【作者】 张利；

【导师】 樊金会；

【作者基本信息】 山东农业大学 ， 园林植物与观赏园艺， 2012， 硕士

【摘要】 本研究以白皮松成熟胚和子叶为外植体，对白皮松不定芽诱导、继代增殖、生根培养进行了系统研究。克隆拟南芥具有不同抗盐机制的基因SOS1和NHX1，分别构建超表达载体。利用所构建载体进行白皮松遗传转化研究，以期提高白皮松的抗盐耐碱性。主要研究结果如下：1.以子叶为外植体时最佳芽诱导培养基为MS+NAA0.1mg/L+6-BA3mg/L+TDZ0.01mg/L；以成熟胚为外植体时最佳芽诱导培养基为MS+NAA0.1mg/L+6-BA2mg/L。2.白皮松芽增殖的最适培养基为MS+NAA0.05mg/L，增殖系数可达6.3，系代2次后芽均高达1.56cm。芽的增殖系数随6-BA浓度的增加而降低。3.添加活性炭对芽增殖作用不明显，但能显著促进芽的伸长，随活性炭浓度增加，增殖系数降低，而≥2cm嫩梢所占比例显著增加。当活性炭超过3g/L时会对芽伸长产生抑制作用。培养基中加入GA3能有效促进芽的伸长，从0mg/L增加到1mg/L时芽均高增长较快，1mg/L到2mg/L之间变化幅度不大，但当超过2mg/L时芽均高有下降的趋势。4.分别对未处理和接种于芽诱导培养基上2、3、5周的白皮松子叶进行石蜡切片分析。结果表明：未处理子叶表皮排列整齐；芽诱导两周后，子叶表皮或包括表皮下一层皮层细胞通过平周分裂形成原始细胞群；培养三周后，由于原始细胞群的进一步分裂，加上单个细胞体积的增大，在子叶表面形成圆形突起；培养五周后原始细胞群突起进一步分裂形成具有明显的细胞学分区的芽原基，芽原基侧方分生组织细胞通过平周分裂形成鳞叶原基；鳞叶原基继续伸长形成鳞叶，同时新的鳞叶原基又从芽顶端的侧生分生组织生成。白皮松子叶不定芽起源于表皮细胞或包括表皮下一层皮层细胞，所以认为外生方式为其不定芽发生的主要途径。5.白皮松子叶Kan本底抗性实验结果表明：7.5mg/L的Kan就能完全抑制子叶的分化。在此基础上根据筛选浓度一般比不能分化的浓度稍高的原则，本实验以Kan10mg/L浓度选择转化细胞。6.不同预培养时间对抗性芽产生的影响结果表明，白皮松子叶预培养0-1d，没有有A抗性芽的产生；预培养3-7d，抗性芽产生的频率从4%增加到8%；考虑到预培养时间过长，分生组织快速分裂会有部分芽分生组织发育为叶原基，而抗生素传递速度较慢，很难扩散到分生组织团内部，会导致假阳性比例增高；因此在以后的转化实验中以4-5天为预培养时间。7.菌液浓度和侵染时间对转化效果的影响表明：菌液浓度较高(OD_600nm＝0.8)时，容易对子叶产生毒害作用，导致其在培养过程中褐化死亡，而且农杆菌生长迅速，在以后的筛选培养过程中很难将其完全脱除，会影响外植体产生抗性芽。当侵染时间超过30min时，软腐的子叶明显增多，极大地降低了其后培养过程中抗性芽的再生率。综合考虑，白皮松子叶转化中，OD_600nm＝0.4时感染30min得到最好效果。8.细菌侵染液和共培养基中AS浓度为100mol/L时，抗性芽的产生频率比对照有明显提高；AS浓度过高（大于200umol/L）时，外植体褐化程度加重，不定芽的分化明显受到抑制。9.采用延迟选择的方式分步筛选抗性芽。在不含Kan的芽诱导培养基上恢复培养一周，然后在含有Kan3mg/L的不定芽诱导培养基上筛选2轮，每轮1周至肉眼能看见不定芽，接着在含有Kan10mg/L的不定芽诱导培养基上筛选2轮，每轮2周。这可以有效减轻外植体对抗生素的过敏反应，使外植体生长状况得到明显改善。更多还原

