杉木不同外植体愈伤组织诱导及植株再生

发布时间：2018-01-12 22:39

  本文关键词：杉木不同外植体愈伤组织诱导及植株再生　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 杉木 组织培养 愈伤组织 再生芽 组织细胞学

【摘要】：杉木(Cunninghamia lanceolata(Lamb.)Hook)是杉科杉木属的常绿针叶高大乔木。由于其生长快、纹理美观、耐腐蚀、便于加工和长纤维等优点,是我国南方主要的造林树种和商品用材树种。随着近年来人工造林面积不断扩大,对杉木良种壮苗的需求日益增加,如何快速扩繁杉木的高世代优良材料和满足杉木造林对高世代良种的需求显的十分迫切。组织培养技术由于具有缩短繁殖过程、节省空间、能周年生产等优点,可以保持杉木无性系母本的优良性状和便于规模化生产,能满足市场对大规模优质杉木苗木的巨大需求,成为当前杉木育苗的重要途径之一。虽然20世纪80年代末国内就开始了杉木组织培养研究,先后利用成熟和未成熟合子胚、子叶、下胚轴、茎段、茎尖等多种外植体开展了杉木再生体系研究,取得了一定成果。但目前在杉木组培离体再生过程中还存在不少问题,主要表现在外植体消毒、不定芽诱导、不定根诱导、组培苗移栽均要分步进行,外植体消毒和不定根诱导较为困难,缺乏杉木组培离体培养的完整技术体系,组培苗成本高等,极大地限制了杉木组培培养技术的大规模应用。特别是由于缺乏成熟的杉木愈伤组织再生体系,极大地限制了通过基因工程手段进行杉木遗传改良的研究。因此,开展杉木愈伤组织再生体系研究对于培育优良杉木良种具有重要现实意义。鉴于此,本论文针对目前杉木愈伤组织诱导和植株再生体系研究中存在的问题,选取以种子发芽的子叶和下胚轴、未成熟胚、组培苗茎段4种外植体为试验材料,通过对杉木不同外植体在消毒、组培苗继代增殖、愈伤组织诱导、愈伤组织继代增殖、愈伤组织再分化和愈伤组织形成与再分化过程中的细胞学观察等方面,进行杉木不同外植体愈伤组织和再生诱导效率的比较研究,探讨不同外植体在组织培养过程中愈伤组织诱导率、愈伤组织增殖率、愈伤组织再分化率的差异性及再生芽的发生方式,筛选出适宜杉木各器官愈伤组织诱导和建立植株再生体系的最适培养基。分析杉木愈伤组织形成和再分化的起源与过程,探讨建立杉木愈伤组织再生体系,为杉木体细胞胚胎再生体系和杉木遗传转化体系提供受体材料和技术支撑。主要研究结论如下:(1)不同杉木外植体的表面灭菌效果存在明显差异。0.1%的Hgcl2处理10min有利于下胚轴、子叶、未成熟胚、茎段4种外植体的灭菌。(2)不同培养基的杉木组培苗继代增殖效果不同。MS+6-BA2.0 mg·L~(-1)+TDZ0.04 mg·L~(-1)+NAA1.0 mg·L~(-1)培养基上的杉木获得较高不定芽分化效果,不定芽平均分化数为28;在MS+6-BA3.0mg·L~(-1)+TDZ0.06 mg·L~(-1)+NAA0.3 mg·L~(-1)培养基上的杉木苗高生长量最大,苗高平均增长15.82mm。综合不同培养基配方的组培苗不定芽分化数和苗高生长量,认为 MS+6-BA2.0 mg·L~(-1)+TDZ0.04 mg·L~(-1)+NAA1.0 mg·L~(-1)培养基是杉木苗继代增殖的适宜培养基。不同光照强度对杉木组培苗继代增殖的影响存在显著差异,其中,3200Lux光照条件下最有利于组培苗的继代增殖。(3)杉木不同外植体在不同培养基上的愈伤组织诱导效果存在明显差异:2,4-D1.0 mg·L~(-1)+NAA2.0mg·L~(-1)+KT0.5 mg·L1 培养基是子叶诱导愈伤组织的适宜培养基,其诱导率达100%;NAA2.0 mg·L-L~(-1)+6-BA2.O mg·L~(-1)+TDZO.O1 mg·L~(-1)培养基是下胚轴诱导愈伤组织的适宜培养基,诱导率达95.8%;2,4-D1.0mg·L~(-1)+6-BA2.O mg·L~(-1)+KT1.O mg·L~(-1)培养基是未成熟胚诱导愈伤组织的适宜培养基,诱导率达 91.9%;2,4-D 1.0 mg·L~(-1)+6-BA2.O mg·L~(-1)+KT0.5 mg·L~(-1) 培养基是茎段诱导愈伤组织的适宜培养基,诱导率为87.98%。子叶、下胚轴在种子发芽第15d时愈伤组织诱导率最高,诱导率分别为97.5%和95%;2,4-D+NAA+KT是四种外植体愈伤组织诱导的最佳激素组合。基本培养基对诱导的愈伤组织质地存在显著影响,MS基本培养基诱导的愈伤组织质量较好,可用于后期再分化,诱导率高达69.46%;DCR基本培养基诱导的愈伤组织质地较密,可用于后期继代增殖,诱导率为65.73%。(4)不同培养基的杉木愈伤组织继代增殖效果存在差异。1/2MS、MS可作为愈伤组织再分化的继代增殖培养基,DCR可作为愈伤组织长期继代增殖的基本培养基。6-BA2.0 mg·L~(-1)+KT1.5 mg·L~(-1)+NAA1.5 mg·L~(-1) 和 6-BA1.5 mg·L~(-1)+KT1.0 mg·L~(-1)+NAA1.0 mg·L~(-1)培养基是愈伤组织继代增殖的适宜培养基。2,4-D有利于降低杉木愈伤组织褐化率,NAA有利于杉木愈伤组织的增殖。当2,4-D浓度为2 mg·L~(-1)时愈伤组织在继代30-45d时转移至新培养基比较适宜;当NAA浓度为1.5 mg·L~(-1)时愈伤组织在继代15-30d期间内转移至新培养基比较合适。杉木愈伤组织的继代增殖适用于暗培养(800Lux)以及低强度光照。接种的愈伤组织直径在11-13mm时最有利于愈伤组织的继代增殖。(5)不同培养基的杉木愈伤组织再分化效果不同:MS+6-BA0.5 mg·L~(-1)+KT1.5 mgL~(-1)是愈伤组织再生芽分化的适宜培养基。质地均匀的愈伤组织再分化率高达89.57%,褐化率为10.43%。愈伤组织在无继代和继代一次后再分化率较高,分别达83.57%和82.37%。(6)杉木生根培养:MS+NAA0.5 mg·L~(-1)+IBA1.0 mg·L~(-1) 生根培养基适用于从愈伤组织上分离杉木再生苗的生根。(7)对杉木愈伤组织形成和再分化不同阶段的细胞学观察发现:不同激素组合下形成的愈伤组织在细胞结构及其物理性质上存在明显差异。有的愈伤组织具有活性可以再分化,有的失去活性无法完成正常发育。同一愈伤组织会同时存在胚性与非胚性愈伤组织,说明杉木是属于愈伤组织发育具有异质性的树种。杉木的不定芽发生有2种方式,可从外植体直接分化形成,亦可通过切口产生的胚性愈伤组织再分化形成。2种发生方式均为外起源。胚性愈伤组织其为浅黄色或米绿色均匀型愈伤组织。
[Abstract]:Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a genus of Taxodiaceae fir evergreen coniferous arbor. Due to its rapid growth, texture appearance, corrosion resistance, easy processing and long fiber and other advantages, is China's southern main afforestation species and commercial timber species. In recent years, with the artificial afforestation area continues to expand, the Chinese fir seed the seedling growing demand, how high generation of fine materials and meet the needs of the rapid expansion of Chinese fir fir afforestation on high generation seed is very urgent. Because the technology can shorten the breeding process of