Optimization Of Somatic Embryogenesis In Picea Likiangensis (Franch.) Pritz And Proteomic Analysis On Somatic Embryos At Developmental Stages

Somatic embryogenesis (SE) is one of the most practical ways in large scale vegetative propagation, which conducts significance both in theoretical research and practical application. However, conifers that play great role in forests have been regarded as the most difficult trees for SE owing to their biological characteristics. SE of Picea trees are easier than other conifers relatively, so they are usually taken as model tree for SE related studies.As an evergreen tree in Picea of Pinaceae, Picea likiangensis (Franch.) Pritz is the only species of the genus growing in Yunnan. It is one of the most important conifers used as timber and garden trees. Employing mature zygotic embryos of Picea likiangensis as explant, the study established and then optimized protocals for SE of the tree. Besides, histological and cytological observation and proteomic analysis on calli and somatic embryos at developmental stages were carried out in the study. The results are shown as followings.1. The optimization of SE were performed from 3 aspects of callus conservation, embryos maturation and synchronization regulation of embryogenesis.Embryogenic callus (EC) were initiated on one-half strength LM medium supplemented with 3.0mg/L 2,4-D and 1.5mg/L 6-BA (pH5.8) and subcultured in the same medium every 10～14 days. Most EC lost their SE capability after subsultured in the callus initiated medium for 6 months. While EC still kept the capability when subcultured 6 months in the medium of one-half strength LM medium supplemented with 1.0mg/L 2,4-D and 1.5mg/L 6-BA.EC developed into cotyledonary somatic embryos through globular embryo, heart-shaped embryo and torpedo-shaped embryo when cultured in maturation medium of one-half strength LM medium supplemented with concentrations of ABA and PEG. The results showed quality and quantity of somatic embryos were obtained on medium with different ABA concentrations. Concentrations of 40mg/L～60mg/L ABA gave the best maturation. The quality and quantity of somatic embryos were also different from the different lines of the same family, even under the same cultures.Synchronical regulation for somatic embryo development was performed through plant growth regulators and culture time. Synchronical regulation for globular embryo was tranferring vigorous EC on the medium of one-half strength LM medium supplemented with 0.2mg/L 2,4-D,60mg/L ABA and 40g/L sugrose, culturing for 15 days and sub-culturing 1-2 times on the same medium. Synchronical regulation for cotyledonary somatic embryo was transfferring synchronical globular embryos on the medium of one-half strength LM medium supplemented with 60mg/L ABA and 40g/L sugrose, culturing 20 days for torpedo-shaped embryos and more 15 days for cotyledonary embryos.2. Morphological and Histocytological observations were conducted on calli and somatic embryos at developmental stages.2 types calli were induced from zygotic embryos of Picea likiangensis. Type I was non-embrygenic callus (NEC) with two shapes of hard-green and grain gathered-yellow. Type II was embryogenic callus (EC) with appearance of soft-transparent filiform. The former type callus grow slowly, withered after subcultures, and can not generate embryos. While the later type grew and propagated rapidly, generated somatic embryos when cultured in maturation media. Contrast to NEC, the cells of EC possessed denser protoplasm, large nucleus, regular shapes and actively cell division. EC showed typical structure of embryogenic suspensor mass for conifers which composed of embryonal head and suspensor system.EC started to develop when cultured on maturation media. The soft-transparent EC became non-transparent and embryonal head grew apparently. From the appearance, embryonal head enlonged and developed to yellow cotyledonary somatic embryos. From cytological aspect, embryogenic cells of EC divided qiuckly, while suspensor cells degenerated gradually. Embryogenic cells began to differentiated on globular stage. With the process of somatic embryogenesis, embryogenic cells arranged more tightly, regularly and polar, vascular tissue and meristem formed.As one of the most popular storage products in plants, distribution and amount of starch could be marker of cell differentiation. There were two starch accumulations during the process of somatic embryogenesis. One was at embryonic cells of EC, the other was at globular embryos before the differentiation of somatic embryos. The distribution and amount reached peak at stage of heart-shaped somatic.3. Protemic analysis on non-embryogenic callus (NEC), embryogenic callus (EC1), globular embryos (EC2), torpedo-shaped embryos (EC3) and cotyledonary embryos (EC4) was carried out in the study.Procedure of protein seperation system was establised, including protein extraction method and. two-dimentional gel electrophoresis.The 5 samples shew rich in proteins. The protein spots for each of 5 samples was 1857 (NEC),1999 (EC1),2344 (EC2),2285 (EC3) and 2205 (EC4) and most proteins were 20～66KD, pH4-7 proteins.Differential proteins and specific proteins changed obviously during somatic embryo development. Analysis on the gel of above 5 samples indicated that most proteins in 5 samples shared similar gel maps while some proteins were found changing, which shown mainly by differentially expressed proteins (3 times or above) and specific proteins.59 proteins of 3 times (above) differentially expressed proteins and 44 specific proteins were detected. The differentially expressed proteins in cotyledonary stage had the siginificant change, up-regulated and specific proteins in torpedo-shaped stage showed the most proportion of the stage, and most of them were low molecular weight proteins.15 out of 44 specific proteins were identified through MALDI-TOP-MS and NCSInr check and 7 were identified. They were Pentatricopeptide (PPR) repeat-containing protein, Beta-hydroxyacyl-ACP dehydratase b, R2R3-MYB transcription factor, Hypothetical protein OsI₀0337 and 3 unknow proteins in Picea sitchensis.The study will lay foundation for vegetative propagation and genetic breeding of Picea likiangensis. The results will provide reference information for further seperating and purating specific proteins related closely to somatic embryogenesis, cloning related genes and revealing embryogenesis mechanism at cytological and molecular level.