The Preliminary Study Of CYP81A6/SaNHX Gene Transformation Into Kenaf

Kenaf (Hibiscus Cannabinus L.) of Malvaceae family is annual bast fiber crop which is an important raw material in linen and paper industry. Kenaf of herbicide-resistant and salt-tolerant, on the one hand, can effectively control weeds and reduce production costs, on the other hand, can take full advantage of saline lake and river beach.The tests detected kenaf pollen in vitro by TTC method and aniline blue staining; observed style after pollination by tissue section; reseached Kenaf seed setting rate and grains per fruit by cutting off style after pollination; observed Embryo by paraffin section method. The results are as follows:(1) In Vitro, the pollen ability dramatically decreased in a short time and disappeared after 7 h; pollen in the medium (sucrose and boric acid) had germinated in 0.5 h, but it was very difficult to continue grow for pollen tubes, and up to 80% in germination rate in the end.(2) Kenaf pollen grains could germinate on the stigma after pollinating for 20min. Pollen tubes went through the style into the ovary after pollinating for 4h; the formation of pollen-tube pathway after pollinating for 6h, and pollen tubes went through the micropyle into the embryo sac and released of contents after 12h; observed the embryo sac state after pollination 18h, when fertilization has been completed.(3) Cutting off style (2.5cm) after pollination, the fruit setting rate and grains per fruit were very low in 0-2h, respectively 10% and 10 grains; the fruit setting rate and grains per fruit were 73% and 20 grains in 4-6h respectively; the majority pollen tubes passed through style into the ovary at this time. The time of pollen tubes through the style identified with the tissue sections'.(4) According to analysis of the tests and field observation, it was 12h after pollination for the the optimal beginning period of foreign gene introduction.Based on the above results, anti-herbicide gene (rice cytochrome P450 gene CYP81A6) and salt-resistant gene (rice grass Na+/H+ anriporter gene SaNHX) were introduced into Fuhong 992 by pollen-tube pathway at the optimal introduction time. The herbicides resistance and salt water tolerance test of kenaf were processed. After obtaining T1 generation, the T1 generation was treated by the critical concentration, and then resistant plants were validated by molecular detection.(5) The appropriate concentration of seedling kenaf (3-4 leaf) against the herbicide (Bentazone) was 1500mg / L. 135 resistant plants were obtained by screening T1 generation, including 59 injection ovary, 76 ovary addition. 32 plants were positive plants by PCR, of which 13 ovary injection, transformation rate was 0.78%; ovary addtion was 19 for 1.26%.(6) The appropriate saline concentration on seedling kenaf (2-3 leaf stage) was 150mmol/l. T1 plants were screened by critical concentration; 39 salt-tolerant plants were obtained.9 positive lines were detected by PCR, of which 4 ovary injection with transformation rate 2.11%, 5 ovary addition with transformation rate 3.45 %.The herbicide-resistant and salt-tolerant offsprings were obtained in the test. Genetic stability will be studied in future. New varieties of dual anti-Kenaf will be obtained by cross breeding.