Tissue Culture And Browning Research Of Medicinal Parasite Plant Cynomorium Songaricum Rupr

Cynomorium songaricum Rupr. is a holoparasitic higher plant. Its succulent stems, which has various biological activities, has been widely used in Traditional Chinese and Mongolian Medicine. Currently, more attentions have been paid on utilization of C. songaricum, following which the demand for C. songaricum is increasing. However, resources of wild C. songaricum is going to be exhausted since serious destruction of C. songaricum’host plant and the environment caused by unreasonable excavation manner, while the technology of artificial cultivation is still not successful yet. Now the dilemma of C. songaricum industry is the contradiction between the huge demand of market and the limited resource of wild C. songaricum. As we know that tissue culture is an effective way to alleviate the contradiction since it is applied to the production of active compositions and the regeneration of a variety of plants, meanwhile, it can provide useful methods to study different mechanisms. Unfortunately, the tissue culture of C. songaricum is rarely reported. Here we report, for the first time, protocols for C. songaricum callus induction, adventitious root and haustorium organogenesis from different explants by screening of conditions of the tissue culture, researching effects of plant growth regulators and observing changes of morphology during the tissue culture. At the same time, we study the reason of the browning during the tissue culture to find the solutions. Therefore, establishment of C. songaricum tissue culture system and research on regulatory mechanisms of germination and primary haustorium organogenesis will provide theory basis for systematic explanation of C. songaricum’s parasitology and theory support for comprehensive development and utilization of C. songaricum. The main results of this study were summarized as follows:1. The optimum medium for callus induction is MS+3.0 mg-L-1 2,4-D+1.0 mg·L-1 6-BA+0.5 g·L-1 AHC+30 g·L-1 sucrose +6.8 g·L-1 agar with the callus formation rate 67%, or MS+2.0 mg·L-1 NAA+1.0 mg·L-1 6-BA+0.5 g·L-1 AHC+30 g·L-1 sucrose+6.8 g·L-1 agar with the callus formation rate 32%.2. The optimum medium for callus proliferation is MS+1.5 mg·L-1 2,4-D+0.5 mg·L-1 6-BA +0.5 g·L-1 AHC+30 g·L-1 sucrose+6.8 g·L-1 agar with callus proliferation coefficient 74%. The optimum medium for adventitious root organogenesis is MS+2.0 mg·L-1 NAA+1.0 mg·L-1 6-BA+0.5 g·L-1 AHC+30 g·L-1 sucrose+6.8 g·L-1 agar with adventitious root organogenesis rate 56%. The number of adventitious root is 5.6 on average,2.3 of which can form callus on the tip.3. The protocols for seed germination of C. songaricum is found. The seed is treated with heat stock on 50℃ for 60min, remove seed testa and culture on B5 medium adding 1 mg·L-1 GA3 for 40 days on 25℃. The germation rate is 8.6%. The optimum medium for callus induction is B5+1.0 mg·L-1 2,4-D+0.5 mg·L-1 KT+1.0 mg·L-1 GA3+0.5 g·L-1 AHC+30 g·L-1 sucrose+6 g·L-1 agar with the callus formation rate 13.7%.4. The optimum medium for primary haustorium organogenesis is B5+0.5 mg·L-1 2,4-D+0.25 mg·L-1 KT+0.5 g·L-1 AHC+30 g·L-1 sucrose+6 g·L-1 agar with primary haustorium organogenesis rate 66.7%. The optimum medium for a large number of primary haustorium organogenesis is B5+1.0 mg-L-1 2,4-D+0.25 mg·L-1 KT+0.5 g·L-1 AHC+30 g·L-1 sucrose+6 g·L-1 agar with the number of primary haustorium 8.3 on average.5. It showed the development of primary haustorium organogenesis from the callus of C. songaricum seeds is similar with primary haustorium formation from adventitious root on in vegetative propagation, they all origins from the root development. At the presence of cytokinins, auxins play the most important roles in the formation of the C. songaricum primary haustorium.6. The browning type and PPO activity have been studied. PPO from C. songaricum showed the highest activity in pH buffer 5.5 at 30℃ pand can keep good stability up to 60℃. The processes of enzymatic browning and nonenzymatic browning both exist in explant. Enzymatic browning plays important role in the prophase of tissue culture, but nonenzymatic browning plays important role in the late stage of tissue culture. The effective method for anti-browning in C. songaricum tissue culture is to control explant size and promote wound healing.