Báo cáo lâm nghiệp: "Walnut somatic embryogenesis: physiological and histological aspects"

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Walnut somatic embryogenesis: physiological and histological aspects D. Cornu Amdlioration des Arbres Forestiers, INRA Ardon, 45160 Olivet, France The aims of this work are: 1) to deter- Introduction mine the developmental and physiological stages able to give rise to somatic Somatic embryogenesis in plant tissue embryogenesis; and, 2) to control histo- cultures has been reported for many spe- logically the true nature of somatic cies. For mass cloning, embryogenesis embryogenesis at an early stage. could be better than organogenesis because it directly produces full plants in- stead of shoots that have to be rooted. Materials and Methods Thus, one major problem in plantlet pro- duction could be bypassed. Moreover, embryogenesis will be a better way to nuts were provided by Mr. Germain The (INRA-Arboriculture, Bordeaux) from a half-sib apply other techniques, such as protoplast family collected on black walnut (NG 23), which fusion or gene transfer. naturally produces a high level of hybrid nuts. Recently, the induction of somatic Nuts (40 at each time) were collected from the end of June to early October at 2-3 week inter- embryogenesis has included more and vals. They were not cold stored and culture woody species, important tree more began not more than 5 days after collection. conifers and hardwoods (for a recent For all experiments, we used media de- review see Tulecke, 1987). For the scribed by Tulecke and McGranahan (1985). Juglandacea family, somatic embryos For the conditioning step, we also used the medium defined by Gupta and Durzan (1986) have been obtained with Juglans regia, J. for spruce. Cotyledon sections were prepared hindsii and Pterocarya (Tulecke and and cultivated as described previously (Cornu, McGranahan, 1985), Catya illinoensis 1988). (Merkle et al., 1987), Juglans nigra, J. For histological studies, suitable tissue major and interspecific hybrids J. nigra x samples were fixed in a formaldehyde-acetic acid-ethanol mixture, dehydrated, embedded in J.regia (Cornu, 1988). As for many other paraffin, sectioned and finally stained with hardwoods, all results obtained with were safranin or fast green. For very soft callus tis- immature seeds and lot of abnormal a sue, small pieces were directly stained with frequently structures embryos or were safranin and observed after squash prepara- observed. tion.
Results and Discussion ible cells in somatic embryogenesis. cannot be certain that Consequently, we culture conditions were optimal for our For all collection times, we have obtained somatic embryos for more generating somatic embryogenesis only at the begin- samples. In terms of growth regulators, ning of August and in one case in Septem- auxins (normally not necessary for walnut ber (Table I) and only on the Tulecke d nu micropropagation) are needed for the McGranahan medium. This reactivity is early conditioning medium (0.01 mg/I of connected with a particular stage of devel- indole butyric acid). This is in accordance opment of the nuts and occurs when the with the results of Label and Cornu cotyledons show a high growth rate before (1988), who found a particularly high they fill the locule. No cultures initiated concentration of indole acetic acid in the from very early embryo stages, endo- liquid endosperm at the corresponding sperms or mature embryos generated stage of development. somatic embryos. This confirms the pre- vious observations obtained on the During the first months of culture, the reactivity of immature nuts of other walnut lines gave compact white embryogenic species (Tulecke and McGranahan, 1985; calluses similar to the original cotyledons. Cornu, 1988). These calluses, were embryogenic but in For Juglans regia the best stage for very limited areas. The adventive somatic obtaining embryogenesis corresponds, in embryos are frequently abnormal and do percentage of dry weight, to the highest not complete their development to plant- content in protein and to the beginning of lets. Progressively, new calluses appear the decrease in soluble sugar and the which are more irregular, soft, crumbly, increase in oil content (Labavitch and Poli- moussey and which grow actively. In these to, 1985). The interaction of the metabolic calluses, we can distinguish different kinds content of cotyledons and the culture of cells: 1) large elongated cells with a medium could, in fact, play a major role in large vacuole; and, 2) small dense cells, the induction or the development of induc- like meristematic cells which gather to-
stages of development of the zygotic macro-calluses. The in micro- gether or embryo could be worthwhile in such inves- structure of the callus depends directly upon the ratio of these 2 types of cells. tigations. The dense calluses show a very high capacity for embryogenesis. They gen- erate large clusters of embryos at different References stages of development (from the globular to the torpedo and even early cotyledona- Becwar M.R., Wann S.R., Johnson M.A., Verha- ry form). At this late stage, embryos could gen S.A., Feirer R.P. & Nagmani R. (1988) be isolated, but to date only 20% complete Development and characterization of in vitro their in vitro development with root and embryogenic systems in conifers. In: Somatic shoot growth. This heterogeneous callus Cell Genetics of Woody Plants. (Ahuja M.R., ed.), Kluwer Academic Publ., pp. 1-18 8 development is similar to that described Cornu D. (1988) Somatic embryogenesis in tis- for conifers (Becwar et aL, 1988) except sue cultures of walnut (Juglans nigra, J. major that elongated cells seem not to be active and hybrids J. nigra x J. regia). In: Somatic in the process of somatic embryogenesis Cell Genetics of Woody Plants. (Ahuja M.R., (suspensor-like function). Finally, this kind ed.), Kluwer Academic Pubi., pp. 45-49 of callus could be used to initiate agitated Gupta P.K. & Durzan D.J. (1986) Plantlet re- liquid cultures, which are easier to handle generation via somatic embryogenesis from subcultured callus of mature embryos of Picea and more efficient for producing better and abies (Norway spruce). In vitro 22, 685-688 homogeneous somatic embryos. Labavitch J.M. & Polito V.S. (1985) Fruit growth and development. !n: Watnut Orchard Manage- ment. University of California, Pubi. no. 21410; pp. 90-94 Conclusion Label P. & Cornu D. (1988) Determination of plant growth substances in liquid endosperm of immature walnut (Juglans nigra) nuts by an ELISA technique. Plant Growth Regul. 7, 209- We have obtained true somatic embryos 2155 in hybrid walnut. Our results indicate that Merkle S.A., Wetzstein H.Y. & Sommer H.E. there may be an optimal period of zygotic (1987) Somatic embryogenesis in tissue cul- embryo development for the generation of tures of pecan. HortScience 22, 128-130 Not all zygotic embryos somatic embryos. Tulecke W. (1987) Somatic embryogenesis in Further investigations are need- respond. woody perennials. In: Cell and Tissue Culture in Forestry, Vol. 2 (Bonga J.M. & Durzan D.J., ed to determine if an optimum physiolo- eds.), Martinus Nijhoff Publ., Dordrecht, pp. 61- gical ’window’ exists for initiating em- 91 bryogenic cultures, or if other factors Tulecke W. & McGranahan G. (1985) Somatic (particularly medium composition) limit the embryogenesis and plant regeneration from cell redetermination of more mature mate- cotyledons of walnut, Juglans regia. Plant Sci. rial. Analysis of liquid endosperm at all 40, 57-63