Biotechnological support for varietal extension of sugarcane

for varietal extension

of sugarcane

F. PAULET, J.-C . G L A S Z M A N N CIRAOCA, BP 5035, 34032 Montpellier Cedex 1, France

Sugarcane axillary bud sprouts. Photo R. Fauconnier

Biotechnical advances

have benefitted

sugarcane varietal

extension and exchange.

An in vitro sugarcane

collection has been set up

to enable quick access

to top agronomic quality

and certified healthy

plant material.

S

ug a rc a n e is m u l t i p l i e d fro m c u ttin g s and each v a r ie t y is a clone. H ybridizatio n to crea­ te new sugarcane varieties is a parti­ cularly long and expensive process. This can be explained on a genetic b a s is . S u g a r c a n e v a r i e t ie s h a v e a h i g h l y c o m p l e x g e n o m e , w i t h 100-130 chromosomes derived from several d iffe re n t ancestral species. Each cross c o m p le te ly destructures the parental traits, and very fe w of th e p ro g e n y have any a g r ic u ltu r a l potential. Evaluating this potential is also complex. The sugarcane growth cycle includes several harvests, w ith an i n i t i a l p la n t stage t h a t o c c u r s a year after the cuttings are planted

o u t. T here are several subse qu en t ratoons, b ut the traits are not very correlated between stages, especially between the plant stage and succes­ s iv e r a t o o n s . M o r e o v e r , p e r f o r ­ mances at the same stage can vary d epending on the e n v iro n m e n t and t h e y e a r . T h is m e a n s t h a t v e r y com p lica ted assessment procedures are required when breeding for high performance clones.

D if f e r e n t p la n t b r e e d in g s ta tio n s w o rld w id e produce varieties that are adapted to their regions. These certi­ fied materials can be of prime interest as progenitors in other variety im pro­ vement programmes. They are some­ t im e s f o u n d to be w e l l a d a p te d

sugarcane

in v itro

culture

Storage of

in vitro plantlets a t 18 °C. Photo CIRAD-CA

to areas b e y o n d those w h e re th e y were originally bred. This means that they can be used for varietal renewal at stations that do not have their o wn s u g a rc a n e b r e e d in g p r o g r a m m e . Such exchange o f d iffic ult-to-b re ed s u g a rc a n e v a r i e t ie s r e p re s e n ts a m ajor advance.

Sugarcane plant material is transpor­ ted as cuttings w h ic h can be carrying diseases o r parasites. Since 1 978, the International Q uarantine Service fo r sugarcane, hosted by the Centre de c o o p é r a t i o n i n t e r n a t i o n a l e en r e c h e r c h e a g r o n o m i q u e p o u r le d é v e l o p p e m e n t ( C I R A D ) in M o n tp e llie r , has been d is p a tc h in g s u g a rc a n e v a r i e t ie s o f i n d u s t r i a l in te re s t, o r m a t e r ia l c lo s e to th is quality, to many countries. All mate­ rial is m onitored over tw o one-year p e r io d s to g u a r a n t e e t h a t it is disease-free. Clones are then sent out as cuttings to potential users (Baudin, 1 9 84 ). The a c t i v it y o f th is service is lim ite d to d is p a tc h in g m a te ria l, i.e. they are not able to keep material after it is released from q ua ra n tin e due to the limited facilities.

An in vitro sugarcane collection, was set u p d o w n s t r e a m f r o m th e Q uarantine Service in an attempt to deal w ith the above-mentioned lim i­ tations (Paulet et al., 1991 ).

In vitro colle ction s already exist for

some other crops such as potato and cassava (Rocca et al., 1989). This is an ideal means of preserving poten­ t i a l l y u s e fu l g e r m p l a s m w i t h o u t req uirin g m uch space, and enables rapid distribution of a broad range of certified healthy plants. This in vitro p la nt material can also be cytopre- served, a useful procedure for very long-term germplasm conservation. In additio n, enzyme electrophoresis and o th e r n e w m o le c u la r b io l o g y to ols are used for rigourous control of the collection.

Production

of in vitro plantlets

In v i t r o p la n t le ts are o b ta in e d by

culture o f axillary buds from healthy p la n t s , as d e s c r ib e d b y S a u v a ire & Galzy (1978).

