發(fā)布時(shí)間：2018-01-12 22:39

  本文關(guān)鍵詞：杉木不同外植體愈傷組織誘導(dǎo)及植株再生　出處：《福建農(nóng)林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 杉木 組織培養(yǎng) 愈傷組織 再生芽 組織細(xì)胞學(xué)

【摘要】：杉木(Cunninghamia lanceolata(Lamb.)Hook)是杉科杉木屬的常綠針葉高大喬木。由于其生長(zhǎng)快、紋理美觀、耐腐蝕、便于加工和長(zhǎng)纖維等優(yōu)點(diǎn),是我國(guó)南方主要的造林樹(shù)種和商品用材樹(shù)種。隨著近年來(lái)人工造林面積不斷擴(kuò)大,對(duì)杉木良種壯苗的需求日益增加,如何快速擴(kuò)繁杉木的高世代優(yōu)良材料和滿足杉木造林對(duì)高世代良種的需求顯的十分迫切。組織培養(yǎng)技術(shù)由于具有縮短繁殖過(guò)程、節(jié)省空間、能周年生產(chǎn)等優(yōu)點(diǎn),可以保持杉木無(wú)性系母本的優(yōu)良性狀和便于規(guī)模化生產(chǎn),能滿足市場(chǎng)對(duì)大規(guī)模優(yōu)質(zhì)杉木苗木的巨大需求,成為當(dāng)前杉木育苗的重要途徑之一。雖然20世紀(jì)80年代末國(guó)內(nèi)就開(kāi)始了杉木組織培養(yǎng)研究,先后利用成熟和未成熟合子胚、子葉、下胚軸、莖段、莖尖等多種外植體開(kāi)展了杉木再生體系研究,取得了一定成果。但目前在杉木組培離體再生過(guò)程中還存在不少問(wèn)題,主要表現(xiàn)在外植體消毒、不定芽誘導(dǎo)、不定根誘導(dǎo)、組培苗移栽均要分步進(jìn)行,外植體消毒和不定根誘導(dǎo)較為困難,缺乏杉木組培離體培養(yǎng)的完整技術(shù)體系,組培苗成本高等,極大地限制了杉木組培培養(yǎng)技術(shù)的大規(guī)模應(yīng)用。特別是由于缺乏成熟的杉木愈傷組織再生體系,極大地限制了通過(guò)基因工程手段進(jìn)行杉木遺傳改良的研究。因此,開(kāi)展杉木愈傷組織再生體系研究對(duì)于培育優(yōu)良杉木良種具有重要現(xiàn)實(shí)意義。鑒于此,本論文針對(duì)目前杉木愈傷組織誘導(dǎo)和植株再生體系研究中存在的問(wèn)題,選取以種子發(fā)芽的子葉和下胚軸、未成熟胚、組培苗莖段4種外植體為試驗(yàn)材料,通過(guò)對(duì)杉木不同外植體在消毒、組培苗繼代增殖、愈傷組織誘導(dǎo)、愈傷組織繼代增殖、愈傷組織再分化和愈傷組織形成與再分化過(guò)程中的細(xì)胞學(xué)觀察等方面,進(jìn)行杉木不同外植體愈傷組織和再生誘導(dǎo)效率的比較研究,探討不同外植體在組織培養(yǎng)過(guò)程中愈傷組織誘導(dǎo)率、愈傷組織增殖率、愈傷組織再分化率的差異性及再生芽的發(fā)生方式,篩選出適宜杉木各器官愈傷組織誘導(dǎo)和建立植株再生體系的最適培養(yǎng)基。分析杉木愈傷組織形成和再分化的起源與過(guò)程,探討建立杉木愈傷組織再生體系,為杉木體細(xì)胞胚胎再生體系和杉木遺傳轉(zhuǎn)化體系提供受體材料和技術(shù)支撐。主要研究結(jié)論如下:(1)不同杉木外植體的表面滅菌效果存在明顯差異。0.1%的Hgcl2處理10min有利于下胚軸、子葉、未成熟胚、莖段4種外植體的滅菌。(2)不同培養(yǎng)基的杉木組培苗繼代增殖效果不同。MS+6-BA2.0 mg·L~(-1)+TDZ0.04 mg·L~(-1)+NAA1.0 mg·L~(-1)培養(yǎng)基上的杉木獲得較高不定芽分化效果,不定芽平均分化數(shù)為28;在MS+6-BA3.0mg·L~(-1)+TDZ0.06 mg·L~(-1)+NAA0.3 mg·L~(-1)培養(yǎng)基上的杉木苗高生長(zhǎng)量最大,苗高平均增長(zhǎng)15.82mm。綜合不同培養(yǎng)基配方的組培苗不定芽分化數(shù)和苗高生長(zhǎng)量,認(rèn)為 MS+6-BA2.0 mg·L~(-1)+TDZ0.04 mg·L~(-1)+NAA1.0 mg·L~(-1)培養(yǎng)基是杉木苗繼代增殖的適宜培養(yǎng)基。