Genetic engineering
and the improvement
of rice and cotton
Insect pests cause considerable damage to certain
crops — forcing farmers to conduct massive chemical
pesticide treatments. Radical changes in plant protection
concepts are thus necessary to reduce negative impacts
on the environment and ecological balances.
Genes encoding entomopathogenic proteins could be
inserted in rice and cotton plants to create
transgenic pest-resistant varieties.
C . PA NN ETIERCIRAD-CA/INRA, Centre de Versailles, 7 8 0 2 6 Versailles Cedex, France
E. G U ID E R D O N I
CIRAD-CA/BIOTROP, BP 5 0 3 5 , 3 4 0 3 2 Montpellier Cedex 1, France
B. H A U
QRAD-CA, BP 5 0 3 5 ,
Research on genetic transformation of cotton for insect resistance was carried out in the Cell Biology laboratory of the Versailles (France) station of INRA, directed by Y. Chupeau. The following scientists were or are involved in the programme:
J. Tourneur (CNRS research scientist) P. Couzi (INRA technician) M . M azier (Ph.D. student 1991-94) V. Dumanois-Le Tan (Ph.D. student 1991-94) M . Giband (CIRAD research scientist) P. Montoro (CIRAD postdoctoral student) In the BIOTROP (CIRAD) laboratory in Montpellier, the following scientists were or are involved in the rice genetic engineering programme:
T. Legavre (CIRAD-GERDAT research scientist) H. Chair (Ph.D. student 1991-94)
N . Mezencev (postgraduate DEA student, Rennes, 1992)
D. Ferrand (postgraduate DESS student, Angers, 1993)
F. Georget (postgraduate DESS student, Angers, 1994)
S. Plchot (postgraduate DEA student, ENSAIA, Nancy, 1994).
CIRAD: Centre de coopération internationale en recherche agronomique
pour le développement, France CNRS: Centre national de la recherche scientifique, France
CSIRO: Commonwealth Scientific and Industrial Organization, Australia INRA: Institut national de la recherche agronomique, France
USDA: United States Department of Agriculture, USA
R
ic e a n d c o t t o n c r o p s h a v e been s u b s ta n tia lly im p ro v e d ( i. e . y i e l d s a n d q u a l i t y ) b y c o n v e n t i o n a l p l a n t b r e e d in g t e c h n i q u e s . H o w e v e r , so m e p r o blems c o n c e rn in g these crops such as r e s is ta n c e to pests ( e s p e c i a l l y insects) are m ore d i f f i c u l t to solve u s in g th es e t e c h n i q u e s . W i t h o u t c o n tr o l, 4 0 % o f the w o r ld 's co tto n c r o p s w o u l d be d e s t r o y e d . Lepidopterans, m a in ly H e lic o v e rp a armigera, Helicoverpa zea, Heliothis v i r e s c e n s and P e c t i n o p h o r a gossypiella, are responsible for 70% o f these losses. In rice, stemborers (also lepidopterans) cause 1 0-30 % of the crop losses, depending on the e x t e n t o f c r o p i n t e n s i f i c a t i o n . Moreover, yield-boosting techniques o f t e n i n v o l v e m a s s iv e use o f p o l l u t i n g c h e m i c a l c o m p o u n d s , w h ic h is not in line w ith sustainable a g r i c u l t u r e o b j e c t i v e s (S A V A R Y & TENG, 1994).C h e m ic a l p e s tic id e tr e a tm e n t was long considered to be the only w ay to control insect pests. H ow e ver, they upset the natural biological balance, are g enerally unselective and often k ill b oth the target pests and th e ir n a tu ra l e n e m ie s . In te n s iv e use o f these c o m p o u n d s can lead to the d e v e lo p m e n t o f resistance in target in s e c ts , w h i c h n e c e s s ita te s an increase in treatm ent dosages. As a result, a lm o s t 3 0 % o f all c h e m ic a l pesticides sold w o r ld w id e are used for cotton crop protection.
A n i n t e g r a t e d p est m a n a g e m e n t strategy has been im p le m e n te d in recent years to reduce the effects of a b u s iv e c h e m i c a l t r e a t m e n t s . It c o m b in e s a ll m e th o d s w i t h c r o p p r o t e c t i o n p o t e n t i a l : c r o p p i n g te chn iqu es, b io lo g ic a l c o n tro l, use o f g e n e tic p la n t tr a its (stud ies on re s is ta n t v a r ie t ie s ) and c h e m ic a l pesticide treatments.
The bacterium Bacillus thuringiensis
and creation of transgenic
insect resistant plants
The B.t. bacterium was first discovered in Japan in 1902 in a silkworm breeding unit. In 1911, it was again isolated in a flour moth population and characterized by Berliner in Thuringen (Germany).
B.t. synthesis of entomopathogenic toxins
B.t. is a gram -positive bacterium that synthesizes entom opathogenic crystalline inclusions during sporulation. The crystalline structure o f the inclusion is an assembly of protoxin subunits, called 5-endotoxins. Most B.t. strains produce several different crystalline proteins. Different strains can contain the same proteins. Cry 8-endotoxins are highly specific.
At least 40 genes encoding protoxins from a w ide range o f B.t. isolates have been isolated and sequenced. These genes were classified by HOFTE & WHITELEY (1989), based on pro te in to x in structural ho m o lo g ie s and s p e c ific itie s . The genes are categorized in four main classes: cryl, cryll, c r y lll and cryIV, each divid e d into subclasses. New toxins are constantly being identified.
The 5-endotoxins are solubilized in the insect's gut (due to the alcaline pH). They are activated by gut proteases that cleave the p ro to xin into a sm aller polyp e p tid e , the toxin . This to xin then binds to the surfaces o f epith e lia l cells in the midgut, inducing lesions that destroy the cells and lead to death of the insect.
Use of B.t. to create insect resistant plants
The chief advantage of this application is that it does not threaten humans, mammals or non-target fauna. The use of B.t. strains in biopesticide spray treatments — for more than 30 years w ith different B.t. strain formulations — has been relatively limited, mainly because of the low field persistence.
The transgenic plant strategy is of considerable interest because it enables toxin production by the plant. The entire plant is therefore protected, especially against insects such as borers that infest plant parts that spraying treatments often cannot reach. The toxin m olecule affects early instar phases o f the insect. The system is environmentally safe since the product is retained within the plant tissues.
Some toxins have been tested against the main cotton and rice pests and the results highlighted interesting genes that could be inserted in the genomes of these two crops (Table 1 ).
Table 1. Toxins active against the main rice and cotton pests.
