Department of Biology, Yale University, 06511, New Haven, CT, USA.
Planta. 1981 Oct;153(1):64-74. doi: 10.1007/BF00385319.
Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 μg d(-1) during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10(-6) M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 μg embryo(-1) d(-1)) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.
甘蓝型油菜幼胚在有和没有各种浓度发芽抑制剂的情况下进行体外培养，并通过比较培养物中贮藏蛋白的积累与种子中的正常积累来监测胚胎发生的进展。从种子提取物中纯化出油菜的两种主要贮藏蛋白（12S 和 1.7S），并用火箭免疫电泳（12S 蛋白）或十二烷基硫酸钠聚丙烯酰胺凝胶电泳（1.7S 蛋白）进行分析。在种子内胚胎发育过程中，当子叶充分发育时（胚干重 0.4mg），首次检测到 12S 和 1.7S 蛋白，并且在最大沉积期间，每种贮藏蛋白的积累速度平均为 26μg/d。当胚含水量开始下降（胚干重 2.7mg）时，1.7S 蛋白的积累停止，但 12S 蛋白的积累一直持续到种子成熟（胚干重 3.6mg）。在胚胎发育结束时，12S 和 1.7S 蛋白分别约占总盐溶性蛋白的 60%和 20%。当胚在第 27 天从种子中取出，正好是贮藏蛋白开始积累的时候，并且被放置在基础培养基上进行培养时，它们在 3 天内过早地发芽，并且氨基酸掺入 12S 贮藏蛋白的量从总掺入量的 3%下降到不到 1%。如果培养基中含有 10(-6)M 的脱落酸（ABA），则将胚放入培养物中时，氨基酸掺入 12S 蛋白的量从总掺入量的 3%增加到 18%，5 天后，积累速率（27.1±2.6μg 胚-1 d-1）与在种子中观察到的最大速率相匹配。高渗透压剂，如 0.29M 蔗糖或甘露醇，添加到基础培养基中，也抑制了早熟发芽，但在 12S-蛋白合成速率与 ABA 培养基的速率相等之前，存在一个滞后期。这些结果表明，种子环境中的某种因素是储存蛋白合成进行所必需的，而 ABA 是一个可能的候选物。
