【作者】 刘小军；

【导师】 王家福；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本研究以枇杷（Erilbotrya japonica Lind I．）为材料，进行了枇杷种质资源离体保存及生理生化的研究。主要包括枇杷快繁体系的建立；枇杷小苗常温限制生长保存和低温保存条件下的生理生化研究；枇杷花粉的超低温保存研究；枇杷种胚超低温保存的脱氢酶活性研究几个方面。 1、枇杷快繁体系的研究 试验中建立了茎尖、幼胚两种枇杷快繁体系。在茎尖快繁体系中，用饱和漂白粉溶液前处理和选择适当大小的外植体，可以有效地防止茎段褐变，提高茎尖培养成活率。培养基BA1.0mg／l+NAA0.5mg／l组合可有效地促进茎尖的发育。 在幼胚培养中，培养基MS+6-BA2.0mg／l+IAA0.5mg／L萌芽效果最好，用培养基MS+BA1.0mg／l+NAA0.2mg／l小苗平均株高较高，而且长势好，所以此种培养基为最适宜的丛生芽增殖培养基。 2、枇杷小苗常温限制生长保存和低温保存的生理生化研究 枇杷离体保存所用培养基为MS基本培养基，低温（11±1℃）保存培养基中加入蔗糖2.5％，琼脂0.75％，常温限制生长所加植物生长抑制剂为多效唑(PP₃₃₃)5 mg／L，蔗糖为2.5％和5％两种，琼脂为0.75％，研究蔗糖和温度对枇杷小苗离体保存的影响。 在对枇杷小苗长达六个月的保存过程中，对与植物逆境生理和代谢调控有关的几个生理指标如呼吸速率、叶绿素和蛋白含量以及多酚氧化酶活性和过氧化物酶活性等进行了检测。 结果表明，在保存过程中，枇杷小苗的呼吸速率被抑制，基本上随保存时间延长而降低；叶绿素的含量则是在保存的第三个月达到最高值，而后下降；可溶蛋白的含量是在保存的第四个月达到最高值，而后下降或是呈一直下降趋势;多酚氧化酶的活性则是呈先升后降趋势，低温对其活性影响较大，而蔗糖含量影响相对较小;过氧化物酶的活性也是一直呈下降趋势，蔗糖对其活性的影响较大。3、批把花粉的超低温保存研究 对经过干燥脱水至不同含水量的批把离体花粉进行了超低温（一196℃）保存实验。结果表明:超低温保存花粉时，花粉的含水量多寡是成败的关键。对于批把而言，选择含水量为13%左右的花粉比较合适。花粉在液氮中保存2h、sh和24h，其发芽率无显著差异。4、批把种胚超低温保存的脱氢酶活性研究 幼胚用60%玻璃化保护剂PVSZ处理，然后进行液氮保存，经解冻处理后，进行脱氢酶活性测定。结果表明，含水量是影响批把种子离体胚超低温保存的重要因素，巧%和20%含水量的脱氢酶活性高，能够较好地保存与维持离体胚活力。冷冻速度对批把离体胚脱氢酶活性影响甚微，而解冻过程则有显著影响。更多还原

【Abstract】 This study explored the germplasm resources preservation and Physiology and Biochemistry of loquat ( Erilbotrya japonica Lind I ) in vitro. It included the establishing of rapid propagation system, Physiology and Biochemistry of limiting the growth with normal temperature and low temperature , Cryopreservation of loquat pollens, dehydrogenase activity of excised embryos of loquat.1. Rapid propagation system of loquatTwo different rapid propagation system of loquat were compared by shoot tip culture, immature embryo culture. In shoot tip culture, it was important to use the saturated bleaching powder to preceding treat and choose the appropriate size explant for avoiding browning. The medium of BA1.0mg/l+NAAO. 5mg/l can increase shoot tip growth effectively. In immature embryo culture, the medium MS+6-BA2.0mg/l+IAAO. 5mg/ lcan promote sprouting effectively. Moreover, when using the medium MS+ BA1.0mg/l+ the NAA0.2mg/l, the buds were higher and grew better, so this medium was the most suitable to multiplication.2. Physiology and biochemistry study of limiting the growth in normal temperature and low temperature preservationPreservation of Loquat used MS medium. In low temperature (11 ± 1 ℃) medium was added cane sugar 2. 5%, agar 0. 75% , in normal temperature for limiting growth the medium was added plant growth depressant PP333 5 mg/l, agar 0. 75%, the cane sugar was 2. 5% and 5% respectively. It is hope to understand the effects of cane sugar and temperature on the seedlings of loquat.In six months preservation of loquat, respiratory rate, chlorophyll, soluble protein, PPO activity and POD activity were determined. They had relation to plant adverse circumstances physiology and metabolic adjustment. It was shown that respiratory rate of the loquat plantlets was lower with preservation time prolonged; Chlorophyll attained the highest value in the third month and declined then; The soluble protein content was related to preservation temperature and cane sugar concentration, it increased before descending or made a slow descent; The PPO activity declined the trend after rising first, the temperature as to its activity influence was bigger, but the cane sugar smaller; the activity of the POD was to descend the trend , however cane sugar as to its influence was bigger, and temperature was smaller .3. Cryopreservation of loquat pollensDehydrated loquat pollens with different water content were stored in super-low temperature(-196℃) .The results indicated that the survival rate of pollen preservated in LN was related to water content. For the loquat, 12. 9% pollen water content was appropriate. The pollens were preserved in LN for 2 h, 8 h, 24 h, its germination rate had no obvious difference than before preservation. 4. Dehydrogenase activity study of excised embryo of loquatThe young embryos were treated with vitrification protector, then preservated in LN. After thawing, dehydrogenase activity was determined. The result showed that the water content of excised embryo was important factor to affect loquat cryopreservation. In excised embryo with 15% and 20% water content the TTCH activity was high, it can conserve and maintenance the excised embryo vitality. At cryopreservation the effect of freezing speed on dehydrogenase activity was smaller, but the effect of thawing was bigger.更多还原