本文選題：草莓　切入點(diǎn)：再生體系　出處：《河北大學(xué)》2017年碩士論文

【摘要】：草莓(Fragaria×ananassa Duch)屬薔薇科草莓屬,是人們?nèi)粘Ｉ钪兄匾乃a(chǎn)品之一。栽培草莓品種多為八倍體,是高度的雜合體,使用常規(guī)育種方法費(fèi)時(shí)費(fèi)力,且育種較難。而且,草莓在生長(zhǎng)過程中,隨著其無性繁殖世代的延續(xù),草莓病毒病會(huì)逐漸積累,嚴(yán)重影響草莓的產(chǎn)量和果實(shí)的品質(zhì),因此,草莓苗一般需要2~3年進(jìn)行一次換苗。本研究為了快速高效的獲得草莓實(shí)生苗和提高草莓試管苗的分化率和增殖系數(shù),并縮短其再生周期,分別以草莓種子、葉片、葉柄和試管苗基部作為外植體,通過改變其培養(yǎng)過程中激素配比及濃度,建立了以試管苗基部為外植體的高效再生體系。為了進(jìn)一步提高其分化效率,將試管苗基部劃分為草莓試管苗葉柄托葉結(jié)合部位和試管苗基部?jī)刹糠肿鳛橥庵搀w,建立了更加快速高效的再生體系。采用SRAP分子標(biāo)記技術(shù)檢測(cè)通過不同途徑獲得的草莓植株的遺傳穩(wěn)定性,探索檢測(cè)子代與母本的相似程度,為離體快速繁殖保存草莓種質(zhì)資源提供了技術(shù)依據(jù)。草莓在生長(zhǎng)過程中同樣會(huì)受到外界環(huán)境的影響。鹽堿地是我國(guó)分布十分廣泛的土壤類型,鹽脅迫會(huì)抑制植株的組織和器官的成長(zhǎng)及發(fā)育。據(jù)統(tǒng)計(jì),鹽害可導(dǎo)致全球農(nóng)作物每年減產(chǎn)20%以上。如何提高草莓的抗鹽性,是目前亟待解決的問題。本試驗(yàn)室吳科瀛研究結(jié)果表明導(dǎo)入Cjhppd基因的煙草抗鹽性顯著提高。本研究建立農(nóng)桿菌介導(dǎo)的草莓試管苗基部為受體的遺傳轉(zhuǎn)化體系,將Cjhppd基因?qū)氩葺仓陱亩@得轉(zhuǎn)基因植株,研究其轉(zhuǎn)基因性狀,為利用基因工程技術(shù)進(jìn)行草莓品質(zhì)改良奠定基礎(chǔ)。主要研究結(jié)果包括:1.建立了高效的草莓再生體系:以草莓試管苗葉柄托葉結(jié)合部位和試管苗基部作為外植體,分化培養(yǎng)基為MS+IBA0.2mg·L-1+6-BA1.5mg·L-1。相較于以葉片、葉柄作為外植體進(jìn)行離體快繁,草莓試管苗葉柄托葉結(jié)合部位和試管苗基部分化均無明顯的“愈傷組織”產(chǎn)生,可直接分化不定芽,且再生周期短。分化率可達(dá)到100%,增殖系數(shù)平均分別為6個(gè)和6.73個(gè)芽體,最高為13個(gè),分割分化芽轉(zhuǎn)移至誘導(dǎo)生根培養(yǎng)基,可形成正常植株,生根率達(dá)90%以上。2.采用CTAB法提取草莓基因組DNA,并建立了草莓適宜的SRAP-PCR反應(yīng)體系。17對(duì)隨機(jī)引物組合可以擴(kuò)增出清晰、穩(wěn)定的條帶,共擴(kuò)增出199條帶,多態(tài)性條帶占比為58.79%,其置信概率為99.99999%,可以用于鑒別任何一種樣品,同時(shí)證明草莓基部分化苗的遺傳穩(wěn)定性較高,為離體快速繁殖保存草莓種質(zhì)資源提供了一定的依據(jù)。3.以gfp基因作為標(biāo)記基因,建立了以草莓試管苗基部為轉(zhuǎn)化受體的農(nóng)桿菌介導(dǎo)的草莓的遺傳轉(zhuǎn)化體系,最高轉(zhuǎn)化率為22.73%。4.利用上述體系將Cjhppd基因?qū)氩葺@得轉(zhuǎn)化植株,試驗(yàn)得到轉(zhuǎn)基因草莓植株。
[Abstract]:Strawberry Fragaria 脳 ananassa Duchbelongs to the genus Rosaceae, which is one of the most important fruit products in people's daily life. The cultivated strawberry varieties are octaploid and highly heterozygous. It takes time and effort to use conventional breeding methods, and breeding is difficult. During the growth of strawberry, with the continuation of its asexual reproduction generation, strawberry virus disease will gradually accumulate, which seriously affects the yield and fruit quality of strawberry. Strawberry seedlings usually need to be changed once for 2 ~ 3 years. In order to obtain strawberry seedlings quickly and efficiently and to improve the differentiation rate and multiplication coefficient of strawberry plantlets in vitro, and to shorten their regeneration cycle, strawberry seeds and leaves were used in this study. As explants, petiole and base of test-tube plantlets were used as explants. By changing the proportion and concentration of hormones in the culture process, an efficient regeneration system was established, which took the base of test-tube seedlings as explants, in order to further improve its differentiation efficiency. The base of the test-tube seedling was divided into two parts: the base of