and mutant (G363 Δ , G492 • ) gametophytes o f C e r a t o p t e r i s .


62


Β


5rr

mM GLYPHOSATE

F i g u r e 2 ( B ) . E f f e c t s o f g l y p h o s a t e on growth i n w i l d type ( θ ) and mutant (G363 Δ G492 • ) gametophytes o f C e r a t o p t e r i s . Measurements o f gametophyte areas were made w i t h B l o q u a n t image a n a l y s i s system a t 18 days f o l l o w i n g sowing. Data a r e shown as the a c t u a l a r e a s o f gametophytes. Each p o i n t r e p r e s e n t s the average o f measurements from 2 0 i n d i v i d u a l s ( ±S.D. i n B ) . f



4.

HIC ΚΟΚ


63

than nonadapted ( s e l e c t e d ) c e l l s . T h i s may be an e f f e c t s i m i l a r t o t h e one documented i n t h e two C e r a t o p t e r i s m u t a n t s . S t u d i e s of g l y p h o s a t e - t o l e r a n t s t r a i n s have shown t h a t EPSP a c t i v i t y i s encoded by t h e aroA l o c u s of S . typhimurium ( 1 2 ) . F u r t h e r s t u d i e s have c l o n e d a g l y p h o s a t e - t o l e r a n t aroA mutant a l l e l e i n t o E . coli and shown t h a t t h e m u t a t i o n a c t s as a dominant gene i n merodiploids. M o l e c u l a r s t u d i e s have shown t h a t t h e mutant a l l e l e c o n t a i n s a s i n g l e b a s e - p a i r change r e s u l t i n g i n a s t r u c t u r a l a l t e r a t i o n o f EPSP, as w e l l as a m u t a t i o n r e s i d i n g i n t h e promoter r e g i o n of t h e operon (_11). T o l e r a n c e i n S^. typhimurium appears t o be a s s o c i a t e d w i t h an i n c r e a s e d a f f i n i t y o f EPSP f o r i t s s u b s t r a t e s , phosphoenolpyruvate and s h i k i m a t e - 3 - p h o s p h a t e ( 1 1 ) . S t u d i e s by S c h u l z et a l . (10) have shown t h a t a g l y p h o s a t e - i n s e n s i t i v e s t r a i n of A . aerogenes c o n t a i n s a g l y p h o s a t e - i n s e n s i t i v e EPSP. G e n e t i c c h a r a c t e r i z a t i o n s of the C e r a t o p t e r i s glyphosatet o l e r a n t mutants have not been c o m p l e t e d . From t h e i n i t i a l dose response d a t a , i t appears t h a t at l e a s t two m u t a t i o n s c o n f e r r i n g d i f f e r e n t l e v e l s o f g l y p h o s a t e t o l e r a n c e have been r e c o v e r e d . F u r t h e r s t u d i e s are needed t o f u l l y c h a r a c t e r i z e t h e e x p r e s s i o n and secondary e f f e c t s of t h e m u t a t i o n s i n both t h e gametophyte and sporophyte g e n e r a t i o n s and t o i n v e s t i g a t e t h e b i o c h e m i c a l b a s i s o f the t r a i t . Conclusions The above examples of the use o f the C e r a t o p t e r i s h a p l o i d s e l e c t i o n system s e r v e t o i l l u s t r a t e i t s u t i l i t y f o r t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f s i n g l e gene m u t a t i