| Barley is the most important crop as wheat, rice and maize, regardless of its planting acreage or production is the 4th great in the world and it is important to the world agricultural production. Many excellent barley cultivars have been developed through genetic engineering which play a important role in improving yield ang quality of barley. However, many hinders still exist in barley biotechnology, The main reason is that cultivarie of transgenic barley remains extremely difficult in somatic embryogenesis and regeneration. Recalcitrant barley cultivars, long tissue culture duration, the unpredictability of tissue culture, and a high degree of genotype dependence are more troublesome in regeneration of barley, and the diversification of barley transformation problem and so on. The study utilized the shoot segments of the seedlings of the barley cultivar E Damai 58288 as the starting material culture in vitro, a high frequency of multiple shoot clumps regeneration system of barley was established. And then, the shoot segments of barley cultivars e damai 9 and E Damai 32122 were transformed using Agrobacterium tumefaciens system with the plasmid pTRAkc-BAR-UT-GFP, using bar gene as the selectable marker. PPT, PCR and Southern blot resistance revealed the integration of transgenes in the T0 and T1 transformants, and 14 plants were identified to the transgenic barley containing the target genes. The main results are presented bellow:With the shoot segments of barley cultivar E Damai 58288 seedlings as explant, we have researched the effect of different mediums to inducing the multiple shoot clumps. The result revealed that it didn’t inducing the multiple shoot clumps when the shoot segments cultured on the MDA medium which contain 2 mg/L 2, 4-D and 2.5 mg/L 6-BA. The high frequency of multiple shoot clumps regeneration system of barley has been established, which the shoot segments as explant and cultured on inducing medium containing 1.0 mg/L TDZ and 1.0 mg/L 6-BA, then transfered to rooting medium containing 1.0 mg/L IBA to rooting, we could obtain the multiple shoot clumps with many side-shoots and strong root. We could regard it as a base of barley trasnformation system with high frequency of regenerating ability.Within Agrobacterium-mediated barley shoot segments transformation. The shoot segments were derived from MS medium culture 3～4 d and then wounded by sterile needle which expose or wound the shoot apical point as far as possible, subsequently, the wounded shoot segments were infected by Agrobacterium tumefaciens GV3101 with plasmid pTRAkc-BAR-UT-GFP and then air-desiccated on sterile filter paper. Then, the inoculated explants were transfered to co-culture medium to co-culture 2～3 d and transfered to resting medium supplement with 500 mg/L Cef to inhibit the growth of Agrobacterium for 7 d and further 30 d selection subculture on rooting-selection-medium containing 2 mg/L PPT at a 10 d interval. The survival resistance plantlets containing root were transfered to flowerpot and growing in greenhouse after jarovization. When the plantlets contained 5～6 leaves, the positive plants were obtained through 200 mg/L PPT daubing identifying, PCR and Southern blot identifying, and then it is ensured that the aim genes were comformed into the barley genome.Factors related with the genetic transformation were investigated respectively. High percentage of positive plants were obtained under the different conditions: osmotic treatment 2 h, ultrasound treatment 15s and so on. This transformation procedure used shoot tips as target, avoiding redifferentiation, high green seedling regeneration rate, low abnormal seedlings within regeneration seedlings, avoiding callus phase leading to fast forming regeneration seedlings and shorten the period, low somaclorlal variation. Furthermore, there is no time limits to obtaining materials, so there are greater area and flexibility when the shoot tips are used as the explants of genetic transformation. |
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