Study Of The Development Of Seed And Endophytes Isolated From Ginkgo Biloba

The development in anatomy of Ginkgo biloba seeds and endophytes in Ginkgobiloba seeds were studied in this paper. The main results are as follows：1. Developmental structure in anatomy of Ginkgo biloba ovules （seeds）1）There was a degraded tissue zone observed at the nucellus base of the infertileovule after pollination. Because of degradation of the nucellus base, the supplement ofwater and nutrient to the female gametophyte or ovule was reduced or terminated, sothat the ovule stoped development.2）It was considered that the pollination drops were from or mainly from theproducts of the tissue degradation which formed the pollen chamber in the top of thenucellus.3）There was a non-cell membranous layer between the endosperm and nucellartissue during the later period of endosperm development. This non-cellular membranouslayer might originate from the thick cell wall or cell membrane of the megaspore. In thedevelopment of the endosperm, nucellar tissue did not disappear and became thecellular membranous layer around the endosperm with the non-cell membrane. Thenucellar tissue membrane around the endosperm was separated from the seed coat(inner seed coat) by the seed cavity at the upper part of seed, and tightly coalesced withthe inner seed coat of the lower half of the seed.4）The integument differentiated into three layers of fleshy outer seed coat, osseousmiddle seed coat and membranous inner seed coat. The upper part of the inner seed coataround the seed micropyle and the seed cavity was a thickening zone containing moresap possibly provided partial nutrition to the development of the endosperm while theinner seed coat was differentiated in the early development stage. The seed cavitypossibly function accumulating water from the seed micropyle and maintaining germination.2. Endophytes in the seed of Ginkgo biloba1）Isolation and identification of the endophytesThere were7strains isolated from Ginkgo biloba seeds, including5strains ofbacteria and2strains of actinomycetes.2）Primary screening of anti-fungal activity of the endophytes in Ginkgo bilobaseedsThe plate confrontation was applied to screen the strains which are of anti-fungalactivity to eight species of pathogenic fungi in the seven endophytes isolated from theseeds. KLBMP3491and KLBMP3496had in a certain degree inhibition for allinstructive pathogens.3) Primary screening of enzyme activity of the endophytes in Ginkgo biloba seeds(1) A strain KLBMP3495with higher fibrinolytic activity and higher cellulaseactivity was chosen from the previously obtained endophyte strains by plate methods,and identified as Bacillus subtilis.(2) The fibrinolytic characteristics of KLBMP3495were of the optimumtemperature of35℃and the optimum pH value of8.0. The cellulase characteristics ofKLBMP3495was of the optimum temperature of55℃and the optimum pH value of7.0. Native-PAGE test showed that the fermentation broth of KLBMP3495had activityof both fibrinolytic enzyme and cellulose enzyme.(3) By single factor and orthogonal methods, the fermentation broth andfermentation conditions of KLBMP3495were tested and optimized with a single factor.The results showed the optimum medium composition for the endophyte to generatefibrinolytic enzyme consisted of1.5%sucrose,0.5%maizena,0.5%KNO3,1.5%soybean powder. The optimum culture conditions were with pH8.0as an initiative,temperature35℃, the rotation speed160r/min, seed inoculation2%,60mL of250mLflask, and fermentation period90h. The optimum composition of fermentation fluidmedium for cellulase activity consisted of1.0%CMC-Na,0.5%maizena,0.3%NH4NO3,2%soybean powder.The optimum fermentation conditions was with pH7.5, temperature40℃, rotation speed160r/min, seed inoculation4%,40mL of250mL flask，and fermentation period75h. Under these conditions, the fibrinolytic activity ofKLBMP3495was increased from320.5U/mL to739.7U/mL and the cellulase activityKLBMP3495was increased from178.11U/mL to609.5U/mL.