Tissue Culture Of Cultivated Coix L.germplasm Resources And The Isolation And Identification Of Endophytic Fungi

Coix lacryma-jobi L.plants belonging to Graminea,are annual or perennial herbs and also important food-medicine homologous plants,which are of high value in nutrition,health-protection and comprehensive utilization value.In recent years,with the increase of the awareness of health protection among the public,the researches on Coix cultivation,germplasm resources conservation and the roles in food and medicine has been on the rise.The Coix are rich in genetic resources and have wide varieties,but high quality new Coix cultivars lacks in present production and its yield and quality have been on the decrease.So far,no systematic research on the tissue culture of Coix cultivars has been seen and the responsiveness of different Coix cultivars and different explants to tissue culture have not been compared and reported and few observations of anthers and the exploration of anther culture have been done.No detailed records and classification of the endophytic fungi occurred in the tissue culture of Coix cultivars has been reported.In this research,aseptic seedlings of 10 Coix cultivars were used as materials.The root segments,aerial root,the upper and lower stem segments,hypocotyls and leaf disks from the aseptic seedlings were used as explants;and the effects of different plant hormone concentrations in the MS medium on the different types of explants in callus induction,differentiation and seedling differentiation were systematically studied and compared.The pollen morphology and the pollen viability of Coix cultivars were observed and compared;the morphological characteristics of its microspore development stages were observed and described;the conditions for in vitro pollen germination were determined;and the anther culture and its callus proliferation system were preliminarily established.The endophytic fungi in the aseptic seedling culture were identified by the isolated fungal cultural colony morphology combined with the PCR amplified fungal internal transcribed spacer（ITS）sequences;the effects of endophytic fungi in Coix were discussed.The main results are as follows:1.The optimum method for obtaining of aseptic seedlings from Coix seeds fortissue cultureFor aseptic seedling culture from Coix seeds,based on the germination rates and contamination rates at the 6 days,10 Coix cultivars were divided into two types,A and B:A type of Coix were less in contamination and the traditional means of disinfection:boiling water or 10%sodium hypochlorite treatment for 20 Min followed with 5 rinses with aseptic water,can be used;B type Coix were more in contamination and were recommended to be disinfected by 0.1%HgCl₂ for 20 Min followed 8 rinses with aseptic water.Still there might have some fungal contaminations.Compared with B type of Coix,A Type had a higher germination rate,lower bacterial and fungal contamination,better growth,and thus are more suitable for tissue culture.2.The optimum conditions for the tissue culture of CoixIn the tissue culture of different Coix explants of both Type A and B,callus was not easy to be induced from root segments and leaf disks.A small amount of calluses could be induced from the upper stem segments and the aerial roots and they had a poor subculture potential.The hypocotyls and the lower stem segments were revealed to be the optimal explants and more suitable for compact,transparent and canary callus induction and callus subculture.The optimum ratio of hormone concentration for the induction of calluses from the lower stem segments of A and B type of Coix was MS+2,4-D 2mg/L+6-BA1mg/L and MS+2,4-D2mg/L+6-BA0.5mg/L respectively;the optimal hormone concentration ratio for the hypocotyl culture of the two types of Coix was MS+2,4-D2mg/L+6-BA1mg/L+500mg/LCH;the most suitable differentiation medium was MS+NAA0.5mg/L+6-BA1.5mg/L+GA2mg/L.It was also found that there was a significant elongation at the early stage of callus induction from stem segments;Test-tube plantlets could be directly induced from the stem nodes.The results indicated that the propagation coefficient and the germplasm resources of Coix seed could be increased by using stem culture.The established tissue culture technique could be used as means to preserve the germplasm resources of Coix and also provide a basis for its rapid propagation.3.The characteristics of pollen grain,pollen vigor,pollen germination in vitro and the culture of anther of CoixThe morphologies of Coix pollen grains in different flowering periods（early,peak and terminal flowering stages）were