Báo cáo lâm nghiệp: "Micropropagation of Eucalyptus dunnii Maid"

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Micropropagation of Eucalyptus dunnii Maid. S. Mendes M.E. Cortezzi Graça Brazil Pesquisa de FlorestaslEME3RAPA, Curitiba, PR, Centro Nacional de plants derived from stem cuttings were used as Introduction explants. Explants were disinfected by 30 min immersion in 0.,°i g ’Benomyl’, immediately 1 I- ’ followed by soaki:ng for 5 min in 1.5% (v/v) com- the Eucalyptus recommended for Among mercial detergent and for 15 min in 1% sodium reforestation in the southern region of Bra- hypochlorite. The disinfectants were removed zil, Eucalyptus dunnii Maid. has been the by 3 successive rinses in autoclaved and bidis- most promising due to its rapid growth, tilled water. stem straightness and frost tolerance. The In the multiplication stage, explants were cul- establishment of tree improvement pro- tured on MS medium (Murashige and Skoog, 1962) containing the following substances grams and extensive plantations, howe- (mgl1): myoinositol (100), nicotinic acid (0.25), ver, have been restrained due to both low pyridoxine.HCI (0.25), thiamine.HCI (0.5), gly- seed production (Graça, 1987) and low cine (2), adenine sulfate (20), sucrose (30,000) rooting capacity when propagated by stem and Difco Bacto-agar (6,000). Growth regulator treatments were 6-benzylaminopurine (BAP) at cuttings. 0.1, 0.5 and 1.0 mg combined with indole-3- 1 I- ’ The high multiplication rates which can butyric acid (IBP,) at 0.01 and 0.1 mg The H. ’ be obtained with micropropagation tech- pH was adjusted to 5.8. Cultures were main- tained under 16 h/8 h light/dark photoperiod niques can assist in overcoming these and 25 ± 2°C; after 30 and 60 d, shoot numbers problems as plant production is rapidly per explant were recorded. increased. For shoot elongation experiments, individual several eucalypt species have Although shoots or clusters were cultured on media containing MS salts (T,) or one-half MS salts been in vitro propagated (Hartney, 1982), (T These treatments were combined with 0.1 ). 2 work with E dunnii has not been reported. mg-l-’GA (T and T respectively) or with 1.0 33 , 4 This paper describes a micropropagation mg GA to give T and T respectively. All 1 I- ’3 5 , 6 technique for rejuvenated E. dunnii. treatments were supplemented with 0.1 mg!l-! BAP and 0.01 m IBA. Further shoot elonga- 1 1- 9’ tion was investigated by comparing the best elongation treatment obtained in the above experiment with the treatments described in Materials and Methods Table I. Elongated shoots (2.0-2.5 cm in length) were subcultured on a medium containing 1/4 x MS Nodal segments of E. dunnii (about 1 cm long) salts at 0.5, 1.0 or 1.5 mg-1- IBA and 10 mg.I- 1 1 containing one node from greenhouse grown
thiamine-HCI to initiate roots. Cultures were explant. Increasing BAP from 0.5 to 1.0 maintained under darkness for the 1st wk, fol- 1 I- ’ mg decreased axillary shoot formation lowed by 16/8 h light/dark photoperiod with light at both auxin concentrations. A similar intensity of 1000 lux. pattern was observed when these shoots All experiments were conducted twice using were subcultured for an additional 30 d randomized design with number of replicates a (Fig. 2). In this subculture, shoot prolifera- per treatment variable to the stage. Cultures tion was greatest at 0.5 mg BAP and 1 I- ’ (unless otherwise stated) were maintained under a 1618 h light/dark photoperiod and 25 ± 0.1 mg IBA. Up to 35 shoots developed 1 I- ’ 2°C. per explant within 60 d. However, at 0.5 1 I- ’ mg BAP, differences observed no were in axillary shoot number between the auxin concentrations. Results During the multiplication stage, not all shoots elongated sufficiently to be rooted. At the end of 30 d, maximum shoot pro- These shoots were then transferred into duction occurred at 0.5 mg BAP and elongation media. Shoot elongation was 1 I- ’ 0.01 mg IBA (Fig. 1). In this treatment, 1 I- ’ greatest when cultured on half-strength an average of 10 shoots developed per MS medium containing 0.1 mg BAP1 I- ’
cation stage was not sufficient for root ini- and 0.01 mg IBA. Additions of GA at 1 I- ’ , 3 tiation. Therefore, an elongation stage both concentrations, did not induce as was needed. much growth as culturing on half-strength MS medium alone. Using this medium, an At the elongation stage, reducing MS increase of 0.4 cm in length was obtained salts to half caused the greatest increase within 15 d. Nevertheless, shoots were in shoot growth. When GA was added to 3 longer when cultured on medium contain- a half- or full-strength MS medium, shoot ing GA compared to those grown on MS , 3 elongation did not improve significantly. medium alone (Fig. 3). Similarly, additions Also, shoots grown on medium containing of activated charcoal and GA nitrogen or , 3 GA were less vigorous and the leaves 3 using different salt formulations and became lanceolate instead of round, as concentrations were not as effective on were those grown without GA This was . 3 shoot elongation as half-strength MS observed in individual and cluster-inocu- medium (Fig. 4). lated shoots. Roots initiated on elongated shoots on The addition of activated charcoal to a 1/4 MS salts containing IBA. A higher per- medium containing GA and other growth 3 centage of shoots formed roots at 1.0 regulators reported to elonga- was cause IBA than at other concentrations. 1 I- ’ mg tion and to the morphological recover characteristics of the species (Franclet and Boulay, 1982). In the present study, the addition of activated charcoal and Discussion and Conclusion growth regulators reduced shoot elon- gation and caused leaf browning. An even greater reduction of shoot growth was also Large-scale micropropagation of E. dunnii observed when shoots were cultured on is feasible due to its high multiplication Gongaives’ medium containing 0.5 mg!l-1 rates. Maximum shoot proliferation was BAP and 1.0 mg-1- IAA. 1 achieved on MS medium containing 0.5 Rooting occurred on 58% of the elon- 1 I- ’ mg BAP and 0.01 mg IBA. Up to 35 1 I- ’ gated shoots at 1.0 mg-1- IBA. This low 1 shoots developed per explant within 60 d. rooting percentage and the poor quality of Shoot length obtained during the multipli- I Oll!
Gongalves A.N. (1983) Reversion to juvenility the root system are the main factors limit- and cloning of Eucalyptus urophylla S.T. Blake ing the successful establishment of E. in cell and tissue culture systems. Silviculture dunnii. 32, 786-787 M.E.C. (1987) Avaliagdo do florescimen- Graça do potencial de produ!Ao de sementes de to e Eucalyptus dunnii Maid. no Brasil. Bol. Pesqui. References Florestall4, 1-12 11 V.J. (1982) Tissue culture of Eucalyp- Hartney Franclet A. & Boulay M. (1982) Micropropaga- tus. Comb. Proc. Int. Plant Propag. Soc. 32, 98- tion on forest resistant eucalypt clones. Aust 109 For. Res. 13, 83-99 T. & Skoog F. (1962) A revised Murashige Gamborg O.L., Miller R.A. & Ojima K. (1968) medium for rapid growth and bioassays with Nutrient requirements of suspension cultures of tobacco tissue cultures. PhysioL Plant. 15, 473- soybean root cells. Exp. Cell Res. 50, 151-158 497