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This work shows that overexpression of the WUS gene from Arabidopsis enhanced the expression of embryogenic competence and triggered organogenesis from some cells of the regenerated embryo-like structures. Agrobacterium-mediated genetic transformation of cotton was described in the late 1980s, but is still time consuming and largely genotype dependant due to poor regeneration. To help solve this bottleneck, we over-expressed the WUSCHEL (WUS) gene, a homeobox transcription factor cloned in Arabidopsis thaliana, known to stimulate organogenesis and/or somatic embryogenesis in Arabidopsis tissues cultured in vitro. The AtWUS gene alone, and AtWUS gene fused to the GFP marker were compared to the GFP gene alone and to an empty construct used as a control. Somatic embryogenesis was improved in WUS expressed calli, as the percentage of explants giving rise to embryogenic tissues was significantly higher (×3) when WUS gene was over-expressed than in the control. An interesting result was that WUS embryogenic lines evolved in green embryo-like structures giving rise to ectopic organogenesis never observed in any of our previous transformation experiments. Using our standard in vitro culture protocol, the overexpression of AtWUS in tissues of a recalcitrant variety did not result in the production of regenerated plants. This achievement will still require the optimization of other non-genetic factors, such as the balance of exogenous phytohormones. However, our results suggest that targeted expression of the WUS gene is a promising strategy to improve gene transfer in recalcitrant cotton cultivars.

This study was conducted over a period of 20 years, to assess the problems involved in developing subcultures over a very long period, of oil palm (Elaeis guineensis Jacq.) somatic embryos which were maintained in vitro on a Murashige and Skoog mineral-based culture medium, without growth regulators. Analysis of the proliferation rate of the embryogenic cultures, along with the survivability of the regenerated plantlets after their transfer into soil and of the flowering of the derived adult palms has been conducted for cultures maintained in vitro during 1 to 20 years. From the ninth year of maintenance, the tissue quality of the somatic embryos gradually began to decline. However, after more than 20 years, 30% of the 20 clones tested still continued to proliferate satisfactorily on the same maintenance medium, keeping their multiplication potential intact. Even though a depressive effect of the age of the lines has been observed on the survival capacity of plants under natural conditions, it is noteworthy that among the clones originating from 20-year-old cultures only eight of them (40%) have exhibited the "mantled" floral abnormality. Different hypotheses concerning the origin of the disruptions observed on the in vitro cultures, plantlets and adult palms that occur over a very long period of in vitro conservation are discussed.

Suspension cultures of oil palm (Elaeis guineensis Jacq.) were established in a medium with 80 or 100 mg.l(-1) 2,4-dichlorophenoxyacetic acid and 1 g.l(-1) activated charcoal, from calli producing embryogenic cells and protruding proembryos. The suspension was composed of meristematic clumps, breaking away and giving rise to new smaller aggregates. Under the best conditions, the initial weight increased about 4 fold in one month. Embryo differentiation was achieved when plating the clumps either directly, or after a phase in hormone-free liquid medium. In the second case, early maturation occurred in liquid medium. Secondary embryogenesis was reduced, and up to 18.1 % embryos formed shoots. Rooted plants were sucessfully transferred to soil.

Requ le 28 janvier 1991 BUFFARD-MOREL, J., VERDEIL, J.-L., et PANNETIER, C. 1992. Embryogenbse somatique du cocotier (Cocos nucifera L.) a partir d'explants foliaires : Ctude histologique. Can. J. Bot. 70 : 735-741. Une Ctude histologique a Ct C rCaliste aux diffkrentes Ctapes de l'embryogenbse somatique du cocotier (Cocos nucifera L.) i partir d'explants foliaires. Elle a permis de localiser les premibres divisions cellulaires au niveau des cellules pCrivasculaires des explants. La croissance des cals nodulaires obtenus est assurCe par la mise en place d'une assise mCristCmatique a fonctionnement de type cambial. La nature racinaire de certaines formations a t t t observCe. Des structures embryogbnes peuvent apparaitre sur les cals nodulaires; entibrement mCristCmatiques au depart, elles ~'Cpidermisent et se multiplient pour la plupart. Leur Cvolution peut conduire i des structures de type embryon aprks fragmentation et contagion mitotique au niveau de l'assise de type cambial. Ce schema general d'obtention de structures embryonnaires peut cependant varier : (i) Soit il y a obtention rapide d'embryons a partir des cals nodulaires primaires. Cette voie a permis la rCgCntration, par embryogenbse secondaire, d'un massif d'embryons (bourgeonnement au p61e racinaire). L'un d'entre eux a dCveloppC une pousse feuillCe et des racines. La plante rCgCnCrCe a Ct C transfCrCe en champ. (i) Soit il y a formation de cals granuleux secondaires et production de f a~o n plus systtmatique de structures embryogbnes. Ces derniers devraient permettre une meilleure maitrise du dCveloppement des structures embryonnaires. Mots c l b : Cocos nucifera L., histologie, embryogenbse somatique, explants foliaires. BUFFARD-MOREL, J., VERDEIL, J.-L., and PANNETIER, C. 1992. Embryogenkse somatique du cocotier (Cocos rz~rcifera L.) a partir d'explants foliaires : Ctude histologique. Can. J. Bot. 70: 735 -74 1.Histological studies were carried out at different stages of somatic embryogenesis of coconut (Cocos nucifera L.) from leaf explants. The primary formations resulted from mitotic divisions of perivascular cells. Differentiation of a cambium-like layer insured the growth of nodular calluses. The root origin of some formations was observed. Embryogenic structures appeared on some nodular calluses maintained in culture; initially they were highly meristematic and often developed an epidermis and divided. Then embryo-like formations could be obtained after fragmentation of the cambium-like layer producing meristematic areas. However, modifications of this scheme could be observed: ( i ) Embryos rapidly developed from a primary callus, and from these embryos, a secondary embryogenesis was induced leading, at the root pole, to a clump of somatic embryos. One of the latter developed a shoot and roots; the regenerated plant was then transferred to the field. (ii) Secondary granulous calluses producing larger quantities of embryogenic structures were formed. This material should prove useful for mastering the technique of developing embryogenic stru...

Summary: The dissolution of cotton fibres has been studied at different development stages before and after the onset of secondary wall deposition in solvents of varying quality. We show that the dissolution of the primary wall is inefficient even in good solvents. In moderately good solvents, the inside of the secondary wall dissolves by fragmentation, whereas the outside of the secondary wall swells. These data demonstrate the existence of a centripetal radial gradient in the dissolution capacity within the fibre, which must be related to age-dependent structural variation in the cell wall layers.

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