Greenhouse maintenance

of stock material

C u t tin g s are ta k e n f r o m c e r t i f i e d healthy plants released from quaran­ tine. The cuttings are sprouted, pla n­ ted and maintained in a greenhouse. P r o p a g a tio n o f p la n ts b y c u ttin g s re ju v e n a te s th e m a te ria l and p r o ­ motes bud development during sub­ sequent in v itro c u ltu re . A ll o f this p l a n t m a t e r i a l is s y s t e m a t i c a l l y treated w it h fu n g ic id e s and p e s ti­ cides alternately on a weekly basis to h in d e r s a p r o p h y tic fung us attacks w h i c h c o u l d j e o p a r d i z e in v i t r o culture.

In vitro

culture

The youngest buds (3-4/cane) were collected fom 4-6 month old green- h o u s e - g r o w n s u g a r c a n e p la n t s ( M o n t p e l l i e r , France). T h e y w e re t h e n c u l t u r e d on MS m e d i u m

(Murashige & Skoog, 1962) in petri dishes at 25°C. They w ere s u b c u l­ tu red separately into c u ltu re tubes after 4-6 weeks w h e n n ew suckers a p p e a r e d . O n c e 10 w e l l - f o r m e d plantlets were available, they were again s u b c u lt u r e d s e p a ra te ly in to culture tubes and stored at 18°C on d ilu te d MS m e d iu m . These in vitro c o n d i t i o n s d e c e le r a t e p l a n t l e t growth, thus reducing the number of culture m anipulations required. The n u t r i e n t m e d i u m le v e l a n d le a f w ithering are monitored regularly to detect clones that should be subcul­ tured first. Subculture intervals vary from 6 months to 2 years, depending on the variety (suckering rates) and depletion of the medium.

Use of in vitro

plantlets

These in vitro plantlets can be used for rapid intro du ction o f new varie­ ties at sugarcane plantations. After a request is filed, cuttings can be made d ir e c t ly or they can u nd ergo rapid micropropagation (Feldmann & Rott, 1991).

Sugarcane bud 15 days after

in vitro subculture. Photo CIRAD-CA

A p e x sprout 1 0 days following cryopreservation. Photo P. Feldmann

state on culture m edium c ontaining no g ro w t h h o rm o n e s . H o w e v e r , a te c h n iq u e is still needed to reduce

in v itro p la n tle t m a n ip u la tio n s and

especially to lim it the need for cell m ultiplication under artificial co n d i­ tions. A cryopreservation te chnique has been developed in experiments w it h v a rie ty Co 641 5 (D e re u d d re , 1994), whereby apices encapsulated in alginate beads are frozen in liquid n itro g e n . The t e c h n iq u e w as s u c ­ cessfully tested on 16 different sugar­ ca ne v a rie tie s , w it h re g e n e r a t io n rates o f 1 4 - 9 1 % ( G o n z a le z A rn a o

e t a ! . , 1 9 9 3 ; Pa ule t e t a l . , 1 9 9 3 ).

Cryogenic preservation theoretically p e rm its in d e f in it e storage o f p la n t material.

Dispatching

in vitro

plantlets

W h e n a given variety is requested, some of the plantlets available at the M o n t p e l li e r in v itr o c o lle c t io n are placed at 25°C for m u ltip lic a tio n . It then takes 3-6 m o n th s to p ro d u c e a b o u t 10 n e w p la n tle ts by natu ral suckerin g. These pla ntle ts are then s u b c u lt u r e d o n to r o o tin g m e d iu m and dispatched. This m u ltip lic a tio n process can be continued if the user has a s u i t a b l e l a b o r a t o r y . T h e w aiting time to receive these in vitro plantlets is thus reduced to 6 months, whereas 2-3 years w o u ld be required to put the requested varieties through a new quarantine cycle.

Performance

of in vitro plantlets

W hen the in vitro plantlet is transfer­ red to soil, it is critical to f o llo w the recommended procedure in order to optim ize regrowth (cf. procedure for transferring in vitro sugarcane plant­

lets to soil). Planted out sugarcane in

vitro plantlets have a particularly dis­

tinct facies, differing from plants pro­ pagated b y c u ttin g s ( F e ld m a n n & Rott, 1991). This d is tin c tiv en es s is v a r i e t y - d e p e n d e n t and g e n e r a l l y expressed by increased tille rin g and

decreased cane diameters; yields are thus close to those obtained w ith cut­ tin gs. These d iffe re n c e s g r a d u a lly d is a p p e a r o v e r th e c ourse o f fie ld propagation.

