黄花苜蓿高频植株再生体系建立的研究

Establishing A Highly Efficient Plant Regeneration System of Medicago falcata L.

黄花苜蓿(Medicago falcata L.)具有较强的抗逆性,是苜蓿育种重要的种质资源。进行黄花苜蓿功能基因研究,有必要建立其高效的植株再生体系。以新疆野生黄花苜蓿的子叶和下胚轴为外植体,以N6大量,MS微量及铁盐,水解酪蛋白2.0 g·L^(-1),维生素VB_19.9 mg·L^(-1),VB_69.5 mg·L^(-1),尼克酸4.5 mg·L^(-1),琼脂8.0g·L^(-1)和蔗糖30 g·L^(-1)等成分为基本培养基,通过比较不同激素配比对黄花苜蓿愈伤诱导率和体细胞胚发生率的影响,优化并建立了黄花苜蓿的高频再生体系。优化的培养基激素配比为:愈伤诱导和保持培养基添加2,4-D3.0 mg·L^(-1)+KT 0.05 mg·L^(-1);分化培养基添加KT 0.4 mg·L^(-1),蔗糖浓度调整为20 g·L^(-1);生根培养基为1/2 MS+蔗糖15 g·L^(-1)+琼脂8.0 g·L^(-1)。在优化培养流程下,新疆野生黄花苜蓿子叶愈伤诱导率可达92%,下胚轴可达95%,体细胞胚发生率可达54%,体细胞胚萌发成苗率为76%。再生周期约为70~80 d。

Medicago falcata is an important germplasm resource for alfalfa breeding because of its strong stress resistance.A highly efficient plant regeneration system is necessary to study the functional genes of M.falcata.The cotyledons and hypocotyls of wild M.falcata from Xinjiang are used as explants to investigate the effects of hormones ratio on callus induction rate and somatic embryos induction rate.A highly efficient plant regeneration system of M.falcata is established in this study.The basic medium includes N6 macronutrients ＋ MSmicronutrients ＋ MS iron ＋ VB1 9.9 mg· L-1＋ VB6 9.5 mg· L-1＋ Nicotinic acid 4.5 mg· L-1＋ CH 2.0 g· L-1＋ agar 8.0 g· L-1,and sucrose 30 g· L-1.For callus induction and maintenance,2,4 D 3.0 mg · L-1 and KT 0.05 mg · L-1 are added.During somatic embryos induction stage,KT 0.4 mg · L-1are added and sucrose is adjusted to 20 g · L-1.Medium for somatic embryo germination and plantlet rooting consists of 1/2 MS ＋ sucrose 15 g · L-1＋ agar 8.0 g · L-1.In this system,the callus induction rate of cotyledon is more than 92%,and that of hypocotyl is more than 95%,while the induction rate of somatic embryos is 54%,and the rate of somatic embryos developing into whole plantlets is 76%.The period of regeneration is about 70～80 days.