tissue culture, space saving, annual production will be advantages such as excellent traits of Chinese fir clones can keep the female parent and easy to scale production. To meet the huge market demand for large scale quality of Chinese fir seedlings, become one of the important ways of the Chinese fir seedling. Although at the end of 1980s China began the organization of Chinese fir Study on the culture, has the use of mature and immature zygotic embryos, cotyledons, hypocotyls, stems, shoot tips and other explants to carry out Chinese fir regeneration system research, has made some achievements. But at present there are still many problems from Chinese fir tissue culture regeneration process, mainly in the sterilization of explants, adventitious bud induction, not root induction and transplanting were carried out step by step, explant and adventitious root induction is difficult, lack of a complete technical system from Chinese fir tissue culture in vitro plantlets, cost higher, greatly limits the large-scale application of Chinese fir tissue culture technology. Especially due to the lack of mature Chinese fir callus regeneration system. Greatly limit the genetic improvement of Chinese fir by means of genetic engineering. Therefore, to carry out research on Chinese fir callus regeneration system is of important practical significance for the cultivation of excellent varieties. In view of Chinese fir This thesis, aiming at the existing induction and regeneration system of Chinese fir in the callus, select the seed germination of cotyledon and hypocotyl, immature embryo, tissue culture seedling stems of 4 explants as experimental material, through the different Chinese fir explants disinfection, subculture proliferation, callus induction, callus tissue subculture, cytological observation etc. during the formation of callus and callus and differentiation in the comparative study of different Chinese fir callus induction and regeneration efficiency, the effect of the different explants on callus induction rate in the process of tissue culture, callus proliferation rate, callus differentiation rate the difference and shoot mode, selected the most suitable medium for induction and establishment of Plant Regeneration System of different organs of Chinese fir callus suitable for callus formation and analysis of Chinese fir. The origin and process of differentiation, to explore the establishment of regeneration system of Chinese fir callus, the recipient material and technical support for Chinese fir somatic embryo regeneration system and genetic transformation system of Chinese fir. The main conclusions are as follows: (1) Hgcl2 10min surface sterilization effect of different Chinese fir explants of.0.1% is significant difference in favor of the hypocotyl, cotyledon sterilization, immature embryos, stem segments of 4 explants. (2) the different medium of Chinese fir seedlings propagation effect of different.MS+6-BA2.0 (-1) mg L~ +TDZ0.04 mg L~ (-1) +NAA1.0 mg L~ (-1) to obtain higher adventitious bud differentiation effect based on the cultivation of Chinese fir, the average differentiation of adventitious buds the number is 28; in MS+6-BA3.0mg L~ (-1) +TDZ0.06 mg L~ (-1) +NAA0.3 mg L~ (-1) cultured on Chinese fir seedling height, seedling height average growth 15.82mm. different medium seedlings number of adventitious bud differentiation and seedling height 鐢熼暱閲，

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