Insect Type of pest Active toxin
Cotton Spodoptera littoralis Helicoverpa armigera Pectinophora gossypiella Cryptophlebia leucotreta phyllophagous lepidopteran carpophagous lepidopteran carpophagous lepidopteran carpophagous lepidopteran CrylC CrylA(b), CrylA(c) CrylA(b), CrylA(c) CrylA(b), CrylA(c) Rice
Chilo suppressalis lepidopteran borer CrylA(a), CrylA(c), CrylB
Integrated c o ntrol program m es are very complicated to set up, requiring e x c e l l e n t t e c h n i c a l s k ills and an o v e ra ll u n d e rs ta n d in g o f the pests and a g r o e c o l o g i c a l s i t u a t i o n . By g e n e tic e n g in e e r i n g t e c h n iq u e s , genes coding for entomopathogenic p r o t e i n s c a n be i n s e r t e d and expressed in pla nts; th e y are n o w being used to create insect resistant varieties.
R e s e a rc h s c i e n t i s t s are a b le to " d i r e c t l y " m o d i f y t h e g e n o m e t h r o u g h g e n e t i c t r a n s f o r m a t i o n t e c h n i q u e s . R e s e a rc h e rs w e r e previously o n ly able to make use of th e v a r i a b i l i t y w i t h i n t h e p la n t species or, if h ig h ly s o p h is tic a te d te c h n iq u e s w ere a v a ila b le , w it h in the genus o f the plant under study. W it h access to this n ew to o l, they n o w have a w id e range o f different p o s s i b i l i t i e s . T he f i r s t fo c u s was to use t h e e n t o m o p a t h o g e n i c properties of the bacterium Bacillus t h u r in g ie n s is B e rlin e r ( c o m m o n ly c a l l e d B .t.) to o b t a i n in s e c t resistance.
Transgenic plants
for the tropics and
subtropics
The first results on B.t. gene transfers in toba cco and to m ato were p u b li shed in 1987. Since then, B.t. genes h ave been tr a n s fe rr e d to v a rio u s o t h e r c r o p s p e c ie s , e .g. c o t t o n , poplar, potato, rice, maize.
Transgenic cotton plants
T h e f i r s t t r a n s g e n i c c o t t o n p la n t s w e r e o b t a i n e d in 1 9 8 7 ( F I R O O Z A B A D Y e t a l. , 1 9 8 7 ; U M B E C K e t a l . ,1 9 8 7 ) th r o u g h tr a n s fo rm a tio n by A g r o b a c t e r iu m t u m e f a c i e n s a nd r e g e n e r a t io n o f t r a n s f o r m e d c e ll s b y s o m a t ic e m b r y o g e n e s i s . P r iv a t e N o r th A m e r ic a n c o m p a n ie s (Agracetus, Calgene, M onsanto) then invested heavily to produce cotton plants that are resistant to certain lepidopterans.Spodoptera littoralis on a cotton leaf.
Photo CIRAD-UREA
M onsanto reported o b ta ining plants w it h hig h resistance to these pests (PERLAK et a i , 1990), but they were not tested in th e f ie ld . A fe w lines th e n s h o w e d so m e " i n s e c t i c i d e " features in the field (JENKINS et al., 1993). These plants w ere o bta in e d through significant m odifica tion s in the n u c le o tid e sequence o f the B.t. gene used, i.e. crylA(b). Indeed, B.t. genes o f b a c t e r i a l o r i g i n are n o t highly expressed in plants.
V e r y i n t e r e s t i n g r e s u lt s w e r e o b ta in e d by Dr. N. T R O L IN D E R 's team (USDA, USA), w h o transferred a g e n e f r o m th e s o il b a c t e r i u m A lc a lig e n e s e u tr o p h u s to p ro d u c e tr a n s g e n ic p la n t s r e s is ta n t to th e h e rb ic id e 2 ,4 - D (2,4 d ic h lo r o p h e - noxyacetic acid). This research was c o n t i n u e d b y C S IR O ( A u s t r a lia ) , w h i c h is a ls o i n v e s t i g a t i n g th e creation of insect resistant varieties in collaboration w ith Monsanto.
Transgenic cotton plants should soon be available on the market. Calgene and Monsanto, w h ic h have finalized agreements w ith Deltapine (a North A m e r i c a n seed c o m p a n y ) , h a v e applied for certification of transgenic c o t t o n v a r i e t ie s w i t h r e s is t a n c e to b r o m o x y n y l o r g ly p h o s a t e (herbicides) or certain lepidopterans fo llo w in g insertion o f B.t. genes. In 1989, CIRAD, in co lla bo ra tion w ith INRA, began transgenic research on c o t t o n at th e IN R A C e ll B i o l o g y laboratory in Versailles (France). Genes o f agronom ic interest can be inserted in the cotton genome by an A. fume/ac/ens-mediated transforma tio n te c h n iq u e . T w o B.t. genes are
involved: the first, crylA(b), encodes a t o x i n t h a t is a c t i v e a g a in s t H. a r m i g e r a a n d C r y p t o p h l e b i a leucotreta; the second, crylC, w hich was isolated and cloned in France at the Institut Pasteur (SANCHIS et a i , 1989), encodes a protein that is very active against Spodoptera littoralis.
C otton regeneration
and transgene expression
Cotton plants are first regenerated in v itro . C o k e r v a rie t y p la nts (C 3 10 , C312, C201) have been regenerated
b y a s o m a t i c e m b r y o g e n e s i s te c h n iq u e 1, based on the results of TROLINDER & G O O D IN (1988). The main focus has been on transfor m a tio n o f v a rie t ie s s h o w in g g o o d regeneration potential, rather than on d e v e lo p in g som atic embryogenesis t e c h n iq u e s and t r a n s f o r m a t io n o f v a r i e t i e s c r e a t e d b y C I R A D . A cco rding to published results, very fe w v a rietie s o th e r th an cvs C oke r have been successfully regenerated. M o r e o v e r , t r a n s g e n e s c a n be t r a n s f e r r e d f r o m t r a n s f o r m e d c v Coker plants to commercial varieties by standard backcrossing techniques. T he m a in p a ra m e te rs in v o l v e d in A. tumefaciens-mediated transforma tion using hypocotyl fragments have been defined.
A re p o rte r gene w ho s e e xpression c a n be r e a d i l y d e t e c t e d by h istochem ical techniques is used at t h is d e v e lo p m e n t phase. T h e gus gene ( e n c o d in g fê-g lucu ro nid ase ), whose expression can be monitored in the presence of a suitable substrate by the fo rm a tio n o f an in d ig o blue c o m p o u n d was used in these early stages. The transformation procedure is n o w f a i r l y w e l l c o n t r o l l e d and th e resu lts are r e p r o d u c i b l e . T r a n s g e n i c p la n t s h a v e bee n o b ta in e d u sing an A. tu m e fa c ie n s strain carrying a plasmid containing a s e le c ta b le gene and the B.t. gene c r y l A ( b ) , w h i c h is a c t i v e a g a in s t H. armigera, a m ajor cotton pest in A frica and Asia. After selfing, these p la n t s w e r e a b le to set seed (PANNETIER et a i , 1994).
