| In order to support birch directional breeding in China and develop an efficient regeneration system for birch genetic transformation, tissue culture of birch (Betula pendula Rroth) superior clones from Germany was studied. In this paper, we screened the optimum regeneration system through studies conducted on explants selection, basal medium composition, hormones kinds and levels, rooting conditions and conditions which plantlets transferred to warm house. Main achievements were shown as follows: key factors affecting the regeneration of birch genotypesBirch mainly regenerates adventitious buds through indirect ways. Different types of explants from sterile plantlets, such as shoot segments, roots and leaves, was suitable for shoot regeneration, respectively. However, genotypes is a limited factor in the establishment of a complete regeneration system. The genotypes 2/86 could be dedifferentiated into callus and further differentiate into buds more easily than Gl, 4N. Moreover, different explants from clone 2/86 need different basal medium. WPM medium, MCM medium and MS medium was most suitable for shoot segments, root explants and leaves, respectively. In addition, the ability of adventitious bud regeneration induced from various explants showed different degrees. In this experiment, compared to root explants, the stem and leafstalk were most easily dedifferentiated and redifferentiatedThe inducing effect of 6-BA was better than that of ZT and TDZ. The better medium was MS macronutrients supplemented with 1.0 mg/L 6-BA+0.3 mg/L KT+0.01 mg/L NAA. The optium concentration of 6-BA was 1.0 mg/L, higher and less concentration of 6-BA could affect the percentage of adventitious bud induction. Moreover, the age of leaves directly influenced the adventitious bud regeneration. The inducing percentage of leaves of young ages is 4.5 time that of leaves in grown age. Multiplication of adventitious budThe results showed that the propagation course of birch adventitious bud and shoot tips could be divided into two stages: multiplication stage and fast-growing stage. In order to get the highest proliferation rate, we employed the method of subculturing rosette buds. At the same time, to ensure shoots grow healthy and strong, the level of CTK in the medium was regulated.Rooting cultureIt has been proposed that there were many factors affected birch clone seedling rooting in vitro. Genotype is an important factor which influence the ability of shoot rooting. The results demonstrateded that 2/86 had the highest rooting ability, while G1 had the lowest rooting ability among all tested materials. 1/2 MS medium was the most suitable for multiplied shoot rooting. For the clone 4N, only NAA was enough to induce multiplied shoot rooting and the suitable concentration of NAA was 0.3 mg/1. Whereas for the clone 1/86 or 2/86, 1/2 MS medium supplemented with 0.1 mg/1 NAA and 0.1 mg/1 IB A was optimum.Percentage of rooting is related to wooden level of Birch seedling, and the more the wooden, the less the percentage of rooting. The sucrose concentration between 1% and 3% haven't apparently effect on rooting rate. But the high level of sucrose resulted in form of the longer root. These results indicated 20g/l sucrose concentration was suitable for shoot rooting. Plantlets transferred into warm houseBirch plantlets in vitro were finally transferred to soil with 1/3 humus soil+1/3 sand+1/3 stand soil or 2/3 humus soil +1/3 stand soil. In order to improve the rate of plantlets survival, we performed different conditions, for example, the days which plantlets cultured in rooting medium, growth status of the rooting plantlets. From our study, we found that the period of adventitious root extend quickly was linked to an increase in plantlets survival.
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