柑桔高效离体再生系统研究

Studies on a direct shoot regeneration system of Citrus sinensis L.Osbeck with a high frequency in vitro.

研究了植物激素、刻伤、放置方式等因子对广柑子叶和上胚轴切段离体再生的影响 ,建立了一个高效的再生系统 .该系统的最佳条件是 :采用实生苗子叶和上胚轴茎段作为外植体 ,MS无机盐 +B5维生素 +5%蔗糖 +0 .8%琼脂 +7.5mg/ L BA+1mg/ L NAA(p H5.7)作为不定梢诱导培养基 ;子叶沿中间纵切为二 ,同时在非切面部分横向刻伤 ,切面朝下与培养基表面接触放置 ;上胚轴则切成 0 .5～0 .8cm长的切段 ,横放于培养基上 .培养条件为恒温 (2 6± 1)℃ ,首先在黑暗下培养 15d,而后转移至16 / 8h光 /暗周期下 ,共经过 70 d培养 ,95%子叶外植体均能发生不定梢 ,平均每个外植体上发生不定梢数为 11.11个 ,96 %的上胚轴切段能发生不定梢 ,平均每个外植体上发生不定梢数为 5个 .这一系统再生频率高 ,操作简便 。

Transgenic plants appear to be a new way to citrus improvement and a powerful tool for studying gene functions. The establishment of an efficient in vitro plant regeneration system suitable for genetic transformation is the first step in this approach. Using cotyledons and epicotyl pieces of one month old seedlings of sweet orange ( Citrus sinensis L. Osbeck) grown in vitro as explants, a direct shoot regeneration system with a high frequency was established. The best results were obtained when cotyledons with the surfaces wounded horizontally by a razor blade were cut longitudinally and epicotyl pieces were placed horizontally on the surface of culture medium. The optimal culture medium is MS salts + B5 vitamins + 5% sucrose + 7.5 mg/L BA + 1 mg/L NAA + 0.8% agar, pH5.7. The best response was obtained when the explants were incubated at (26±1)℃ for 15 days in darkness, followed by 45 days on a 16/8 h light/dark cycle. The best regeneration system tested allowed the attainment of adventitious shoot from 95% and 96% of the cotyledon and epicotyl explants, respectively. An average of 11.11 well differentiated shoots per explant was obtained in cotyledon explants, and in epicotyl explants the yield was 5.