| Flax (Linum usitatissimum L.) is one of important economic crops, from flax stalk we can obtain high-grade fiber for textiles, and from seeds we can obtain high-grand oil which have rich unsaturated fatty acids. Heilongjiang province is one main production area of fibre flax in China. Along with the improvement of people's living standard, it is gradually important to develop flax production. Two varieties, H14(domestic) and Opaline(foreign) were selected as test materials to study on organization cytology, characteristics of physiology and biochemistry, changes of endogenous hormones content and RAPD markers during organogenesis and somatic embryogenesis of flax, and explain its occurrence mechanism. Research results are as follows.1. Culture conditions for organogenesis in flax were optimized, we obtained optimal sterilization time of aseptic plantlet with different genotype, aseptic plantlet with 10-15d had good activity. Furthermore, flax hypocotyl was optimal material for callus induction, and the better position is horizontal onto the medium. The prime medium of initial callus induction was MS+4mg/L IAA +2mg/L KT, and the best medium of adventitious buds induction during organogenesis was B5+2.5mg/L KT +1mg/L IAA. The best rooting induction medium was 1/2B5+0.3mg/L IBA. Induction duration was 20d or so, and initial callus were weak green, compact and stiff. The adventitious buds induction period was about 30d. IAA and KT were key factor to induce bud of flax callus. There are different in growth of callus among genotypes. 1.5g/L activate carbon could lower the browning ratio of callus.2. For somatic embryogenesis,the initial callus induction of flax needed high concentration 2,4-D and low concentration 6-BA. but overabundance 2,4-D could inhibit induction and growth of callus, the prime induction medium was MS+2mg/L 2,4-D +0.5mg/L 6-BA. The induction medium of somatic embryogenic was MS+0.5mg/L NAA +0.5mg/L 6-BA. Mostly embryogenic callus were yellow-white, granule, incompact and wetting surface. Genotype had evident effects on somatic embryogenesis. We only gained globular and heart-shaped embryo in H14. Changes of light had not effect on somatic embryogenesis in flax.3. It was showed by cytology method that flax embryogenic callus consisted of regular shaped cells with dense cytoplasm, large nucleus while the nonembryogenic callus cells with large volume, small nucleus and shin cytoplasm. Embryogenic cell mass, 2-cell, 3-cell, proembryogenic masses, initial spherical embryo and hearted embryo were observed, but Somatic embryogenesis had not further development. It showed that the medial cortex had starch grains distribution in PAS reaction from cross section of H14 hypocotyls. Starch grains distribution in palisade tissue cells of cross section of OP cotyledon were observed. There were plenty of starch grains in outside cell of initial callus of H14 and OP. Embryogenic cell of H14 and OP accumulated large number of starch grains which might supply energy for cell division and differentiation. 4. It was showed by transmission electron microscope that the initial callus cells had large central vacuole, which squeezed its cytoplasm to a thin layer around the brim of cell. Nuclear distributed the corner of cells because of being squeezed by large vacuole, however we found the presence of nuclear. Compared embryogenic callus with initial callus its cell wall became thick, it had dense cytoplasm which was full of the whole cell, big nucleus with obvious nucleolus. Many starch grains and chloroplasts including starch grains distributed on the cytoplasm area of cell membrane. The cells of nonembryogenic callus were almost occupied by central large vacuole, and its cytoplasm was extruded. Nucleus and other organelles were hardly observed. Browning organelle had breakdown and had not organelle at all. As showed as followed by Scanning electron microscope that flax initial callus owned dense surfaces and there was little gap among cells, which shared silk-shaped attachment in the cell surface. On the contrary, embryogenic callus had smooth surfaces, and the embryogenic cells were closely conjuncted to form cell masses in which loosely connection and large empty were observed. The cells of cell masses were regular- shaped, and many of them exposed on the surface of embryogenic callus. Nonembryogenic callus and browning callus surface shape was irregular and covered with lamellar. They had different shape with large volume.5. The content of CAT, POD, soluble protein, nucleic acids and soluble sugar in embryogenic callus were higher than in nonembryogenic callus, these substance changes of content and components may be relevanted to maintain of embryogenic and hearty splitting and differentiation of cells. The content of CAT and soluble sugar was maximum in aseptic plantlet, the content of CAT, nucleic acids, soluble protein and soluble sugar callus were all lowest in browning tissue, which showed the physiological state of browning callus. The POD content was highest in initial callus tissue, this was relevant to mechanical damage. The SOD content was high in the aseptic plantlet and adventitious buds, and lowest in initial callus, this was relevant closely to antioxidant ability. The content of soluble sugar ect were gradually increased in proembryogenic and spherical embryogenic, which were prepared for substance and energy of somatic embryogenesis.6. The results of SDS-PAGE indicated that the protein staining of nonembryogenic callus was very light, and the metabolism of protein was very weak. The main protein components of each stage of flax somatic embryogenesis were very similar, which means the stability of somatic embryogenic components. The protein scope of each stage for H14 were 24-94kD, and 20-100kD for OP. It presented 9 POD isozyme strips during the initial somatic embryogenesis, 6 of those were presented continuously during the initial somatic embryogenesis, which were used for the biochemical markers of the initial somatic embryogenesis. The amylase isozyme strips were less in the initial somatic embryogenesis, nonembryogenic and browning callus, and more in nonembryogenic and browning callus, the number of amylase isozyme strips varied with different varieties. The EST isozyme strips color varied from deep to light during the stages of flax initial somatic, which is opposite to the variety of POD isozyme strips and starch isozyme strips.7. The level and the balance of endogenous hormones can adjust to flax morphogenesis. The content of IAA in embryogenic callus was higher than in nonembryogenic callus, and it maintained high level in proembryogenic period and globular embryogenic period, its content was lowest in browning callus. The content of ABA was highest in explant, its content was less in proembryogenic period and globular embryogenic period, low level of ABA was benefited to the embryogenesis. The peak value of ZR content present to initial callus, the ZR content of embryogenic callus was lower than in nonembryogenic callus, its content was the lowest in browning callus. The IAA/ZR ratio of embryogenic callus was far higher than in nonembryogenic callus, it may be benefit to the expression of embryogenic capability.8. The number of flax chromosome were 2n=32, the variation rate was 2.5% after 5 subculture, and 4.3% after 9 subculture in this test. It had quantity variation and the chromosome structure variation. According to RAPD markers, it was found that one strip had lost in browning callus compared with aseptic plantlet and other periods in the amplification product of S10,S21,S24 and S48, it was discerned that the genome DNA of flax had changed during subculture(add to browning callus).
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