| Zanthoxylum bungeanum is an important economic species in China.It has higher medicinal and edible value.Since the living standard of continual improve,the commodities of zanthoxylum have been to diversity,the industrial structure need to upgrade,however,it is rather disproportional compare to the expected zanthoxylum germplasm of the enormous consuming market.One of the best practical practices for solving the problem is to develop new excellent cultivars.Currently,Zanthoxylum genetic improvement mainly relies on natural variation and hybridization,but zanthoxylum belongs to apomictic plants,which makes Zanthoxylum germplasm enhancement and breeding work has been difficult.The technique of protoplast isolation,culture and fusion,which can enrich the germplasm resources,have shown great potential in breeding.Therefore it is significant to study protoplast culture and fusion in order to create new germplasm and improve genetic characteristics of cultivars in zanthoxylum.In this study,in order to meet the requirements of protoplast culture,research has been carried out of three aspects:First,embryo culture of Zanthoxylum in Hancheng county;second,using leaves of Zanthoxylum bungeanum as experimental material,protoplast isolation and purificiation of Zanthoxylum in Hancheng county,for establishing protoplast isolation technology;third,taken protoplast from plantlets leaves of Zanthoxylum in Hancheng county,as materials to study protoplast culture technology system.The main results are as follows:1.A Preliminary Study on Embryo Culture in Zanthoxylum bungeanumSeeds of Zanthoxylum bungeanum were taken as experimental material for studying the effects of different conditions on embryo culture.The results showed that seeds of Zanthoxylum bungeanum were soaked in 75%alcohol for 30s,then being sterilized into 0.1%HgCl2 for 10 min.The mature embryos were inoculated vertically in the 1/2MS medium for induction.The optimum medium for inducing the adventitious bud was MS+6-BA 1.0mg·L-1+NAA 0.1 mg·L-1+sucrose 30 g·L-1+agar 6 g·L-1,with the highest frequency of65.44%.And on the medium of MS+6-BA 0.5 mg·L-1+IBA 0.1 mg·L-1+sucrose 30g·L-1+agar 6 g·L-1,the adventitious could be multiplied rapidly.The rooting culture medium was 1/4MS+IBA 0.4 mg·L-1+sucrose 30 g·L-1+agar 6 g·L-1.2.Protoplast isolation and purification of Zanthoxylum bungeanumUsing leaves of Zanthoxylum bungeanum as experimental material.Through experiment,studying the effects of pretreatments,leaf age,osmoticum concentration and sources leaves on protoplast separating,and protoplast yield and protoplast viability were measured by using hemocytometer counting and FDA staining.The results showed that there were significant difference of protoplastyield and viability between leaves in 4℃and in dark for pretreatment.The mesophyll protoplasts from the aseptic seedling of less than 35d was better,and the young leaves of less than 60d were suitable for preparing protoplasts.3.Protoplast culture of Zanthoxylum bungeanumPlantlets leaves of Zanthoxylum bungeanum were taken as experimental material for studying the effects of the medium,the method,the composition of hormone,the protoplasts density for protoplast culture.The results showed that the protoplasts were cultured on the medium of modified WPM+0.5mg/L 2,4-D+1.0mg/L 6-BA+1.0mg/L NAA.Cell division was found in 3-4 days.Cell division took place 3-5 times after 2 weeks culture.Cell colony was found in 28 days and microcalli in 60 days.When regenerated mini calli grew to 0.5cm,the protoplast culture medium was needed to change into the adventitious bud medium.Only4 adventitoius buds were obtained in this experiment. |
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