Current status of

the in vitro collection

The CIRAD in vitro sugarcane c o lle c ­ t i o n in M o n t p e l l i e r (F ra n c e ) w as f o u n d e d in 1 9 8 2 . T he c o l l e c t i o n p r e s e n tly in c lu d e s 363 v a r ie t ie s , most o f w h ic h are established and stored at 18°C. Im p ro v e m e n ts are being investigated to deal w it h the increased size of the collection.

Varieties in the collection

The collection includes 363 varieties available upon auth orizatio n o f the corresponding breeding institutions (cf. list of sugarcane varieties stored at the in vitro collection).

Genetic stability and

cryopreservation technique

It seems that stored w h o le plant tis­ sues (i.e. w it h o u t d e d iffe re n tia tio n — buds, plantlets, etc.) can a p rio r i be maintained in a genetically stable

Controlling genetic

variation

To avoid varietal mislabellings, it is essential to be able to detect possible c o n f u s io n s t h a t c o u l d o c c u r as a result o f the many steps between the tim e the c lo n e arrives as a c u ttin g and its release as an in vitro plantlet. Enzyme electrophoresis can be used to assess clonal identity. A p ilot study carried out w ith 62 sugarcane varie­ ties revealed the presence o f 21 poly­ m orphic bands in some o f the varie­ ties, w h i l e abse nt in o the rs . Each v a r i e t y w as s e p a ra te ly c o m p a r e d w ith each of the other varieties (i.e. 1 891 c o m p a ris o n s ) and o n l y t w o pairs could not be distinguished; the band patterns o b ta ine d for each o f the other 58 varieties were unique. By t h i s t e c h n i q u e , e n z y m e s are e x tra cte d fro m y o u n g fresh leaves c u t f r o m an in v i t r o p l a n t l e t . C haracterization by restriction frag­ m en t length p o ly m o r p h is m (RFLP) D N A analysis is m ore c o m p le x but also much more precise; a fingerprint c a n th u s be o b t a i n e d f o r e a c h variety. RFLP analysis, in contrast to is o e n z y m a t ic te c h n iq u e s , can be p e r f o r m e d on d r i e d le a v e s . T h is m e a n s t h a t c l o n e s f r o m t h e M o n t p e llie r in v itro c o lle c tio n can be c o m p a r e d at a n y t i m e w i t h others preserved under tropical field conditions.

sugarcane

in v itro

culture

Conclusion

Biotechnology is sometimes used to create n e w u n iq u e p la n t varietie s, e . g . g e n e t i c t r a n s f o r m a t i o n . B i o t e c h n o l o g i c a l t e c h n i q u e s are e m p l o y e d to c e r t i f y th e g e n e t i c purity of germplasm conserved in the C IR A D s u g a r c a n e c o l l e c t i o n in M o n tp e llie r . Hence, hundreds and even thousands o f varieties can be sto re d in a s m a ll space — t o t a l l y p r o t e c t e d fr o m insects, diseases, clim atic hazards, w ith very little risk o f g e n e t i c d r i f t o r a c c i d e n t a l between-variety mixing. This c o lle c ­ tio n is o f m ajor interest for varietal e x te n s io n and s h o u ld p ro v e to be a key c o m p o n e n t fo r in te rn a tio n a l sugarcane germplasm conservation.

Genetic mapping of sugarcane varieties

Several steps are required fo r varietal characterization by restriction fragm ent length po lym orp hism (RFLP) D N A analysis (nuclear D N A is considered here, not cytoplasm ic D N A ):

- D N A extractio n;

- D N A digestion, i.e. restriction enzym e digestion o f D N A to cleave at w e ll-d e fin e d sites, thus fo rm in g fragments of different lengths;

- agarose gel electrophoresis separation o f fragments by length; - transfer of separated fragments on to nylon membranes;

- m olecular hyb rid iz a tio n w ith specific D N A probes (pre-labelled w ith phosphorus 32 fo r instance): each probe detects and binds to fragments on the m em brane that contain com p lem e ntary sequences;

- recognized fragments are then revealed by placin g the m em brane in contact w ith an a utoradiographic film w h ic h is then printed by the probe radioactivity;

Each clo ne is thus characterized by a set o f bands representing its fingerprint (photo). M an y different probes are available. W ith o n ly five probes, it is possible to distinguish more than 10 0 0 0 ran do m ly chosen varieties.

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Banding profiles obtained for 1 5 sugarcane varieties. For each variety, D N A w as digested by the Hind III restriction enzym e, fragments w ere separated by electrophoresis, and hybridization w as performed with the U M C 3 9 probe.

