Báo cáo khoa học: "In vitro propagation of interspecific hybrids"
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- in Alnus In vitro of interspecific hybrids propagation H. Sbay, J. Guillot, P. Danthu D. Prat Laboratoire de Genetique des Populations d i4rbres Forestiers, ENGREF, 14, Girardet, F-54042 rue Nancy, France used: A. glutinosa, A. cordata, A. incana and Introduction A. rubra. The best performing and plastic hybrids (Prat, 1988) were studied: A. glutinosa Alnus species show promise for afforesta- x A. incana (GI), A. rubra x A. glutinosa (RG), A. cordata x A. glutinosa (CG) and A. cordata x tion and wood production, particularly on A. incana (CI).. ) poor soils, since they are fast-growing and Shoots cut from selected trees were soaked nitrogen-fixing trees. This allows mixed in fungicide (Benlate, 0.15%) for 24 h and then plantations with benefits to the main disinfected with calcium hypochlorite (7% for accompanying forest species by nitrogen 10 min) and kept on nutritive medium con- supply. The genus Alnus includes some taining sucrose for 1 day. Afterwards, shoots were disinfected with a mercuric chloride solu- fast-growing species adapted to various tion (0.1 %, for 10 min.). Nodes were separated ecological situations (Martin, 1985). Gene- in an anti-oxidative solution (2.8 mM dithiothrei- tic improvement programs are being de- tol, 2.8 mM cysteine hydrochloride, 2.8 mM veloped to produce effective clonal varie- citrulline, 2.5 mM sodium ascorbate and 0.1% ties able to grow under various ecological polyvinyl pyrrolidone 40 000) to avoid the browning of explants, and finally put into culture conditions. Controlled hybridizations (in- medium. Some aspects of in vitro culture were traspecific and interspecific) were carried tested to improve the techniques. out to obtain improved progenies from which trees will be selected. Field trials show good performance of interspecific hybrids (Prat, 1988). The latter should be Results propagated to confirm their superiority and then be distributed afterwards as selected clones. In vitro micropropagation is Basal culture medium for in vitro culture applied because of the poor development of cuttings. Three media were compared for the growth of shoots: woody plant medium (WPM, Lloyd and McCown, 1980), Mura- Materials and Methods shige and Skoog (1962) medium (MS), and Quoirin and Lepoivre (1977) medium (QL) supplemented with WPM micronu- Early selection of trees (at age 4 yr) was carried trient and addenda. Glucose (15 g ), I I- ’ out in progeny trials; 4 progenitor species were
- indolebutyric acid (IBA, 2.5 pM) and ben- glucose, fructose, galactose, sucrose, zylaminopurine (BAP, 2.5,uM) were added mannitol and sorbitol. Carbohydrates were to the semi-solid media. added to complete WPM supplemented with IBA (1.0 !M) and agar. Each tested clone (CI, CG and RG) grew the best on WPM. RG clones The most extensive growth and num- showed the least growth. The level of IBA bers of roots and leaves (Table I) were was reduced to 0.5 pM to avoid callus for- observed in media containing either glu- mation at the explant basis. The suppres- cose, galactose or fructose. Sucrose was sion of BAP allowed multiplication by elon- not the best carbohydrate source. The gation. height increment at the end of the experi- ment (2 mo) was significantly higher when the carbohydrate source was fructose. For Effects of carbohydrate source all other characteristics, the glucose (15 g medium was never )-containing 1 I- ’ different from the treatment inducing the Optimum carbohydrate source was re- ported by Crémiere et al. (1987) to vary by best performance. Glucose (15 g was ) 1 I- ’ species. Two clones (CI and GI) were test- thus the carbohydrate source retained, but ed with various carbohydrate sources: fructose (15 5 g.I-.I ) might be also retained.
- Effects of activated charcoal Conclusion The amount of activated charcoal (resus- Plants from in vitro multiplication were grown in the nursery and followed the pended after autoclaving) was tested up to 40 g!l-!. Shoot elongation and weight same development as seedlings, without plagiotropy. Clones may be produced from increment were stimulated by activated charcoal in the range 5-20 interspecific selected hybrids by in vitro g!l-1 for both culture, as was previously described for tested clones (CI and GI). pure species (Tremblay et al., 1986; Cré- The effects of sedimentation and auto- miere et al., 1987). Gi, RG, CI and CG claving of activated charcoal were also clones will soon be subjected to clonal analyzed. The supernatant had no effect trials, prior to afforestation with selected on the growth of shoots. The significantly clones. largest growth and numbers of roots and leaves were observed when activated charcoal (5 g.¡- was resuspended after ) 1 autoclaving the media. References The addition of gibberellic acid (GA , 3 1.5 pM) to activated charcoal had no Cremiere L., Sbay H. & Prat D. (1987) In vitro culture of Alnus species. Acta Hortic. 212, 543- effect on shoot elongation. Without acti- 546 vated charcoal, GA caused a high death 3 Lloyd G. & McCown B. (1980) Commercially- rate of explants. feasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot-tip culture. Proc. Int Plant Prop. Soc. 30, 421-427 Acclimatization to greenhouse conditions Martin B. (1985) Les aulnes. AFOCEL- ARMEF Info. For6t 268, 177-191 Murashige T. & Skoog F. (1962) A revised Rhizogenesis of shoots was induced in medium for rapid growth and bioassays with vitro by IBA (0.1-10.0 ,uM) without acti- tobacco tissue cultures. Physiol. Plant. 15, 473- vated charcoal. More than 95% of the 497 shoots from CI and Gi clones were rooted Prat D. (1988) Interet de I’hybridation interspéci- within 2 wk. fique et de la multiplication vegetative: le cas de I’aulne. In: Actes 2 Colloque Sciences et e Rooted plants were then transferred into Industries du Bois. Tome 1, Arbolor, Nancy, pp. the greenhouse on a double-layer substra- 161-168 tum (a layer of vermiculite on a layer of Quoirin M. & Lepoivre P. (1977) Etude de fertilized peat and pine bark) allowing fast- milieux adapt6s aux cultures in vitro de Pru- nus. Acta Hortic. 78, 437-442 er growth of progressively acclimated Tremblay M.F., Perinet P. & Lalonde M. (1986) plants. Unrooted plants did not grow; Tissue culture of Alnus spp. with regard to sym- auxin application at the time of transfer bioses. In: Biotechnology in Agriculture and into the greenhouse did not induce Forestry, Trees vol. I. (Bajaj Y.P.S., ed.), Sprin- enough roots. ger-Verlag, Berlin, pp. 87-100
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