o n s f o r h e r b i c i d e t o l e r a n c e . U s i n g t h i s s y s t e m , i t i s p o s s i b l e t o i n i t i a t e s e l e c t i o n s and determine i f p u t a t i v e mutants have a s t a b l e g e n e t i c b a s i s w i t h i n a p e r i o d o f l e s s than f o u r months. D u r i n g t h i s t i m e l a r g e numbers of p u t a t i v e mutants can be generated and i n i t i a l l y c h a r a c t e r i z e d t o i d e n t i f y p o t e n t i a l l y d i f f e r e n t mutations that confer various levels of tolerance. As s u c h , t h e system i s v a l u a b l e f o r thoroughly i n v e s t i g a t i n g the g e n e t i c b a s i s of s e l e c t e d m u t a t i o n s . In a d d i t i o n , because of t h e u t i l i t y o f the gametophyte g e n e r a t i o n f o r c o n d u c t i n g growth s t u d i e s under c o n t r o l l e d c o n d i t i o n s , i t i s p o s s i b l e t o r a p i d l y a s s e s s t h e e f f e c t s of p a r t i c u l a r r e s i s t a n c e t r a i t s on t h e growth and development o f t h e o r g a n i s m . These s t u d i e s can be s u b s e q u e n t l y extended t o t h e s p o r o p h y t e g e n e r a t i o n and conducted under greenhouse conditions. T h i s c a p a b i l i t y can be e s p e c i a l l y i m p o r t a n t when s e l e c t i o n s are b e i n g c a r r i e d out t o i d e n t i f y m u t a t i o n s t h a t might be o f p a r t i c u l a r use i n crop improvement. For instance, p r e l i m i n a r y s t u d i e s w i t h t h e g l y p h o s a t e - t o l e r a n t mutants d e s c r i b e d above i n d i c a t e t h a t t h e m u t a t i o n s c o n f e r r i n g t o l e r a n c e may a l s o suppress gametophyte g r o w t h . I f a s i m i l a r e f f e c t i s observed i n t h e s p o r o p h y t e g e n e r a t i o n , i t would appear t h a t t o l e r a n c e a s s o c i a t e d w i t h t h e s e p a r t i c u l a r m u t a t i o n s i s not d e s i r a b l e from a g e n e t i c e n g i n e e r i n g p e r s p e c t i v e . The l o s s of growth p o t e n t i a l i n t h e g l y p h o s a t e - t o l e r a n t c a r r o t c e l l l i n e s (31) may i n d i c a t e a s i m i l a r s i t u a t i o n . S t r a i n s o f S . t y p h i m u r i u m and E. c o l i . t h a t c o n t a i n a g l y p h o s a t e - t o l e r a n t aroA a l l e l e e x h i b i t a 15 p e r c e n t lower growth r a t e on minimal medium than i s o g e n i c s t r a i n s c o n t a i n i n g e i t h e r a w i l d - t y p e a l l e l e or both w i l d - t y p e and mutant a l l e l e s (12). In c o n t r a s t , t o l e r a n c e i n t h e A . aerogenes mutant d i d not e f f e c t growth p o t e n t i a l , as expressed by the mean d o u b l i n g t i m e , i n comparison w i t h w i l d - t y p e c u l t u r e s ( 1 0 ) . I t i s , of c o u r s e ,



