Abstract
Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr) is a native conifer in North China with great economic and ecological values. Somatic embryogenesis (SE) is a powerful tool for the mass clonal propagation in plants. In this study, we described a high-efficiency SE system via indirect pathways and investigated the effect of genotype, culture conditions and phytohormones on SE. Immature zygotic embryos (IZEs) of L. principis-rupprechtii Mayr were used as explant materials. In the induction stage, embryogenic tissues (ETs) were induced on mLV medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 6-benzylaminopurine (6-BA). The initiation frequencies showed significant differences (p < 0.05) among 20 genotypes of open-pollinated mother trees with the highest induction frequency reaching 30%. For tissue proliferation, proliferation in liquid medium was more efficient compared with proliferation in semi-solid medium, providing a multiplication rate of 3.12 in an 8-day subculture period. As a necessary exogenous plant growth regulator (PGR) for somatic embryo maturation in conifers, abscisic acid (ABA) was optimized at 16 mg L−1 in this system. Next, an orthogonal test on osmotic pressure factors showed 50 g L−1 sucrose, 7 g L−1 phytagel and 75 g L−1 polyethylene glycol (PEG) was the optimal combination for somatic embryo maturation in L. principis-rupprechtii Mayr. Moreover, the dispersion culture method provided a more efficient somatic embryo maturation, up to 545 per gram of fresh weight (FW). Finally, 2 g L−1 of active charcoal (AC) was found to increase the somatic embryo germination rate to 63.46%. The improved protocol of SE will serve as a foundation for establishing mass propagation and genetic transformation of L. principis-rupprechtii Mayr.
Highlights
Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr.), a deciduous conifer, widely grows in middle and high elevations of Northern China
After inoculating explants in the induction medium, we observed a continuous induction of embryogenic tissues (ETs), which could be classified into four types (Figure 1)
The second type involved tissues that were initially brown, and after 1–2 subcultures, new ETs gradually grew on the surface of the brown tissues (Figure 1b)
Summary
Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr.), a deciduous conifer, widely grows in middle and high elevations of Northern China. Somatic embryogenesis (SE) is a more effective and genetically stable method for rapid propagation of plants than other vegetative propagation [3], and it has been the most important development for plant tissue culture, which involves the in vitro production of plants or embryos from single haploid or diploid somatic cells without cell fusion [4]. It is much like its zygotic counterparts, with obvious bipolar structures [5]. It is for mass propagation and for enabling the implementation of biotechnological tools that can be used to increase the productivity and wood quality of plantation forestry [6]
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