Smith D L, Krikorian A D
Dept of Biochemistry and Cell Biology, State Univ. of New York, Stony Brook 11794-5215.
Physiol Plant. 1990;80:329-36.
A mixed culture comprised of both embryonic globules and nonembryogenic callus was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D- containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 mM NH4+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages. Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2,4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of non-embryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.
采用成熟的方法，从胡萝卜品种“ Scarlet Nantes”的幼苗下胚轴在含2,4 - D的培养基上获得了由胚性小球和非胚性愈伤组织组成的混合培养物。然后将混合培养物转移至pH接近4的无激素培养基上，并进行连续继代培养。该培养基含有1 mM NH4 +作为唯一氮源。以这种方式培养时，胚性小球能够增殖而不发育到后期胚胎阶段。非胚性愈伤组织无法存活。在这种低pH无激素培养基上对胚性小球进行连续培养，得到了完全由球形前期原胚（PGSPs）组成的培养物。PGSP培养物已如此连续增殖维持了近2年，而未丧失胚性潜力。当在pH 5.8缓冲的相同无激素培养基上培养时，这些无激素维持的PGSPs会继续发育到后期胚胎阶段。我们表明，pH接近4的无激素培养基在维持胡萝卜2,4 - D启动的胚性细胞以可接受的生长速率增殖而不发育到后期胚胎阶段的能力上，可以替代2,4 - D。该程序提供了选择性条件，不允许非胚性细胞生长，同时为胚性细胞增殖提供了适宜的环境，在研究驯化方面应具有重要价值。
