辣椒抗枯萎病体细胞变异无性系筛选技术研究
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摘要
本研究以五个辣椒(Capsicum annuum L.)品种(系)为材料,针对不同基因型、苗龄、外植体种类、激素组合等因素对辣椒植株离体再生的影响进行了较为系统的研究,建立了辣椒高效植株再生体系。同时以辣椒枯萎病原菌培养滤液作为粗毒素,对毒素的生物活性进行了测定,研究了毒素对辣椒种子发芽、幼苗生长及胚根伸长的毒害作用。在此基础上,本研究以辣椒枯萎病粗毒素为选择剂,通过连续筛选,建立了辣椒抗枯萎病体细胞变异无性系筛选体系,获得了一批抗枯萎病的辣椒体细胞突变体植株。本研究获得了以下结果:
     1.从“农城椒2号”、“农城椒3号”、“0127”、“0159”、“0171”等五个辣椒品种(系)中筛选出“农城椒2号”、“0127”、“0171”三个再生能力较强的基因型。确定了辣椒离体再生最适外植体为子叶,最适苗龄为12~14d。筛选出高效不定芽分化培养基(MS+3%蔗糖+0.6%琼脂+5.0mg/L BA+0.5~1.0mg/L IAA),不定芽诱导率可达98%。诱导出的不定芽转入MS+3%蔗糖+0.6%琼脂+3.0mg/L BA+1.0mg/L IAA+2.0mg/L GA_3+10.0mg/L AgNO_3芽伸长培养基,不定芽伸长率最高可达47.1%。不定芽伸长后在MS+3%蔗糖+0.6%琼脂+0.2mg/L IAA+0.1mg/L NAA生根培养基上诱导不定根。待其长成发达根系后移栽大田成苗,建立了辣椒高效植株再生体系。
     2.用查彼克液体产毒培养基培养辣椒枯萎病病原菌,取其培养滤液作为粗毒素,测试了毒素液对“农城椒2号”、“0127”、“0171”三个基因型种子发芽、幼苗生长、胚根伸长的作用。结果表明,辣椒枯萎病病原菌粗毒素具有一定的热稳定性,能够强烈抑制辣椒种子发芽,抑制辣椒幼苗生长,对种子胚根的伸长也具有毒害作用,且随着毒素处理浓度的升高而增强。在同一毒素浓度下,不同品种(系)受粗毒素抑制程度有所不同,毒素对“0127”幼苗生长的抑制作用显著强于其对“农城椒2号”、“0171”的抑制作用,说明“0127”对辣椒枯萎病的抗性在一定程度上比“农城椒2号”、“0171”的抗性弱。
     3.用“农城椒2号”、“0127”的子叶作为外植体诱导产生愈伤组织,以辣椒枯萎病粗毒素作为选择剂,筛选辣椒抗枯萎病细胞变异无性系。结果表明:不同辣椒品种(系)愈伤组织对毒素有不同的忍耐力,辣椒对枯萎病的抗性在一定程度上能在离体条件下表现出来:辣椒枯萎病病原菌粗毒素对辣椒子叶愈伤组织诱导、愈伤组织的生长增殖及不定芽分化具有显著的抑制作用,随着粗毒素浓度的增加,抑制作用增强;辣椒枯萎病病原菌粗毒素对不定芽分化、生长的毒害作用更强。
     4.本研究共获得辣椒抗枯萎病再生植株32株,其细胞无性系经抗毒素特性鉴定分析,表明经过粗毒素连续筛选得到的细胞变异无性系对辣椒枯萎病具有稳定抗性。
This study established the program of a high efficient in vitro regeneration procedure of five pepper cultivars. The effect of genotypes, explants, hormones, and days of seedlings on the tissue culture in pepper was investigated systematically. Furthermore, the study had a research on the degree of damage of the crude toxin derived from capsicum wilt pathogen to the germination of seeds, growth of seedlings, and elongation of radicel in pepper. On the basis of these steps, the study selected some cell mutant lines resistant capsicum wilt in pepper by use of the crude toxin, and obtained several plants regenerated in vitro. Some conclusions were drawn as following.
    1. Three cultivars of pepper (Capsicum annuum L.), 'Nongchengjiao 2', '0127', and '0171', were screened out of five cultivars for their high capacity of regeneration. The experimental results showed that the cotyledon explants taken from 12-day-old seedlings displayed the highest shoot-buds regeneration capacity. As far as culture medium was concerned, the buds induced well on MS+3% sucrose+0.6% agar + 5. 0mg/L BA+0.5-1.0mg/L IAA, which made the shoot buds differentiation frequency of 98%. Moreover, 45.7% of induced buds elongated well on the medium of MS+3% sucrose+0.6% agar+3.0mg/L BA+1.0mg/L IAA+2.0mg/L GA3+10.0mg/L AgNO3. The regenerated plants were transplanted into nutrient soil and developed into normal plants. So, an efficient program for plant regeneration from explants of pepper had been developed.
    2. The crude toxin was obtained by means of liquid culture of the capsicum wilt pathogen with Czapek's liquid medium. Using the crude toxin, the study investigated the effect of capsicum wilt on the germination of seeds, the growth of seedlings, and the elongation of radicel in three cultivars of pepper, 'Nongchengjiao 2', '0127', and '0171'. The experimental results showed that the crude toxin of capsicum wilt pathogen had the capacity of thermal stability, and can not only extremely inhibit both the germination of seeds and the growth of seedlings, but also do great harm to the elongation of radicel. Under the same concentration of crude toxin, the degree of damage was different from varied genotypes. The crude toxin inhibit the growth of seedlings of cultivar '0127' more extremely than that of cultivar 'Nongchengjiao 2' and '0171', which indicated that the resistance of cultivar '0127' to capsicum wilt was less than cultivar 'Nongchengjiao 2' and '0171' to some extent.
    3. Using crude toxin, some cell mutant lines resistant to capsicum wilt were filtrated in vitro by means of callus induced by cotyledon explants in two pepper cultivars, 'Nongchengjiao 2' and '0127'. The experimental results meant that the resistance to capsicum wilt can be showed in vitro because of different capacity of disease resistance between varied genotypes. The crude toxin had sharply negative effect on both the inducement and the growth of callus. Also, it done great harm to the regeneration of buds differentiated from the callus. Compared with the degree of the damage to callus, the crude toxin inhibited both the differentiation frequency and the growth of the buds more strongly.
    4. Several plants resistant to capsicum wilt were obtained in vitro. It was proved that their cell mutant lines had a stable resistibility to capsicum wilt through the identification and analysis.
引文
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