Chapter 3 In Vitro Plant Cultures for Herbicide Prescreening
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J. Gressel Department of Plant Genetics, The Weizmann Institute of Science, Rehovot, IL-76100, Israel
Cell cultures are ideal axenic physiological systems to study herbicide action without problems of cuticular transfer or complications of translocation. Still, not all metabolic systems function in all cells at all times in the cell cycle. Inhibitors of photosynthesis are often inactive in non green cells, and root-active herbicides may be degraded in green cells. Nutritional components in the medium may interfere with herbicide action. The criteria needed to develop universal pre-screens which show activity for all herbicides are discussed and evaluated. It is far easier to develop "dedicated" pre-screens which are used to measure single types of activity or single chemical types of herbicide. All results with in vitro systems should be validated with whole plants.
T h e major part of this chapter ostensibly discusses the use of in vitro systems for herbicide screening. T h e importance of this discussion exceeds t h a t of screening, as the basic question addressed is the a p p l i c a b i l i t y of in vitro systems for use i n a l l herbicide research. If in vitro systems do n o t parallel the plant response, there are great l i m i t a t i o n s t o their use i n metabolic studies i n selecting for herbicide resistant crops, herbicide physiology studies, as well as screening. T h i s chapter discusses advantages a n d l i m i t a t i o n s , successes a n d failures of using in vitro c u l t i v a t e d callus, suspension culture a n d in vitro m i n i plant systems for universal a n d dedicated pre-screens. V a r i o u s aspects of such systems have recently been reviewed {1-3). A t least one commercial l a b o r a t o r y offers a n in vitro pre-screen system (4). The Need. Innovative pesticide producers t y p i c a l l y screen five t o t w e n t y t h o u s a n d chemicals per year per c o m p a n y f o r h e r b i c i d a l a c t i v i t y . S u c h " p r i m a r y " screens usually contain 5 t o 10 plant species treated b o t h pre a n d post emergence at one t o three rates. T h e last published cost analysis for such a screen w a s 13 years ago a n d w a s 0097-6156/87/0334-0041 $06.00/0 © 1987 American Chemical Society
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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
$200 per c o m p o u n d (5). Less t h a n 5 % of the compounds tested are p r o m o t e d to the next level of screening. T h e greenhouse space a n d the 4-8 weeks needed to o b t a i n answers clearly call for quicker, cheaper pre-screens w h i c h can eliminate inactive compounds. P r e screens allow for synthesis of m i l l i g r a m s instead of the grams of each analog to be screened. A n in vitro pre-screen costing a t e n t h the cost of greenhouse screen need eliminate o n l y 1 0 % of the inactive compounds to break-even financially. U s u a l l y 4 0 - 6 0 % of the inactive compounds are eliminated b y in vitro pre-screening. In one series of thiocarbamates reported, o n l y 3 5 % of the whole p l a n t inactive compounds c o u l d be eliminated w h e n a 6 7 % i n h i b i t i o n criterion was usedfS). T h e most i m p o r t a n t criterion for a universal in vitro herbicide prescreen is t h a t a n active herbicide not be missed. T h i s is not to say t h a t whole plant screens have not missed active herbicides. C o m p o u n d s have been tested a n d rejected as inactive t h a t competitors later f o u n d a n d developed i n t o major herbicides. Some i n d u s t r i a l whole-plant p r i m a r y screens were of too short d u r a t i o n to detect a n d assess the potential of herbicides such as glyphosate. A s i n d u s t r i a l chemists are intent i n discovering " n e w - c h e m i s t r y " i.e. new leads i n t o novel herbicide chemistry - the imperative operational c r i t e r i o n s h o u l d be restated as: an in vitro pre-screen must " n o t miss new chemical leads". T h e m a i n thesis of this review is to show t h a t in vitro systems are a c t u a l l y better able to meet this modified criterion t h a n whole p l a n t systems. Advantages of in vitro systems. C o n s i d e r i n g that a pre-screen is used to elucidate phytotoxic or phytostatic compounds w h i c h are potential herbicides, they have the following advantages: 1. C e l l cultures are axenic; they lack microorganisms of soil a n d leaf surface w h i c h might biodegrade a phytotoxic c o m p o u n d . T h i s can be a double-edged sword; they also lack microorganisms t h a t m a y activate a herbicide, although such are not yet k n o w n . 