草坪植物马蹄金的器官培养及原生质体培养研究

【作者】 严寒；

【导师】 田志宏；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2001， 硕士

【摘要】 马蹄金（Dichondra repens Forst.）是一种优良的地被兼观赏草坪草种。马蹄金的器官和原生质体培养在生物技术育种中作为品种快繁、变异体筛选以及体细胞杂交和遗传转化的主要技术基础，必将引起人们的广泛关注和高度重视，但目前尚无明确的报道。本文以国内外大面积推广栽培的马蹄金为材料，对其子叶、下胚轴、叶片及原生质体培养进行了研究，建立了较为系统的器官和原生质体培养体系，为开辟这一优良的草坪植物的育种新途径提供了重要的理论基础和技术基础。现将本文的主要研究结果分述如下： 1．以马蹄金的无菌苗的子叶、下胚轴和叶片为外植体，研究了不同的激素组合对愈伤的诱导的影响，其中子叶以1.0mg/L 2.4-D+0.2mg/L6-BA或0.2mg/L KT组合处理，下胚轴以1.0mg/L2.4-D+0.2mg/L 6-BA或0.2mg/L KT、0.5mg/L NAA+0.5mg/L 6-BA组合处理，叶片以1.5mg/L 2.4-D+0.2mg/L KT、1.0mg/L 2.4-D+0.2mg/L 6-BA的激素组合处理效果最好。出愈率均为100％，而且生长势也很好。在子叶和下胚轴的诱导中，添加0.5mg/L NAA还可以直接诱导出不定根。同时研究得出，MS、B₅培养基是马蹄金子叶、下胚轴和叶片外植体诱导愈伤的理想培养基。 2．研究了不同的激素组合对愈伤分化的影响，子叶的愈伤在MS附加O.1mg/L 2.4-D、2mg/L 6-BA和3mg/L AgNO₃的培养基上分化效果最好，分化率达20.7％，而下胚轴在各种激素浓度组合下均未见分化。 3．采用光照条件下12d龄的子叶，在含纤维素酶（Cellulose R-10）2％、果胶酶（Pectinase）0.8％的酶解液中，甘露醇为0.5M的渗透压条件下，酶解9h的原生质体的产量最高，活力最强。 4．研究了不同类型的培养基、不同的培养密度和培养基中不同的激素组合对原生质体的生长发育的影响，结果表明：以1.0×10⁵/ml的密度培养在完全去掉NH₄NO₃的KM₈P培养基中，附加0.5mg/L 2.4-D、0.5mg/L6-BA和0.5mg／L NAA时，对原生质体的分裂和愈伤的形成极为有利。草坪植物马蹄金的器官培养及原生质体培养研究 5.参试的四种培养方式以液体浅层培养法的效果最佳，其原生质体恢复分裂早，分裂频率高达203%，植板率最高为1.33%，固体平板培养尽管能使原生质体分裂，但分裂的细胞不能持续分裂形成小愈伤，同时伴随有出芽分裂现象。 6.在原生质体来源的愈伤的分化中，比较了不同的激素组合对实生苗再生的影响，其中以o.Zmg/LZ，4一n+4mg/L6一BA和o.Zmg/L NAA+4mg/L6一BA的两种激素组合效果较好，分化率分别为22.2%和20.0%。再生的无根苗在附加0.2m岁L的Ms培养基上即可形成完整的植株。更多还原

【Abstract】 Creeping dichondra (Dichondra repens Forest.) may be considered as a kind of excellent turfgrass and ground cover plants. It’s organ culture and protoplast culture, being an important basic technique of rapid clonal propagation , selection of mutative plants , somatic hybridzation and genetic transformation, would be attached extensive attention, but still no definite report up to now. This article used "creeping dichondra", a cultivar being cultivated home and abroad, as material and studied it’s cotyledon, hypocotyl, young leaf and protoplast culture and systematically established a series of technique of organs and protoplasts, which will provide important theoretic and technical bases for biotechnology breeding of creeping dichondra. The main results are as follows:1. By studies on effects of different hormone combinations on the induction of calli from different explants, the results indicated the combinations of 1.0 mg/L 2.4-D + 0.2mg/L 6-BA or 0.2mg/L KT were the best for cotyledons, 1.0 mg/L 2.4-D + 0.2mg/L 6-BA or 0.2mg/L KT and 0.5mg/L NAA + 0.5mg/L 6-BA for hypocotyls, 1.5mg/L 2.4-D + 0.2mg/L KT and 1.0mg/L 2.4-D + 0.2mg/L 6-BA for young leaves with 100 % of callus ratio and good growth. For cotyledon and hypocotyls, the adventitious roots could be induced when 0.5mg/L NAA existed. It also could be find that MS was perfect culture medium of the induction of calli of the cotyledon, hypocotyls, young leaf.2. The results of different hormone combinations on the differentiation of calli have been studied. The best condition was MS with 0.1mg/L 2.4-D, 2.0mg/L 6-BA and 3.0mg/L AgNO3, in which the ratio of differentiation was 20.7%. But the calli from hypocotyls could not differentiate through fair and foul.3. The highest protoplast yield and best activity was obtained from the solution of Cellulose R-10 2.0%, Pectinase 0.8% and 0.5M Mannitol with the treatment of 9h for the 12d cotyledon in the light.4. The results of different culture media, the culture density and the concentration and hormone combination on the division and growth of protoplast have been studied. Under the condition of 1.0 105/ml and KM8P without NH4NO3, 0.5mg/L 2.4-D, 0.5mg/L NAA and 0.5mg/L 6-BA is the best and beneficial of the division of protoplasts and the growth of calli.5. The thin layer method is very good in four of culture methods, in which the protoplasts divided early and had the highest frequency of division of 20.3% and the higest frequency of plating of 1.33%. For the solid embedded method, the protoplasts also divided early, but not divided continuatively into micro-calli, at the same time, bud division appeared.6. The effects of different hormone combinations on the differentiation of calli from protoplasts also have been discussed. Accordingly, the two hormone combinations of 0.2mg/L 2.4-D+ 4.0mg/L 6-BA and 0.2mg/L NAA + 4.0mg/L 6-BA had the frequency of differentiation of 22.2%, 20.0%, the seedlings without roots could become rooted on the MS of 0.2mg/L NAA.更多还原