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目的:获得高山红景天同源四倍体新材料。方法:用高山红景天愈伤组织原生质体为材料,进行了40%PEG6000介导的原生质体融合,对融合后原生质体进行了低密度、低融点琼脂包埋培养。按原生质体大小对融合原生质体进行了活体标记,建立了起源于单个原生质体的单细胞姊妹系,获得同源四倍体材料。结果:新生子细胞、中期细胞及其原生质体的直径RD与RM均与公式RD=0.793 7RM大致相符。未融合的二倍体、两原生质体融合产物直径的变化范围分别为16.7μm≤R<21.3μm,21.0μm≤R′<26.8μm,两者直径范围存在部分重叠。融合原生质体培养时,接种密度以1×104个/mL为宜,在此密度条件下植板率为20.3%,单细胞起源的姊妹系微克隆生长迅速,易于进行标记、彼此分离并继续培养;染色体计数的结果表明,二倍体、四倍体单细胞姊妹系再生植株染色体数目分别为26,52,不同节位叶片经流式细胞仪检测了DNA含量,证实没有嵌合体的存在。结论:本研究为高山红景天多倍体育种提供了科学依据。
Objective: To obtain the homologous tetraploid new material of Rhodiola sachalinensis. Methods: Protoplast from Rhodiola sachalinensis was used as the material, 40% PEG6000 mediated protoplast fusion was carried out, and the protoplasts were fused with low density and low melting point agar. The fusion protoplasts were in vivo labeled according to the size of the protoplasts, and a single-cell sister line derived from a single protoplast was established to obtain autotetraploid materials. Results: The diameters RD and RM of neonate, metaphase cells and their protoplasts were almost in accordance with the formula RD = 0.793 7RM. Unfixed diploid body, two protoplasts fusion product diameter range of 16.7μm≤R <21.3μm, 21.0μm≤R ’<26.8μm, the diameter range of the two there is a partial overlap. Inoculation of protoplasts with inoculum density of 1 × 10 4 / mL is appropriate, with a plating rate of 20.3% at this density. Single-cell-derived sister microcolonies grow rapidly and are easily labeled and separated from each other and continue to be cultured The results of chromosome counting showed that the chromosome numbers of diploid and tetraploid single sister sister plants were 26 and 52, respectively. The DNA content of leaves of different nodes was detected by flow cytometry, which confirmed the absence of chimera. Conclusion: This study provided a scientific basis for breeding Rhodiola sachalinensis polyploidy.