2. T h e y lack a cuticle. T h e c u t i c u l a r penetration barrier prevents ascertaining whether m a n y new chemical groups are toxic. T h i s barrier can later be overcome by synthesis of analogs of active structures or b y better f o r m u l a t i o n . T h e knowledge t h a t a chemical is toxic if it reaches the plant cell is p r o b a b l y the most i m p o r t a n t lead available f r o m cell cultures that whole p l a n t screens l a c k . T h e absence of cuticle a n d short diffusion distances i n cell cultures, provide a possibility for performing r a p i d kinetic, physiological a n d biochemical studies (6,7). 3. O n l y s m a l l amounts of chemical are neeaed. M o d e r n synthetic chemistry allows derivative synthesis of analogs a n d verification of their structure i n s m a l l batches. If m i l l i g r a m quantities can show potential t o x i c i t y w h y synthesize grams? 4. Space requirements a n d labor are reduced. One t e c h n i c i a n , w i t h racks of tubes or cluster dishes can screen a few h u n d r e d compounds per week. C e l l cultures are pipetted, simple metabolic tests can be used for assessment a n d m a n y steps can-be automated. 5. T i m e . T h e longest in vitro pre-screen can give the chemist an answer w i t h i n a week, if a c o m p o u n d has potential. T h i s r a p i d feedback eliminates the frustrations of w a i t i n g m o n t h s t i l l the latest synthesis is given p r e l i m i n a r y evaluation.
3.
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6.
GRESSEL
Plant Cultures for Herbicide Prescreening
In vitro screens w i t h metabolic measurements can provide more accurate n u m e r i c a l d a t a for Q S A R (quantitative structure a c t i v i t y relation) analyses.
Disadvantages. There are cases where there m a y be no advantage to in vitro systems: 1. In vitro pre-screens do not show up m a n y compounds w i t h plant g r o w t h regulator ( P G R ) p o t e n t i a l . M a n y companies now consider P G R s as uneconomical a n d this m a y a c t u a l l y be an advantage. 2. C o m p o u n d s can be missed unless extreme care is t a k e n i n designing a universal screen. 3. Q S A R d a t a are derived o n l y for the cell level. Separate Q S A R d a t a m u s t be obtained for penetration at the whole p l a n t level. T h i s too can be a n advantage. Balance. T h e major use of in vitro systems is clearly to "weed o u t " t o t a l l y inactive compounds. T h e y are not very useful for " l o o k - a l i k e " chemistry a r o u n d existing active herbicides except where Q S A R d a t a at the cellular level are needed. T h e n one needs not use a n " u n i v e r s a l " pre-screen b u t a " d e d i c a t e d " one for the t y p i c a l p a r t i c u l a r chemistry i n v o l v e d . T h e m a i n use for the " u n i v e r s a l " pre-screen is screening f r o m " r a n d o m " syntheses a n d from directed syntheses for new chemistry. D e d i c a t e d screens s h o u l d be used for " b i o r a t i o n a l " syntheses; syntheses designed to i n h i b i t a specific metabolic p a t h w a y i n the p l a n t . S t i l l , a c o m p o u n d synthesized to i n h i b i t one p a t h w a y m a y instead i n h i b i t another... a n d thus, s h o u l d also be tested i n a universal pre-screen. Developing
Universal
and "Dedicated"
Pre-Screens
