| Trifolium repens L. is a member of genus Trifolium, Leguminosae, which is also known as white clover. This plant is one of high-yield and quality forage. Because of the advantages of strong aggressiveness and extensive management, long green period, and good ornamental effect, it is often used as the slope greening plants. The application of T. repens is limited to a certain extent because of its seedling narrow ecological adaptation, weak drought tolerance, and not salinity. Some achievements have been made in the research on T. repens in transgenic, antiviral resistance, physiological stress etc. Our research team has got the new transgenic materials of T. repens with drought and salinity tolerance better than the common variety ‘HAIFA’. This study established the efficient tissue culture system of T. repens L. from the embryogenesis callus re-dedifferentiation. The artificial seed production technology of T. repens was researched. This study also lays the theoretical foundation for the preservation and propagation of new materials of transgenic T. repens, and provides technical support for using artificial seeds of drought resistant and salt tolerant transgenic T. repens in soil spray sowing to green the slope. The main results obtained are as follows。(1)Callus was induced from stem, shoot tip, leaf and petiole as explants. The results showed that the callus from petiole grew fast with loose texture. The callus induction was significantly better than that of the other three kinds of explants. There is the highest rate up to 90% using petiole as explants to MS+2mg/L 2,4-D+0.5mg/L 6-BA as the medium for callus induction. It’s the highest rate of callus proliferation when embryogenesis callus was grown in MS+0.5mg/L NAA+0.5mg/L 6-BA cultured media for 21 days.(2) There are great differences between embryonic and non-embryonic callus in the appearance of features, histological analysis and other aspects of regeneration. From the exterior features, the texture of embryonic callus was firmer, with raised surface particles, yellow or yellow-green colors. However, the texture of non-embryonic callus was loose, powdery surface, soaked, white surface color, with green interior. That the embryogenic cells were tightly packed in embryogenic callus was found by the way of paraffin observation. The embryonic cells were smaller than the surrounding cells, stain deeper, large volume of embryonic cell nuclei, located in the central of the cell. Non-embryonic cell structure callus were loose, irregular, lighter staining, smaller nuclei, biased side cells, and nucleus cannot be seen in some cells.(3) In the differentiation medium of MS+0.5mg/L 6-BA+0.5mg/L NAA, the induction rate of adventitious buds was 70%. The rooting rate was higher and more lateral roots growing out when rooting medium of 0.5mg/L 6-BA+0.6mg/L IBA was added. The rooting rate was low and less lateral roots in MS medium without the hormone.(4) In order to apply Sedum sarmentosum Bunge in the slope greening by spraying, MS+4%sodium alginate+1mg/L 6-BA+0.1mg/L GA3+0.5mg/L NAA was used as artificial endosperm, the spherical and elastic artificial seeds were achieved after being dropped into the solution of 2% CaC12 for l5 min,the germination rate of the artificial seeds was 95% after 20 days, and the rooting rate was 90% after 30 days, the seedling rate was 60% in a Petri dish germination. This formula was a suitable combination of artificial seeds for Sedum sarmentosum Bunge.(5) The artificial seeds were spherical, moderately hard and elastic when the stems with auxiliary buds were selected as the artificial embryo, using 4% sodium alginate+0.5mg/L 6-BA+0.1mg/L GA3+0.6mg/L IBA as artificial endosperm, with 2%CaCl2 aqueous exchange 15 min, and the germination rate of these artificial seeds is more than 70% and rooting rate is more than 90%. It will have a certain inhibitory effect on the germination of artificial seeds when the active carbon was added in the artificial endosperm. |
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