The Research On ISSR Molecularmarker And Tissue Culture Of New Varieties Of Lonicera Macranthoides

Lonicera macranthoides Hand. Mazz, also named large flower honeysuckle or large silver flower, is a perennial and semi-evergreen shrub plant and belongs to the family Caprifoliaceae. It produces the highest chlorogenic acid content among all the cultivars of honeysuckles and is the main cultivar of medicinal honeysuckle in Hunan, Chongqing and Guizhou. The honeysuckle industry in China has made considerabel development, however, problems such as lack of thoroughbred, cultivars complexity, low dried flowers yield, low chlorogenic acid content, backward in propagation technology, long cycles of seedling and low propagation coefficient still exist, which restricts the healthy and sustainable development of honeysuckle industry. In this paper, Lonicera macranthoides cv'Jincuilei','Yincuilei'and'Baiyun'has been studied, also on their ISSR molecular markers and rapid tissue culture. The change and regulatory mechanisms of endogenous hormones and polyamines of these new varieties have been systematically analyzed in the process of tissue proliferation and trooting. The purpose of the research is that new varieties of Lonicera macranthoides will be identified effectively from the molecular level, Establish a efficient tissue culture and rapid propagation system of new varieties, and thus facilitate sustainable development of honeysuckle industry in our country. The main results of the research are as follows:(1) Research on ISSR molecular marker in new varieties of Lonicera macranthoides. Based on the genomic DNA extracted from the leaves of Lonicera macranthoides, the factors influencing ISSR were optimized and the effect of6factors such as annealing temperature, Taq DNA polymerase dosage, DNA templates concentration, primer concentration, dNTPs concentration and Mg2+concentration on ISSR amplification were tested. A reaction system and amplified procedure suitable for Lonicera macranthoides were established. Using these optimal amplification conditions,10stable and repeatable ISSR primers were selected from total100primers. The genome DNA of3new varieties of Lonicera macranthoides and19medicinal honeysuckle varieties were amplified with the10ISSR primers and108bands were amplified totally, including96polymorphism bands with polymorphism rate of88.9%, indicating medicinal honeysuckle plant has the abundant genetic diversity. DNA fingerprinting of3new varieties of Lonicera macranthoides were constructed, Three new varieties and other medicinal honeysuckle plant varieties were identified from the molecular level. The systematical cluster analysis conducted with UPGMA and PCA (principal component analysis) showed that22medicinal honeysuckle varieties could be classified into2groups, the first group is Lonicera japonica Thunb., the second one is L.macranthoides Hand. Mazz, and each group could be divided into north cultivars and south cultivars. Genetic differences between varieties of honeysuckle were correlated with their geographical differences.(2) Research on tissue culture of new varieties of Lonicera macranthoides. Taking Lonicera macranthoides cv'Jincuilei','Yincuilei'and'Baiyun'as test materials, a serial of single factor, double factors and orthogonal tests were designed, and experiments were carried out on explant choice, germicidal treatment, preparation of minimal medium, plant growth regulators and its ratio, Biotin D concentration and activated carbon concentration, culture temperature, transplanting matrix and containers, rooted seedling disinfection, production cost reducing measures. Sterile system of tissue culture was established, and suitable initial mediums were obtained:MS+6-BA1.0mg·L-1+NAA0.1mg-L'1for 'Jincuilei'and'Yincuilei', MS+6-BA1.0mg·L-1+IBA0.1mg·L-1for'Baiyun'; suitable subculture mediums were screened out:improved MS+6-BA0.5mg·L-1+NAA0.1mg·L-1+Biotin D1.0mg·L-1for'Jincuilei', improved MS+6-BA0.5mg·L-1+NAA0.05mg·L-1+Biotin D1.0mg·L-1for'Yincuilei'and improved MS+6-BA1.0mg·L-1+IBA0.1mg·L-1+Biotin D0.5mg·L-1for'Baiyun', the average multiplication coefficient was4.65. It was found that appropriate low temperature was better for inducing Lonicera macranthoides tissue culture seedlings rooting, and the preference temperature is20℃. Suitable rooting mediums for the three varieties were screened out:1/2MS+IBA3.0mg·L-1+activated carbon200mg·L-1, the average rooting rate was97.4%. Meanwhile, using leaves of sterilized'Jincuilei'tissue culture seedlings as test material, callus and adventitious buds were induced. The optimum medium for leaves callus and adventitious buds inducing was B5+6-BA1.0mg·L-1+2,4-D0.5mg·L-1and B5++KT0.2mg·L-1+NAA1.0m·L-1, respectively, and the inductivity was86.7%and73%, respectively. The technique of "single plant one-step transplanting in plastic cups" was studied out and the suitable transplanting matrix was a mixture of yellow soil, rice hull ash and silver sand with the ratio of1:2:2, the average transplanting survival rate reached95%. Production