| The gene of pumpkin is greatly heterozygous,and it would take too much time and cost a great deal of labor to obtain pure lines by self-crossing.Therefore,the study of inducing regeneration plants through ovule and ovaries culture is of great significance.The haploid plants of Cucurbitaceae was obtained mainly by in vitro culture of ovary or ovule.In this study,unfertilized ovule was cultured in oscillating liquid culture medium for days and then transferred to solid culture medium,which is a new way of in vitro culture of pumpkin.The effects of disinfection method,oscillation speed,liquid culture time,hormone,sucrose concentration,plantlet transplantation and so forth were studied and the process of ovule development observed,based on MS medium without coagulant.The following results have been achieved:1.It is a better way to extract ovules from ovary disinfected in Mercuric Chloride solution for liquid culture to induce callus,of which growth is robust and fine.The callus tissue of ovule cultured in liquid medium was derived from the integument,the embyoid is derived directly or indirectly from the interior of the ovule.2.150 rpm is relatively suitable vibrating speed for liquid culture of unfertilized ovule of pumpkin,which could keep the callus tissue size from augmenting rapidly as well as maintain the integrity of the ovule,surpassing 100 rpm and 200 rpm.3.Hormone has a great effect on ovule enlargement and callus producing.The hormone combination of 0.5 mg/L 6-BA+0.25 mg/L NAA+1.0 mg/L 2,4-D was best for liquid culture,in which the callus rate reach 91.54% after culture for 5 days.5 d is the best liquid culture time,better than 3 d and 7 d.4.The callus rate of Cucurbita moschata Duch.ex Poir.exceeded the one of Cucurbita maxima Duch.ex Lam..After cultured in liquid medium for 5 days,‘Huang lang' reached 94.37%,which exceeded ‘Yong an 2' by 9.65%.5.It was easier to obtain embryoid for pumpkin ovule to be cultured in liquid MS+0.5 mg/L NAA + 0.05 mg/L TDZ for 5 days and then transferred to modified Nistch +2.0 mg/L 6-BA +0.1 mg/L NAA.6.40 g/L is the best sucrose concentration in liquid culture medium,which is better than 30 or 60 g/L.And the callus rate of ‘Yong an 2'? ‘Huang lang' and ‘Mi ben' was 91.28%?95.15% and 99.00% respectively.7.It is a better way for unfertilized ovule of ‘Mi ben' to reach a high embryoid rate,being 9.52%,after cultured in liquid MS+0.5 mg/L NAA + 0.05 mg/L TDZ+40 g/L sucrose+ 7.5 mg/L colchicine for 5 days and subsequently cultured in solid modified Nistch +2.0 mg/L 6-BA +0.1 mg/L NAA for 25 days.8.It is a better way of plantlet transplantation to wash and soak the root in 2.5 g/L carbendazim solution after breaking the culture bottle,then rinsing them with water,transferred them to float tray filled with substrate disinfected by a little carbendazim.After that,the plantlets should be watered every day by a small amount of water.In short,it is concluded that the culture technical system of unfertilized pumpkin ovule is as following: After ovaries of female flowers 1 day before flowering were collected in the afternoon,they should be disinfected and cut into 2 to 3 mm thin slices with a scalpel,from which the ovule were carefully picked out with a small tweezer and placed in liquid MS medium +0.5 mg/L NAA +0.05 mg/L TDZ +40 g/L sucrose + 7.5 mg/L colchicine.After cultured for 5 days at oscillation speed of 150 rpm,the ovule were transferred to solid modified Nistch medium + 2.0 mg/L 6-BA + 0.1 mg/L NAA +30 g/L sucrose + 500 mg/L glutamine + 6.8 g/L agar with ovule side rather than callus side inserted in the medium.Subsequently they were cultured in modified Nistch medium +30 g/L sucrose + 500 mg/L casein hydrolysate + 6.8 g/L agar.After culture for 11 weeks,the embryoid can be induced.After the seedlings were formed,the ploidy were identified by flow cytometry.There were 8 haploid plantlets.Plantlets were transplanted after disinfection for twice. |
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