| Lonicera japonica Thunb, a perennial and half an evergreen vine plant, is belong to Lonicera of Capriloliaceae and also called two treasure flower or honeysuckle. It has been well known as heat and detoxifying medicine since ancient times. At present, there are significant benefits to the treatment of febrile disease, colds, sore throat, diarrhea and other symptoms, and has been a greater role in the anti-cancer, anti-virus, using the active ingredients of green acid (chlorogenic acid), chlorogenic acid (iso-chlorogenic acid) from Lonicera japonica Thunb.The regeneration system of Lonicera japonica Thunb in vitro was studied to lay foundation for rapid propagation and large-scale production; Aiming to provide theoretical basis for obtaining polyploids of Lonicera japonica Thunb, which contain higher medicinal value, the method of combining of tissue culture and polypioid breeding was used; Aiming to provide scientific basis for cultivation and further promotion of Lonicera japonica Thunb, the method of directional screening salt-tolerant mutant by chemical mutagenesis techniques and culture in vitro was used.This study used the leaves of Lonicera japonica Thunb as the explant to study on the system of regeneration; Polyploid and salt-tolerant variant by chemical induction of somatic cells of Lonicera japonica Thunb. The main study works and results were as follows:1The regeneration system establishment of Lonicera japonica Thunb in vitroThe leaves of. Lonicera japonica Thunb were used as explants and the influences of2,4-D and other plant growth regulators on callus induction and regeneration were studied. The results showed that the optimum media were MB+1.0mg/L2,4-D+0.5mg/L KT for callus induction, with an induction rate of91.8%; MB+2.0mg/L6-BA+0.1mg/L IBA for adventitious bud differentiation, with a regeneration rate of93.3%; and1/2MB+2.0mg/L IBA+0.2mg/L IAA for plantlet rooting, with a rooting rate of83.3%. Most (89.3%) of the plantlets survived after transplantation.2Study on polyploid induction and its identification by chemical induction of somatic cells of Lonicera japonica Thunb.Callus were induced by the leaves of Lonicera japonica Thunb as the explants. The methods, both soaked culture and mixed culture, were used to induce polyploidy. The most effective method to induce polyploid of Lonicera japonica Thunb was obtained by comparing the induction rate. The results showed that using soaked culture had the higher induction rate. The highest induction rate of polyploid,30.5%, was obtained from the callus soaked in the liquid medium supplemented with0.1%colchicine15h on a rotator. The highest induction rate of polyploidy,21.2%, was obtained from the callus cultured in the medium supplemented with0.1%colchicine15d. The polyploidy plant of Lonicera japonica Thunb was obtained with the chromosome number of2n=4x=36. The polyploidy plant exhibited some morphological and cytological variation, including the larger guard cell size of stomata, more chloroplasts in stomata guard cell, the leaf index decreased and growing slowly. The mix-fold phenomenon was also found among treatment plants.3Selection of salt-tolerant variant o by chemical induction of somatic cells of Lonicera japonica Thunb and study on its physiology characteristicsThe leaves of Lonicera japonica Thunb in vitro were treated with EMS. With the increase of processing time and concentration, the differentiation and survival rate of Lonicera japonica Thunb leaves decreased. The semi-lethal concentration of EMS (0.6%) and processing time (6h) were selected. Lonicera japonica Thunb leaves treated with the selected EMS concentration and processing time were selected at0.8%salinity stress. Mutant with stronger resistence was gained. The result of the identification showed the selected mutant has more superoxide dismutase (SOD), peroxidase (POD) activity, proline protein content and less malondialdehyde (MDA) content, than the control, indicating that mutant of Lonicera japonica Thunb has more sale tolerance than the control. |
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