【作者】 徐洪伟；

【导师】 陆静梅；

【作者基本信息】 东北师范大学 ， 植物学， 2007， 博士

【摘要】 本文利用发根农杆菌遗传转化玉米，通过毛状根的诱导、毛状根的分化培养获得再生植株。毛状根再生植株在形态学上表现根系发达，节根分节较多，侧根发达，生长迅速。以毛状根再生植株为材料，以H99、吉单101和四单8为对照，着眼于抗旱性的复杂性、系统性和整体性，分别从植物形态学、植物生理学、植物发育学等角度对玉米抗旱性鉴定指标进行了系统研究。在研究结果的数量分析过程中借引了灰色系统理论及灰色关联度分析法，以及隶属函数值法等数学统计分析方法对玉米抗旱性鉴定指标进行评价。结果表明，由于毛状根再生植株强大的根系，即使在水分胁迫下形态学指标、发育指标、生理指标上仍能表现出很好的抗旱性。1．以玉米自交系H99为材料，幼叶、茎段和愈伤组织为外植体，利用A4、R1601菌株和ATCC15834菌株进行侵染，通过对外植体的选择、外植体最适生长条件的优化、农杆菌侵染条件的优化、除菌条件的优化、毛状根生长条件的优化，建立玉米的毛状根培养系统，获得毛状根。转化的毛状根在不含激素的培养基上快速生长，并表现出典型的毛状根生长性状。研究表明，ATCC15834和A4菌株侵染率高，只有愈伤组织被诱导产生毛状根。培养条件：发根农杆菌菌液浓度在A₆₀₀=1.0；乙酰丁香酮浓度为50μmol／L；侵染时间为25分钟；共培养时间为3天；共培养的培养基的pH值为6.0；抑菌培养基添加“羧苄青霉素150 mg／l+头孢霉素100 mg／l”的培养条件最佳。2．将毛状根再生植株进行分化诱导，在不同激素配比的分化培养基上，对照的种子苗根褐化死亡。毛状根在3号培养基上形成愈伤组织并一次成苗。3．PCR检测和PCR-Southern分子杂交检测结果表明，所获得的玉米毛状根和毛状根再生植株在500bp处有与rol C基因相同大小条带存在。PCR-Southern分子杂交检测进一步证明了该条带是rol C基因，玉米毛状根和毛状根再生植株的基因组中整合了来源于Ri质粒的rol C基因。4．水分胁迫下，不同生长发育时期的玉米根系的生长发育受抑、生理活性下降。表现在根长、根数、根体积、根干重、根系的总吸收而积、根系的活跃吸收而积等指标在干旱情况下明显下降。抗旱指数与各指标的关联度分析表明，根干重、根数、干物质胁迫指数和单株叶面积与抗旱相关性最大。不同生育时期水分胁迫下供试玉米株高、单株鲜重、单株干重、单株叶面积、产量及产量构成因子均有不同程度下降。5．不同生育时期水分胁迫下使叶绿素含量、光合速率、蒸腾速率、细胞间隙CO₂浓度、气孔导度都有不同程度降低。水分胁迫下叶绿素a和叶绿素b均有降低，总叶绿素含量的变化与叶绿素a含量的变化基本一致。气孔导度与净光合速率具有相关性。6．水分胁迫使玉米植株的整株根系水导降低，毛状根再生植株的根系水导下降了13.2％，H99下降了84.7％降幅最大，四单8下降了34.7％，吉单101下降了37.4％。降幅次序是H99＞吉单101＞四单8＞毛状根再生植株。总之，在水分胁迫下毛状根再生植株与正常水分情况下相比各项指标略有下降。但是，与对照品种相比均维持在较高水平，差别显著（p＜0.05）。毛状根再生植株强大的根系，即使在水分胁迫下仍能保证植株生长发育过程中的水分代谢。因此，对水分胁迫的敏感指数较低，表现对抗旱的很好适应。更多还原

【Abstract】 This paper reports hairy roots induced by Agrobacterium rhizogenes and production ofregenerative plants from hairy root cultures in Maize. The root lines of regenerative plantsgrew rapidly, showing more branching roots and lateral roots. In the dissertation, consideringthe complexity of drought-resistance, comparing to some lines of H99, Jidan 101, and Sidan 8,the morphological, physiological, and developmental characteristics of drought-resistance inregenerative plants of Maize （Zea Mays L.） were systemically studied and compared to. Theindexes systems of drought-resistance identification were established quantitative analyzingwith methods of grey correlation degree, subordinate function values and systematic analysisof clustering. The high drought-resistance on the morphological, developmental, andphysiological characteristics under water stress were showed in strong root lines ofregenerative plants from hairy root cultures in Maize.1. The explants from maize H99 seedlings and embryonic calli were induced by threestrains of virulent A. rhizogenes, R1601, ATCC15834, and A4 in this study. Afteroptimization of the explants, growth condition, Agrobacterium infection process,and hairy roots growth condition, the hairy roots cultured in hormone-free mediashowed the vigorous growth with typical hairy root features. In this study, we foundthat A. rhizogenes （A₄ and ATCC15834） concentration (A₆₀₀=1.0), the time ofsubmerged leaf disks in the bacterial suspension （25 min）, long-term co-cultivation（3-d）, pH-6.0, 50 u mol/L acetosyring concentration, and medium containingcefotaxime （100 mg/l） and carbenicillin （150 mg/l） for removing bacteria weresuperior for transforming the Ri plasmid in Maize calli only.2. In terms of plant regeneration from hairy roots, different ratios of hormone exhibitedvarying degrees of response. Hairy roots induced by strains ATCC15834 and A4 inmedia No.3 developed into plantlets directly. In contrast, no response at all was inthe control.3. The PCR analysis showed that amplification with the rol C primers showed a 500 bpband for the transformed roots, no band was observed for the untransformed roots.PCR-Southem hybridization further proved that rolC gene were integrated intogenome of hairy roots and regenerated plantlets.4. The growth and physiological activity of the root lines of Maize were inhibited ordecreased at different development stages, such as length, number, volume, dryweight, total absorbing area, and active absorbing area decreased evidently underwater stress. The quantitative analyzing with methods of grey correlation degree showed that the root dry weight, root number, and leaf areas of single plant wasgreatly related to drought-resistance. The height, fresh weight of single plant, dryweight of single plant, leaf areas of single plant, yield of single plant and yieldcomponents were reduced under water stress at different development stages.5. The content of chlorophyll （chlorophyll a and chlorophyll b）, photosYnthesis rate,transpiration rate, intercellular CO2 concentrations, and stomatal conductance wasall reduced under water stress at different development stages. The decreased valuesof total chlorophyll were proportional to the decrease of chlorophyll a. Meanwhile,the stomatal conductance was related to the net photosYnthesis rate.6. Under water stress condition, whole root hydraulic conductivity of Maize wasdecreased, the results showed there were different decrease extents at differentMaize lines, such as the regeneration plant was increase by 13.2%, 84.7% H99,34.7% Sidan 8, and 37.4% Jidan 101. The order of decrease extent was H99＞Jidan101＞Sidan 8＞the regeneration plant from hairy root culture in Maize.In short, the indicators of regeneration plants from hairy root culture declined slightlyunder water stress. However, compared to the control lines, varieties will remain at arelatively high level, the difference was significant （p＜0.05）. The regenerative plants fromhairy root culture in Maize with high root lines can support its moisture metabolism wellunder water stress during growth and developmental stages. So, the regeneration plantsshowed that there were lower sensitivity Indexes and higher adaptability fordrought-resistance under water stress during its development stages.更多还原