Somatic Embryogenesis And Biochemical Characteristics In Quercus Variabilis Bl.

Oriental oak (Quercus variabilis) is one of the most valuable forest species with multiple uses. Besides yielding timber, the tree is a primary industrial source of cork. The acorn cups serve as a source of tannin extract. The logs are used for fuel, burning charcoal and the cultivation of mushroom and could contribute to rural development. It is known that Q. variabilis populations have been decreasing in China mainly due to overexploiting in the past decades, which have resulted in a lot of genetic resources loss,as well as ecological and economic benefit decrease. Therefore,it is necessary to development the technologies for conservation and genetic improvement of this species. Owing to seeds being recalcitrant for storage,late fruiting age,low seed yield,long seed period and only production abundance seeds each 3 to 5 years,which makes against to establish seed orchard. It is difficult for conventional vegetative propagation such as cutting. Additionally adult materials is not easily to be used for organogenesis due to physiological aging. All of these become the limit factors of gene preservation , rapidly propagation and genetic improvement of this species. Biotechnology based on somatic embryogenesis provide an alterability method of micropropagation,genetic improvement and for germplasm conservation of forest species. Thus it is practical importance in development somatic embryogenesis technique for Q. variabilis.This research is the first all-around to investigate the somatic embryogenesis of Q. variabilis. A set of system of regeneration plantlets was developed. The effective media and conditions for somatic embryos induction,maturation and germination were screened. With that regeneration plantlets were obtained. The genetic stability of embryogenic callus and germination plantlets were investigated. The origin and ontogenesis processes of somatic embryo of Q. variabilis were revealed. By means of analyzing the biochemical changes in the course of somatic embryogenesis,the regulation effects of exogenetic factors were showed. The results of paper filled a gap about somatic embryogenesis of Quercus spp. in China, and also provides an available new way for gene preservation,rapidly propagation selected material and genetic improvement for Q. variabilis. The results are as follows:1. Somatic embryos could be induced from zygotic embryos,two month old seedlings leaf and stem segments of Q. variabilis., High induction frequency of 90% was achieved on MS medium supplemented with 0.5 mg/L 6-BA and 2,4-D, whereas rate on hormone-free medium was only 16.7% for zygotic embryos collected in middle of July. By adding 6-BA in medium individually, somatic embryogenesis appeared directly on the zygotic embryos without detectable callus. Secondary embryogenesis appeared with 2,4-D in medium individual or combination with 6-BA. The genotypes of mother trees had great an impact on the inducing rate. Proliferation of somatic embryos occurred from the explant through primary somatic embryogenesis or proliferation of secondary somatic embryos from already formed somatic embryos through repetitive embryogenesis. The best results were obtained on 1 mg/L 6-BA and 0.25 mg/L NAA.2. The morphological Appearance of mature somatic embryos would be described as cotyledons thick, milky or ivory white, pale yellow or green, hypolotyl developed into radicle and about 5-8 mm long in diameter. With 3% sucrose-treated embryos resulted in 15.6% maturation and 7.58% germination. Maturation treatments with ABA was in favor of somatic embryos maturation. The superior mature medium was by adding 0.5 mg/L ABA and 0.5 mg/L PVP together in basal medium with 0.5% sucrose,with that obtained 70% maturation. The rate of germination reached 65.9% on 1/2 MS basal medium with 0.5 mg/L 6- BA and 0.25 mg/L IBA,the rate of conversion 9.4%.3.The chromosome complement of 2n=2x=24 metacentric and submetacentric chromosomes was generally observed. By analyzing chromosomes of embryogenic callus tissue cells with 8 months continuous subculture demonstrated that chromosome structure and number were relatively steady,in which there were 97.2% diploid, 1.87% tetraploid and 0.93% haploid. Aneuploids and chromosomes structure variation were not found. Nuclei in some meristematic cells derived from subsurface layer of hypocotyl of somatic embryos were lamellar or elliptic. Trinuclear cell was found in mature somatic embryos occasionally. No chromosome number varation was observed in germination somatic embryos.4. The histological examnation showed that the origin of somatic embryos was single epidermal or subepidermal cells. Somatic embryos displayed a standard development pattern,from globular ones to heart-like and torpedo-shaped ones and finally to embryo with two cotyledons,which was similar to the development of zygotic embryo in vivo. Directly adventive somatic embryo formed from elongated hypocotyl of immature zygotic embryo. The adventive change occurred from no callus epidermal cell. Single cell origin with densely stained cytoplasm and nucleus was observed. Therefore,the embryogeneic masses could used to inoculate with Agrobacterium tumefaciens for genetic transformation and get no mosaic plantlets based on singe cell origin. 5. The soluble sugars,protein contents and antioxides enzyme activities of embryogenic callus were clearly higher than that of non-embryogenic callus. The low antioxides enzyme activities were detected for matured somatic embryos cultured on sucrose-treated medium. The soluble sugars content of mature embryos markedly increased (3.08 to 4.81 mg·g/FM)while sucrose concentration changed from 3% to 5%. High sucrose concentration (5% and 8%)enhanced the embryos soluble protein contents. The soluble sugars content increased by adding 0.5mg/L ABA to medium,while high ABA levels was no favourable the soluble sugars accumulation. In the same time soluble protein contents of embryos also increased by adding different levels ABA. The study showed there was a close relationship between the rate of maturation and germination and matured embryos soluble sugars contents. All of above demostrate theoretically that maturation medium with 5% sucrose as carbon source and adding 0.5 mg/L ABA is in favor of somatic embryos maturation of Q. variabilis.