Optimization Of Somatic Embryogenesis In Torreya Grandis‘Merrillii’ And Microscopic Observation On Somatic Embryos At Developmental Stages

Torreya grandis ‘Merrillii’ is a nut tree species particular to China for multiple uses including for the production of fruit, oil, medicine, timber, greening and ornament. So far there has been a heavy market demand for grafted seedlings for the species has high economic benefits and a long economic life. Establishment of a somatic embryogenesis protocol for Torreya grandis ‘Merrillii’ to meet the market demand and to provide a technical support to germplasm conservation, establishment of a genetic transformation protocol, and industrialized seedling breeding.With immature embryos of Torreya grandis ‘Merrillii’ as explants, both single factor and multi-factor orthogonal designs were adopted to stusy effects of cell lines, pretreatment of explants by different methods(high osmotic pressure at a low temperature and drying treatment), basic media(SH, LM, DCR, BM, B5 and WPM), sucrose concentrations, complex additives(including Gln and CH), plant growth regulators(NAA, 2,4-D, 6-BA, ABA and GA), osmotic adjustment substances(PEG6000), metal ions(AgNO3), concentrations of active carbon, culture modes(solid or liquid culture), and environmental conditions(dark culture) on in vitro embryo culture, induction and proliferation of embryonic callus, and maturing and germination of somatic embryos..As a result, culture conditions for the induction and proliferation of embryogenic callus were optimized; media and culture modes for maturing and germination of somatic embryos were screened; and physiological and biochemical changes inside somatic embryos under different treatments(POD, CAT, soluble proteins and soluble sugars)were explored. Histological and cytological observations of somatic embryogenesis were conducted with conventional Paraffin section technology and observation of early embryonic callus by iodine-iodide kalium staining, based on which a relative complete elucidation of origin and development of somatic embryons was performed in Torreya grandis ‘Merrillii’.The main results obtained are presented as follows:1.The germination rate of immature embryos can reach 65% when cultured in the medium of SH + NAA 0.1 mg/L + AC 500 mg/L + sucrose 2% with dark condition for 72 hours, then cultured in light for 15 days.2.The results showed that medium of SH+NAA 0.1 mg/L+AC 500 mg/L+sucrose3%+Gln 0.5 g/L was valid for inducing embryonic callus.The callus induction rate can reach 71% when treated with hypertonic solution for 16 hours under 24 ℃before culture, and most of embryonic callus is ivory, which can differentiate into clumps clava embryos at the same time.Then with the solid medium 1/2SH+2,4-D 2 mg/L(or NAA 1 mg/L)+6-BA 0.5 mg/L,the proliferation of embryonic callus can reached highest of 2.09.When subculture in the mature medium of SH+ABA 10 mg/L+PEG6000 40-80 g/L+AgNO3 5-10 mg/L+AC 500 mg/L+surose3% for 2-3months, some somatic embryos can matured.Before germinate the embryos were subjected to a drying process for 6d.Then cultured in the medium of 1/2SH+BA 0.1 mg/L+NAA 0.1 mg/L+AC 400 mg/L+GA 1.5 mg/L with light condition for 1months.Test and analysis results indicate that different cell lines have some differences in callus proliferation rate and embryogenesis rate.4 cell lines were select from 69 cell lines which in high embryogenesis rate.The LM medium have the highest callus proliferation rate with 7.88, WPM medium have the highest embryogenesis rate with average 218.89 per dishes.3.The morphology and histology of somatic embryogenesis were observed by iodine-iodide kalium staining and Paraffin technology.The results showed that the embryonic callus originated from the symmetry division of single cell and cell mass in the epidermal or cortex of the hypocotyl.The callus consist of two types of cell.One is embryogenic cells with densely cytoplasmic, another is highly vacuolated and elongated cells.Those two group cells form the proembryogenic masses(PEMs), as PEMI, PEMⅡ, PEMⅢ.The PEM cultured Ⅲin the basic media without Auxin, then develop formed embryogenic suspensor mass, and transferred to mature media.The proembryo undergo globular, club-shaped, heart, torpedo and cotyledon embryo.When matured somatic embryo were transferred into SH media, radicle elongated, and germ developed by the grow of needles.The degenerate cleavage polyembryony in the suspensor of zygotic can recovery growth by cultured in vitro.Secondary somatic embryos were regenerated on primary ones.