N o single system can be universal i n showing uf> the h e r b i c i d a l potential of a l l compounds. N o in vitro system using cells or m i n i plants represents a l l tissues a n d all species. Herbicides can be tissue specific a n d species selective a n d different herbicides act at different rates. T h i s requires t h a t any universal pre-screen has t o be m u l t i specific, multi-tissue a n d m u l t i - t i m e . M a n y of the factors for consideration i n developing such screens m a y superficially seem m u n d a n e or clearly apparent. B a s e d o n failures, this is o n l y so i n retrospect. Evaluating Plant-Culture Parallelism. A s there are m a n y differences between s p r a y i n g a plant post-emergence o r the soil pre-emergence a n d treating in vitro, it is rather h a r d to directly correlate the two systems. T h e c o m m o n procedure for evaluating the parallelism is to treat cultures a n d plants w i t h a wide v a r i e t y of herbicides, other pesticides and some non-toxic compounds. T w o methodologies can be used to assess the parallelism: (1) rough c r i t e r i a to show t o x i c i t y ; (2) statistical r a n k i n g procedures. T h e rough c r i t e r i a indicate t h a t if a c o m p o u n d is above a certain level of t o x i c i t y , then a c o m p o u n d s h o u l d be screened. U s i n g a singleculture procedure w i t h non-photosynthetic cells, 3 of 19 k n o w n herbicides d i d not meet the p r o m o t i o n c r i t e r i a (8). W h e n a twoculture system was used, all herbicides tested w o u l d be p r o m o t e d (^). a n d T a b l e V I I i n (1).
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R a n k i n g procedures allow for more careful evaluation of p a r a l l e l i s m . A series of compounds are p u t t h r o u g h p l a n t a n d in vitro tests^ at fixed concentrations a n d they are r a n k e d i n b o t h as to their relative activities (Figure 1). In any given system there can be false )ositives; those compounds t h a t are more active in vitro t h a n i n plants F i g u r e l a ) , usually because of the lack of the cuticle. False positives provide previously u n k n o w n i n f o r m a t i o n o n potential p h y t o t o x i c i t y . M a n y non-systemic fungicides give positive responses i n cell cultures (1,4)- False negative compounds (Figure l a ) show no a c t i v i t y i n culture b u t are active o n plants. W h e n a single culture system is used, there can be a good overall correlation despite false negatives (Figure l c ) . S t i l l , a false negative is a " m i s s e d " herbicide, a n d as such is unallowable. F o r this reason, multi-system pre-screens have been developed (1,4) to preclude missing compounds. M u c h better correlation coefficients are usually o b t a i n e d w i t h single system dedicated pre-screens. These entail Q S A R relationships using related compounds o n a single p l a n t in situ vs. in vitro pre-screen (Figure Id), or use crop varieties h a v i n g differential tolerance to a single herbicide (Figure l b ) .
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Î
Choosing the Biological Systems. F r o m the preceding discussion it is clear t h a t more t h a n one biological system must be chosen. Considerations for the ease of p i p e t t i n g a n d h a n d l i n g have brought algae to m i n d . One such screen used E r l e n m e y e r flasks a n d required long incubations (11). E a r l i e r literature (12). clearly shows t h a t algae are efficient at showing a c t i v i t y of photosynthesis i n h i b i t i n g herbicides such as p r o m e t r y n a n d f l u o m e t u r o n (Table I). A l g a e are clearly inadequate at showing a c t i v i t y of phenoxy-type herbicides such as 2,4-D. M o r e tonnage of 2,4-D type herbicide is still used t h a n other herbicides, so algae cannot be used as the sole test o r g a n i s m . C y a n o b a c t e r i a (blue-green algae) are even less representative of plants. T h e y lack a higher p l a n t type nucleus, a n d thus d i n i t r o a n i l i n e type herbicides w h i c h prevent the t u b u l i n f o r m a t i o n necessary for nuclear d i v i s i o n are inactive (Table I). T a b l e I. Effect of Selected Herbicides o n A l g a l G r o w t h species
"Prometryn (triazine)
Chlorella (green) Anabaena (blue-green)
93 100
"Tlûometuron (phenylurea
ATachlôr (chloracetamide)
"Trofluralin (dinitroaniline)
~\% TnEiBltîôn ôî growth"" rate) 82 59 41 100
Sôurce:~Representative d a t a condense
