Optimize On In Vitro Culture Of Unpollinated Ovule In Pumpkin(Cucurbita Moschata D.)

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 replacem-ent. In this study,'Shi Yan NO.1'was materials, MS+sucrose(30.00g·L^-1)+agar(6.00g·L^-1) +2,4-D(1.00mg·L^-1)+NAA(0.25mg·L^-1)+6-BA(0.50mg·L^-1) was the bas-ic medium,and based on this medium,the effects of KT pretreatment and AgNO₃ stimulation on the induction of unfertilized ovules was investigated. Meanwhile,the development state of embryo sac during four important period was observed by paraffin section method and the chromosome number of root tip was investigated by carbol fuchsin squash method. The aim of this study is to reveal the developmental mechanism of embryo of pumpkin and establishbasis for breeding of new pumpkin cultivar. The main results of our resear-ch are showed as follows:1. MS+sucrose(30.00g·L^-1)+agar(6.00g·L^-1)+2,4-D(1.00mg·L^-1)+NAA(0.25mg·L-1)+6-BA(0.50mg·L^-1)is better induction medium for unfertilized ovules embry-oid of pumpkin.The induction rate of embryoid was 3.3%. MS+sucrose (30.00g·L^-1)+agar (6.00g·L^-1)and MS+sucrose(30.00g·L^-1)+agar(6.00g·L^-1)+activated carb-on (1.00g·L^-1) was better suitable for differentiation and regeneration medium.2. The study found that adventitious buds can develop into normal plants, but the ratio is very low,only 0.3%⁶.7%,while the ratio of embryoids develo-ping into normal plants was more than 70%.3. 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.4. The ovary of the three days before blossoming was in mononuclear embryo sac period;the ovary of the two days before blossoming was in binu-cleate embryo sac period;the ovary of the day before blossoming was in tetra-nuclear embryo sac period;the ovary of blossoming day was in mature embryo sac period with seven cells and eight nuclei. Unfertilized ovules of flower bl-ossoming day(0d) was best stage for in vitro culture. Embryoid rate and seedl-ing rate were the highest,and were higher than the other two periods(-2d,-1d).5. 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'.