PhotoJ.-C. Glaszmann

m e d iu m in s te rile sealed p la s tic c u lt u r e tubes. A fte r s u b c u lt u r in g , t h e y are ke pt at 2 5 - 3 0 ° C f o r 3 -4 w e e k s b e fo re b e in g dispatched.

Removal from sterile conditions

This can be done once the rooted in vitro p la ntle ts have 5-6 roots th a t are 2-3 cm long. U n c a p th e c u lt u r e tubes, c a r e f u lly remove the plants, rinse the roots in water, clip off the leaves to reduce evaporation and stimulate bud sprouting.

Fungicide treatment

Dip the plantlets in a solution w ith Benlate (1 g/l fo rm u la te d product) or A lie tte (1 g/l formulated product).

Plantlets in Jiffy 7 under a minigreenhouse.

Rooted in vitro plantlets ready for planting in soil

Dipping the plantlets in a fungicide solution.

Transplanting

In v itro p lantlets are transferred to jif f y 7

(compressed peat pellets). First, soak the peat pellets for 10 min w ith the above des­ cribed fungicide solution. Pierce the pellet and s lip th e ro o ts i n t o t h e h o le . C lo s e the h ole and p ile th e peat up a ro un d the plantlet.

Transplanting plantlets in Jiffy 7.

Soil planting

conditions

Place the ready-to-plant in vitro p la n tle ts o u t o f th e w in d and sun. C o v e r th e Jiffy 7 p e lle ts w i t h c l e a r p l a s t i c to h i n d e r drying and leave them covered until 2-3 new leaves are produ­ ced. The plantlets can be misted

if any w ithering is noted. Water the plantlets daily, w ith a leaf- t y p e f e r t i l i z e r s u p p l e m e n t (e.g. M a i r o l , 2 g/l fo rm u la te d product) after 2-3 weeks. Spray t h e p l a n t l e t s w e e k l y w i t h B a y le to n 5 (1 g/l f o r m u la t e d p r o d u c t ) to c o n t r o l f u n g a l attacks.

Field planting

T h is ca n be d o n e a f t e r 4^6 w ee ks if th e p la n tle ts are ■grow ing v ig o u r o u s ly . D ip the J iffy 7 p e l l e t a nd p l a n t l e t in B a y le to n (1 g /l), p re p a re th e p la n t in g site, d ig h o le s large enough to plant the peat pellets. D o n o t d a m a g e th e p la n t in g b lo ck or roots. Place the Jiffy 7 p e l l e t s in th e s o il d i r e c t l y .

C lip the leaves slightly to reduce drying. If the pla ntle ts are not very v ig o u ro u s after 6 weeks in Jiffy 7, transfer them to pots with more substrate to supplement the depleted Jiffy 7. Treat the plantlets w it h a systemic insecticide (Curater).

The transfer to soil should be c arried o u t w he n the soil temperature is above 1 8°C. H igh atm ospheric h u m id ity is favourable. The plantlets should be misted periodically, even if the fields are irrigated.

Photos P. Feldmann Greenhouse plantlets, 6-8 weeks old,

ready for planting.

Sugarcane varieties

in the

in vitro

collection

Laboratoire de culture C IR A D -C A , BP 5 0 3 5 in vitro,

3 4 0 3 2 M o n tp e llie r C e d e x 1, France.