B.t. genes inserted in th e g e n o m e o f th e t r a n s g e n i c p l a n t h a v e to be h i g h l y e x p r e s s e d in o r d e r to pro vid e sufficient pro tectio n against ins e c t pests. H o w e v e r , as a lre a d y
1. S o m a tic e m b ry o g e n e s is : organ fragments (here a piece o f h yp o c o ty l) are c u lt u r e d on a s u ita b le n u tr ie n t m e d iu m , re s u ltin g in fo r m a tio n o f disorganized cell clusters called "c a lIi". Some c a llu s c e lls th e n g iv e rise to somatic embryos through a process that is fa irly similar to embryo formation from fertilized zygotes.
Transformed cotton plant. Photo C. Pannetier
The bacterium
Agrobacterium
tumefaciens
A. t u m e f a c ie n s tr a n s fe rs p a r t o f its g e n e tic i n f o r m a t io n to the plantSubsequent to injuries, A. tumefaciens b a c te ria in d u c e tu m o rs a ro u n d the crown of the plant. This disease is cal led crown gall. In 1974, a correlation was established between the disease and the presence of a high molecular w e ig h t p la s m id in th e b a c te r iu m , te rm e d th e Ti ( " t u m o r in d u c in g " ) plasmid. Tumoral tissue nuclear D N A contains a Ti plasmid D N A fragment, i.e . T - D N A (" tra n s fe rr e d D N A " ) (F ig u re i). Ti plasmids in c lu d e genes th a t are re s p o n s ib le fo r b a c te r ia l virulence (Figure 2).
A fte r tra n sfe r, genes c a rrie d by the T - D N A are expressed in th e p la n t. These are o n c o g e n e s th a t c o n tr o l synthesis o f auxin s and c y to k in in s , which induce continuous uncontrolled p roliferation o f plant cells and tum or formation. They also synthesize opines, used by bacteria as a growth substrate.
A. tum efaciens features used to
t r a n s fe r in t e r e s t in g genes to cultivated plants
An interesting feature of A. tumefaciens is its a b il it y to tra n s fe r genes. This p ro m p te d th e idea o f u t i l i z i n g the feature to transfer in te re stin g genes attached to T -D N A . V arious vectors w e re c re a te d , p a r t ic u l a r ly th o se in v o lv in g a binary system (Figure 3). Two plasmids replace the Ti plasmid. T he f ir s t o n e , o fte n c a lle d th e autonom ous vector, carries a T -D N A lim ite d by boundaries, w ith in w h ic h genes to be transferred to the plant are c o n ta in e d . The se c o n d " d is a r m e d Ti p la s m id " (w ith T - D N A d e le te d ), carries the "tra n s -a c tin g " v iru le n c e function, i.e. enabling T -D N A transfer from the autonomous vector.
Plant cell Transformed plant cell
Figure 1. The bacterium Agrobacterium tumefaciens transfers some of its genetic information to the plant.
T - D N A (for transferred D N A ) carries oncogenes ( O N C ) , i.e. o p in e synthesis genes (OPS).
It is limited b y "boundaries": right bo rd er (RB) a n d left b o rd er (LB), com posed o f a 2 5 nucleotide sequence.
The region between these tw o boundaries is transferred.
O n the plasm id, there a re also VIR genes, w hich e n a b le T - D N A transfer, with O PS genes controlling o p in e catabolism . O R I is the origin o f replication.
RB
Figure 2. Diagram of the Ti plasmid.
Figure 3. Diagram of the binary system.
RB
Both plasmids c arry a n origin o f replication, w hich controls their multiplication in A . tumefaciens o r E. coli. This bacterium is used to construct the region to be inserted in the plan t genom e.
Anthonomus grandis on a cotton flower bud.
Photo CIRAD-UREA
m e n t i o n e d , th e s e g e n e s are o f bacterial origin and poorly expressed in p la n t s . T w o s tr a t e g ie s w e r e d e v e l o p e d to i m p r o v e B .t. g e n e e x p r e s s i o n : m o d i f i c a t i o n s o f th e n u c le o tid e sequence and inse rtio n upstream o f th e gene o f interest o f sequences that provide more efficient t r a n s la t io n (M A ZIE R e t al., 199 4). Potentially transformed cotton plants h ave been p r o d u c e d w i t h T - D N A inserted from a vector containing the T o b a c c o M o s a ic V i r u s " o m e g a leader" sequence, w h ic h p otentially c o u l d e n h a n c e t h e t r a n s l a t i o n e f f i c i e n c y o f g en es p la c e d downstream.
Studies w ere undertaken to m o d if y the nucleotide sequence o f the gene e n c o d i n g th e t o x i n t h a t is a c t i v e a g a in s t 5. l i t t o r a l is. T o b a c c o — a c o m m o n m o d e l p la n t in c e ll and m o le c u la r b io l o g y la b o r a to r ie s —
was initially used to test the effects o f g en etic m o d if ic a tio n s . C o m p le te ly s y n t h e t i c genes — r e c o n s t r u c te d fro m an ideal n u c le o tid e sequence w i t h o u t a n y c h a n g e in th e a m in o acid sequence — have been inserted in the cotton genome.
C ircum venting insect resistance
B.t. gene tra n s fe r and s u b s e q u e n t e x p r e s s i o n in t h e g e n o m e o f commercial varieties are essential for th e ir fu tu re u t i li z a t i o n . The aim o f research c o n d u c te d at C IR A D and IN R A is to o b t a in su s ta in e d p la n t p r o t e c t i o n a g a in s t pests. It is important to avoid the develo pm ent of insect resistance towards the toxin synthesized by the transgenic plant. V a r i o u s s tr a t e g ie s h a v e b e e n designed to reduce such risks. They are generally based on low ering the
Construction of a plant expression vector
A chim eric gene had to be constructed so that the foreign gene (often called the transgene) c o u ld be expressed in the plant (Figure 4).
The prom otor can be constitutive, i.e. induce gene expression throughout the plant and at all stages of development, or specific to an organ or tissue, or even be inducible by wounding, etc.
This c h im e r ic gene is in s e rte d in a plasm id (autonomous c irc u la r DNA), w h ic h in tu r n is in s e rte d in the b a c te r iu m E s c h e ric h ia c o li. The resulting transformation vector can be m ultiplied by culturing the bacterium. It can th e n be tra n s fe rr e d to A. tu m e fa c ie n s o r used in p la s m id form for so-called direct transformation
(2) (1) (3) Regulating Coding Terminal region sequence region
■ ■■H
D N AFigure 4. Chimeric gene structure.
te c h n iq u e s : p r o t o p la s t o r e m b ry o electroporation, particle acceleration, etc.
