| Bupleurum chinense DC.is a perennial herb belonging to the BupleurumL genus of Umbelliferae family.It’s officially recommended as one of the source of Radix Bupleuri(in Chinese,Chaihu)by "Pharmacopoeia of People’s Republic of China"(2015 edition).B.chinense DC.,one of the herbs in traditional Chinese medicine(TCM).The usage of Radix Bupleuri is in the forefront of all the TCM.Polyploid plants have huge features which can improve the medicinal component content of the plants.In this study,Chuanbeichai No.1 which is new commercial variety of B.chinense DC.was used as experimental material.The condition of generation proliferating which shorten the growth peroid of B.chinense DC.in greenhouse was studied and it shorten the time of polyploification resources applied in Radix Bupleuri breeding as well.By using the best generation proliferating conditions,Chuanbeichai No.1 were induced with treating the shoot tip meristem and soaking method(whole plant with colchicine).The effects of different treatment conditions and time on survival rate、mutation rate and chlorophyll content were studied.By compareing the character difference of mutation group and diploid plants,finding optimal conditions for chromosome preparation of root-tip in B.chinense DC.,the chromosome polyploidy of mutation group were confirmed which were the foundation for further study of polyploid Radix Bupleuri.The results of the study are as follows:1.The conditions which reduce the growth peroid of B.chinense DC.The conditions in greenhouse for generation proliferating which shorten the growth peroid of B.chinense DC.were:50%nutritional soil + 50%sand to germination,more than 200 cm2 growth area,25 ℃,a photoperiod of 16 hours,and moderate water supply.By using this method,2.46 generation per annum of B.chinense DC.which greatly shorten the growth peroid can be obtained.It provides technical support for obtain the fertile progenies of polyploid radix bupleuri as well.2.The effects of different treatment conditions of B.chinense DC.For the study of colchicine induced methods,Soak method is more suitable than treating the shoot tip meristem.Under this method,by only using colchicines,the average mutation rate of Radix Bupleuri is 41.11%;two combination were both the best treated combination which gained the highest mutation rate of 66.67%:4 hours treatment time by using 0.1%colchicine concentration and 5 hours treatment time by using 0.4%colchicine concentration.While by adding gibberellin in colchicine,the best treated combination which is 3 hours treatment time by using 0.2%colchicine concentration gained the highest mutation rate.By adding gibberellins in colchicine,the average mutation rate of Radix Bupleuri is 26.11%.Compareing the average mutation rate of Radix Bupleuri,the colchicines without gibberellin is more appropriate in creating polyploid Radix Bupleuri.Treatment times and concentrations have significant influence on the chlorophyll content of B.chinense DC..The highest chlorophyll content is 55.58 SPAD under the treatment time:3 hours and 0.5%colchicine concentration.While with adding gibberellin in colchicine,the highest chlorophyll content is 67.14 SPAD under the treatment time:5 hours and 0.4%colchicine concentration.Those were all significantly higher than the control group(19.99 SPAD).Adding gibberellin in colchicine increased the chlorophyll content of B.chinense DC..3.Some of the biological indexes have significant differencebetween treated combination and the control groupThe mutation group compared to control group:the length of leaf shorten 42.85%,the width of leaf increased 116.67%,the leaf index is only 26.63%of control,the plant height shorten 30.88%,the stem diameter increased 9.43%and the internodal length shorten significantly.The seeds of mutation group began to root and germinate is earlier,it’s rooting rate higher than control,the same as rooting index,germination rate,germination potential and germination index.The stoma number of unit area in lower epidermis of mutation group was decreasing.The volume of stomata guard cell was expanding and the number of chloroplasts was increasing.4.The results of flow cytometryAccording to the detection of flow cytometry,the cell DNA comparative content of the progenies in mutation group was increased one time comparing to control group.And some chimeras of the progenies of mutation group were founded.5.Root-tip chromosomes preparation conditions and chromosome counting of B.chinense DC.Under the different conditions for makingchromosome slices of root-tip in B.chinense DC..The slicesmicroscopy result shows the optimum making conditions were as follows:root pruning time between 9-10 am and 4-5 pm,the length of root is 0.5-1 cm,treating 4 hours by N2O,40 min enzymolysis time.According to the cytological study,the chromosome number in the control group are 2n=2x=12,but there are 3 kinds of chromosome number in the mutation group:2n=2x=12,2n=4x=24,2n-2=22.The results show that the polyploid Radix Bupleuri which was induced by colchicine were successfully obtained. |