cost can be significantly reduced when using plain boiled water and white granulated sugar instead of distilled water and sucrose, which increased productivity effect of factory nursery of Lonicera macranthoides new varieties. (3) Research on cutting propagation technique of Lonicera macranthoides new varieties bred by tissue culture seedlings. Using Lonicera macranthoides'Jincuilei'as test material, experiments were carried out to study on the influence to propagation of Lonicera macranthoides new varieties bred by tissue culture seedlings with different cutting propagation matrix, cutting propagation period, plant growth regulators, concentration of ABT6and soak period. The results showed that the proper cutting propagation matrix was a mixture of peat soil and pearlite with the ratio of1:3at volume, the appropriate cutting propagation period was in May and September, the suitable plant growth regulator was300mg·L-1ABT6in what the shoots soak for120min. The average survival ratio of cutting propagation reached95%, which substantially reduced the production cost of tissue culture of Lonicera macranthoides new varieties.(4) Research on endogenous hormones changes in process of tissue culture of Lonicera macranthoides new varieties. Using Lonicera macranthoides'Jincuilei'in vitro seedlings as test material, the endogenous hormones content changes were analyzed during in vitro seedling proliferation and differentiation, rooting and callus and adventitious buds inducing, and the influences of6-BA concentration on in vitro seedling subculture and IBA on rooting were also studied. The results showed that:①Subculture proliferation and differentiation of Lonicera macranthoides was mainly regulated and controlled by endogenous ZR, ABA and GA, and was benefited by a higher concentration of ZR and GA and lower concentration of ABA. An appropriate high concentration of IAA also indistinctively benefits proliferation and differentiation of plexus buds. A higher content of IPA is positively related to explant cultivation initiation of Lonicera macranthoides,but less closely with proliferation and differentiation. Higher GA/ZR value is dominant in endogenous hormones equilibrium relation, which benefits seedling cultivation proliferation, differentiation and growth initiation. However, it needs lower GA/ZR value in flourishing period, as well as GA/ABA value.②Endogenous hormones play different roles in the process of adventitious roots formation and growth of Lonicera macranthoides. The former is positively related by a high concentration of IAA, ABA and a lower concentration of GA, IP A, ZR, yet the latter is positively by a high concentration of IAA, GA, ZR, IP A and a lower concentration of ABA. Endogenous hormones equilibrium relation also takes part in the process of adventitious roots formation and growth of Lonicera macranthoides synergeticly. A lower (ZR+IPA)/IAA value and a higher IAA/ABA and ABA/GA value benefits their formation, and a higher (ZR+IPA)/IAA value and a lower ABA/GA value benefits their growth.③Endogenous hormones play different roles in the process of formation and growth of callus and adventitious buds induction of Lonicera macranthoides. Low concentration of GA, IAA, ZR, ABA is positively related to callus inducing, which is benefited by high concentration of GA,IAA and ZR. Adventitious buds inducing is mainly regulated by endogenous IAA and ABA, and low level content of endogenous IAA,ABA,ZR and GA promotes the formation of adventitious buds from callus.(5) Research on polyamines change in the process of tissue culture of Lonicera macranthoides new varieties. Using Lonicera macranthoides'Jincuilei'in vitro seedlings as test material, the polyamines content changes were analyzed during in vitro seedling proliferation,differentiation and rooting, and the influences of6-BA concentration on polyamines content during in vitro seedling subculture and of IBA concentration on polyamines content during in vitro seedling rooting were studied. The results showed:①Endogenous polyamines plays different roles in the process of in vitro seedling proliferation and differentiation of Lonicera macranthoides, which is positively related by Spd and Put content and Put/Spm value, but less closely with Spm content, Put/Spd value and Put/(Spd+Spm) value. A highter Spd and Put content, Put/Spd value benefits proliferation and differentiation and growth initiation of in vitro seedling, However, it needs a lower Put and Spd content and Put/Spm value in flourishing period.②Put,Spd, Spm content and their equilibrium relation play different roles in the process of in vitro seedling root primordium proliferation and differentiation of of Lonicera macranthoides. A high level of Spd and Put content, Put/Spm value and Put/(Spd+Spm) value benefits their differentiation and formation, yet a low level of those boost their growth. A low level of Spd content and Put/Spm value is not only positively related to differentiation of seedling cultivation root primordium, but also to their growth.