In the transformation vector, the gene of interest is often associated w ith one or several selectable reporter genes (also in th e fo rm o f c h im e r ic genes). T hey enable re la tiv e ly sim ple selection or detection of transformed cells or tissues. The selectable genes are often antibiotic (or h e rb ic id e ) resistance genes. An E. c o li gene encoding R-glucuronidase (gus gene) is a common reporter gene. Its presence in the plant genome can be e a s ily d e te c te d by h is to c h e m ic a l techniques (in the presence of a suitable s u b s tra te , X g lu c , an in d ig o b lu e precipitate is formed).
Chimeric genes are schematically divided into three parts:
- a codin g sequence corresponding to the sequence o f D N A , w hich is transcribed into messenger RNA, which in turn
is translated into a protein (1 ); - a prom otor region, located upstream o f the codin g sequence, is com posed o f several sequences, one o f w hich is recognized b y the R N A polymerase, thus indu cing the onset o f D N A transcription (2); - a te rminator sequence (3).
Agrobacterium
tumefaciens-
mediated
transformation
of plants
The transformation process
Transformation o f cotton is described here to exemplify the general process. An organ fragment (hypocotyl fragment for cotton) is placed in contact with the bacterium A. tumefaciens containing a binary system — this is the inoculation stage. The plant tissue is cultured for a specific period o f time in vitro (under aseptic conditions On modified nutrient medium) — this is the coculture stage. A n t i b io t ic s are th e n a d d e d to th e culture medium, the first one prevents bacterial growth and the second selects th e tra n s fo rm e d p la n t c e lls . A selecta b le gene is inserted in the p la s m id to g e th e r w it h th e gene o f interest; generally this gene provides a n t i b io t ic re s is ta n c e . O n l y c e lls c o n ta in in g this a n tib io tic resistance gene can undergo d iv is io n in a cell medium containing the selective agent ( u s u a lly k a n a m y c in ). T he gene o f interest is also inserted in these cells.
Em bryogenesis in tr a n s fo r m e d cotton
W h e n r e g u la r ly s u b c u ltu re d on m e d iu m c o n ta in in g p la n t horm ones and the selective agent, the transformed cells give rise to a callus. In certain c u ltu re m e d iu m and e n v iro n m e n ta l (lighting, temperature, etc.) conditions, neoformation of plants from this callus can be o b ta in e d by c o n v e n tio n a l m ic r o p r o p a g a t io n te c h n iq u e s . For c o tt o n , th e c a llu s g ive s rise to a so-called em bryo g e n ic tissue w ith in w h ic h som atic em bryos d e ve lo p . In turn, these embryos give rise to young p la n tle ts w h ic h are s u b s e q u e n tly tra n s fe rre d to th e g re e n h o u s e (see c o lo u r plates). M o le c u la r and biochemical analysis are then carried o u t to d e te rm in e th e c o p y n u m b e r o f in s e rte d genes, and th e le v e l o f expression of this gene in the plant.
s e l e c t i o n p re s s u r e b y l i m i t i n g insect/toxin contacts: refuge plants2, t r a n s g e n i c / n o n - t r a n s g e n i c p la n t r o t a t i o n s , p la n t s e x p r e s s in g th e t o x i n o n l y in c e r ta in o rg a n s , etc. (MACCAUGHEY & W H A LO N , 1992). The strategy used involves associa tin g t w o genes th at code for toxins w i t h d i f f e r e n t m o d e s o f a c t i o n . H e n c e , an i n s e c t m i g h t d e v e l o p r e s is ta n c e to o n e o f t h e t o x i n s , b u t it w i l l r e m a in s u s c e p t i b l e to the second.
Studies were thus launched to assess th e a s s oc iatio n o f genes e n c o d in g
B.t. t o x i n s a nd g en es e n c o d i n g protease in h ib ito rs . These protease i n h i b i t o r s ( H I L D E R e t a l. , 1 9 8 7 ; R Y A N , 1 9 9 0 ) i n h i b i t th e in s e c t's digestive proteases and consequently h in d e r its g r o w t h ; t h e i r m od es o f action therefore differ from those of B.t. t o x in s and th e y have a m u c h w i d e r h o s t ra n g e . T h e r e s u lt s o f in s e c t b io a s s a y s , i.e . b l e n d i n g protease i n h ib it o r s in to a n u tr ie n t m e d iu m , h ig h lig h t e d t h e ir effects on a c a rp o p h a g o u s l e p id o p te r a n , H. a r m ig e r a , a n d o n a b e e t le , A n t h o n o m u s g r a n d i s (LE T A N - D U M A N O IS , 1994). These tests were conducted in the CIRAD entomology laboratory.
M ore than 100 em bryogenic cotton lines h a v in g inte g ra te d a protease inhibitor gene were obtained; dozens o f t r a n s fo r m e d p la n t c lo n e s have been transferred to the greenhouse. M o le c u la r and bio chemical analysis of these plants are under way, along w it h p r e li m in a r y tests on insects. T h e i n i t i a l r e s u lt s i n d i c a t e t h a t hig h q u a n t it ie s o f th e p r o te in are s y n t h e s i z e d b y t h e p l a n t , in th e leaves and bolls.
Transgenic rice
Few p r i v a t e p la n t b i o t e c h n o l o g y c o m p a n ie s , a p a rt fr o m A g ra c e tu s (USA) and Japan T o b a c c o , M it s u i T o a ts u a n d P la n te c h R es e a rc h
2. Refuge p la n ts : are u n tra n s fo rm e d p la n ts th a t are g ro w n w it h o r c lo s e to tra n s g e n ic p la n ts (e x p re s s in g the entomopathogenic toxin).
In s titu te (Japan), have invested in g e n e t i c e n g i n e e r i n g o f r ic e . T he fo l lo w in g results w ere o b ta ine d by p u b l i c l a b o r a t o r i e s , w i t h m a j o r f u n d i n g f r o m th e la r g e s - s c a le Rockefeller F oundatio n program m e that began in 1985.
The first genes o f interest for insertion in ric e are those th a t c o n fe r resis t a n c e to in s e c ts (B .t. e n d o t o x i n genes, pla nt protease inhibitors and lectins), viruses, bacteria, fungi and herbicides. Transgenic rice varieties have already been produced w h ic h harbour a synthetic crylA(b) gene, a s n o w d r o p le c t in gene, p o ta to and cowpea protease inhibitor genes, the capsid protein of the stripe virus, and that of the spherical and rod-shaped tungro viruses and phosphin othricin and s u lfo n y lu re a resistance genes. H o w e v e r , v e ry fe w o f these tra n s genic varieties have been field tested. T h e g e n e t r a n s f e r s yste m is n o w o p e r a t i o n a l f o r r ic e e v e n t h o u g h there are still problem s c o nc e rn in g other cereals — nevertheless regular progress is being made in this area. M e d i u m - and lo n g - t e r m research studies a im at in s e rtin g genes that could improve grain quality, increase tolerance to drought, salinity, anoxia and cold into the rice genome.
