| In order to obtain the best way to induce un-pollinated ovary and ovule in pumpkin, we used"NO.1"as test material which mainly studied the influencing factors of pumpkin in vitro gynogenesis ,such as heat pretreatment time, 2, 4-D concentration, hormone combination and while the process embryo sac development was observed by the technology of paraffin section. We expected to lay a theoretical and practical foundation on pumpkin in vitro gynogenesis. The result showed as:1. The heat pretreatment in dark was essential for in vitro gynogenesis. The best pretreatment was under 35℃for 6 days on pumpkin in vitro culture of un-pollinated ovary, which had big differences compared with 5 days heat pretreatments and 7days pretreatment(p<0.01). There showed no difference between different development embryo sac, it proved that different development embryo sac has similar sensitivity under heat-inducible. The result of in vitro culture unpollinated ovule in pumpkin showed that: 5 days under this treatment turning green ratio reached 80% and embryoid induction rate reached 15%.was the best heat pretreatment period. The result showed that at 35℃heat treatment for 6 days can effectively improve the embryoid rate. In a word, the best heat pretreatment time was 6days on pumpkin in vitro culture un-pollinated ovary or 5 days on pumpkin in vitro culture un-pollinated ovule.2. The first step of inducting medium for pumpkin in vitro gynogenesis is to compare the influence of 2, 4-D in different concentration to Inducement in Vitro, the result are as follows: 3.5 mg.L-1,4.0 mg.L-1 and 4.5mg.L-1of the 2, 4-D concentration can stimulate the growth of vitro gynogenesis effectively. Embryoid induction rate reached highest level when 2, 4-D concentration was 3.5mg.L-1, and the embryoid induction rates of the three stages embryo sac were above 4.5%. The next was successively 4.0mg.L-1 and 4.5mg.L-1. Cotyledon embryo was inducted on the medium with the three 2, 4-D concentration at pre-flowering 0.5d, and the number was four, two and one. On pumpkin in vitro culture un-pollinated ovule, medium supplemented with 1.0 mg.L-1 2, 4-D and 1.5mg.L-1 2, 4-D can effectively induct gynogenesis. In a word, the amount of adding 2, 4-D to vitro gynogenesis should be different according to different ways of growth of vitro gynogenesis.3. We tried to screening the best hormone combination on MS basic medium. The result showed that: 4.0mg.L-12, 4-D + 0.5mg.L-1 NAA + 0.5 mg.L-16-BA was the optimal compounding for inducting unpollinated ovary in vitro gynogenesis. The best hormone was 1.0mg.L-12, 4-D+0.25mg.L-1NAA+1.0mg.L-16-BA, which induced 6 cotyledon embryos from unpollinated ovule. Comparatively speaking, the medium supplemented with higher concentration 2, 4-Dand NAA and lower concentration 6-BA can effectively induce embryo from pumpkin unpollinated ovary. But inducing embryoid from pumpkin nupollinated ovule needed the medium supplemented with lower concentration 2, 4-D and NAA and higher concentration 6-BA.4. Sucrose and glucose could be used as carbon source of pumpkin un-pollinated ovary in vitro culture, and used to induct gynogenesis. The medium containing 30mg.L-1 sucrose inducted embryoid of the highest rate, comparing induced embryoid between difference sucrose concentrations. Between the induced effects of different glucose concentration, the best induced effect was 40mg.L-1 glucose. Comparing the highest induction rate between different carbon sources, 8.52% was sucrose's, 4.44% was glucose's. The inducted effect of sucrose was better than glucose's. We studied the induct effect of in vitro culture pumpkin unpollinated ovule with the medium containing sucrose. The best sucrose concentration was 25mg.L-1, which embryoid induction rate was 83.3% and 7 cotyledon embryo were inducted.5. The embryo sac development process was observed by technology of paraffin section. The result showed that: at pre-flowering 3 d, embryo sac was at mononuclear stage; at pre-flowering 2 d, embryo sac was at binuclear stage; it became tetranucleate embryo sac, at pre-flowering 1 d; at pre-flowering 0.5 d, eight nuclears embryo sac cell could be observed in ovule.
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