Studies On Hypocotyl Adventitious Bud Regeneration And Tetraploid Induction Of Black Locust(Robinia Pseudoacacia L.)

This experiment established a plant regeneration system using hypocotyl of black locust at first. And then further chromosome doubling by treating hypocotyls and cotyledons with colchicine in vitro were carried out relying on their regeneration system. Finally, the morphological and anatomic characteristics of induced tetraploidy and diploidy plants were compared.The main results are as follows:1. The highest hypocotyl regeneration rate (68.52%) was obtained when using hypocotyl near the radical, placed horizontally and cultured on MS basal medium supplemented with 1.5 mg·L-1 6-BA,0.05 mg·L-1 NAA and 3% sugar. An average of 5.56 shoots per explant was obtained. The most suitable medium for root regeneration were on MS basal medium supplemented with 0.20 mg·L-1 IBA and 3% sugar and 100% of them were successfully rooted.2. We totally obtained 43 genotypes of homozygous tetraploid black locust and 2 genotypes of mixoploid black locust by inducing chromosome doubling using hypocotyl segments and cotyledons as explants in vitro. The highest efficiency of tetraploid induction was 53.33% by treating hypocotyl explants with 70 mg·L-1 colchicine for 72 hours and 37.5% by treating cotyledons explants with 70 mg·L-1 colchicine for 48 hours.3. We compared morphological and anatomical characteristics of tetraploidy and diploidy plants.The results showed that the length, width and density of stomata, the number of chloroplasts in leaf guard cells and chlorophyll content of tetraploid black locust was 1.77 times,2.00 times,0.69 times,1.92 times and 1.40 times compared to diploid black locust. The stomatal length and width on leaf surfaces and the number of chloroplasts in leaf guard cells were stable and reliable markers for the rapid screening of tetraploids at early stage.