Producing transgenic rice plants
and regeneration problems
The first transgenic rice plants were o b ta in e d by d ir e c t gene transfer to p ro to p la s ts in te m p e ra te j a p ó n i c a v a rie tie s (T a ip e i3 0 9 , N ip p o n b a re , Yamahoushi) in 1988 (ZH A N G et a i, 1 988), 4 years after the first reports o n o b t a i n i n g t r a n s g e n i c d ic o t s through protoplast or A. tumefaciens mediated transform ation. Indeed, it was o nly in 1985 that regeneration of p r o t o p l a s t - d e r i v e d j a p ó n i c a rice p la n t s w a s f u l l y m a s te r e d . The technique was then applied to indica rice varieties (IR54, IR72) w h ic h are generally more difficult to regenerate (PENG et al., 199 2); h o w e v e r, the technique was not very efficient with th ese g e n o ty p e s . T h e re are o fte n problems o f male sterility and ploidy levels in regenerated transgenic rice
Embryogénie calli with developing cotton somatic embryos. Photo C. Pannetier
Transformed callus on a cotton hypocotyl fragment, developing on selection medium.
Photo C. Pannetier
Chilo suppressalis damage in a rice field.
Photo M . Betbeder
Cross-section of the stem of a young transformed rice plant showing constitutive expression of the gus gene in cells from different rolled leaves.
Photo E. Guiderdoni
Expression of the gus gene in immature rice embryo scutellum cells following microparticle bombardment.
Photo F. Georget Young cotton plantlet developed from a somatic embryo.
Photo C. Pannetier
Expression of the gus gene in young regenerated rice plantlets.
Purified preparation of rice protoplasts, obtained through enzyme treatment of cell suspensions, prior
to polyethylene glycol treatment. Photo E. Guiderdoni
Examples of molecular hybridisation with DNA of five rice plants. Hybridisation is performed with a radioactive probe composed of a sequence from the transferred gene, providing evidence that the transgene has been inserted in the rice genome. This example involves a resistance gene against ammonium glufosinate herbicides.
Photo H. Chair
plants. M o re v a riatio ns (m o rp h o lo g ic a l m o d if ic a t i o n s ) o c c u r in th e progeny as compared to protoplast- d erive d plants that do not undergo any transform ation treatment. These p la n t s a lr e a d y s h o w d i f f e r e n c e s w h e n c o m p a r e d to n o r m a l l y g e r m in a t e d p la n ts (D A V E Y e t al., 1 9 9 1 ). A l t h o u g h th e re h ave been m any p u b lic a tio n s on this process, very few laboratories have used it to develop efficient systems for routine mass p r o d u c t i o n o f t r u e - t o - t y p e transgenic rice plants.
G e n e transfer techniques
for rice
In 1991, Agracetus o b ta ine d trans g e n ic ric e p la nts b y m ic r o p a r t ic le acceleration of exogenous D N A into im m ature embryos o f j a p ó n ic a and in d ic a varieties (CH R ISTO U et al., 1 9 9 1 ) . In o t h e r l a b o r a t o r i e s , t r a n s fo rm a t io n s b y th is te c h n iq u e w e r e a ls o c a r r i e d o u t w i t h c e ll suspensions and e m bryogenic calli. The statement that this te chn iqu e is universal is not q uite correct, since some im m ature embryos o f various r ic e v a r i e t i e s c a n n o t r e g e n e r a t e plants and, in any case, it w ou ld not be p o s s i b l e t h r o u g h a u n i q u e process.
O v e r a l l, th is is p r o b a b ly the m ost prom ising transfo rm atio n te chnique fo r rice and cereals. A lth o u g h the process is protected by many patents, it is used by an increasing number of laboratories. It seems better than the protoplast transformation te chnique since it a v o id s m a n y o f th e abo ve described variations, i.e. first genera tio n plants are generally fertile and
m o r p h o lo g ic a lly norm al. D u rin g in v i t r o c u l t u r e , a b n o r m a l i t i e s in reg en erated p la nts are s o m e tim e s c a u s e d b y th e lo n g r e g e n e r a t io n p h a s e — w h i c h is s h o r t e r f r o m e m b r y o s th a n p r o t o p l a s t s . It is d iffic ult to determine w h ic h of these methods is superior in terms o f their efficiencies in producing true-to-type tra n s g e n ic p la n ts s in c e th e y have n e v e r b e e n c o m p a r e d u s in g th e same g e n o ty p e o r u n d e r th e same laboratory conditions.
In 1 9 9 3 , a T a iw a n rese arch team ( C H A N e t a l., 1 9 9 3 ) a n d la t e r a Japanese te a m (HIEI e t a l., 1 99 4) o b ta in e d m o le c u la r e v id e n c e th a t /4. t u m e f a c i e n s c a n be used to t r a n s f o r m r ic e . T h e e f f i c i e n c y is d e p e n d e n t o n t h e r e g e n e r a t io n potential of the genotype.
Transfer of insect
borer resistance
to M e d iterran e an rice
In 1 9 9 0 , C IR A D la u n c h e d a gene tran s fe r p r o je c t fo r M e d ite rra n e a n and rainfed rice varieties. The aim is to p r o d u c e r ic e r e s is t a n t to th e A s ia n a n d E u r o p e a n s t e m b o r e r , C h ilo suppressalis, a p y ra lid borer, w h i c h c a u s e s c r o p losses o f 7 - 1 5 q u i n t a l s / h a in C a m a r g u e ( F r a n c e ), a n d to t h e A f r i c a n b o re rs M a l i a r p h a s e p a ra te lla and C h i l o z a c c o n iu s . T he s tu d ie s are b e ing c a rrie d o u t in the BIO TRO P research unit of CIRAD (Montpellier, France).
Rice varieties exist that are naturally t o l e r a n t to b orers, b u t th is t r a i t is under polygenic control. This hinders c o n v e n tio n a l gene transfers and, in a d d it io n , it is n o t c e rta in th a t the tolerance w ill remain stable after the ric e v a r i e t y is d is t r i b u t e d w id e ly . The p ro je c t involves transfer o f B.t. endotoxin genes, but initial w ork has b e e n s p e c i f i c a l l y f o c u s e d on M e d it e r r a n e a n r ic e v a r ie t ie s and C. s u p p r e s s a l i s . T h e t e c h n i q u e s d e v e lo p e d (active to x in id e n t ific a t i o n , t r a n s f o r m a t i o n ) c o u l d be r e a d i l y e x t e n d e d to t r o p i c a l rice varieties and pests.
A f f i n i t y a n d t o x i c i t y a n a ly s is r e v e a le d th a t t o x i n s e n c o d e d by c r y lA (c ) , c ry lA (a ) and c r y lB genes w e r e t h e m o s t a c t i v e a g a in s t C. suppressalis larvae (FIUZA, 1995). N a tiv e genes o f these th ree toxins were cloned and synthetic genes are being prepared, or have already been obtained, in collaboration w ith Prof. ALTOSAAR's laboratory (University of Ottawa, Canada).