the leaf petiole and the base of the test-tube seedling as explants. A more rapid and efficient regeneration system was established. The genetic stability of strawberry plants obtained from different ways was detected by SRAP molecular marker technique. It provides a technical basis for rapid propagation and preservation of strawberry germplasm resources in vitro. Strawberry will also be affected by the external environment during its growth. Saline-alkali land is a widely distributed soil type in China. Salt stress inhibits the growth and development of plant tissues and organs. According to statistics, salt damage can lead to a reduction of more than 20 percent a year in global crops. The results of Wu Keying study in our laboratory showed that the salt tolerance of tobacco introduced into Cjhppd gene was significantly improved. A genetic transformation system was established in which the base of strawberry tube seedlings mediated by Agrobacterium tubuloides was a receptor. The transgenic plants were obtained by introducing Cjhppd gene into strawberry plants, and the transgenic characters were studied. The main results were as follows: 1. An efficient strawberry regeneration system was established. The explants were based on the binding site of leaf petiole and the base of test-tube seedling of strawberry test-tube seedling. The differentiation medium was MS IBA0.2mg L-1 6-BA1.5mg L-1.Compared with leaves, petiole was used as explant for rapid propagation in vitro. There was no obvious "callus" in the base differentiation of petiole and basal part of petiole of strawberry plantlets, and adventitious buds could be directly differentiated. The regeneration period is short, the differentiation rate can reach 100%, the average multiplication coefficient is 6 and 6.73 buds respectively, the highest is 13. The differentiation bud is transferred to the induction rooting medium, and the normal plants can be formed. The rooting rate was over 90% .2.The genomic DNA of strawberry was extracted by CTAB method, and the suitable SRAP-PCR reaction system. 17 pairs of random primer combinations could amplify clear and stable bands and amplify a total of 199 bands. The percentage of polymorphic bands is 58.79 and its confidence probability is 99.999999.It can be used to identify any kind of samples and to prove that the basal differentiation seedlings of strawberry have high genetic stability. The results showed that the genetic transformation system of strawberry mediated by Agrobacterium tumefaciens was established by using gfp gene as marker gene and in vitro rapid propagation and preservation of strawberry germplasm resources. The highest conversion rate was 22.73. 4. The transgenic strawberry plants were obtained by introducing Cjhppd gene into strawberry.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S668.4

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