| Withing more and more researchs on multifoliolate alfalfa, it as a good quality of alfalfa materials, a lot of research results and applying value have been found. Such as nutrition value with greater than3leaflets and the same as palatability.The research established multifoliolate alfalfa suspension system by exploring the optimum condition and influence factors,In order to fast and efficient accessed to multifoliolate alfalfa plants,comprehensive and accurate analysis of the research from Cellular level,which was multifoliolate alfalfa growth and development.Growth characteristics and quality and mutant selection.The experiment take the second generation obtained by Grandeur as materials.Experiment factors are NAA、6-B、KT、2,4-D、Inoculum density and subculture times,respectively,each factor set six different levels.Through uniform design found the optimal culture conditions of multifoliolate alfalfa suspension system, and measured morphological indices and physiological indices under different treatments,including Suspension cell color, Fresh weight of suspended cells, Dry weight of suspended cells,Relative amount of growth of cell suspension cultural, suspension cell vitality,PCV, pH and so on.In addition, researcher based on multifoliolate alfalfa suspension system, Study on heat resistance of multifoliolate alfalfa further, exploring the high temperature impact on multifoliolate suspension culture. this provided the foundation of multifoliolate alfalfa cells in response to heat stress and physiological mechanism of adaptability, and what is possible that select a heat resistance multifoliolate alfalfa variety for JiangHuai area.The main research findings are as follows.1. The best multifoliolate alfalfa suspension system was0.5mg·L-1NAA+2.0mg·L6-BA+0.2mg·L-1KT+2.5mg·L-1+2,4-D+0.6g(Inoculum density)+10d (subculture times). In the condition of the fresh weight and dry weight of suspension cell reach the highest,PCV content was also greater.pH of multifoliolate alfalfa suspension cells ranged from5.34to6.73.To sum up,at the early stage of suspension cells was decreases tendency,however,after gradually reducing started to rise again.The optimum pH of different treatments each were not identical, the treatment No.3suspension cell had optimal pH of6.04and5.87.in this condition,The fresh and dry weight of suspended cells can achieve greater value.2. KT、2,4-D、Inoculum density and subculture times had inhibitory influence on fresh weight of suspended cells;KT and Inoculum density showed inhibitory effect on the dry weight of suspended cells,just as the KT and2,4-D control PCVcontents of suspended cells.6-BA、2,4-D and subculture times can stimulates the increase of dry weight of suspended cells, as6-BA and Inoculum density promote PCV contents of suspended cells.3. Using TTC meathod meatures absorbance values, we should set wavelength of616nm,put to use phosphate buffer and0.6%TTC.Different treatments showed two kind of trends,one was increasing-decreasing,the other one was decreasing-increasing.Treatment No.3was belong to the former.4. By observing the culture period for14d multifoliolate alfalfa suspension cell growth process, suspending liquid from clear to muddy to brown yellow color, Smell was also from tasteless to peculiar smell, Suspension cell growth from less to more, Finally, there was a lot of Suspension cell mass on the bottom.Through painting dry and wet growth curve found2～6d for latent phase,6～10d for logarithmic growth phase, after10d into Stagnate phase. The treatment No.3cell subculture of5d～12d cells in the vigorous growth stage,it was time to develop protoplast and screening of successive transfer culture.The highest callus rate of treatment No.3was59%, fresh weight was also higher than others,The differentiation rate was58%,but,the pollution rate of suspension cell in the forming process was higher, more than50%were contaminated.5. To some extent,high temperature can promote growth rate of suspension cell,the reason may be associated with the high temperature can boost cell division. High temperature was closely related to browning cell suspension system of multifoliolate alfalfa. With the increase of temperature,the browning rate was on the rise.The turning point temperature35℃has been measured.The semilethal temperature was40℃.At high temperature45℃,suspended cells severe browning and it showed that serious phenomenon of cell death. High temperature can accelerate the content of proline of cell suspension system of multifoliolate alfalfa,Due to high temperature damage cell of multifoliolate alfalfa, which lead increase the number of membrane permeability and active oxygen of cell to make mechanism to protect themselvse.At the high temperature, Not only the peak of prolin was higher than room temperature,but also there ahead of time,it means that cells were cultured by high temperature5d, proline content can reach maximum. In the high temperature-stress, at first, the suspension cell vitality had a little change, starting from the3d was declining, cultivate suspension cell vitality began to recover in a few days, and showed a trend of rising.6. In the experiment, multifoliolate alfalfa seeds was inoculated by1/2MS culture medium which can the growth of hypocotyl,With the purpose of got good explants and improved the quality of callus and cell suspension system of multifoliolate alfalfa. |
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