A t the b e g in n in g o f this p ro je c t, a t e c h n i q u e i n v o l v i n g d i r e c t gene transfer to p ro to p la s ts was m a in ly
used fo r g e n e tic tra n s fo rm a t io n o f e lite M e d it e r ra n e a n rice va rietie s. A system w as d e v e lo p e d fo r p la n t regeneration from em bryogenic cell s u s p e n s io n -d e riv e d p ro to p la s ts o f A rie te , M ia ra and T h a íb o n n e t rice varieties (G U ID E R D O N I & CHAIR , 199 2). V a r ia tio n s o b s e rv e d in th e field in the progeny o f these plants w e r e f o u n d to be g e n o t y p e d e p e n d e n t — in te rm s o f rate , am plitude and directio n — but they c o u l d s t i l l be used in b r e e d in g schemes (MEZENCEV et a i , 1995). A p ro to p la s t tr a n s fo r m a t io n stu dy was also carried out, p ro m p ting the d e v e lo p m e n t o f an e f f i c i e n t gene transfer technique. Genes could thus be inserted in protoplasts after either of the fo llo w in g treatments: chemical p o l y e t h y l e n e g l y c o l t r e a t m e n t o r d i s r u p t i o n w i t h an e l e c t r i c cu rren t (electroporation). C hem ical treatment was chosen for the project based on the results of com parative studies (C H A IR , 1 99 5). Instead o f using antibio tic resistance, selection o f transformed tissues was obtained via re s is ta n c e to p h o s p h i n o t r i c i n (Basta herbicide) through expression
o f t h e p h o s p h i n o t r i c i n a c e t y l tr a n s fe r a s e g e n e ( o r b a r g e n e d e r i v e d f r o m t h e S t r e p t o m y c e s h y g r o s c o p i c u s b a c t e r iu m ) . C o n s t i t u t i v e p ro m o to rs w e r e id e n t if ie d that enable high expression in rice.
An e ffic ie n t system was created to produce transgenic cv Miara, Ariete and T h a íb o n n e t plants at a rate o f 0.5-1 p la n t s / 1 0 0 0 0 0 p ro to p la s ts p r o c e s s e d . H o w e v e r , th e r e are s t ill m a n y p r o b le m s In o b t a i n i n g t r u e - t o - t y p e p la n ts , e.g. a lb i n i s m associated w ith the use o f protoplasts from m lc ro s p o re -d e rlv e d c a lli, and o b t e n t i o n o f h ig h e r th a n n o r m a l p lo id y levels (2n). Promising results, w h i c h c o u l d s o lv e s om e o f these p r o b l e m s , h a v e b e e n o b t a i n e d t h r o u g h th e use o f p r o to p la s t s o f s o m a tic o r i g i n , and m ic r o p a r t i c l e acceleration of D N A into im m ature embryos (GEORGET, 1994).
The effects of constitutive expression of a native gene and a synthetic B.t. gene on pyral borer resistance in a s u s c e p tib le v a rie t y and a t o le r a n t variety w ill soon be compared.
Direct gene transfer techniques
Electroporation
E le c tro p o ra tio n was firs t d e v e lo p e d fo r a n im a l c e lls and b a c te r ia . It w as s u b s e q u e n tly a p p lie d to p la n t c e lls fro m w h ic h th e c e ll w a lls had been e n z y m a t ic a l ly re m o v e d , i.e. protoplasts. It was considered essential to remove the cell walls to a llo w DNA to e n te r th e c e lls . H o w e v e r , re c e n t success in obta in in g transgenic plants fro m z y g o tic e m b ry o s and c a llu s fragm ents, pretreated o r not w ith an enzymatic solution, suggests that genes c o u ld be in s e rte d th r o u g h in ta c t c e ll w a lls u sin g h ig h p o w e r v o lta g e and capacitance electroporation. In practice, protoplasts are suspended in a saline buffer solution in the presence of plasmid DNA. They are then placed in a tank between electroporator terminals w h ic h deliver a charge o f capacitance (expressed in (jFarads) and v o lta g e (in v o lts ) fo r a s p e c if ic tim e (in the
millisecond range). The electric field thus created depolarizes phospholipid layers o f th e p ro to p la s t plasma m e m brane, inducing temporary holes through which DN A molecules can enter the cells. Polyethylene glycol treatment P ro to p la s ts , fir s t su sp e n d e d in a s a lin e b u ffe r s o lu tio n , are p la ce d in a p o ly e th y le n e g ly c o l s o lu tio n (high m o le c u la r w e ig h t c o m p o u n d ) , w it h b iv a le n t cations (M g2+ o r Ca2+) and p la s m id D N A . The c a tio n s p r o v id e bridges between D N A and the plasma mem brane (both negatively charged). The p o ly e t h y le n e g ly c o l fuses and destabilizes the plasmalemma, allowing DNA to enter the cells.
Microparticle acceleration
Plasmid D N A , p re c ip ita te d on metal (tungsten or gold) microparticles under the action of alcohol or salts, is deposited on a macroprojectile (an aluminum foil
disk o r a p la stic screen) o r in a d ro p o f w a te r, d e p e n d in g on th e ty p e o f particle gun used. W ith an a lu m in iu m fo il d e vice , pressure exp a n sio n after th e e x p lo s io n o f p o w d e r o r n e u tra l c o m p re s s e d , gas p ro p u ls e s th e m a c r o p r o je c t ile , w h ic h is th e n stopped by a stopping plate, w h ile the microprojectiles continue on towards the target. W ith the plastic screen type, a flow of helium carries the microparticles directly towards the target. W ith a drop o f w a te r, an e le c t r ic arc is c re a te d between electrodes, w he re the w ate r drop is deposited; the particle gun causes its v a p o riz a tio n , thus p ro p u ls in g the microparticles. Microparticle propulsion is performed in a chamber containing the target and in w hich a partial vacuum is created to facilitate propulsion. For rice, the target can be a spread sam ple o f em bryo g e n ic cell suspension, cal I i or embryo scutellum, or microspores.
Chilo suppressalis larvae in a rice sucker.
Photo J. Escoute
Thereafter, fu rthe r c o m b in a tio n s of d i f f e r e n t s y n t h e t i c g en es o r an e n d o t o x i n g e n e a n d a p ro te a s e i n h i b i t o r g e n e , c o n t r o l l e d b y constitutive or regulated promotors, could be constructed and inserted in the rice genome.
