| Three tobacco (Nicotiana tabacum L.) cultivars, SR-1, W38, GEXING 1 were used for isolating zygotes. An efficient and simple protocol was optimized for isolating not only zygote protoplast but also zygotes with different cases of remaining cell wall. Living isolated zygotes of tobacco were cultured in vitro by microculture technique. As a result, an in vitro zygotic embryogenesis system was build up, in which single tobacco zygotes could develop into fertile plants via embryogenesis or organogenesis under optimal condition. Our result revealed that zygote cell wall and its directional expansion play critical roles in zygotic embryogenesis in vitro. Besides, zygotes were induced todevelop into fertile plants through embryogenesis by means of a simple and stable ovule culture technique. A single-cell-squashing technique was also established for the observation the dynamic distribution of organelle DNA in tobacco zygotes and early embryos. The main results are summarized as follows:1. An efficient and simple protocol was optimized for isolating not only zygote protoplasts but also zygotes with different cell wall remaining. Zygotes at 108 HAP(hour after pollination) were classified into four different types according to the ratio of their vertical and transverse length. The ratio in Zl zygotes was 1.0, and Z2, Z3, Z4 was 1.2, 1.5, and 2.0 respectively. Different types of zygotes were used for detailed comparative study.2. Living zygotes of tobacco SR-1 were isolated and cultured in vitro by microculture technique. Fertile plants were regenerated from the calli derived from cultured zygotes via organogenesis. Ovules of 120 HAP were collected and used as feeder. MS combined with KM8p was selected as basic medium. Zygotes isolated at HAP 108 turned out to be suitable material for in vitro culture. Over 80% such zygotes could divide and around 10% of them could grow into calli and regenerate fertile plants.3. Z4 zygotes at 108 HAP was isolated through short enzymatic maceration or mechanical dissection; its ratio of longitudinal length and transverse length was 2.0. Different media and culture methods wereapplied and compared. In terms of formation of mature embryos, M3 was better than M5, but with regards to formation mature embryo capability, M5 was better than M3. The optimal method for inducing zygotic embryogenesis in vitro was method D. As different media were used in Minicell and Petri-dish with feeder system, the zygotes did not show evident deviation from division pattern of embryo in vivo. The optimal protocol for mimic early division mode of embryo in vivo was method B. Developmental patterns of early stage, such as mode of first division, division pattern of basal and apical cell, number and fate of suspensor were all studied in vitro. According to the results of our experiment, the suitable conditions for tobacco zygotes embryogenesis in vitro were summarized which could bear out feasibility of zygotic embryogenesis in vitro, especially the early events, which play critical roles in embryogenesis.4. Some important parameters related to zygotic development, including area, perimeter, transverse length, vertical length and the ratio of transverse length and vertical length were investigated during zygote direction expansion. All of the five indexes increased smoothing at early stage of zygote development. A late stage of zygote directional expansion, all the indexes, except transverse length, very quickly increased.5. Developmental fate of the four different types of zygotes at 108HAP (Z1-Z4) in their further development was traced and the earlydivision mode was noticed. It was proved that zygote cell wall played an important role in the determination of cell development fate, and affected early division mode during embryogenesis in vitro. Only the Z4 zygotes with quite complete cell wall could fulfill embryogenesis conforming to the developmental mode in vivo.6. A single-cell-squashing technique was established for the observation of organelle DNA distribution...
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