Study on Persimmon Breeding by Means of Tissue-Cell Culture Systems

柿子组织细胞培养系统育种研究

• 批准号: 05454054
• 负责人: SUGIURA Akira
• 金额: $ 4.16万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454054/
• 关键词: Somaclonal variation; Isozyme; Cell fusion; Somatic hybrid; RAPD analysis; Unreduced pollen; Chromosome number; 完全甘ガキ; 3倍体胚; 染色体数; カルス; プロトプラスト; アガロースビーズ培養; 再生植物体

Using tissue and cell culture systems for persimmon cultivar improvement, we tried to induce somaclonal variation, to develop somatic hybrids by cell fusion and to obtain 3n plants through rescue culture of immature embryos resulting from pollination with unreduced pollen. The results obtained are summarized as follows :(1)gamma ray irradiation and PFP treatment were applied to Jiro callus and plantlets were regenerated from the affected celli. The regenerants were compared with the original Jiro plants morphologically and by isozyme analyzes, however, no differences were observed between them. More mutagens need to be tested to induce somaclonal variation in persimmons.(2) Callus protoplasts of cvs. Jiro and Suruga were fused electrically and cultured for plant regeneration. Regenerants were obtained from 127 callus lines, 22 of which were shown to have twice the nuclear DNA content of the parental calli. RAPD analysis and chromasome counting revealed that the regenerants were somatic hybrids between the two cultivars and had dodecaploid chromosome number of around 2n=180, which is twice the number of parental plants.(3) Pollen size distribution was examined with several persimmon cultivars. Giant pollen, distinctly larger than the normal pollen, were observed in all the cultivars but the ratio of giant pollen to total pollen varied among cultivars, ranging from about 10% for Zenjimaru and Amayotsumizo, about 2% for Okugosho to less than 1% for the rest cultivars. Nuclear DNA analysis revealed that those giant pollen is unreduced 2n pollen. Unreduced giant pollen was sorted by 62mum nylon mesh filter and used for pollination ; giant pollen of Zenjimaru and Okugosho were crossed for Jiro and Fuyu flowers, respectively. The resultant immature embryos were dissected and cultured in vitro.Chromosome counting demonstrated that the plantlets thus obtained were all nonaploid (2n=9x=135) and their nuclear size was clearly larger than that of hexaploid parents.

利用组织培养和细胞培养技术进行柿品种改良，诱导体细胞无性系变异，通过细胞融合培育体细胞杂种，并对未减数花粉授粉产生的幼胚进行挽救培养，获得3 n植株。主要研究结果如下：（1）对次郎愈伤组织进行γ射线照射和PFP处理，获得了再生植株。对再生植株进行形态学和同工酶分析，结果表明再生植株与原代次郎植株无明显差异。需要更多的诱变剂来诱导柿子的体细胞无性系变异。(2)黄瓜愈伤组织原生质体。以次郎和骏河为材料，进行电融合和培养再生植株。从127个愈伤组织系中获得了再生体，其中22个被证明具有双亲愈伤组织的核DNA含量的两倍。RAPD分析和染色体计数结果表明，再生植株为两品种间的体细胞杂种，染色体数目约为2n=180，是亲本的2倍。(3)研究了几个柿品种花粉粒大小的分布。大花粉明显大于正常花粉，在所有品种中观察到，但大花粉占总花粉的比例因品种而异，从Zenjimaru和Amayotsuizo的10%左右，Okugosho的2%左右到其余品种的不到1%。核DNA分析表明，这些巨大的花粉是未减数的2n花粉。用62 μ m尼龙网过滤器分选未还原的巨大花粉并用于授粉; Zenjimaru和Okugosho的巨大花粉分别与次郎花和冬玉花杂交。对所获得的幼胚进行离体培养，染色体计数表明，所获得的植株均为九倍体（2n= 9 x =135），其细胞核明显大于六倍体亲本。