This study investigated developmental changes in cold stressed microspores of Indica rice variety At 303. After 3 d at 10°C, approximately 76% of microspores were in the late uni-nucleate stage. Even after 5 d, 49% of viable cells remained in the late uni-nucleate stage without advancing to the bi-nucleate stage. In comparison, microspores undergoing normal gametogenesis in planta progressed rapidly from uni-nucleate to bi-nucleate stage eventually forming tri-nucleate pollen during this period. Thus, cold stress prevented normal microspore development and retained cells in the late uni-nucleate stage which is the most favorable stage for in vitro induction of sporophytic development in rice. Uni-nucleate microspores subjected to cold stress showed a characteristic pattern consisting of several minute vacuoles surrounding a centrally positioned nucleus, which can be interpreted as an early indicator of sporophytic determination in Indica rice microspores. During in vitro culture phase, freshly plated yellow anthers became brown. After 4 wk in culture, 51% of the anthers had discolored. Significantly, all yellow anthers contained only non-viable cells whereas 10% of the brown anthers had few viable cells. Some microspores in brown anthers underwent division on callus induction medium. The first division was symmetrical and occurred after 2 wk. The second division occurred after 4 wk and resulted in four-celled structures. Anther-derived callus was either compact or friable. Histo-differentiation occurred mostly from compact callus. Cell clusters, each delimited by a protoderm, were observed in histological sections of callus grown for 2 to 4 wk on regeneration medium. Within a cellular unit, two heterogeneous cell populations were arranged in concentric rings with larger cells in the center and smaller cells towards the periphery. However, an apical-basal polarity that is present in embryo-like structures was not observed. Therefore, it may be surmised that in Indica rice, regeneration from anther-derived callus takes place not by the formation of somatic embryos but by direct organogenesis.

本研究调查了籼稻品种 At 303 冷胁迫小孢子的发育变化。在 10°C 下 3 天后，大约 76% 的小孢子处于单核后期。即使在 5 天后，仍有 49% 的活细胞保持在单核晚期，而没有进入双核阶段。相比之下，在植物中进行正常配子发生的小孢子从单核到双核阶段迅速发展，最终在此期间形成三核花粉。因此，冷胁迫阻止了正常的小孢子发育并在单核晚期阶段保留了细胞，这是体外培养最有利的阶段。诱导水稻孢子体发育。受冷胁迫的单核小孢子表现出由围绕中心定位的核的几个微小液泡组成的特征模式，这可以解释为籼稻小孢子孢子体测定的早期指标。在体外期间培养阶段，新鲜种植的黄色花药变成棕色。培养4周后，51%的花药变色。值得注意的是，所有黄色花药仅包含非活细胞，而 10% 的棕色花药几乎没有活细胞。棕色花药中的一些小孢子在愈伤组织诱导培养基上分裂。第一次分裂是对称的，发生在 2 周后。第二次分裂发生在 4 周后，产生了四细胞结构。花药衍生的愈伤组织紧密或易碎。组织分化主要来自致密愈伤组织。在再生培养基上生长 2 至 4 周的愈伤组织的组织切片中观察到每个由原胚层界定的细胞簇。在一个细胞单元内，两个异质细胞群排列在同心环中，较大的细胞在中心，较小的细胞在外围。然而，未观察到胚胎样结构中存在的顶端-基底极性。因此，可以推测在籼稻中，花药衍生愈伤组织的再生不是通过体细胞胚的形成而是通过直接的器官发生发生的。