Prospects
Genetic transformation enables plant breeders to overcome limitations due to barriers between species. After the d is c o v e r y o f th is t e c h n iq u e in the early 1980s, there were great hopes for an explosion of new varieties with i n t e r e s t i n g t r a i t s in th e 1 9 9 0 s . P r e l i m i n a r y s t u d ie s f o c u s e d on introducing characters of high market v a lu e (e.g. h e r b i c i d e a nd in s e c t resistance). It to o k q u ite some tim e before genetic engin eering had any m a r k e d i m p a c t in t h i s f i e l d , b u t transgenic varieties should soon be
a v a ila b le on the market. H ow e v er, f o r s e v e r a l t r a n s g e n i c v a r i e t ie s especia lly w ith respect to herbicide resistance, there are current discus sions on some technical problems. In programmes under w ay at CIRAD, th e first step in c rea ting transgenic plants has been to d e v e lo p reliable a n d r e p r o d u c i b l e g e n e t r a n s f e r t e c h n i q u e s . In s e c t r e s is ta n c e is t h e m a in g o a l o f v a r i e t a l im p ro v e m e n t. C lo se c o lla b o r a t io n b e tw e e n b io lo g is ts , e n to m o lo g is ts and p la n t b re e d e rs is n e e d e d fo r th e e f f e c t i v e use o f t r a n s g e n i c p la nts c o n t a i n i n g genes e n c o d in g entomopathogenic proteins. Various s tr a t e g ie s , s p e c i f i c a l l y a im e d at l i m i t i n g th e d e v e l o p m e n t o f resistance in p la nt pests, should be d e s ig n e d f o r th e s e t r a n s g e n i c v a r ie t ie s b e fo re t h e i r w id e s p r e a d distribution.
Continuous research on new sources o f resistance is essential for efficient lo n g - t e r m c o n t r o l . S tudies on the e f f i c i e n c i e s o f v a r i o u s p r o t e i n s (protease inhibitors, lectins, etc.) are p re s e n tly u n d e r w a y . Insect genes t h a t c o u l d be used to u p s e t t h e i r d e v e lo p m e n t is a p rom ising area of study (STEWART, 1991).
The d e v e lo p m e n t o f resista nce to insect pests remains a prime research f o c u s . N e v e r t h e le s s , g e n e t i c engineering offers considerable new p o te ntia l, and studies are, and w il l be, c a r r ie d o u t to in v e s t ig a t e a ll cro p p in g aspects, e.g. on resistance to abiotic stresses (salinity, tempera ture, etc.), creation o f F1 hybrids, and improvement of cotton fibre and rice grain qualities. There are great gene pools in w ild -type varieties that have not yet been tapped. W ild species of c o t t o n h a v e m a n y u s e fu l t r a it s : b a c te ria l disease resistance, male s t e r i l i t y , d r o u g h t re s is ta n c e , technological features, insect resis tance, etc. However, there have been v e r y f e w s u c c e s s fu l in s e r t io n s o f th e s e t r a i t s i n t o c u l t i v a t e d v a r ie t ie s to c re a te n e w v a rie t ie s . Biotechnology c ould extend current horizons by enabling plant breeders to utilize this high genetic diversity. Access to these new techniques is an
Choices of transformation techniques
W hy is A. tumefaciens not used for rice?
Rice, as is the case with other cereals, resists A. tu m e fa c ie n s in fe c tio n . The fact that tumors do not form from differentiated tissues of these plants is not related to the agrobacteria, but rather to the inability of most cereal cells to proliferate. In 1986, Grimsley and collaborators demonstrated that A. tumefaciens could insert its T-DNA into the nucleus of a cereal cell. The lack of tumor formation from the inva ded cereal cell is due to a difference in the injury responses of monocot and d ic o t c e lls : the fo r m e r reacts by degenerating and the latter produces scar tissue. The inefficiency of gene transfers in grass species co u ld be explained by an absence of suitable c o n d it io n s fo r d iv is io n in c e lls th a t have been in fe c te d w it h an agrobacterium.
Following initial studies by a Taiwan research team, Japanese scientists of the Japan Tobacco company were ab le to s u c c e s s fu lly c o c u ltu r e proliferating embryogenic cells with agrobacteria, clearly demonstrating
the p o s s ib ility o f la rg e -s c a le production of transgenic rice plants. Direct gene transfer techniques for rice
In 1 9 8 8 , th e firs t tra n s g e n ic rice plants were obtained by direct gene transfer techniques involving physical ( e le c tr o p o r a tio n ) o r c h e m ic a l (p o ly e th y le n e g lyco l) treatm ent o f protoplasts. The regeneration system firs t has to be f u l l y mastered. This a p p ro a c h was fo u n d to be c o m p lic a te d to im p le m e n t, o n ly a p p lic a b le to a fe w g e n o ty p e s , sh o w e d a h ig h risk o f o b ta in in g unwanted variations, because of the long in vitro culture steps with total cell isolation. Callus cell suspension and embryo scutellum transformation systems were developed to limit these drawbacks. Transgenic rice plants were thus obtained in 1991, and these tr a n s fo r m a tio n te c h n iq u e s w ere w id e ly adopted for transgenic rice production. Interesting results were also re c e n tly o b ta in e d w ith other techniques, e.g. electroporation o f em bryo cells and treatm ent w ith a pulsed electric field.
i m p o r t a n t c h a l l e n g e f o r less developed countries. M any potential a p p l i c a t i o n s w i l l be h i g h l y beneficial, especially for developing low -in pu t cropping systems. Despite d e l a y i n g th e e x p e c t e d b e n e f it s , analysis on the impacts of transgenic v a r i e t i e s ( c r o p p i n g p r a c t i c e s , e n v i r o n m e n t a l i m p a c t , e tc .) are im perative before th eir agricultural e x te n s io n . Research s h o u ld also address the needs of less developed c o u n t r i e s , a n d c o l l a b o r a t i v e p r o g r a m m e s c o u l d be set up to p ro v id e access to im p ro v e d p la n t material but also to the technology.
Cotton boll damaged by Helicoverpa armigera.
Photo CIRAD-UREA
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Abstract... Resumen... Résumé...
C. PANNETIER, E. GUIDERDONI, B. HAU — Geneticengineering and the improvement of rice and cotton.
The tran sfe r of genes coding fo r entom opalhogenic proteins makes it possible to create rice and cotton varieties resistant to insect pests. The first focus is the use of genes coding for "insecticide" toxins of the bacteria
B a c illu s th u r in g ie n s is . B .t. genes have thus been
introduced in various plants: cotton, poplar, potato, rice and m aize. The fir s t tra n s fo rm a tio n o f cotton was achieved in 1987 via A gro ba cte rium tum efaciens and regeneration by somatic embryogenesis. Insecticide properties were confirmed in the field by several lines after substantial modification of the nucleotide sequence of the B.t. gene used, since B.t. genes of bacterial origin are v e ry w e a k ly expressed in p lan ts. CIRAD, in co llab oration w ith INRA, p erform s research on the transformation of cotton via Agrobacterium tumefaciens for resistance to Heliotis arm igera (B.t. gene, crylA(b)) and to Spodoptera littoralis [B.t. gene, crylQ . However, use of plants synthesizing a single B.t. toxin may cause the appearance o f resistance in the ta rg e t insects. Different strategies can be used to prevent the occurrence of such resistance. That studied by the CIRAD/INRA team consists o f c o m b in in g tw o genes coding fo r entom opathogenic proteins w ith d iffe re n t modes of action: B.t. toxins and protease inhibitors. Transgenic cotton plants incorporating a B.t. gene or expressing a p rotease in h ib ito r gene have been o b ta in e d and set seed. Rice tr a n s fo rm a tio n was achieved o n ly 4 years a fte r the first dicot transgenic plants were obtained. However, regenerated transgenic rices have more variations than true-to-type plants. Application of resistance to borers ( C hilo supp ressalis, M a lia r p h a
separatella and Chilo zacconius) in Mediterranean rice
varieties has been undertaken by CIRAD. The transfer method involves protoplast transformation. Overall, the application of genetic engineering to cultivated plants has been slower than expected, but transgenic varieties will soon be available on the market. Analysis of the impact fo r cropping techniques and the environm ent should precede the release of transgenic varieties for cropping. Keywords: cotton, rice, breeding, biotechnology, Bacillus
thuringiensis, insect pest control, toxin, protease inhibitor.
