| Tung tree(Vernicia fordii),a genus of the Euphorbiaceae family,is an important woody industrial oil species in China.However,the lack of seeds with good quality has seriously restricted the development of the tung tree industry.Nowadays,haploid breeding and molecular breeding has already been proved to be one of the most important way to breed high-yielding and highly resistant oilseed tungsten seeds.Our group has developed Agrobacterium-mediated transgenic technology in the root of tung tree,but has not yet achieved to construct a complete genetic transformation system through root regeneration.In this paper,we investigated the effects of different pretreatment methods,different basic media and hormone ratios on the morphogenesis of tung tree under in vitro culture by using ovules,radicles and roots as the explants,and finally obtained haploid guava tissue using ovules as the explants and regenerated plants using radicles as the explants.The study laid an important foundation for the ploidy breeding and molecular breeding of tung tree,and the main findings are as follows.1.The optimal pretreatment and time for ovule guilt induction was confirmed,and ovules could be induced to three different states of callus and embryoid.The highest callus induction rate of 92.59%and the lowest ovule browning rate of 6.17%were achieved after 2-day pre-chilling treatment,indicating the short-term pre-chilling treatment was beneficial for the callus induction of ovule.The browning rate of ovules after the pre-heating treatment was significantly higher than that of the pre-chilling treatment and the control group without prechilling,and the callus induction rate was significantly lower than that of the precooled treatment and the control group.Therefore,2-day pre-cooling treatment at 4℃ is the best way for pretreatment of in vitro culture of ovule.2.The optimal basic medium and hormone ratios for ovule callus induction was confirmed,and ovules could be directly induced to produce adventitious shoots.In ovule callus induction,several basic media were effective in the following order:MS>1/2 MS>WPM>white.The optimal basic medium and hormone ratios for ovule callus induction was confirmed by orthogonal tests:MS+0.04 mg/L TDZ+1.0 mg/L 6-BA+0.1 mg/L NAA+0.1 mg/L IAA with induction rate of 96.30%.153 callus were examined for ploidy by flow cytometry,and 1 haploid,142 diploid,6 tetraploid,and 4 mixed ploid were obtained.The ovules were found to generate adventitious shoots directly after 20 d of culture in 1/2 MS+0.06 mg/L TDZ+2.0 mg/L 6-BA+ 0.05 mg/L NAA+0.1 mg/L IAA medium,which bypass callus formation,although the roots could not be grown further due to weak growth capacity,it proved the potential of the ovules to produce adventitious shoots directly by induction.3.The system of tung tree radicle regeneration was established.The best hormone combination for the induction of adventitious shoots by ovules was determined MS+6BA 1.0 mg/L and GA3 2.0 mg/L.The induction rate was 68.24%and the average number of shoots was 1.54.The best medium for the proliferation of adventitious shoots of tung tree roots was 1/2 MS+1.0 mg/L 6-BA+0.1 mg/L IBA+1.0 mg/L GA3.The most suitable medium for adventitious shoot growth was 1/2 MS+0.5 mg/L NAA+1.0 mg/L GA3.Robust adventitious shoots were transferred to WPM-based medium with 1.0 mg/L NAA+2.0 mg/L GA3 rooting medium for rooting culture,and the rooting rate could reach more than 80%after 30 days.4.In vitro culture of tung tree roots was explored.The best pretreatment for root induction and regeneration of tung tree was determined using primary and fibrous rootsof live tung tree seedlings as explants:washing powder for 30 min+75%alcohol for 1 min+0.1%ascending mercury for 45 min.The best formulation for callus induction was 0.1 mg/L 2-ip+2.0 mg/L 6-BA+0.05 mg/L ZT+2.0 mg/L GA3.Probably,the callus failed to induce further adventitious shoots because of reduced root activity due to sterilization. |
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