Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

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.

Keywords

Korean pine / Somatic embryogenesis / Embryogenic callus / Megagametophytes / Embryonic maintenance

References

[1] Ahn CH, Tull AR, Montello PM, Merkle SA. A clonal propagation system for atlantic white cedar (chamaecyparis thyoides) via somatic embryogenesis without the use of plant growth regulators. Plant Cell Tissue Organ Cult, 2017, 1: 97-101.

[2] Cao Y, Shen HL, Li J. Primary study on callus induction of Pinus koraiensis. For Sci Technol, 2009, 34(3): 1-4. (in Chinese)

[3] Gao F, Shen HL, Liu CP, Wang Y, Zhang P, Yang L. Culture condition optimization and explant selection for callus induction from mature embryo of Pinus koraiensis. J Nanjing For Univ (Nat Sci Ed), 2017, 41(03): 43-50. (in Chinese)

[4] Humánez A, Blasco M, Brisa C, Segura J, Arrillaga I. Somatic embryogenesis from different tissues of spanish populations of maritime pine. Plant Cell Tissue Organ Cult, 2013, 111(3): 373-383.

[5] Klimaszewska K, Hargreaves C, Lelu-Walter MA, Trontin JF. Advances in conifer somatic embryogenesis since year 2000. In vitro embryogenesis in higher plants, 2016, New York: Springer 131 161

[6] Lelu-Walter MA, Thompson D, Harvengt L, Sanchez L, Toribio M, Pâques LE. Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genet Genomes, 2013, 9(4): 883-899.

[7] Lelu-Walter MA, Gautier F, Eliášová Kateřina, Sanchez L, Teyssier C, Lomenech AM, Metté CL, Hargreaves C, Trontin JF, Reeves C. High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in pseudotsuga menziesii [mirb.]. Plant Cell Tissue Organ Cult, 2018, 132(1): 137-155.

[8] Liang Y, Shen HL, Liu CP, Yang L, Zhang P. Comparison of methods for extracting high-throughput sequencing RNA from Korean pine seeds. J For Res, 2016, 27(1): 33-40.

[9] Liu BG, Li CH, Zhang HG. Screning of influence factors on maintenance and proliferation of embryogenic callus of Picea koraiensis. J North East For Univ, 2010, 7(38): 56-60.

[10] Niskanen AM, Lu J, Seitz S, Keinonen K, Von Weissenberg K, Pappinen A. Effect of parent genotype on somatic embryogenesis in scots pine (pinus sylvestris). Tree Physiol, 2004, 24(11): 1259-1265.

[11] Nunes S, Marum L, Farinha N, Pereira VT, Almeida T, Sousa D. Somatic embryogenesis of hybrid pinus elliottii var. elliottii × p caribaea var. hondurensis and ploidy assessment of somatic plants. Plant Cell Tissue Organ Cult, 2018, 132(1): 71-84.

[12] Pullman GS, Bucalo K. Pine somatic embryogenesis using zygotic embryos as explants. Methods Mol Biol, 2011, 710(710): 267-291.

[13] Salaj T, Matusova R, Salaj J. Conifer somatic embryogenesis-an efficient plant regeneration system for theoretical studies and mass propagation. Dendrobiology, 2015, 74: 69-76.

[14] Santa-Catarina C, Silveira V, Guerra MP, Steiner N, Macedo AF, Floh EI. The use of somatic embryogenesis for mass clonal propagation and biochemical and physiological studies in woody plants. New For, 2012, 43(4): 429-440.

[15] Shen XH, Jiang XN, Yill SP, Mahedrappa MK. Somatic Embryogenisis (SE) in Korean pine (pinus. Koraiensis sieb. et zucc). J Chengdu Univ (Nat Sci), 2005, 24(1): 11-14. (in Chinese)

[16] Steiner N, Farias-Soares FL, Éder C, Schmidt Pereira MLT, Scheid B, Rogge-Renner GD. Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of araucaria angustifolia, (bert.) o. kuntze. Protoplasma, 2016, 253(2): 487-501.

[17] Tret’yakova IN, Shuvaev DN. Somatic embryogenesis in pinus pumila and productivity of embryogenic lines during long-term cultivation in vitro. Russ J Dev Biol, 2015, 46(5): 276-285.

[18] Trontin JF, Aronen T, Hargreaves C, Montalbán IA, Klimaszewska K (2016) International effort to induce somatic embryogenesis in adult pine trees. Vegetative propagation of forest trees, pp 211‒260

[19] Wang Y, Zhao TT, Yang W. Proper condition for callus induction from mature zygotic embryo explants of Pinus koraiensis. For Eng, 2015, 2: 5-7. (in Chinese)

[20] Yang XY, Zhang XL. Regulation of somatic embryogenesis in higher plants. Crit Rev Plant Sci, 2010, 29(1): 36-57.

[21] Zeng FC, Zhang XL, Cheng L, Hu LS, Zhu LF, Cao JL, Guo XP. A draft gene regulatory network for cellular totipotency reprogramming during plant somatic embryogenesis. Genomics, 2007, 90(5): 620-628.

[22] Zuzana V, Kateřina E, Lucie F, Martin V. The role of auxins in somatic embryogenesis of abies alba. Cent Eur J Biol, 2011, 6(4): 587-596.