examined microscopically and compared for the first time.Large differences exist in the lengths of tassels,stigmas,anthers and caryopses at different flowering stages;and the sizes of the pollen grains were different and shapes ranged from ellipsoidal to spherical,and distinct uninucleate and binucleate pollen grains of various ratios and a single germinating hole could be seen.It was determined that the pollen vigor at the peak flowering stage is the highest.The pollen vigor of Guangdong,Guangxi and Hebei at the peak flowering stage were higher than those of Sichanxiaobaike,Punongyihao and XingrenxiaobaikeIt was also observed in vitro that different Coix pollens have different optimum germination media.The optimum pollen germinating solution for Guangdong Coix,Guangxi Coix and Hebei Coix was determined to be 15%sucrose+0.01%boric acid at25℃,while the other was 10%sucrose+0.01%boric acid;they all showed a maximum germination when cultured at 25℃for 5-7 h.The observation of the pollen grains of the early flowering stages showed that its development is continuous,with uninucleate,binucleate and trinucleate stages coexist in different ratios;the same piece of anther have microspores of different developmental stages.Microspores at the late uninucleate stage is suitable for anther tissue culture,the pollen is spherical with small vacuoles.During Coix anther culture,in the same culture medium,calluses and embryoids coexist.The anther?s callus induction rate was in significant difference among different Coix cultivars.Calluses could be successfully induced from Xingrenxiaobaike,Zheyiyihao,Guangdong Coix,Guangxi Coix,Fujian Coix,Hebei Coix and Liaoning Boke Coix in the medium of MS+2,4-D 1²mg/L+KT1.5 mg/L.And Some Calluses of Coix could be successfully differentiated into short cluster buds in the medium of MS+IAA0.5mg/L+KT2 mg/L.The established culture conditions for pollen germination in vitro and for anther culture laid a good basis for further induction and breeding of the haploid seedling of Coix.4.The Isolation and identification of Coix endophytic fungiThe fungi appeared in the MS medium only after 5-6 days?aseptic Coix seedling culture from caryopses were pick and transferred to fresh PDA medium and continue culture for several days prior to observation.The characteristics of the frontal and opposite of colonies and the microscopic characteristics of its mycelia and microspores were photographed and described in detail.The ITS of the isolated individual fungus was obtained by PCR with primers ITS1 and ITS4 and its genomic DNA as template,and sequenced.And the obtained ITS sequences were subject to BLAST analysis for sequence identities and similarities.All together 9 species of fungi were isolated and identified by colony morphological comparison combined with the PCR amplified ITS sequences-based molecular analysis of the isolated Coix endophytic fungi:Penicillium expansum,Penicillium Polonicum,Cladosporium cladosporioides,Aspergillus ruber,Alternaria alternate,Aspergillus flavus,Aspergillus oryzae,Fusarium acuminatum,Mucor racemosus and Fusarium.Among the 9 species of the identified fungi,Penicillium expansum,Aspergillus oryzae,Cladosporium cladosporioides and Mucor can produce a variety of composite enzymes which have great application in fields of microbial chemistry,food science and fermentation;Penicillium,Aspergillus flavus,Fusarium and Alternaria alternate can produce corresponding toxins which are harmful to plants,animals and humans.These results can be used not only as a reference for the exact prevention of pollution in the tissue culture of Coix,for example,the addition of corresponding specific antibiotics for aseptic culture.It can also enrich the endophytic fungi resource pool of Gramineae plants and provide new ideas for the improvement,cultivation,post-harvest caryopsis storage of Coix cultivars,and the development of new natural drugs based on Coix,among many others.In summary,this research established a systematic tissue culture technique that could be used for the preservation of the germplasm resources of the job’s tears（Coix lacryma-jobi L）and provided a basis for its rapid propagation.The preliminarily established culture system for Coix anther in vitro and its callus proliferation was the basis for further haploid breeding and other basic studies of Coix.The isolation and identification of 9 species of endophytic fungi of Coix will enrich the endophytic fungi resource database of Gramineae and provide us new ideas as to the development of new natural drugs and the improvement of the health-enhancing and medicinal values of the Coix.