B37172 Co31 2 CP52-43 CP72-356 L60-25 Ph i 156226 RB705007 T u c 6 8 -1 8

B4362 Co331 CP52-48 CP72-370 L62-96 P hil58260 RB705146 T u c 6 8 -1 9

B46364 C o 4 1 9 CP57-603 CP72-1 312 L65-69 Ph i I6553 RB705375 T u c 6 9 -1 17

B47258 Co421 CP59-73 CP73-351 L66-97 Ph i I6559 RB735220 T u c 7 1 -5

B49119 Co449 CP60-01 C P 73-1547 L72-85 Ph i I6607 RB735275 Tuc72-1 6

B 5 1 129 Co462 CP62-258 CP74-383 LF63-863 POJ2878 RB748022 Tuc72-23

B52107 Co527 CP63-306 CP74-2005 LF66-291 8 PR1007 RD7410 Tuc74-1

B60125 Co740 CP63-588 CP77-414 M 3 1 /4 5 PR1016 RP148-70 T uc74-6

B63118 Co775 CP65-357 CRA6026 M 56 9 /6 9 PR61632 S17 T u c 7 4 -1 0

B63119 Co785 CP66-31 5 EAK7076 N11 R397 SIP58-1 36 Tuc74-24

B64277 Co842 CP66-346 FR80234 N12 R469 SP70-1005 T uc74-26 C 3 34-64 C0 IOOI CP67-41 2 FR80236 N14 R472 SP70-1078 T uc74-34 C323-68 C o l 148 CP 67-413 FR80412 N15 R526 SP70-1081 T uc74-46 CB36-24 C0 I I 57 CP670 IAC4865 N 16 R566 SP70-1 284 Tuc75-1 CB40-1 3 C0 I I 77 CP68-350 IA C 51205 N 17 R567 SP70-1423 Tuc75-2 CB41-76 C0 I 202 CP68-1022 IA C 52 150 N18 R568 SP70-1478 T uc75-3 CB45-1 55 C0 I 23 O CP68-1026 IAC58480 N19 R570 SP70-3225 U C W 5 4 6 5 CB46-47 C062175 CP68-1067 IAC64257 N5 22 19 R572 SP70-3370 W I8 2 7 7 7 CB47-1 5 C o6304 C P 6 8-1154 Ja59-03 N 5 3 2 1 6 R70367 SP71-799 CB47-355 C o 6 4 1 5 CP69-1059 Ja64-11 N A 5 6-7 9 R7417 SP71-6163 CB 49-260 C0 6 8 O6 C P 7 0-1133 Ja64-15 N C o 3 1 0 R75631 SP72-4928

CB53-98 CoS443 CP72-353 Ja64-20 N C o334 RB7096 SP75-179

CB56-1 26 CoS510 CP72-355 KW T57-423 N C o376 RB70194 Tuc67 -27

B6504 B7695 B7882 B 7 9 1 18 H 5 6 -2 7 8 M 6 5 7 /6 6 M Y 64 -26 Q 1 2 6 B6623 B76102 B78130 B79130 H 5 6-48 48 M 3 0 3 5 /6 6 NI1 Q 1 2 7 B69379 B76113 B78178 B79226 H 5 7-51 74 M 69 5 /6 9 NIF3 Q 1 2 9 B69566 B76132 B78224 B8007 H 5 9-37 75 MEX64-1 214 N IN 2 Q 1 3 0 B69758 B76146 B78237 B8008 H62-4671 M E X 65-1424 Q 7 5 Q 1 3 4 B70442 B76181 B78242 B8066 H 6 5-70 52 M E X 66-1247 Q 8 4 Q 13 5 B70462 B76196 B78244 B8093 H 6 6-49 27 M E X 68-200 Q 9 0 Q 1 3 7 B70482 B76247 B78245 B80276 H 6 8 - 1 1 58 M EX69-290 Q 95 Q 1 3 8 B70520 B76398 B78249 B80361 H 6 8-22 35 MEX70-485 Q 9 6 RK65-37 B70531 B7784 B78266 B80689 H 6 9-82 35 M 0 I4 5 -O3 Q 10 2 SES14 B70532 B7795 B78299 BJ7013 H 6 9 -9 1 03 M Q 7 2 -1 1 7 5 Q 10 3 SES231 B72191 B77123 B78358 F146 H 7 0-14 4 M Q 7 2 -4 0 0 5 Q 1 0 8 US56-1 5-8 B74254 B77126 B78360 F151 H 7 2-85 97 M Q 7 2 -5 0 0 6 Q 1 0 9 G aloa B74359 B77392 B78366 F156 H 7 3 - 6 1 10 M Q 7 2 -5 0 8 9 Q 1 1 0 Kaba B74477 B77415 B78436 F160 H7 5-87 76 M Q 7 6 -2 3 Q111 M ali B75300 B77565 B78482 F161 H 7 7-66 94 M Q 7 6 -5 3 Q 11 3 Mana B75412 B77639 B78560 F167 H 7 8-72 34 M Y 53 -53 Q 1 1 4 O n o B75519 B77740 B78604 F175 HJ57-41 MY53-1 73 Q 11 5 Tabongo B75524 B7802 B78628 F176 M 5 74/62 M Y 54 -62 Q 1 1 7 Triton B75527 B7814 B78697 F178 M 2 3 7/62 M Y 5 4 -1 29 Q 1 2 0 Trojan B75532 B7852 B78700 H 3 7 -1 933 M 21 73 /6 3 M Y 55 -14 Q121 Uba Naguin B7639 B7877 B7997 H 5 0-72 09 M 3 76/64 M Y 57 -15 Q 1 2 4 Agriculture et développement Special issue- December 1 9 9 4

