双单倍体南瓜后代子叶离体再生植株研究

Study on in vitroregeneration of double haploid plants offspring of pumpkin(Cucurbita moschata Duch.) with cotyledon explants

【目的】探索一种快速扩繁南瓜双单倍体植株的技术,以扩大双单倍体植株的数量,提高其育种效率。【方法】以双单倍体南瓜后代子叶为外植体,在MS培养基中分别添加不同质量浓度的6-BA(1.0,4.0mg/L)和NAA(0.05,0.1,0.2,0.5,1.0mg/L),筛选最佳组合;在此基础上,继续添加不同质量浓度的2,4-D(0.5,1.0,1.5,2.0mg/L),筛选3种激素的最佳组合。在MS+6-BA 4.0mg/L+NAA 0.2mg/L的不定芽诱导培养基上,研究其对不同苗龄子叶的诱导效果。将不定芽接种在含不同质量浓度NAA(0.05,0.1,0.2,0.5mg/L)的MS生根培养基上,比较生根率和根系生长势。【结果】在诱芽培养基中,添加2种激素时,以6-BA 4.0mg/L+NAA 0.2mg/L的MS培养基诱导率最高,为66.67%,分化系数为3.89;添加3种激素时,以6-BA 4.0mg/L+NAA 0.2mg/L+2,4-D1.5mg/L的MS培养基诱导率最高,达80.95%,分化系数为4.12。当子叶展开后苗龄为3d时,不定芽诱导率相对较大,为77.42%。不定芽在MS+0.1mg/L NAA培养基上生根效果最好,根系生长健壮,生根率达100%。【结论】双单倍体南瓜后代子叶离体再生的适宜激素质量浓度组合为MS+6-BA 4.0mg/L+NAA 0.2mg/L+2,4-D 1.5mg/L;以展开后3d苗龄的子叶作为外植体诱导效果最好;MS+0.1mg/L NAA是不定芽诱导生根的最适培养基。

[Objective] We explored a highly efficient plantlet regeneration technology on double hap- loid plants of pumpkin to get a great quantity of double haploid plants to promote the efficiency in pumpkin breeding. [Method] Taking the cotyledon of double haploid offspring of pumpkin as the explants, we inocu- lated on several types of MS-based media which were added 6-BA （1.0,4.0 mg/L） and NAA （0.05,0.1, 0.2,0.5,1.0 mg/L） in different concentrations to select the optimal concentration combination;Based on this, we continued to study adding different concentrations of 2,4-D （0.5,1.0,1.5,2.0 mg/L）,and screen three hormone optimal combination. The effect of the different ages of cotyledon explants in MS＋6-BA 4.0 mg/L＋NAA 0.2 mg/L adventitious bud inductive medium was studied. Adventitious buds were inoculated on MS medium supplement with different concentrations （0.05,0.1,0.2,0.5 mg/L） of NAA. The condi-tion of rooting rate and growth vigour of root was analyzed. [Result] In the bud induction medium,adding two hormones MS＋6-BA 4.0 mg/L＋NAA 0.2 mg/L induced the highest rate 66.67% ,and the differenti- ation coefficient was 3. 89;Adding three kinds of hormones MS＋ 6-BA 4.0 mg/L-＋NAA 0. 2 mg/L＋ 2,4-D 1.5 mg/L induced the highest rate of 80.95 %, differentiation coefficient was 4.12. Adventitious bud induction rate was bigger, 77.42O/oo, when the age of cotyledon was 3 days after unfolding. The effect of adding 0.1 mg/L NAA in MS medium on rooting culture was better, the root can grow strongly, rooting rate of adventitious bud reached 100%. [Conclusion] The suitable hormone concentration combination for in vitro regeneration of the cotyledon of double haploid plants offspring is MS＋6-BA 4.0 mg/L＋NAA 0.2 mg/L＋2,4-D 1.5 mg/L. 3-day-old cotyledon after unfolding as the explants expresses the best induc- tion effect. MS＋0.1 mg/L NAA is the suitable medium for root induction.