Establishment of leaf regeneration system for European plum

Abstract:【Objective】To construct a stable and efficient regeneration system for plum, the leaves from European plum (Prunus domestica) were used as explants to study the optimum conditions for adventitious shoot regeneration, proliferation, growth and rooting. Using the leaves as explants has the advantages of convenient collection, abundant materials and low cost.【Methods】Leaves from branches of four P. domestica cultivars Startovaya, Victoria, France and Bluebyrd were surface-sterilized in 0.5% sodium hypochlorite (NaClO) solution for 10 min and rinsed thrice in sterile distilled water, and then surface- sterilized in 0.1% mercuric chloride (HgCl2) solution for 6 min and again rinsed 5-6 times with sterile distilled water. The main vein of leaves was slightly cut and plated to Murashige and Skoog (MS) media supplemented with different concentrations of 6-Benzylaminopurine (6-BA) and Indole-3- butyric acid (IBA) for regeneration. Leaves from in vitro cultured seedlings after subculture for 30 days were cut in the same way and placed on the woody plant (WPM) media supplemented with different concentrations of Thidiazuron (TDZ) and 2, 4- Dichlorophenoxyacetic acid (2, 4- D) for regeneration. The regenerated shoots from 40-day-old cultures were placed on MS media supplemented with different concentrations of 6-BA and IBA for proliferation. The small individual shoots from proliferation culture were transferred to MS media supplemented with different concentrations of 6-BA and IBA for stem elongation. Then the elongated adventitious shoots with 3-4 leaves were transferred for rooting. All of each experiment repeated for three times.【Results】For four P. domestica cultivars, the adventitious shoots were only regenerated from Victoria with a regeneration rate of 5.4%, and then the following researches were carried out for P. domestica cultivar Victoria. Firstly, WPM medium was used as basal culture medium and the effect of plant growth regulators, different explant and dark incubation time on adventitious shoot regeneration was investigated. The concentrations of TDZ and 2, 4-D were 2.0 mg·L-1 and 0.2 mg · L^{- 1} , which were best for adventitious shoot regeneration and the regeneration rate reached 18%. The intact leaves rather than petioles, middle part of leaves and leaves tip from the middle part of tissue culture seedling were the best explants for adventitious shoot regeneration. The regeneration rate was further raised up to 25.6%. The five different dark incubation times were set and the intact leaves incubated in dark for 14 days had the highest adventitious shoot regeneration rates (33.4%). Secondly, MS medium was used as basal culture medium and the effect of plant growth regulators on adventitious shoot proliferation was investigated. When the concentration of 6-BA was 3 mg·L^-1 , it had a higher proliferation rate than the other two concentrations (2 mg · L^{- 1} and 1 mg · L^{- 1} ) and the state of regenerative shoot was yellowing, curly and vitrified. When the concentration of 6-BA was 2 mg·L^-1 , a small quantity of regenerative shoot was vitrification. When the concentration of 6-BA was 1 mg·L^-1 , the state of regenerative shoot was yellowing. Thirdly, MS medium was used as basal culture medium and the effect of different concentrations of 6-BA was investigated on the growth of regenerative shoot. When the concentrations of 6-BA and IBA were 0.2 mg·L^-1 and 0.1 mg·L^-1 , the stem of regenerative shoot was robust and grew faster than others. Finally, the effect of different concentrations of IBA was analyzed on rooting rate, root length and rooting coefficient. There were no roots from regenerative shoot without IBA addition. When the concentration of IBA was 0.5 mg·L^-1 , the highest rooting rate achieved 74% with an average root length of 14.7 cm and highest rooting coefficient of 4.69.【Conclusion】A stable regeneration system for European plum using leaves was established. The concrete steps include: the mature leaves in mid part were inoculated on WPM media supplemented with 2.0 mg·L^-1 TDZ and 0.2 mg·L^-1 2, 4-D with dark-incubated for 14 days, and the highest frequencies of adventitious shoot regeneration was 33.4% . The optimal adventitious shoot proliferation medium was MS media supplemented with 2.0 mg ·L^-1 6-BA and 0.1 mg·L^-1 IBA, and the proliferation rate was 7.03. The optimal growth medium was MS media supplemented with 0.2 mg · L^{- 1} 6-BA and 0.1 mg · L^{- 1} IBA. The highest rooting rate of 74% was observed on MS media supplemented with 0.5 mg · L^{- 1} IBA. Then the seedling survival rate was 83.3% after seedlings were transplanted into the pots.