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Báo cáo khoa học: "Walnut (Juglans regia L.) micropropagation"

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Walnut (Juglans regia L.) micropropagation M.A. Revilla, J. Majada R. Rodriguez Fisiologia Vegetal, Universidad de Oviedo, Espana Lab. ed for 5 min in 0.5% N solution followed by CIO A Introduction 5 min in 75% ethanol and 3 rinses in sterile distilled water. Embryonic axes were cultured for 8 wk before excising the nodal segments. problems involved in wal- One of the main Juvenile material was taken from 2-3 mo old micropropagation by tissue culture nut plantlets germinated under greenhouse condi- techniques is the low rate of multiplication. tions that had been sprayed every 15 d with a This can be due to the slow growth of the solution of 0.04 g/l kasugamicin (Lainco), regenerated shoots leading to extended 0.97 g/l zineb (Agrocros) and 0.38 g/l cupric culture periods and thus resulting in the oxychloride (Agrocros). Before taking the explants from the juvenile material, the plantlets appearance of latent contamination in the were sprayed 2 or 3 times, every 5 d, with a culture (Somers et al., 1982; Driver and solution of 100 mg/i BAP (benzylaminopurine) Kuniyuki, 1984; McGranahan ef al., 1986). and 50 mg/i GA (gibberellin) (McGranahan et 3 To overcome this problem, we have tried al., 1988) to induce vigorous growth. to culture nodal segments from embryonic The medium used was MS (Murashige and and juvenile material in a double-phase Skoog, 1962), supplemented with 30% sucrose, 0.7% agar and different combinations of BAP system, which has been shown to in- (1-5 mg/1), IBA (indole butyric acid, 0.1 mg/1), production of axillary shoots crease IAA (indole acetic acid, 0.05 mg/1) and GA 3 (Viseur, 1985), and to include in the cul- (0.1-1 mg/1). The culture conditions were 16 h ture medium antibiotic mixtures to prevent photoperiod and 25°C. To decrease explant exudation, they were transferred onto fresh bacterial contamination (Phillips et al., medium 1, 3, 5 and 8 d after culture (Driver and 1981; Young et al., 1984). This research is Kuniyuki, 1984; McGranahan et al., 1988). For being conducted to optimize the micropro- double-phase culture, sterile liquid medium was pagation techniques for walnut (Juglans poured over explants after their transfer onto regia L.). the solidified medium. The liquid phase always covered the solid medium surface. Rooting was initiated by dipping the shoot base into the liquid medium containing IBA (2 mg/1) for 24 h and transferring it onto solidifi- Materials and Methods ed medium containing 1 °!° activated charcoal. The addition of some antibiotic mixtures to the culture medium allowed the recovery of a Experiments have been made with embryonic high percentage of contaminated explants. The and juvenile nodal segments of walnut. levels of antibiotics tested were as follows: Embryonic axes were excised from seeds pre- cefotaxim 25-75 mg/l, tetracycline 25 mg/l, viously imbibed for 24 h in water and disinfect-
rifampicin 6 mg/l, streptomycin 1-2 mg/I, ampi- double-phase and plant growth stimula- cillin 25mg/I. Antibiotic solutions were filter ste- tion, will improve the proliferation rate in rilized. walnut. Sixty percent of the shoots regenerated from embryonic material produced roots. Similar rooting conditions were used by Results and Discussion Meynier (1985) for hybrid walnut. Micropropagation Effect of antibiotics shoot cultures on The pretreatments given to the plant The best growth regulator combinations material together with the surface steriliza- for micropropagation of J. regia L. from tion of the explants allowed the recovery, nodal segments of embryonic or juvenile after 15 d of culture, of 85% of the material were 1.0 mg/i BAP and 0.1 mg/l explants. However, latent endogenous IBA. The same results were shown for contamination appeared after 1 or 2 mo of Juglans nigra (Somers et aL, 1982) and culture resuming in only 5% final recovery Paradox (Driver and Kuniyuki, 1984). of the explants. Higher BAP concentrations, for short cul- periods, produced morphological ture In an attempt to solve this problem, anti- modifications in leaves and shoots, and biotic mixtures were added to the initial finally induced vitrification. The application culture medium or to the fresh medium of 0.1 mg/I GA induced greater elonga- 3 used for transfers. tion in the embryonary shoots but had no In the first case, the addition of cefotax- effect on the juvenile shoots. im (25 mg/I), tetracycline (25 mg/I), rifam- The establishment of the explants in picin (6 mg/I) and streptomycin (2 mg/I) (mixture A) allowed the recovery, after 9 double-phase cultures increased the wk of culture, of 50% of the explants. The micropropagation rate for both types of use of cefotaxim (25 mg/I), tetracycline plant material (Table I). Similar results were observed for juvenile material when (25 mg/I) and ampicillin (25 mg/I) (mixture the plantlets in the greenhouse were stim- B) reduced the recovery to 38%. Higher ulated with growth regulator solutions. concentrations of cefotaxim induced Presumably, the use of both treatments, necrosis and death of all the explants.
after 12 wk of culture References Explants transferred to a medium without antibiotics showed new growth of bacterial contamination. Driver J.A. & Kuniyuki A.H. (1984) In vitro prop- Therefore, the bacteriostatic effect pre- agation of Paradox walnut Juglans hindsii x Juglans regia rootstock. HortScience 19, 507- dominated one. No the bactericidal over 509 morphological differences observed were McGranahan G.H., Leslie C.A. & Driver J.A. between control and antibiotic-treated (1988) In vitro propagation of mature Persian explants. walnut cultivars. HortScience 23, 220 Experiments made with contaminated McGranahan G.H., Tuleke W., Arulsekar S. & Hansen J.J. (1986) Intergeneric hybridization in explants, already established in a medium the Juglandaceae: Pterocarya sp x Juglans without antibiotics, showed that, when regia. J. Am. Soc. Hortic. Sci. 111, 627-630 these were subcultured in fresh medium Meynier V. (1985) Mise en culture in vitro de with antibiotics (mixture A), 60% of the m6rist6mes de noyers hybrides. C.R. Acad. explants could be recovered. In walnut, as Sci. Paris Ser. /// 301, 261-264 in other woody species, such as apple, Murashige T. & Skoog F. (1962) A revised rhododendron and Douglas fir, the intro- medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473- duction of antibiotics into the medium pre- 497 vents bacterial contamination (Young et Phillips R., Arnott S.M. & Kaplan S.E. (1981) al., 1984). Antibiotics in plant tissue culture: rifampicin From this research, it can be concluded effectively controls bacterial contaminations without affecting the growth of short term that the conditions under which the source explant cultures of Helianthus tuberosus. Plant plant is grown and the treatments given to Sci. Lett. 21, 235-240 the explant in culture are important for Somers P.W., Van Sambeek J.W., Preece J.E., success in walnut micropropagation. We Gaffney G. & Myers O. (1982) In vitro expect that the combined actions of the micropropagation of black walnut. Proc. 7th North Am. For. Biol. University of Kentucky phytosanitary treatment of the plants in Press, Lexington, pp. 224-230 the greenhouse or in the field, the applica- Viseur J. (1985) Micropropagation of pear, tion of growth regulator solutions, the cul- Pyrus communis L., in a double-phase culture ture of explants in a double-phase system medium. Acta Hortic. 212, 117-124 and the addition of antibiotics, will greatly Young P.M., Hutchins A.S. & Canfield M.L. aid the establishment of an efficient micro- (1984) Use of antibiotics to control bacteria in propagation method from selected mature shoot cultures of woody plants. Plant. Sci. Lett. walnut trees. 34, 203-209

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