大豆整个子叶节外植体再生体系的建立及与子叶节、胚尖再生体系的比较

High Frequency Plant Regeneration from Whole Cotyledonary Node Explants and Comparison with Cotyledonary Node and Embryonic Tip Regeneration System in Soybean [Glycine max(L.)Merrill]

大豆再生较为困难，难以满足基因工程的需要。为建立一种快速高效的再生体系，大豆整个子叶节作为外植体进行再生。成熟大豆种子在MSB，添加BA0．4mg·L^-1的培养基中萌发5～7d后，切取子叶节外植体，含有BA或CPPU添加一定浓度的IBA的培养基用于筛选芽诱导培养基，确定了芽诱导的最适培养基条件为MSB，添加BA3．0mg·L^-1和IBA0．2mg·L^-1，将再生芽放入MSB5培养基中伸长至3cm左右后，放入MSB5添加IBA0．5mg·L^-1的培养基中生根，生根的小苗炼苗后移栽。大豆整个子叶节再生体系芽再生频率可以达到94．7％，平均每个外植体可以得到28个芽。将此再生体系与传统的大豆子叶节再生体系、近几年应用较多的大豆胚尖再生体系在再生频率、出芽数目、芽伸长情况以及再生周期等方面进行比较，结果表明：大豆整个子叶节再生体系在外植体再生频率以及出芽数量上优于其它两种体系。

Soybean [ Glycine max （L.）Merrill] in vitro regeneration had been proved to be difficult, which limited the application of gene engineering on soybean. A more efficient and successful regeneration system for soybean was reported here using whole cotyledonary node as the explant. Whole cotyledonary node explants were obtained from aseptic seedlings cultured on MSB5 medium supplemented with N^6- benzyladenine （BA） at the concentration of 0.4 mg·L^-1 for 5- 7 d. MSB5 medium containing different concentration and combinations of BA or N - （ 2 - chloro- 4- pyridyl） - N ＇- phenylure a （ CPPU ） with indole- 3 - butyric acid（IBA）were selected for shoot regeneration in whole cotyledonary node regeneration system and MSBsmedium containing 3.0 mg·L^-1 BA and 0.2 mg·L^-1 IBA was proved to be optimum. The regenerated shoots were elongated on the MSB5 medium to about 3 cm and rooted on MSB5 medium containing IBA 0.5 mg ·L^-1. Plantlets with well - developed roots were hardened and planted in greenhouse to maturity. Using of this system, shoot regeneration frequency could reach 94.7% and one explant could regenerate 28 shoots on average. In comparison with traditional cotyledonary node and embryonic tip regeneration system, whole cotyledonary node regeneration system was superior in shoot regeneration frequency and number of shoot.