【作者】 陈彦；

【导师】 周坚；

【作者基本信息】 南京林业大学 ， 植物学， 2006， 博士

【摘要】 本文在研究紫薇的生物学特性的基础上，将小盐芥总DNA通过花粉管通道导入紫薇，获得耐盐转化幼苗。 (1)生殖生物学特性。苯胺蓝染色法显示，从授粉到花粉萌发不足0.5h，开花后24h花粉管进入子房进行受精，由此确定花粉管通道导入外源基因的最佳时机是开花后22～24h。此外，紫薇花粉量大，萌发率高，大量花粉管穿过花柱进入子房，子房内胚珠多数，为外源DNA的进入提供更多的通道和机遇。 (2)花粉活力。碘-碘化钾法和过氧化物酶法可以根据染色深浅准确判断花粉活力的强弱。 (3)种子萌发和育苗。水和化学试剂浸种都能够促进平皿中紫薇种子萌发，其中45℃温水恒温4h、共浸泡24h的种子萌发率最高。相同处理的种子在基质中的出苗率均低于平皿中的发芽率。 (4)盐胁迫对种子萌发及幼苗的影Ⅱ向。0.1％、0.2％、0.4％、0.6％、0.8％、1.0％NaCl溶液抑制紫薇种子萌发。0.1％NaCl是3～4cm高幼苗的最适胁迫。 (5)DNA提取及RAPD-PCR反应条件的初步优化。SDS-高盐低pH法适合提取盐生植物DNA；用CTAB浸提前先用缓冲溶液洗去多糖适合紫薇叶片DNA的提取，且65℃温育0.5h、5％PVPP效果最佳。RAPD-PCR的关键因素是模板的有效浓度。 (6)小盐芥总DNA导入紫薇。利用直接滴加和切花柱后滴加共获得8106粒转化种子。0.3％NaCl溶液浇灌1个月后，共得到12棵耐盐幼苗。更多还原

【Abstract】 The salt-tolerant transgenic seedlings of L.indica L. were obtained by the technique of pollen tube pathway on the base of studies on its biological characteristics.(1) Habit of reproductive biology. There was not more than 0.5 h from pollination to pollen germination on stigma. It was the optimal time for exogenous DNA to be introduced into ovule when fertilization was completed at 24th h of post floral. In addition, there were many advantages in exogenous DNA introduction, such as a huge amount of pollen dropping on stigma, high germination rate of pollen, a huge bundle of pollen tubes going through style, several ovules in every locul.(2) Pollen activity. Iodine-potassium iodide method was most suitable for the determination of pollen activity of L.indica L.(3)Seed germination and raising seedlings. The seed germination on water-saturated filter paper in petri dish was promoted by soaking in water and chemical agents, respectively. seed immersion in 45℃ water for 4 h followed by a 20-hour continue soaking was the optimal pretreatment. The epigeal germination rate of seeds on medium (turf, vermiculite, perlite 2:1:1) was much lower than the germination rate of seeds in petri dish with the same pretreatment.(4) Salt tolerance of seeds and seedlings. There was inhibition in germination when seed was stressed by NaCl of 0.1%、 0.2%、 0.4%、 0.6%、 0.8%、 1.0%. 0.1% NaCl was suitable for the growth of 3～4 cm high seedlings.(5) DNA extraction and preliminary selection of reaction conditions for RAPD-PCR. SDS-high salt at low pH method was suitable for DNA extraction from halophyte leaves. Higher quantity and quality of genomic DNA of L.indica L. was obtained by the regular CTAB method after polysaccharides was removed when 0.5 h incubation time and 5% PVPP were used. The concentration of template DNA was critical to RAPD-PCR.(6) Introduction of genomic DNA of salt cress into crape-myrtle. 8106 putative transgenic seeds were obtained by the technique of direct dropwise and dropwise after servering style. 12 seedlings survived the 1-month irrigation of 0.3% NaCl.更多还原