Callus Induction,Proliferation And Metabolomics Analysis Of Phyllostachys Edulits Seed

Phyllostachys edulis is the most widely distributed bamboo species in China,with important economic and ecological value,but its biological characteristics make it difficult to carry out traditional hybrid breeding.Therefore,the establishment of a stable and efficient regeneration system has an important role in genetic improvement of P.edulis base on genetic transformation.However,at present,there are still problems such as difficulty in explant extraction,high pollution rate,and low inductive rate of callus in the regeneration system of P.edulis,which seriously hinders the development of P.edulis biological breeding technology.In this study,we used P.edulis seeds as explants,we sought ways to reduce the pollution rate of P.edulis seeds from multiple angles,explored the effects of different plant growth regulatory substance combinations,different nitrogen forms（ammonium nitrogen,nitrate nitrogen）ratios and nitrogen source concentrations on the induction and proliferation of callus seed,and analyzed the effects of nitrogen morphology ratios on the metabolites in the induction of callus tissue and organogenesis of P.edulis seeds..The conclusions are as follows.1.Sterilized vermiculite,sterilized filter paper,sterilized desiccated cotton and gauze were used as media for aseptic germination of P.edulis seeds in order to induce healing tissues with germinated sterile seedling stem tips and other parts,and the results showed that sterilized vermiculite was more suitable for aseptic germination of P.edulis seeds,but sterile seedling stem tips and other parts were all contaminated during healing tissue induction.Therefore,this study directly used P.edulis seeds as explants for healing tissue induction and proliferation culture.It was found that reducing the number of inoculated explants in a single culture vessel could effectively reduce the contamination rate of P.eduliso seeds,and by using small size culture vessels with one seed inoculated per vessel could ensure low contamination rate while avoiding wastage of culture medium.On this basis,the contamination rate of P.edulis seeds treated with 50%84 disinfection solution for 10 min+10%sodium hypochlorite for 20 min was lower and the initiation rate was higher than that of 10%sodium hypochlorite disinfection for 20 min and 50%84disinfection solution for 20 min treatment.2.The results of L₉（3⁴）orthogonal test with different cytokinin types（KT,ZT,BA）and concentrations(0.1,0.5,1 mg·L^-1)and 2,4-D concentrations(2,4,8 mg·L^-1)showed that the differences in seed initiation rate of P.edulis under different treatments were small,and the effects of 2,4-D concentration and different cytokinin concentrations on the induction rate and germination rate of healing tissue were greater than those of cytokinin The highest healing tissue induction rate of P.edulis seeds was achieved with 2,4-D concentration of 8 mg·L^-1,cytokinin concentration of 1 mg·L^-1and cytokinin of ZT,while the highest germination rate of P.edulis seeds was achieved with 2,4-D concentration of 2mg·L^-1,cytokinin concentration of 0.5 mg·L^-1and cytokinin of BA.Therefore,the best formula for the induction of P.edulis seeds was MS+1 mg·L^-1ZT+8 mg·L^-12,4-D.The induced healing tissues were transferred to MS+4 mg·L^-12,4-D medium for 30 d,and then selected uniformly colored and textured healing tissues were transferred to different proliferation medium.The results showed that the highest proliferation rate was achieved in MS medium without the addition of plant growth regulating substances,and there was no browning,while browning,waterlogging and death occurred to different degrees in the proliferation medium with the addition of NAA,2,4-D and ZT.3.The developmental status of P.edulis seeds in the induction medium with different ammonium-nitrate ratios（total nitrogen concentration of 60 mmol/L）varied greatly,with three states of induction of healing tissue,sprouting into roots and shoots,and the presence of both healing tissue and roots and shoots.The seed initiation rate and healing tissue induction rate of P.edulis were the highest at NH₄⁺/NO₃^-ratio of 40/20,85.02%and67.87%,respectively,and the lowest at NH₄⁺/NO₃^-ratio of 0/60,72.06%and 11.41%,respectively;the germination rate was the highest at NH₄⁺/NO₃^-ratio of 30/30,70.83%,and the lowest at NH₄⁺/NO₃^-ratio of 60 mmol/L.NH₄⁺/NO₃^-ratio of 60/0 was the lowest at48.25%.The healing tissues obtained from the induction medium were transferred to the same treatment medium for 30 d.The effect of different ammonium-nitrate ratios on the proliferation of P.edulis healing tissues was further investigated,and the results showed that the proliferation rate of healing tissues was highest at NH₄⁺/NO₃^-ratio of 30/30,250.86%,and lowest at NH₄⁺/NO₃^-ratio of 0/60,48.54%.4.With the increase of nitrogen concentration in the medium（NH₄⁺/NO₃^-ratio of40/20 in the fixed medium）,the seed initiation rate,healing tissue induction rate,germination rate and healing tissue proliferation rate of P.edulis increased and then decreased.The seed initiation rate and healing tissue induction rate of P.edulis were highest at 30 mmol·L^-1（89.29%and 82.14%,respectively）and lowest at 180 mmol·L^-1（58.70%and 29.76%,respectively）,while the seed germination rate and healing tissue proliferation rate of P.edulis were highest at 90 mmol·L^-1（63.33%and 181.35%,respectively）.The seed germination rate and healing tissue proliferation rate were the highest at 90 mmol·L^-1with 63.33%and 181.35%,respectively,and the lowest at 0mmol·L^-1with 18.85%and 52.41%,respectively.5.A total of 30 samples of different NH₄⁺/NO₃^-treatment induced calluses P.edulis seeds were detected by LC-MS,and a total of 641 metabolites were identified.Screening of differential metabolites found that amino acids and their derivatives,most lipids,and some benzene and and their derivatives were up-regulated in the group with higher inductivity of callus,and downregulation occurred in flavonoid compounds,coumarins and their derivatives.The differential metabolites were mainly involved in metabolic pathways such as phenyl propane biosynthesis,biosynthesis of secondary metabolites,flavonoid biosynthesis,phytohormone signaling,amino acid biosynthesis,and lipid metabolism.Among them,asparagine,biocytin,jasmonic acid,12-oxophytodienoic acid,salicylic acid,popcornin,isomudustin,and apigenin were the key metabolites.It is hypothesized that during the process of healing tissue induction in P.edulis seeds,a large amount of amino acids and their derivatives accumulate to provide energy and substrate for healing tissue formation,while interacting with flavonoids and their derivatives,lipids,etc.to promote the synthesis of phytohormones such as jasmonic acid and salicylic acid to promote healing tissue formation.