Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Abstract

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo (SE) yield, unsynchronized SE development, and a high percentage of deformed SEs. We aimed to improve F. mandshurica SE production by synchronizing SE development, improving SE quality, and inducing root formation to obtain complete regenerated plants. Cotyledons of immature zygotic embryos of F. mandshurica were induced to form callus and then SEs. The SE induction percentage from explants differed among 32 mother trees, and the one with the highest SE induction percentage (29.8%) was used for further experiments. The highest callus induction percentage was 94.2% on ½-strength Murashige and Skoog medium (MS½) supplemented with 0.15 mg·L⁻¹ naphthalene acetic acid. The highest callus proliferation coefficient (240.5) was obtained on McCown’s Woody Plant Medium containing 0.1 mg·L⁻¹ 6-benzyl adenine and 0.15 mg·L⁻¹ 2, 4-dichlorophenoxyacetic acid. The highest number of SEs (1020.5 g⁻¹ fresh weight) was obtained on MS½ medium supplemented with 1 mg·L⁻¹ 6-benzyladenine. The highest number of cotyledon embryos (397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L⁻¹ abscisic acid and then applying a drying treatment. The cotyledon embryos were milky white, uniformly sized (average length 4.7 mm), and 80% of them were normal. The SE rooting percentage on ½MS medium containing 0.01 mg·L⁻¹ NAA was 37.5%. Overall, the germination percentage of SEs was 26.4%, and complete regenerated plants were obtained after transplanting and acclimation. These results provide more possibilities for the preservation and breeding of F. mandshurica.

Keywords

Fraxinus mandshurica / Somatic embryogenesis / Callus induction / Cell differentiation / Plant regeneration

References

[1] Bernula D, Benkő P, Kaszler N, