North China Larch Somatic Embryogenesis And Genetic Transformation System Established

In this thesis, by using 3l1-A regression design, the experimental system of somaticembryogenesis in Larix princis-Rupprechtii including induction media, proliferationmedia, maturation media and germination media, was studied. Transformationexperiments with established embryogenic cultures were carried out. The effect ofnapkthalene acetic acid (NAA) and 2,4-dichlorophenoxy acetic acid(2,4-D) on thesubculture and proliferation of embryogenic calli, capable of producing early stageembryos, were explored as well as development and rooting of embryos. Using maltose toreplace sucrose in the maturation medium influenced maturation and rooting of embryos.Ou the basis of formation and developmental characteristics of embryogenic callus, wecollected 10 differeut kinds of embryogenic (EC) and non-embryogenic calli (NEC) toanalyze their physiological and biochemical background. The technical systems ofinduction, maturation and rooting of somatic embryos, from embryogenic callus of Larixprincipis-Rupprechtii were established. Furthermore, the transformation with a truncatedgene of Bacmes thuringensts (B.t) and a geue for drought resistance from Loblolly pine (lp3)were carried out The key techuiques of transformation were tested and discussed.The results showed that, it was successful to take immature zygotic embryos of Larixas explants. The medium for induction of embryogenic calli, which is accordiug to Guptaaud Durzan (1987- 1/2SPE, here S) was used. For proliferation and maturation, thismedium was enricbed with boron (S+B). Germination media based on Douglas-fircotyledou revised medium (DCR) or Woody plant medium (WPM) were better than S+Bmedium. Main factors for induction of embryogenic calli were 2,4-D, 6-benzyl aminopurine (BA) and kinetin (KT). The best combination of them was 1.29mg/L 2,4-D.0.39mg/L BA and 0.58mg/L KT calculated by data of a 311-A regression analysis. Thiscombination with the basal S medium could get a maximum of l3.93mg/L freshweight ofinduced embryogenic callus five weeks after establishing immature zygotic embryos. Onthe basis of experimental data, we established the quadratic equation between freshweightof induced embryogenic callus and 2,4-D, BA and KT as the following: y=1.1246+5.9344x₁ + 23.3774x₂ + 22.7825x₃ ─ 2.5755x₁x₂ + 1.4763x₁x₃ ─ 10.l607x₂x₃ ─ 2.2311x₁²â”€l7.937lx₂²â”€17.8356x₃² (R²=0.9849). We also analyzed main effects and mutual effects ofVIIImain factors by the quadratic equation.In order to improve quality of somatic embryos and rooting frcquency, 2,4D wasreplaced by naphthalene acetic acid (NAA), which improved tbe quality of eariy stageembryos, and thus improved the fOrmatiou of high quality somatic embryos. Subcultureson this maturation medium, coutaining 3o/. maltose to replace sucrose, together witb 4o/oPEG4000 and 0.l% activated cbarcoaI (AC), the maturation frequency of somatic embryoswas not lower (106.7 embryos per gram EC), but the quality of somatic embryos wasimproved distinctly sucb as the bypocotyls axis was longer and the rooting frequeucy wasup to 57.89o/o. The root system formed grew normal. WPM medium as germinationmedium for somatic embryos, containing phloroglucinol (PG) 6mglL was the bestconcerning embryo germination and rooting.Based on the appearance of callus growth and bebaviour (structure, color etc.) ofLarir embryogenic callus, the physiological and biochemical background in tbese differentkinds of embryogenic and non-embI'yogenic caIIi were aua1yzed. This incIuded callustreatcd by ABA as well. The contents of amino acids, saccharides, hydroxybeuzene acid,phytohormones as well as differeut ions were tested. R6suItS indicated that the averageconteut of l8 amino acid8 of non-embryogenic calli was higher than embryogenic calli.Especially' the contentS of Arginine, Lysine and V8line in the non-embryogeuic calli were3-4 times higher thau tbat iu embryogenic callus. The contentS of amylose andtrisaccha...