| ABSTRACT:Pumpkin（Cucurbita moschata D.）, which has higher nutritioal and ornamental value is an important economic vegetable crop in our country. While the pumpkin varieties is a litter and very slow replacement. Haploid breeding has advantages because of the high purity of doubled haploid plants. These plants are ture breeding for all the characters and the selection efficiency is greatly enhanced. It requires only single selection generation thereby improving breeding efficiency. 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, KT pretreatment and AgNO3 stimulation, 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 main conclusions as follow: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 unpollinated 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 6 days on pumpkin in vitro culture unpollinated ovary or 5 days on pumpkin in vitro culture unpollinated 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: 3.5mg·L-1、4.0mg·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 in vitro gynogenesis.3. We tried to screening the best hormone combination on MS basic medium. The result showed that: 4.0 mg·L-1 2,4-D+0.5 mg·L-1 NAA+0.5 mg·L-1 6-BA was the optimal compounding for inducting unpollinated ovary in vitro gynogenesis. The best hormone was 1.0 mg·L-1 2,4-D+0.25 mg·L-1 NAA+1.0 mg·L-1 6-BA, which induced 6 cotyledon embryos from unpollinated ovule.Comparatively speaking, the medium supplemented with higher concentration 2,4-D and 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 30 mg·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 40 mg·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 25 mg·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 preflowering 3d, embryo sac was at mononuclear stage; at preflowering 2d, embryo sac was at binuclear stage; it became tetranucleate embryo sac, at preflowering 1d; at pre-flowering 0.5d, eight nuclears embryo sac cell could be observed in ovule. AgNO3 stimulation on pumpkin unfertilized ovule promoted the occurre-nce of adventitious buds.16mg·L-1 was the best concentration for the ovules. KT pretreatment on pumpkin unfertilized ovule inhibited the occurrence of adv-entitious buds. The inhibition was the most obvious in the current unfertilized ovule. The occurrence rate of adventitious buds was reduced from 50% to 3.3%. The inhibition on other two inoculation period of unfertilized ovule was lower than 0 day. KT pretreatment and AgNO3 stimulation had some inhibitio-n on the induction of unfertilized ovules and seedlings.6. The ovary of the three days before blossoming was in mononuclear embryo sac period;the ovary of the two days before blossoming was in binucleate embryo sac period;the ovary of the day before blossoming was in tetranuclear embryo sac period;the ovary of blossoming day was in mature embryo sac period with seven cells and eight nuclei. Unfertilized ovules of flower blossoming day （0d） was best stage for in vitro culture. Embryoid rate and seedling rate were the highest,and were higher than the other two periods （-2d,-1d）.7. Drawn the materials at 10 am,use Kano fixative to fix for 24h after 4℃cold water treating for 24h,and use 1mol·L-1 hydrochloric acid in 60℃w-ater bath dissociation for 7 minutes,at last use modified carbol fuchsin to sta-in for 10min,microscope. The cell chromosomes treated by this method were clear and easy to observe and count. The number of chromosome were 44 by observing‘Shi Yan NO.1’.8. Trifluralin is a herbicide .Trifluralin have function of making double chromosomes. In the experiment the pumpkin shoot apices were treated with different concentration of triflualin and colchicine.The results showed that triflualin was an efficient agent for induction of pumpkin chromosome doubling with shoot apices, the suitable concentration was 0.006%.Colchicine could be efficiently replaced by trifleralin. Triflualin concentration for tetraploid induction is very low and effective. The price of trifleralin is also low. It is low toxicity and less harmful to human and environment. Flow cytometry was used to determine the ploidy of pumpkin plantlets induced after trifleralin and colchicine treatment in shoot apices. |
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