High-efficiency regeneration from leaf explants of apple rootstock Luzhen 1

Abstract: 【Objective】Apple rootstocks are used to influence precocity, tree size, fruit quality, yield efficiency, mineral uptake and disease related to replanting, and to withstand adverse environmental conditions. Dwarfing rootstocks play an important role in developing modern orchards. New apple rootstock cultivar Luzhen 1 is a semi-dwarfing rootstock which was selected and released by Shandong Institute of Pomology. This new rootstock has similar effect on tree size to M.26. It can be propagated clonally and has good grafting compatibility with many scion cultivars. The purpose of this paper is to establish a system of high-efficiency plant regeneration from leaf explants of Luzhen 1 and to provide a method for commercial propagation of the plant and genetic improvement using biotechnological methods.【Methods】In mid-to-late April, semi-lignified healthy shoots of Luzhen 1 were used as explants for culture of in vitro sterile plantlets. The leaves from the aseptic plantlets were cultured to induce ad-ventitious shoots. Through the above experiments, the effects of carbon source and type and concentration of cytokinin on leaf regeneration were studied. Further, the effects of basic medium and sucrose concentration on rooting of adventitious shoots were also studied.【Results】The axillary buds on semilignified shoots broke and extended to form aseptic green plantlets, after culturing on the axillary bud initiation medium for 3 weeks. These plantlets grew well on the secondary proliferation medium with good proliferation and elongation growth, and the monthly multiplication ratio was above 5.0, indicating easy multiplication and propagation of Luzhen 1. In the two carbon sources tested, D-sorbitol and sucrose were both effective in inducing adventitious bud. Except that the adventitious bud regeneration rate on D-sorbitol was significantly higher than that on sucrose at a low cytokinin (TDZ) concentration of 0.6 mg ·L-1 , there was no significant difference in the adventitious bud regeneration rate between the two carbon sources in other treatments, both of which were as high as 90%. For the average number of adventitious buds per leaf, there was no significant difference between the two carbon sources under the same type and concentration of cytokinin. These results indicate that the carbon source is not strictly required for adventitious bud regeneration from the isolated leaves of the new rootstock cultivar. Under the same carbon source, the adventitious bud regeneration rate and average bud number per leaf were the lowest when TDZ was at 0.6 mg · L- 1 , and the results were consistent for the two carbon sources. When the carbon source in the medium was D-sorbitol, there was no significant difference in the regeneration rate of adventitious buds between TDZ and BA, and between different concentrations. However, the average number of adventitious buds per leaf at 1 mg · L- 1 concentration of TDZ was significantly higher than that at 0.6 mg·L-1 , while there was no significant difference between the two concentrations of BA. When the carbon source in the medium was sucrose, the regeneration rate of TDZ at 0.6 mg·L-1 was significantly lower than that of other treatments, but the average number of adventitious buds per leaf was not significantly different among different treatments. On adventitious-bud induction medium, cytokinin BA was more effective than TDZ in inducing the elongated and growing adventitious shoots, while the adventitious buds induced by TDZ needed to be transferred to the proliferation medium without TDZ in order to obtain such adventitious shoots. The best medium for inducing adventitious buds from leaves was MS medium with 1 mg ·L-1 TDZ, 0.3 mg ·L-1 IBA and 30 g ·L-1 sucrose. After root induction culture for 12 days, the emergence of early adventitious root protrusions could be observed. After culture for 18 days, the formation of short roots became obvious, with final root formation rates above 88% . After culture for 22 days, no new adventitious roots were induced and root elongation growth could be observed. All the eight rooting media tested could successfully induce rooting from the adventitious shoots, but the rooting rate varied greatly, while the average number of roots per shoot had no significant difference. Independent of the concentration of IBA, the basic medium of 1/4 MS with the lower sucrose (20 g · L- 1 ) was more effective to increase rooting rate and the number of roots per shoot than that with a higher sucrose (30 g·L-1 ). In addition to 0.3 mg·L-1 IBA, rooting rate on 20 g·L-1 sucrose was significantly higher than that on 30 g · L- 1 sucrose. On the basic medium of 1/2 MS, there was no significant difference in rooting rate and number of roots per plant between the two sucrose concentrations or between the two IBA concentrations. The rooting rate of the four treatments could be higher than 70%, indicating that, on the basic medium of 1/2 MS, there was no strict requirement on sucrose or IBA concentration for the adventitious rooting in Luzhen 1. The results indicated that adventitious shoots of Luzhen 1 root easily. The optimal rooting medium was 1/4 MS supplemented with 0.3- 0.5 mg ·L-1 IBA and 20 g ·L-1 sucrose. The rooting rate was over 93% and the average number of roots per plant was 5.9.【Conclusions】It is relatively easy to induce bud regeneration from leaf explants andin vitro rooting from the adventitious shoots in the new semi-dwarfing apple rootstock cultivar Luzhen 1. Based on this study, the optimal medium for adventitious bud induction of Luzhen 1 is MS+1 mg·L-1 TDZ+0.3 mg·L-1 IBA+30 g·L-1 sucrose, and the best medium for in vitro rooting from the adventitious shoots is 1/4MS+0.3-0.5 mg·L-1 IBA+20 g·L-1 sucrose.