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将香榧(Torreya grandis‘Merrillii’)的未成熟合子胚置于SH+0.1 mg·L-1 NAA+500 mg·L-1AC+3%蔗糖+0.5 g·L-1 Gln培养基上暗培养45 d,诱导产生半透明颗粒状胚性愈伤组织;将胚性愈伤组织转入SH+20 g·L-1 PEG+10 mg·L-1 ABA培养基中暗培养3个月诱导体细胞胚。采用碘—碘化钾染色和石蜡切片技术对体胚起源、形态发育与细胞组织学进行了观察。结果表明:胚性愈伤组织起源于合子胚胚轴表皮或皮层细胞的对称分裂。胚性愈伤组织含有两类细胞,一种是细胞质浓厚、细胞核大、体积小的圆形胚性细胞,另一种是高度液泡化拉长的细胞。胚性愈伤组织包含由这两种细胞构成的原胚团Ⅰ、原胚团Ⅱ和原胚团Ⅲ,以及一些游离细胞。原胚团Ⅲ在无植物生长调节剂的SH基本培养基上形成原胚,原胚接入成熟培养基,历经球形、棒状、心形、鱼雷形胚后发育成子叶胚。将子叶胚转入萌发培养基后胚根伸长,胚芽发育长出针叶,形成完整的再生植株。同时在离体培养中合子胚胚柄处退化的裂生多胚也能重新发育形成胚体。在初生体胚发育过程中表面常伴有次生体细胞胚的形成。
Immature zygotic embryos from Torreya grandis ’Merrillii’ were cultured on SH + 0.1 mg · L-1 NAA + 500 mg · L-1 AC + 3% sucrose + 0.5 g · L-1 Gln medium for 45 d, inducing translucent granular embryogenic callus; embryogenic callus was transferred to SH + 20 g · L-1 PEG + 10 mg · L-1 ABA medium for 3 months to induce somatic cells Embryo. Iodine - potassium iodide staining and paraffin sectioning were used to observe the origin, morphology and histology of somatic embryos. The results showed that the embryogenic callus originated from the symmetrical division of zygotic epidermis or cortex cells. Embryogenic callus contains two types of cells, one is a large cytoplasm, large nucleus, small round embryonic cells, the other is a highly vacuolar elongated cells. The embryogenic callus contains progenitor I, procollagen II and procollagen III, as well as some free cells, composed of these two types of cells. Progenome Ⅲ formed embryos on SH basal medium without plant growth regulators, and the proembryos were inserted into mature medium and developed into cotyledons after spheroidal, rod-shaped, heart-shaped and torpedo-shaped embryos. The cotyledon embryo transferred to the germination medium after elongation, germination and growth of needles, forming a complete regeneration of plants. At the same time, the degenerated schizonts and polyembryos in the embryo of zygotic embryos can be re-developed to form embryoid bodies. The surface is often accompanied by the formation of secondary somatic embryos during primary somatic embryogenesis.