不同光照強(qiáng)度對(duì)杉木組培苗繼代增殖的影響存在顯著差異,其中,3200Lux光照條件下最有利于組培苗的繼代增殖。(3)杉木不同外植體在不同培養(yǎng)基上的愈傷組織誘導(dǎo)效果存在明顯差異:2,4-D1.0 mg·L~(-1)+NAA2.0mg·L~(-1)+KT0.5 mg·L1 培養(yǎng)基是子葉誘導(dǎo)愈傷組織的適宜培養(yǎng)基,其誘導(dǎo)率達(dá)100%;NAA2.0 mg·L-L~(-1)+6-BA2.O mg·L~(-1)+TDZO.O1 mg·L~(-1)培養(yǎng)基是下胚軸誘導(dǎo)愈傷組織的適宜培養(yǎng)基,誘導(dǎo)率達(dá)95.8%;2,4-D1.0mg·L~(-1)+6-BA2.O mg·L~(-1)+KT1.O mg·L~(-1)培養(yǎng)基是未成熟胚誘導(dǎo)愈傷組織的適宜培養(yǎng)基,誘導(dǎo)率達(dá) 91.9%;2,4-D 1.0 mg·L~(-1)+6-BA2.O mg·L~(-1)+KT0.5 mg·L~(-1) 培養(yǎng)基是莖段誘導(dǎo)愈傷組織的適宜培養(yǎng)基,誘導(dǎo)率為87.98%。子葉、下胚軸在種子發(fā)芽第15d時(shí)愈傷組織誘導(dǎo)率最高,誘導(dǎo)率分別為97.5%和95%;2,4-D+NAA+KT是四種外植體愈傷組織誘導(dǎo)的最佳激素組合。基本培養(yǎng)基對(duì)誘導(dǎo)的愈傷組織質(zhì)地存在顯著影響,MS基本培養(yǎng)基誘導(dǎo)的愈傷組織質(zhì)量較好,可用于后期再分化,誘導(dǎo)率高達(dá)69.46%;DCR基本培養(yǎng)基誘導(dǎo)的愈傷組織質(zhì)地較密,可用于后期繼代增殖,誘導(dǎo)率為65.73%。(4)不同培養(yǎng)基的杉木愈傷組織繼代增殖效果存在差異。1/2MS、MS可作為愈傷組織再分化的繼代增殖培養(yǎng)基,DCR可作為愈傷組織長(zhǎng)期繼代增殖的基本培養(yǎng)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)培養(yǎng)基是愈傷組織繼代增殖的適宜培養(yǎng)基。2,4-D有利于降低杉木愈傷組織褐化率,NAA有利于杉木愈傷組織的增殖。當(dāng)2,4-D濃度為2 mg·L~(-1)時(shí)愈傷組織在繼代30-45d時(shí)轉(zhuǎn)移至新培養(yǎng)基比較適宜;當(dāng)NAA濃度為1.5 mg·L~(-1)時(shí)愈傷組織在繼代15-30d期間內(nèi)轉(zhuǎn)移至新培養(yǎng)基比較合適。杉木愈傷組織的繼代增殖適用于暗培養(yǎng)(800Lux)以及低強(qiáng)度光照。接種的愈傷組織直徑在11-13mm時(shí)最有利于愈傷組織的繼代增殖。(5)不同培養(yǎng)基的杉木愈傷組織再分化效果不同:MS+6-BA0.5 mg·L~(-1)+KT1.5 mgL~(-1)是愈傷組織再生芽分化的適宜培養(yǎng)基。質(zhì)地均勻的愈傷組織再分化率高達(dá)89.57%,褐化率為10.43%。愈傷組織在無(wú)繼代和繼代一次后再分化率較高,分別達(dá)83.57%和82.37%。(6)杉木生根培養(yǎng):MS+NAA0.5 mg·L~(-1)+IBA1.0 mg·L~(-1) 生根培養(yǎng)基適用于從愈傷組織上分離杉木再生苗的生根。(7)對(duì)杉木愈傷組織形成和再分化不同階段的細(xì)胞學(xué)觀察發(fā)現(xiàn):不同激素組合下形成的愈傷組織在細(xì)胞結(jié)構(gòu)及其物理性質(zhì)上存在明顯差異。有的愈傷組織具有活性可以再分化,有的失去活性無(wú)法完成正常發(fā)育。同一愈傷組織會(huì)同時(shí)存在胚性與非胚性愈傷組織,說(shuō)明杉木是屬于愈傷組織發(fā)育具有異質(zhì)性的樹(shù)種。杉木的不定芽發(fā)生有2種方式,可從外植體直接分化形成,亦可通過(guò)切口產(chǎn)生的胚性愈傷組織再分化形成。2種發(fā)生方式均為外起源。胚性愈傷組織其為淺黃色或米綠色均勻型愈傷組織。
[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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