Abstract
The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm. Callus can be induced from immature embryos of Korean pine (Pinus koraiensis Sieb. et Zucc.; Pinaceae) as explants, but there are problems, such as low proliferation efficiency, loss of embryogenicity, poor vigor; thus, best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear. To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation, we varied hormone concentration, subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages, then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design. The results showed that the family origin and collection date of explants significantly affected callus induction (induction rate reached 1.67%). Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L−1 6-benzyl adenine, 1 mg L−1 naphthaleneacetic acid, 30 g L−1 sucrose, 500 mg L−1, l-glutamine, 500 mg L−1 casein hydrolysis and 6.5 g L−1 agar. In addition, the combination of 2,4-dichlorophenoxyacetic acid + 6-benzyl adenine also had a better proliferative effect on callus. The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different. Transfer to new medium every 13–15 days not only maintained robust callus vigor, but also yielded a larger proliferation coefficient. The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.
Highlights
Somatic embryogenesis has become the main mode for vegetative reproduction because somatic embryos develop both apical meristem and root meristems (Lelu-Walter et al 2018)
The explants were inoculated on the callus induction medium for about 30 days, and callus was ejected from the micropyle end (Fig. 1b)
As shown by morphologic and microscopic examination, the proembryonic stage was best as explant material in terms of callus induction for P. koraiensis
Summary
Somatic embryogenesis has become the main mode for vegetative reproduction because somatic embryos develop both apical meristem and root meristems (Lelu-Walter et al 2018). The use of somatic embryogenesis and the cryopreservation of embryogenic callus enables large-scale propagation of excellent seed resources (Lelu-Walter et al 2013; Tret’yakova and Shuvaev 2015; Ahn et al 2017). For this reason, obtaining a sufficient supply of high quality callus is a critical step for somatic embryogenesis. The main factors affecting the maintenance and proliferation of embryonic callus include the nutrient and hormonal composition of the medium, growth temperature and humidity, subculture cycle and genetic background of the embryonic callus. A combination of auxin and cytokinin is usually used for embryonic callus maintenance and proliferation of conifer species (Zuzana et al 2011; Klimaszewska et al 2016; Nunes et al 2018)
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