Adventitious shoot regeneration from the leaves of in vitro grown ‘Zhongli 1’ pear (Pyrus spp.)

Abstract

The pear (Pyrus spp.) is one of the most important temperate fruit crops. The technique of adventitious shoot regeneration from leaves is considered to be one of the shortcuts in the research on pear genetic modification and cellular engineering, which, however, has not been widely used. As the regeneration frequency of pear leaves is usually very low, the research on adventitious shoot regeneration from pear leaves is eagerly needed. In this experiment, the factors affecting shoot and bud regeneration from the leaves of ‘Zhongli 1’ pear were studied, and an efficient protocol for shoot regeneration was established. The results showed that different types of basic media, different combinations of plant growth regulators, leaf placement on medium, periods of dark culture and the use of silver nitrate (AgNO₃) on culture media all significantly affected the adventitious shoot regeneration frequency of ‘Zhongli 1’ pear. The details are as follows: (1) Among three kinds of basic media, NN69 was better for ‘Zhongli 1’ shoot regeneration, followed by half (½) MS, while full MS had no effect on shoot regeneration; (2) Thidiazuron (TDZ) was better than 6-benzylaminopurine (6-BA) for ‘Zhongli 1’ regeneration, with an optimal concentration of 1.5 mg·L^-1, and the regeneration rate under this concentration could reach 85%, with 2.72 buds per leaf. 0.5 mg·L^-1 indole-3-butyric acid (IBA), which induced a higher regeneration frequency, was a better choice for pear regeneration compared with 0.3 mg·L^-1 naphthaleneacetic acid (NAA). Among the different combinations of plant growth regulators, TDZ + IBA was better for inducing high regeneration frequency; (3) The abaxial surface of leaves touching the medium was beneficial for leaves to uptake nutrients from the medium, and because of that, the regeneration frequency of leaves was significantly higher than that of leaves touching the medium with their adaxial surfaces (obverse side of leaf); (4) Dark culture was necessary for bud regeneration, and the best duration for dark culture of ‘Zhongli 1’ pear was 21 days; (5) The addition of 1.0 mg·L^-1 AgNO₃ into the culture medium could promote adventitious shoot regeneration significantly. A high adventitious shoot regeneration frequency was obtained in this research, which will be beneficial for further research on efficient and stable in vitro plant regeneration systems and genetic modification of pear.

Key words： ‘Zhongli 1’ pear; adventitious shoot regeneration; growth regulators; tissue culture; in vitro

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