【Abstract】 Mature embryo and cotyledon of Pinus bungeana as explants, were studied on Pinusbungeana adventitious bud induction, subculture multiplication and rooting. The gene SOS1and NHX1of Arabidopsis thaliana with different salt tolerance mechanisms were cloned andconstructed the overexpress vectors separately. The overexpress vectors were used to studythe P. bungeana genetic transformation, In order to increase the salt resistance alkaline-resisting features.The main results were summarized as follows:1. The best shoot induction medium for cotyledon was MS+NAA0.1mg/L+6-BA3mg/L+TDZ0.01mg/L,the best shoot induction medium for mature embryo was MS+NAA0.1mg/L+6-BA2mg/L.2. The optimum shoot proliferation medium was MS+NAA0.05mg/L, multiplicationcoefficient up to6.3,bud average height as high as1.56cm after culture two times. Budproliferation coefficient is reduced with increasing the concentrations of6-BA.3. With the concentration of activated carbon increasing, multiplication coefficient isreduced, and2cm young shoots proportion increased significantly, it’s no obvious effect forshoot proliferation, but can significantly promote bud elongation. When it more than3g/L,the bud elongation will be inhibited. GA3can effectively promote bud elongation, along withthe GA3from0mg/L to1.5mg/L, buds are growing faster,1mg/L to2mg/L changesslightly but when more than2mg/L, there is a decrease trend of the bud average height.4. To did not handle and vaccination respectively in bud induction medium on2,3,5weeks of coatedpinus P.bungeana cotyledons paraffin section for analysis.Two weeks afterbud induction cells, including epidermal cells or subepidermal a layer of cortical cells throughpericlinal divisions to form original cells group; Training after three weeks, due to the furtherdivision of the original cells group, coupled with a single cell volume increases the formationof circular protrusions in the cotyledon surface;Training after five weeks,original cells groupfurther split have obvious formation Cytological partition of bud primordia,the budprimordia lateral meristematic tissue cells through periclinal divisions formed scale leafprimordia;Scale leaf primordia continued elongation of the formation of scale leaves, whilethe bud primordia lateral meristematic tissue cells formed new scale leaf primordia; P.bungeana cotyledons in vitro shoot originated in the Epidermal cells or subepidermal alayer of cortical cells, so that exogenous way for the main way of adventitious buds.5. P. bungeana cotyledons Kan background resistance experimental results show that7.5mg/L Kan will be able to completely inhibit the differentiation of cotyledon. On this basis,according to the screening concentration generally slightly higher than the concentration cannot differentiation the principle, in this experiment10mg/L Kan was used to select thetransformed cells.6. The different pre-culture time influence the generation of resistant bud results showthat the P. bungeana cotyledons pre-cultured0-1d, do not produce any resistance buds; pre-cultured for3-7d, the frequency of resistant shoots have increased from4%to18%;Takinginto account the pre-culture for too long, the meristem quickly divide and part of the budmeristem develop into leaf primordia, while the antibiotic to pass slower and difficult tospread to within the meristem group, which will lead to increased false-positive ratio;subsequently, transformation experiments pre-cultured time of4-5days.7. Bacteria liquid concentration and infection time on the effects of the transformationshow that: bacterial concentration is higher(OD600nm＝0.8), it is easy to produce toxic effectson the cotyledons, lead to its in the process of cultivation Browning died, and the rapidgrowth of Agrobacterium, in the later screening culture is difficult to completely removal willaffect the explants produce resistant shoots. When the infection time more than30min, thecotyledons of soft rot increased significantly, and greatly reduces the regeneration rate ofsubsequent cultivation process of resistant shoots. Comprehensive consideration, P. bungeanacotyledons transformation, infection in the OD600nm=0.4under30min to get the best effect.8. AS concentration in Bacteria infection fluid and Cocultivation medium for100umol/L, the frequency of the resistant shoots more than the control have increasedobviously.AS too high concentration (more than200umol/L), explant Browning level deepen,iadventitious buds was significantly inhibited.9. By the delayed choice of step-by-step screening resistant shoots. Recovery culturedone week in the bud induction medium without Kan, then in contain3mg/L Kan of inadventitious bud induction medium screening two rounds, each round one week to the nakedeye can see adventitious buds. Then in contain10mg/L Kan of in adventitious bud inductionmedium screening two rounds, each round one week. This can effectively reduce the explantsallergic reactions to antibiotics, the growth of explants situation improved significantly.更多还原