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Abstract... Resumen... Résumé

F. PAULET, J.-C . G L A S Z M A N N - Biotechnological support for varietal extension of sugarcane. B io tech no lo gica l a d va n ce s h a v e b e n e fit t e d su g a rc a n e va rie tal extensio n an d ex ch ang e. An in vitro sugarcane collection has no w b e en set up as a s u p p le m e n t to the "International Q ua rantine Service" org an ized by CIRAD in M o n tp e llie r (F r a n c e ). In vitro su cker r e g e n e r a tio n and latency conservation o f the resulting plantlets has been developed. Almost 4 0 0 varieties or clones ( 1 0 specimens o f e a c h ) h a v e no w been sto red. This in vitro collection does not ta k e much space an d enables distribution of a broad ron ge o f he althy plants rapidly. This in vitro plant m aterial can also be cryopreserved, a useful procedure for genetic resource co ns erva tio n. E n zy m e electrophoresis an d o th e r m o le c u la r b io lo g y tools (RF LP ) a r e used to control the collection, thus g u aranteein g its reliability. Key words: Saccharum, sugarcane, do ne, in vitro culture, m olecular biology, e n zy m e electrophoresis, cryopreserva- tion, in vitro collection, quarantine.

F. PAULET, J.-C. G LA S ZM A N N - Las biotecnologías como ayuda a la difusión de variedades de caña de azúcar.

La diffusion y los intercambios de variedades de caña de azúcar sacan provecho hoy de los progresos realizados en biotecnología. Como com plem ento del servicio de "cuaren­ t e n a i n t e r n a c i o n a l " o r g a n i z a d o p o r e l C IR A D en M o n t p e llie r , se ha e m p r e n d id o la constitución de un a colección de caña de azúcar in vitro. Se ha llevado a cabo la regeneración in vitro de ye m as axilares y la conserva­ ción en letargo de las plántulas obtenidas. Actualm ente se tienen alm acen ad as cerca de 4 0 0 variedades (o clones), con 1 0 muestras por va rie da d. Esta "vitroteca" ocupa un espacio reducido y pe rm ite suministrar ráp id am e n te plan­ tas sanas pa ra una am plia g a m a de m ate ria l. Esta fo rm a

in vitro tam b ié n posibilita la aplicación de la crioconserva- ción, que es útil para la preservación de recursos genéti­ cos. La utilización de la electroforesis de enzim as y de las n u e v a s h e r r a m ie n t a s de la b io lo g ía m o le c u la r (RFLP) refu erza el control y la fiabilidad de la colección. Palabras clave: Saccharum, caña de azúcar, clon, cultivo

in vitro, bio lo g ía m o le c u la r, e lec trofore sis e n z im á tíc a , crioconservacíón, vitroteca, cuarentena.

F. PAULET, J.-C. GLASZMANN - Les biotechnologies en soutien à la diffusion variétale chez la canne à sucre.

D iffu s io n et éc ha nge s v o r ié t a u x chez lo t a n n e à sucre bénéficient au jourd 'hu i des progrès en biotechnologie. En co m plém ent du service de « qu ara ntaine intern ationale » organisé par le CIRAD à Montpellier, il a été entrepris de constituer un e collection de conne à sucre in vitro. La régénération in vitro de bourgeons axillaires et la conser­ vation en vie ralentie des plantules obtenues ont été mises au point. Près de 4 0 0 variétés (ou clones) sont m ain tenant sto c k é e s à ra is o n d e 1 0 é c h a n t illo n s c h a c u n e . C ette « vitrothèque » occupe un espace réduit et pe rm et de fo u r­ nir rap id em e nt des plants sains pour une large g a m m e de m a té r ie l. Celte f o r m e in vitro ren d possible é g a le m e n t l'application de la cryoconservation, utile pour la préser­ vatio n des ressources génétiques. Le recours à l'électro- phorèse d 'enzym es et ou x n o uv eau x outils de la biologie m oléculaire (RFLP) vient renforcer le contrôle et la fiabili­ té de la collection.

Mots-clés : Saccharum, canne à sucre, clone, cu ltu re in vitro, biologie m oléculaire, électrophorèse en zym atiq u e , cryoconservation, vitrothèque, qu ara ntaine .