红江橙胚乳愈伤组织诱导和三倍体植株再生

CALLUS INDUCTION AND TRIPLOID PLANT REGENERATION FROM ENDOSPERM OF ’HONGJIANG’SWEET ORANGE

以'红江橙'(Citrus sinensis (L.) Osb.)自然授粉12-16周的幼果为材料、剥取细胞形成中后期的胚乳进行培养。结果表明:低温处理的幼果的胚乳形成愈伤组织的频率有所提高;来自预培养在 MT 分别附加 NAA、GA、BA 的三种培养基中2-6天的胚乳,愈伤组织诱导率明显提高,其中以含 NAA 条件下预培养2天的诱导效果最好,诱导率达22.9%。 2,4-D 是诱导胚乳愈伤组织的主要因素,它与 BA、CH 配合效果更好,诱导率高达33.3%。胚乳愈伤组织先培养于 MT+BA 或 GA_3中30天后,转至不同氮源或激素条件下 20天左右再转回相应的 MT+BA 或 GA_3中,形成胚状体和芽原基。芽原基在单独 BA 或与 GA_3配合条件下形成芽,胚状体在单独 GA_3或再加 BA 的条件下发育成植株。胚乳植株有79.4%的细胞染色体数目2n=26-27,证明再生植株为三倍体。胚乳植株通过嫁接,首次成功地移植于土壤中生长。

Endosperm at late stage of cell formation, excised from open-pollinated fruit of sweet orange (Citrus sinensis Osbeck. cv.'Hongjiang'), was suitable for culture in vitro. The results indicated that 2,4-D was necessary for callus' induction, and supplemented with BA, CH in medium was more effective; the percentage of induction was as high as 33.3%. Endosperm tissues, excised from the fruits treated with low temperature (4—7℃) for 16 and 19 days, and from the young seeds precultured on MT basal medium respectively with NAA, GA_3, BA for 2—6 days, also stimulated to callus formation. When endosperm callus was transfered to the differentiation medium, embryoids and shoot-buds only developed in a sequence of culture conditions. Callus was first cultured on MT + BA/GA_3, then transfered to different media with various nitrogen or hormone concentration, and finally transferred back to the first culture medium. Shoots were regenerated from shoot-buds in the medium in presence of hormones with only BA or with GA_3. The whole plants were regenerated from embryoids in presence of GAs or with BA. Analysis of endosperm plantlets showed that 79.4% of the observed celld have chromosome number of 2n = 26 — 27, nearly the triploid number of 2n = 3x = 27. Through grafting on lemon seedlings as rootstocks in vitro, plantlets were growing successfully in soil.