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i m p o s s i b l e t o assess t h e p r o b a b l e e f f e c t s o f such genes i n a multicellular green plant until after their i n c o r p o r a t i o n and expression. Other attempts t o employ homosporous f e r n s f o r t h e r e c o v e r y o f s p e c i f i c m u t a t i o n s have been r e p o r t e d . C a r l s o n (33) used t h e f e r n s Osmunda cinnamomea and Todea b a r b a r a i n a s t u d y i n v o l v i n g i n d i r e c t s e l e c t i o n f o r a u x o t r o p h i c gametophyte m u t a n t s . Howl and and Boyd (34) s e l e c t e d f o r a l t e r e d photomorphogenic responses t o r e d l i g h t Τ η gametophytes o f P t e r i d i u m a q u i l i n u m . Both o f t h e s e s t u d i e s , however, were l i m i t e d by t h e i n a b i l i t y t o c u l t u r e t h e s e l e c t i o n s through an e n t i r e s e x u a l c y c l e and were t h e r e f o r e unable t o c o n f i r m a g e n e t i c b a s i s f o r t h e p u t a t i v e mutants t h a t were r e c o v e r e d . Few g e n e t i c s t u d i e s have used f e r n s as e x p e r i m e n t a l o r g a n i s m s . T h i s may be a s s o c i a t e d w i t h t h e w i d e l y h e l d view t h a t a l l homosporous f e r n s a r e d e r i v e d from p o l y p l o i d s ( i . e . p a l e o p o l y p l o i d s , see 3 5 ) , c o n t a i n numerous d u p l i c a t e d l o c i and are g e n e t i c a l l y complex ( 3 6 ) . However, e x p e r i e n c e w i t h s e l e c t i o n s u s i n g C e r a t o p t e r i s , a l o n g w i t h d a t a from r e c e n t s t u d i e s o f isozyme v a r i a t i o n w i t h i n o t h e r f e r n s (37,38) show t h a t e x t a n t d i p l o i d f e r n s g e n e r a l l y behave as g e n e t i c d i p l o i d s and n o t as p o l y p l o i d s . Therefore, ferns w i t h a p p r o p r i a t e l i f e c y c l e f e a t u r e s , such as t h o s e i l l u s t r a t e d by C e r a t o p t e r i s , c a n be o f c o n s i d e r a b l e use e x p e r i m e n t a l l y . In a d d i t i o n t o s e l e c t i o n s f o r h e r b i c i d e t o l e r a n t genotypes w i t h C e r a t o p t e r i s , t h e s e l e c t i o n system has a l s o been u t i l i z e d t o s e l e c t f o r a wide v a r i e t y o f o t h e r t r a i t s , i n c l u d i n g i n s e n s i t i v i t y t o t h e p l a n t hormone a b s c i s i c a c i d and t o l e r a n c e t o sodium c h l o r i d e , L - a z e t i d i n e - 2 c a r b o x y l a t e , h y d r o x y p r o l i n e , 5 - f l u r o d e o x y u r i d i n e and 2 - a m i n o e t h y l - L c y s t e i n e . The c o n t i n u e d use o f t h i s system can p r o v i d e many a d d i t i o n a l o p p o r t u n i t i e s f o r t h e s e l e c t i o n and r e c o v e r y o f a v a r i e t y o f m u t a t i o n s for research a p p l i c a t i o n s i n plant molecular biology, herbicide c h e m i s t r y and g e n e t i c e n g i n e e r i n g . As i n any s y s t e m , t h e r e a r e l i m i t a t i o n s - f o r o n e , C e r a t o p t e r i s has n o t been e s t a b l i s h e d as and d o e s n ' t appear t o have much promise as a major a g r i c u l t u r a l c r o p ! However, t h e s u c c e s s t o d a t e w i t h a wide v a r i e t y o f s e l e c t i o n a g e n t s , a l o n g w i t h t h e g e n e r a l u t i l i t y o f t h e system as i l l u s t r a t e d w i t h t h e above examples, i n d i c a t e t h a t t h e system p r o v i d e s c e r t a i n e x p e r i m e n t a l advantages f o r i n v i t r o m u t a t i o n s e l e c t i o n and c h a r a c t e r i z a t i o n . The c o n t i n u e d development and a p p l i c a t i o n o f m o l e c u l a r t e c h n i q u e s can a l l o w t h e p r a c t i c a l u t i l i z a t i o n o f s e l e c t e d m u t a t i o n s from t h i s and o t h e r s e l e c t i o n systems i n both b a s i c and a p p l i e d s t u d i e s . Acknowledgments I thank my c o l l e a g u e s O t t o Schwarz, L i z a C a r r o l l and Thomas Warne f o r a d v i c e and encouragement and Gary H u n t z i n g e r and J e n n i f e r P a n t e r f o r technical assistance. F i n a n c i a l s u p p o r t from M a r t i n M a r i e t t a C o r p . , N P I , N a t i o n a l S c i e n c e F o u n d a t i o n G r a n t DCB-85-11273 and The O f f i c e o f Research and Graduate S t u d i e s a t The U n i v e r s i t y o f Tennessee i s g r a t e f u l l y acknowledged.

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RECEIVED June11,1986


Applications of In Vitro Selection Systems - American Chemical Society

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