Optimization Technical Studies On Peanut Tissue Culture

The peanut is a good economic crops. Using biological technology to insertexogenous good genes into peanuts is a effective method for improving quality withpeanut. tissue culture that is such a kind of high efficient biotechnology means. Usingtissue culture technology building high frequency plant regeneration system is peanutspeanut breeding key, but because the plant regeneration rate is low, drawing season islimited, and peanut in vitro culture with larger genotype dependence, regeneration andvarieties of greater difference between the constraints, it is difficult to obtain highefficiency, stable and continuous regeneration plant. Explore and improve the highfrequency plant regeneration system has important theoretical and practicalapplication value.The four peanut breed as the goal, studying from the choice of the ways ofsterilization, different choice of explant, uncertain bud induction, antibiotics to inhibitbacterial contamination, seedling cultivation, rooting culture and hardeningtransplanting, in order to optimize the peanut plant regeneration system for thepurpose, screening and optimization peanuts high frequency plant regeneration systemconstruction conditions, the establishment and optimization of peanut high frequencyplant regeneration system, for peanuts genetic transformation lay the foundation forthe further research. So as the study found:1. Suitable method of peanut seed sterilization is: under sterile conditions, dip in75%alcohol for0.5-1min, then in10%NaClO for10min for surface disinfection, andfinally wash with sterile water3-4times, each for3-5min. This method have a goodsterilization effect, causing less damage to the seed.2. inducing of the somatic buds2.1Different genotypes induce abventitious buds:all kind of genotypes cansuccessfully induce abventitious buds.In the basic induction medium B3N1,LuHua14and Huayu20are the highest rat of abventitious buds, reaching76.65%and75.81%,respectively.Huayu16and8130are the lowest reaching52.2%and45.80%,respectively.2.2Different explant to induce abventitious buds:The study found that the bestexplant for the peanut to induce abventitious buds was the germinated6d embryosnicleaflet,whose rate of the induction of abventitious buds was significantly higher thanother explants. Germinated3d embryonic leaflet,with length of1-2mm, is not easy tocut, and prone to bacterial contamination and browning and the rate of abventitiousbuds induction was72.5%.As for germinated9d embryonic leaflet, the leavsgradually opened, bud differentiation capacity gradually reduced and abventitiousbuds induction rate was68.75%. Epicotyl,hypocotyl and cotyledons also induced asmall amount of abventitious buds with induced rate of3.75%,5%and7.5%respectively.2.3The effect of different concentration of plant growth regulators on adventitiousshoot from peanut leaflets: In basic induction medium （B₃N₁）,the abventitious budsinduction rate of germinated6d HuaYu20embryonic leaflet,dealt by2mg/LAgNO₃,was up to85%,while vitrification as low as22.86%. N1induction medium isof high frequency in the four genotypes, generally more than80%.In N1mediumHuaYu20abventitious buds induction is the highest rat of89.2%. The medium of theT-series treated by the different concentrations of TDZ and NAA and6-BA shows acommon performance in the induction experiment of HuaYu20embryonic leaflet,with the highest induction rate of only73.91%, worse than N1medium.So Tseries are not the best induced medium for HuaYu20embryonic leaflet.3.The regeneration plant seedling and root training3.1In vitro shoot differentiation training: differentiation ratio of abventitious buds ishighest in the callus differentiation medium C6（5mg/L6-BA,4mg/LGA）,reaching86.67%, which can be used as the best differentiation medium for HuaYu20adventitious shoot elongation.3.2Use of paclobutrazol in strong sprout and rootage culture: Paclobutrazol caneffectively inhibit the plant height,the formation of strong seedlings,effectively solveto the problem of slender and thin seedling stems in grouped-culture seedlings.Whenthe concentration is higher, grouped-culture seedlings grow robust,but severelydwarfing.S0it is not conducive to strong seedling.In peanut rooting medium,added0.2mg/L paclobutrazol effect was best with well-developed root system,the highsurvival rate of transplanted seedlings.When paclobutrazol concentration exceeds0.2mg/L in rooting medium,the propagation coefficient and survival ratereduced.Therefore,in the process of peanut tissue culture,the plantlets robust,highpropagation coefficient and the survival rate of transplant should take into account.3.3The inhibitory effect of antibiotics on bacteria: Adding medical antibiotics of acertain concentration to peanuts tissue cultured seedlings subculture medium caneffectively inhibit bacterial infection of the medium.The inhibitory effect of Penicillinsodium for polluted tissue culture seedling is the most significant in five kinds ofantibiotics in the study.60mg/L Penicillin sodium can inhibit bacterium at the rate of100%.It has no significant effect on normal growth in tissue culture,so can be chosento use in peanuts tissue culture.However,addition of kanamycin and rifampicinsignificantly affects chlorophyll synthesis of tissue culture seedling.So notrecommended to use.3.4Root training: taking root in IR2inductive medium is good, rooting rate is high,up to95.04%with mang and long roots as well as amall callus in rooting parts,Aftertransplanting,the survival rate is also up to47.31%.4. Hardening seedlings and transplantingCompared with survival rate of different ground substance transplant,the highestsurvival rate of regeneration seedling is up to81.67%,by ground substance ofsand/vermiculite/soil=1:1:1.