C. PANNETIER, E. GUIDERDONI, B. HAU — Ingeniería genética y mejora del arroz y del algodón. La tran sfe re nc ia de genes que codifican proteinas entomopatógenas permite crear variedades de arroz y de algodón que resisten a algunos insectos dañinos. El primer eje desarrollado es la utilización de genes que codifican toxinas "insecticidas" de la bacteria Bacillus
thuringiensis. De este modo, se han introducido genes de B.t. en diferentes especies: algodón, álamo, patata, arroz
y maiz. Las primeras obtenciones de plantas de algodón tra n s fo rm a d a s d a ta n de 1 9 8 7 , a p a r tir de la transformación mediante Agrobacterium tumefaciens y de la regeneración por embriogénesis somática. Algunas cepas confirmaron en el campo propiedades insecticidas tras im p o rta n te s m od ific a c io n e s de la secuencia nudeótida del gen B.t. utilizado, pues los genes de B.t., de origen bacteriano, se expresan muy debilmente en un contexto vegetal. CIRAD, en colaboración con el Instituto francés de Investigación Agrícola (INRA) está realizando investigaciones acerca de la transformación del algodón mediante Agrobacterium tumefaciens para la resistencia a
Heliotis arm igera (gen de B.t. crylA(b)) y a Spodoptera littoralis (gen de B.t. crylc). Sin embargo, la utilización de
plantas que sintetizan una sola toxina de B.t. puede provocar la aparición de resistencia en los insectos destinatarios. Pueden desarrollarse diferentes estrategias para evitar la aparición de esta resistencia. La estudiada por el equipo CIRAD/INRA consiste en asociar dos genes que codifican proteinas entomopatógenas de modos de acción diferentes: las toxinas de B.t. y los inhibidores de proteasis. Se obtuvieron algodoneros transgénicos que hablan integrado un gen de B.t. o que expresaban un gen inhibidor de proteasis y que produjeron semillas. Más de cuatro años después de las primeras obtenciones de dicotiledóneas transgénicas, se p ro du jeron arroces transgénicos. No obstante, los arroces transgénicos regenerados presentan a menudo variaciones respecto a las plantas conformes. CIRAD ha comenzado la aplicación a la transferencia de resistencia a los barreneros ( Chilo
suppressalis, M aliarpha separatella, Chilo zacconius) en
los arroces mediterráneos. El método de transferencia utiliza la transformación de los protoplastos. En general, la aplicación de la ingeniería genética a las plantas cultivadas ha sido menos rápida que lo previsto, pero la llegada en el mercado de variedades transgénicas es inm inente. La difusión en cultivo de las variedades transgénicas deberá ir precedida del análisis de las consecuencias para las técnicas de cultivo y el medio ambiente.
Palabras clave: algodón, arroz, genética, biotecnologia,
Bacillus thuringiensis, lucha contra los insectos, toxina,
inhibidor de proteasis.
C. PANNETIER, E. GUIDERDONI, B. HAU — Génie génétique et amélioration du riz et du cotonnier. Le tr a n s fe r t de gènes co da nt p o u r des p ro té in e s entomopathogènes permet la création de variétés de riz et de cotonnier résistant à certains insectes ravageurs. Le premier axe développé est l'utilisation de gènes codant pour des toxines « insecticides » de la bactérie Bacillus
thuringiensis. Des gènes de B.t. ont été ainsi introduits
dans différentes espèces : cotonnier, peuplier, pomme de terre, riz, maïs. Les premières obtentions de cotonniers transformés datent de 1987, à partir de la transformation via A grobacterium tumefaciens et de la régénération par embryogenèse somatique. Quelques lignées ont confirmé au champ des propriétés insecticides, après d'importantes modifications de la séquence nudéotidique du gène de B.t. utilisé, car ces gènes d'origine bactérienne, s'expriment très fa ib le m e n t dans un co n te xte vé g é ta l. Le CIRAD, en collaboration avec l'INRA, conduit des recherches sur la transformation du cotonnier via A. tumefaciens pour la résistance à Heliotis a rm ig era— gène de B.t., crylA(bJ— et à Spodoptera littora lis— gène de B.t., crylC. Mais, l'uti lisation de plantes synth étisan t une seule to xin e de
B .t. peu t e n tra în e r l'a p p a ritio n de résistance chez
les insectes ciblés. Différentes stratégies peuvent être développées pour éviter l'apparition de cette résistance. Celle qui est étudiée par l'équipe CIRAD/INRA consiste à associer deux gènes codant pour des protéines entomopa thogènes à modes d'action différents : les toxines de B.t. et les in h ib ite u rs de p rotéases. Des coto nn iers transgéniques ayant intégré un gène de B.t. ou exprimant un gène d 'in h ib ite u r de pro téa se o n t é g a le m e n t été o btenus et o nt p ro d u it des g ra in e s. Plus de quatre ans après les premières obtentions de dicotylédones transgéniques, des riz transgéniques ont été produits. Cependant, les riz transgéniques régénérés présentent souvent des variations par rapport aux plantes conformes. L'application au transfert de résistance aux foreurs des tiges IC hilo suppressalis, M a lia rp h a sepa rate lla , Chilo
zacconius) pour les riz méditerranéens et tropicaux est
engagée par le CIRAD. La méthode de transfert utilise la tran sfo rm a tion des protoplostes. Dans l'ensemble, l'application du génie génétique aux plantes cultivées a été moins rapide que prévue, mais l'arrivée sur le marché de variétés transgéniques est imminente. La vulgarisation des variétés transgéniques devra être précédée de l'analyse de l'im p a c t de cette in tro d u c tio n sur les techniques culturales et sur l'environnement.
Mots-clés : cotonnier, riz, génétique, biotechnologie,
Bacillus thuringiensis, lutte contre les insectes, toxine,
inhibiteur de protéase.
