Báo cáo lâm nghiệp: "A micropropagation system for Eucalyptus dunnii x Eucalyptus sp"

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A micropropagation system for Eucalyptus dunnii x Eucalyptus sp. M. Fantini Jr. M.E. Cortezzi 2 Graça 1 Agro-Florestal, Teldmaco Borda, PR, and Klabin do Parana 2 Pesquisa de FlorestaslCNPF-EMBRAPA, PR, Brazt7 Centro Nacional de in an open greenhouse. After leaf Introduction growing removal, segments were surface-sterilized in 1% NaCIO plus 2! drops of Tween-20/100 ml for A Eucalyptus dunnii hybrid was first no- 15 min followed by 3 rinses in autoclaved dis- ticed during seedling production in 1984. tilled and deionized water. The basal medium This spontaneous hybrid originated from a for initiation consisted of Murashige and Skoog seed production area of E. dunnii and its (1962) salts plus vitamins as described by Gamborg and W’etter (1975), 3% sucrose with characteristics, such as leaf color, shape, BAP (benzylaminopurine) and IBA (indole wax content and stem color :.;ere distinct butyric acid) at 0.1 mg The pH of the H. ’ from the type. Although the hybrid growth medium was adjusted to 5.7 prior to the addi- potential cannot yet be determined, early tion of 6 g of Eiacto-agar. At the multiplication 1 I- ’ tree seiection revealed 2 important fea- stage, the medium combinations of BAP and KIN (kinetin) (0.1, 0.5 and 1.0 mg!l-!) and IBA tures: 1) tolerance to frost comparable to (0.01, 0.05 and 0.1 mg!l-!) were used. For the that of the parental species, which is a shoot elongation experiment, single shoots or major factor to consider in establishing clusters of shoots were placed on a medium Eucalyptus in the southern region of Bra- containing the treatments shown in Table I. Cul- zil; 2) the hybrid can be easily propagated tures in each treatment were incubated under by stem cuttings, as opposed to E. dunnii. 16 h/8 h tight/dark photoperiod (around 1200 lux), at 25°C. After 60 d in culture, shoot height While this method is successful, in vitro recorded. Fioots initiated Knop was were on propagation would reduce propagation White (1954) medium supplemen- (1985) or on stock requirements and would also be a ted with IBA (0.5, 1.0 and 1.5 mg!l-!) and ribo- rapid method for mass clonal propagation. flavin (0 and 5 1!’1-1). All experiments (unless The development of a system to micro- otherwise stated) were completely randomized with a variable number of replicates in each propagate Eucalyptus dunnii x Eucatyp- stage. Prior to transfer to greenhouse condi- tus sp. was the objective of this study. tions, the plantlets in the culture vessels were established in 50 cm polypropylene containers 3 with a mixture of vermiculite and sterile soil Materials and Methods (1:2.3, v/v) under high humidity in the green- house. After 2 wk in this environment, the hu- midity was gradually reduced to ambient condi- Nodal segments of E. dunnii Eucalyptus sp. x tions. collected from rooted cuttings actively were
Results observed among these treatments, was single additions were not as effective in Optimum shoot multiplication rate was promoting shoot development as they obtained on the medium containing 0.5 were when combined (T6). On the other mg BAP and 0.05 mg IBA (Table II). 1 l- ’ 1 I- ’ hand, increasing the BAP level (T7) re- A rate of 8 shoots per explant was devel- sulted in shoots shorter than those sub- oped within 30 d. When KIN was used, to T6. Shoots cultured in the dark jected multiplication rates were lower than those were more elongated than those grown with BAP. Nevertheless, the same optimal under 16 h light photoperiod (Fig. 1). In- levels as those determined with BAP were oculation procedures affected shoot grow- found with KIN (6:1 )(Table 11). th only in the treatments in which all pro- moters were used (Fig. 2). Under those Shoot elongation was influenced by the treatments (T6 and T7), single shoot ino- (Figs. 1 and 2). When only MS treatment culation produced longer shoots compa- medium was used, shoot elongation did red to clusters. Despite this treatment, not occur (Fig. 1 The single addition of shoots in cluster gave a higher yield of AC, GA BAP or IBA or both at 0.01 , 3 less developed shoots, but these were still 1 I- ’ mg to the MS medium, increased shoot of adequate size for rooting. length. Although no significant difference
Rooting occurred in all treatments but it 90% after 2 wk in generated plantlets was greatest on Knop medium with 1.0 the under high humidity. greenhouse was 1 mg-1- IBA (Fig. 3). On this medium, root- ing was 82% compared to 60% observed White medium at the same IBA on Discussion and Conclusion concentration. Rooting was also stimu- lated by an IBA concentration up to 1.0 mg!l-1. As the concentration of IBA in- Micropropagation is a viable system for creased beyond 1.0 mg root formation , 1 I- ’ propagation of E. dunnii x Eucalyp- mass decreased. Similarly, rooting was also tus sp. High shoot multiplication rates reduced when riboflavin (5.0 mg.J- was ) 1 were obtained with 0.5 mg BAP or KIN, 1 - ’I included in the medium. Survival of re- combined with 0.05 mg!l-! IBA. In this
combination, BAP treatment and promoted the development of an was more effective in promoting shoot proliferation undesireable root system with fewer roots, than KIN was. At the elongation stage, the which were more brittle than those without addition of AC, GA BAP (0.05 mg ) 1 I- ’ , 3 riboflavin in all treatment combinations. and IBA (0.05 mg in the MS medium I-1) ’ The success of the establishment of the produced the best shoot growth. Although regenerated plantlets (90% survival) under incubation in the dark increased shoot greenhouse conditions indicates, once length, light incubated shoots grew more more, the potential of this method to pro- readily than dark incubated ones. Similar- pagate E dunniix Eucalyptus sp. hybrids. ly, elongation was better when shoots were inoculated on medium individually as opposed to clusters. Inoculation of clus- ters, however, yielded many elongated References shoots with an adequate root system. The best percentage of rooting was obtained De Fossard R.A. In: Tissue Culture Pro- (1981) on Knop medium with IBA at 1.0 m H. ’ pagation. Harold L. Lyon Arboretum Lecture - Shoots rooted better on Knop than White University of Hawaii - Lecture no. 10, pp. 39 medium, regardless of the IBA level. Addi- Gamborg O.L. & Wetter L.R. (1975) In: Plant tion of riboflavin to the rooting medium has Tissue Culture Methods. National Research Council of Canada, Saskatoon, p. 91 been reported to improve root formation through the modification of root morpholo- Knop W. (1965) Quantitative untersuchungen Ober den ernahrugsprozess der pflanzen. gy. Fewer and longer roots were produced Landwirtsch Vers.-St. 7, 93-107 in the presence of riboflavin and these Murashige T. & Skoog F. (1962) A revised roots developed towards the medium, medium for rapid growth and bioassays with whereas those initiated in the absence of tobacco tissue culture. Physiol. Plant. 15, 473- riboflavin grew on the surface of the 497 medium (De Fossard, 1981). In the pres- White P.R. (1943) In: A Handbook of Plant Tis- ent study, the addition of riboflavin re- sue Culture. The Jaques Cattel Press, Tempe, duced the rooting capacity of the explants AR