印度南瓜离体再生与遗传转化体系的优化

发布时间：2018-01-26 10:51

  本文关键词： 南瓜 外植体 愈伤组织 子叶节离体再生 遗传转化 农杆菌介导　出处：《河南科技学院》2017年硕士论文　论文类型：学位论文

【摘要】：南瓜(Cucurbita spp.)种质资源丰富,用途广泛,在世界范围内普遍种植。随着南瓜资源产业化利用的不断深入,筛选出抗逆性强的新品种已成为当前主要的育种目标,而现有的南瓜资源已经很难完全满足新品种选育的不同需求。传统的育种方法不仅周期长,难度系数大,而且遗传性状难以稳定,越来越多的育种工作者借助基因工程进行种质创新以提高南瓜的抗逆性。建立高效稳定的南瓜离体再生体系是开展南瓜基因工程的重要前提,也为进一步开展基因功能研究和遗传转化等相关育种工作奠定基础。本研究以印度南瓜‘北观’(C.maxima‘Beiguan’)自交系为试验材料,研究不同外植体愈伤组织的诱导及愈伤组织继代培养的条件,探究利用诱导的愈伤组织建立植株再生体系的方法;研究了以南瓜子叶节为外植体的再生体系建立中无菌苗的培养方式、苗龄、子叶节外植体大小、不同植物激素浓度配比等因素对南瓜离体再生的影响,优化了南瓜的离体再生体系;同时研究了南瓜子叶节再生中不同抑菌剂浓度、不同抗生素浓度、超声波辅助农杆菌介导法等因素对南瓜遗传转化的影响,优化了南瓜的遗传转化体系,确立了南瓜的最佳再生和农杆菌介导StNHX1基因转化南瓜的条件,以期为南瓜遗传转化研究奠定基础。研究的主要内容和结果如下:(1)南瓜愈伤组织的诱导及其建立再生体系的探究:以印度南瓜‘北观’无菌幼苗为试验材料,其下胚轴、子叶、真叶、茎段作为外植体均可诱导出愈伤组织,其中子叶和茎段较易诱导出愈伤组织;愈伤组织在MS+1.0 mg/L 6-BA(或2.0 mg/L KT)+0.2mg/L NAA培养基上进行继代培养效果较好,可获得质量较好的愈伤组织。将诱导出的愈伤组织转接到含有不同植物激素种类、浓度组合的培养基以诱导愈伤组织的再生,但均未诱导出不定芽。(2)南瓜子叶节离体再生体系的优化:将剥去种壳的种子,先在超净工作台上用75%酒精消毒30s,再用0.1%升汞消毒9min,无菌水冲洗4-5次,再用无菌水浸泡5h后,接种于MS培养基中,28℃暗培养,待胚根长至0.5cm左右时,再暗培养24h,然后进行光照培养,可获得优质的无菌苗;通过筛选,以5d苗龄的子叶节(1/4子叶+2 mm下胚轴)作为外植体,以MS+1.5 mg/L 6-BA+4 mg/L AgNO3为子叶节诱导培养基,以MS+0.5 mg/L 6-BA+0.05 mg/L GA3为不定芽伸长培养基,以MS+0.1 mg/L NAA为生根培养基,可以作为南瓜子叶节再生的最佳培养体系。(3)南瓜子叶节的遗传转化条件的优化:用农杆菌EHA105菌株侵染南瓜子叶节,培养基中添加500mg/L的抑菌剂羧苄青霉素,可有效抑制农杆菌过度增殖,且不会对外植体分化产生抑制作用;筛选抗生素双丙氨膦的最适浓度为5mg/L。当应用超声波辅助农杆菌介导法转化南瓜子叶节时,超声波60W功率,处理3min,可在外植体损伤最轻的情况下,较大程度的提高转化率。
[Abstract]:Cucurbita spp.is rich in germplasm resources and widely used in the world. Screening new varieties with strong resistance to stress has become the main breeding target at present, but the existing pumpkin resources have been difficult to fully meet the different needs of breeding new varieties. The traditional breeding methods not only have a long period, but also have a large coefficient of difficulty. And hereditary traits are difficult to stabilize. More and more breeders are using genetic engineering to carry out germplasm innovation to improve the resistance of pumpkin. It is an important prerequisite to develop pumpkin genetic engineering to establish an efficient and stable pumpkin regeneration system in vitro. This study also laid a foundation for further research on gene function and genetic transformation. In this study, the inbred line of Indian pumpkin'C. Maxima Beiguanli 'was used as experimental material. To study the induction of callus from different explants and the conditions of callus subculture, and to explore the method of establishing plant regeneration system by callus induction. The effects of culture methods, seedling age, cotyledon explant size and hormone concentration on regeneration of pumpkin cotyledons were studied. The regeneration system of pumpkin in vitro was optimized. At the same time, the effects of different concentrations of antimicrobial agents, different concentrations of antibiotics and ultrasonic assisted Agrobacterium tumefaciens on the genetic transformation of pumpkin were studied, and the genetic transformation system of pumpkin was optimized. The optimal regeneration of pumpkin and the conditions of Agrobacterium tumefaciens mediated transformation of StNHX1 gene into pumpkin were established. In order to lay a foundation for the study of genetic transformation of pumpkin, the main contents and results of the study are as follows: 1). Study on Callus Induction and Regeneration system of Pumpkin: the sterile Seedling of Indian Pumpkin 'Beiguan' was used as experimental material. Its hypocotyls, cotyledons, true leaves and stem segments can be used as explants to induce callus, among which cotyledons and stem segments are easy to induce callus. The callus was subcultured on MS 1.0 mg/L 6-BA (or 2.0 mg/L KT) 0.2 mg / L NAA medium. Good quality callus could be obtained. The callus could be transferred to the medium containing different plant hormones and concentration to induce callus regeneration. But none induced adventitious bud. 2) Optimization of in vitro regeneration system of pumpkin cotyledon: peeling off the seeds of the seed shell, sterilizing with 75% alcohol for 30s on the super-clean table. After sterilizing with 0.1% dimercuric acid for 9 minutes, washing with sterile water for 4-5 times, then soaking in sterile water for 5 hours, inoculating in MS medium at 28 鈩，

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