Acquisition of plants regenerated from embryo sac of unfertilized ovary in muskmelon

Abstract:【Objective】To provide new materials and technical support for the theoretical study on plant genetics and practical breeding, the influencing factors of haploid formation induced by unfertilized ovary of muskmelon were studied.【Methods】Three muskmelon varieties were used as experimental materials, and unfertilized ovary melons were cultured in vitro on the MS medium at 25 °C with 16 h/8 h (day/night) and 2 000 lx light intensity in a culture chamber. The influence of different genotypes, culti- vation seasons (spring and summer sowing), heat shock times (2 d, 3 d and 4 d), inoculation modes (slitting and crosscutting) and hormones (TDZ, KT and IAA) were compared on embryoid induction, and al- so the impact of different hormones (KT, 6-BA, GA3) on embryoid occurrence was investigated. The ploidy of regeneration plants was identified by the flow cytometer, and judged by the peak value, with the diploid plant serving as the control.【Results】Embryo induction rate was significant difference among three different genotypes, and the embryoid induction frequency of‘Jiangjunyu’was the highest (38.3%), next to it was‘Qianlongmi’(28.3%), and the lowest embryoid induction frequency was found with‘Zhongyun 18’(18.3%). The embryoid induction frequency of the material sowed in sum- mer was higher than that sowed in spring, and the performance of the three melon materials was consis-tent. Among them, the embryoid induction frequency of‘Jiangjunyu’sowed in summer was the highest (38.3%), followed by‘Qianlongmi’and‘Zhongyun18’(28.3% and 18.3%, respectively). The highest induction rate of‘Jiangjunyu’sowed in spring was 6.7%, followed by‘Zhongyun 18’(3.3%), and the embryoid induction frequency of‘Qianlongmi’was zero. Ovary inoculation materials were treated for 3 days in the dark at 35 °C, and the embryo induction rate was higher than that for 2 and 4 days. Three embryoids were produced per bottle on average, followed by 2 days of heat shock with an average of one embryoid produced per bottle，and no induced embryoids were obtained after 4 days of heat shock, and the embryoid was prone to vitrification. Transverse or longitudinal cutting of ovary had no signifi- cant influence on embryoid induction. The embryoid frequency of transverse cutting was 33.3%, and the embryoid induction rate of longitudinal cutting was slightly lower than that of transverse cutting, which was 28.3%. However, more abnormal buds produced easily during vertical cutting, which affect- ed the formation of embryoids. Under the condition of adding 0.03 mg · L- 1 TDZ alone in MS medium, the embryoid induction rate achieved up to 25.0%. When the concentration was 0.06 mg·L-1, the embry- oid induction rate was the lowest (5.0%), the green ovule grew very slowly, gradually faded and turned yellow.Onthebasisof0.03mg·L-1 TDZ,0.2mg·L-1 KT,0.01mg·L-1 IAAand15%coconutjuicewere added, and the embryoid induction rate was the highest (46.7%). The results showed that the induction rate of embryoid was higher as the hormone concentrations increased. The germ elongated rapidly on the MS medium with 0.2 mg·L-1 KT, the bud elongated fast but got yellow with 0.2 mg·L-1 6-BA, and when GA3 concentration was 200 mg · L- 1, the bud elongation was slow and vitrification took place. Rootsgrewfasterandstrongerwhen0.1mg·L-1 IAAwasaddedinthe1/2MSmedium.DNAflowcy- tometry was used to identify the ploidy of regenerated plants obtained from megaspore. The results dem- onstrated that the group of regenerated plants was complex, which included haploid, diploid, triploid and mixed-ploid plant types. The proportion of diploid plants in the three varieties‘Jiangjunyu’‘Qian- longmi’and‘Zhongyun18’was 64.3%, 80% and 50.0%, respectively, and the proportion of haploid plants was 21.4%, 20% and 12.5%, respectively. Triploid and chimera were only found in‘Jiangjunyu’and‘Zhongyun18’, with the proportions of triploid being 7.1% and 12.5%, respectively, and their chimera proportions were 7.1% and 25%, respectively.【Conclusion】Different genotypes of melon materi- als had a great influence on the induction rate of embryoid. The embryoid induction rate of plants in different sowing periods varied significantly. The induction frequency of summer sowing materials was higher than that of spring sowing materials. Heat shock pretreatment could initiate the development of female nucleus, and the best effect was achieved in 3 days after the treatment of in vitro ovary by dark heatshockat35°C.Theoptimalembryoidinductionmedium was MS+0.03mg·L-1 TDZ+0.2mg·L-1KT+0.01mg·L-1 IAA+15% coconutjuice, which show edtheembryoidinductionratewasup to 46.7%. The group of regenerated plants was complex, which included haploid plant with weak growth, diploid plant with strong growth and normal fertility, triploid plant with normal growth but abnormal fertility and mixed ploid plant types.