【作者】 郑先波；

【导师】 夏国海；

【作者基本信息】 河南农业大学 ， 果树学， 2003， 硕士

【摘要】 重点对三倍体无籽西瓜成熟胚无菌萌发，愈伤组织诱导、继代、分化以及植株再生等进行了系统的研究，建立了高效愈伤组织扩繁体系，为无籽西瓜体细胞胚诱导、体细胞遗传变异特性及细胞工程研究奠定了基础。主要试验结果如下： 1．无籽西瓜成熟胚消毒的最佳方案是先用75％酒精预消毒30s，然后用10％漂白粉+0.5％NaClO消毒30 min，再用无菌水冲洗3～5次。消毒胚平放于蛭石或琼脂培养基中，先暗培养3d再转至培养室，胚萌发很快，无菌苗生长健壮。无籽西瓜不同品种与成熟胚的萌发生长没有必然的联系，只要给予适宜的光、温、水、气条件，不同品种的成熟胚均可正常萌发。 2．适宜诱导愈伤组织的外植体是2～6d苗龄的无菌苗子叶和下胚轴，最佳培养基组合为MS+NAA 1.0mg／L+BA0.5 mg/L。无籽西瓜愈伤组织的产生首先需要黑暗条件的启动，其扩增与生长分化则需在光下进行；接种后直接进行光照处理，抑制愈伤组织形成。品种与愈伤组织诱导关系密切，在C₂₄（MS+NAA 1.0mg/L+BA0.5mg/L）培养基中“黑蜜5号”是最易产生愈伤组织的品种，可作为无籽西瓜体细胞诱导等方面研究的理想材料。 3．不同糖源对无籽西瓜下胚轴愈伤组织诱导的作用存在显著差异，其中蔗糖效果最好，葡萄糖次之，乳糖和麦芽糖差异不明显，而山梨糖效果最差。蔗糖浓度影响无籽西瓜下胚轴愈伤组织的诱导，浓度超过3％时诱导率呈下降趋势；但适当提高蔗糖浓度有利于愈伤组织的进一步分化，能促进颗粒状愈伤组织的形成。从诱导率和愈伤组织质地综合考虑，蔗糖浓度以5％为佳。加速愈伤组织增殖时，可以适当降低蔗糖浓度，蔗糖浓度2％最有利于“黑蜜5号”无籽西瓜愈伤组织的继代扩增。；＆．愈伤组织芽分化的最佳培养基组合是MS＋BA4．oms／L”NAno·sins／， 芽分化率高达 7 5％。丛状芽诱导的理想培养基组合是 MS＋BAZ．oms／L＋IBAO．sins／L和MS＋BAZ．0。s／L＋IBAI．0。s／L，丛芽增 殖倍数均超过8，有效芽率高达93．52％以上，基部没有愈伤组织产生。 MS培养基中附加 0．Zing／L，0．sing／L，1．omg／L的 KT均可明显促进矮 弱芽苗的生长发育卜 5．无根芽苗在附加 IAAO．4 ms＊或 IBAO．3 ms／L的 1／ZMS培养基 中生根都很理想，生根率均达到100见 平均根数在4．38条以上，且 无根芽苗基部没有愈伤组织产生。生根苗先闭瓶炼苗6～sd，后移栽到 经过消毒的自然蛙石＋草炭门：2八：V)中，移栽成活率可达 97．5？以上。更多还原

【Abstract】 This work was mainly studied germinating of triploid Citrullus lanatus on germ-free condition, callus induction, subculture, differentiation and plantlet regeneration. The high efficiency system for callus amplification of triploid Citrullus lanatus was established. All the results were beneficial to somatic embryogenic cell induction, character of somaclonal variation and cell engineering of triploid Citrullus lanatus. The results indicated:The seeds of triploid Citrullus lanatus were surface sterilized as follows: (1) Rinsing with 75% alcohol for 30s. (2) Sterilizing with 10% bleaching powder and 0.5% NaClO for 30min and washing with sterile distilled water for 5min (3 to 5 times). The best germination occurred on the condition of the seeds in vermiculite media or agar media. The seeds were cultured in dark for three days and then transferred into culture room. After seven days, the sound seedlings were sprouted. The germination of seeds on germ-free condition had no relation to variety genotype. They would germinate normally if only the seeds in suitable conditions of light, temperature, water and air.The best explants were hypocotyl and cotyledon of the seedlings which were two to six days old. The most suitable medium for callus induction to triploid Citrullus lanatus was MS+NAA 1.0mg/L+BA 0.5mg/L. It would take one week in darkness that the callusogenesis of the explants was switched on, then the callus amplification and its further differentiation must be in light. Significant differences of callus inductive rate and index of induction were found among the various genotypes ofexplants cultured in C24 medium. In C24(MS+NAA l.Omg/L+BA 0.5mg/L) medium, "Heimi 5 Hao" was the easiest one to induce callus. It would be a perfect material for somatic embryogenic cell induction of triploid Citrullus lanatus.Enormous differences of callus inductive rate and index of induction were found among the different sugar source. Sucrose was the best one, glucose came second. Lactose, maltose and sorbose were disadvantageous to the induction and growth of callus. Considerable differences of callus inductive rate and index of induction were found among the different sucrose concentration too. When its concentration was above 3%, the rate of induction was descent. But when the concentration was increased properly, it was advantageous to its further differentiation. As far as the rate and the texture of callus were concerned, the most suitable sucrose concentration was 5%.In order to increase the speed of callus propagation, sucrose concentration would be lowered properly. As for "Heimi 5 Hao", the proper concentration was 2%. The most suitable media for inducing adventitious buds from callus was MS+BA4.0mg/L+NAA0.5mg/L. The percentage of adventitious buds was 75% on the media and the adventitious buds developed normally. The mutiplication coefficient of adventitious buds was exceed eight when the adventitious buds were cultured in the media of MS+BA2.0mg/L+IBA0.5mg/L(or MS+BA4.0mg/L+IBA1.0mg/L). The efficient buds percentage was 93.52% and there were not any callus. The adventitious buds stretched fast in the medium of MS+KT (0.2mg/L or 0.5mg/Lor 1.0mg/L).The stretched shoots rooted easily in the medium of 1/2MS +IAA0.4mg/L (or IBA0.3mg/L). The rate of root induction was 100% and there were not any callus.Most of the rooted plantlets of triploid seedless watermelon survived with a survival rate of 97.5% with the method as follows: six to eight days supplemental lighting treatment outside laboratory firstly, then transfer them in sterile distilledvermiculite + trurfy soil (1/2 V:V) mixture. And the microplants were strong.更多还原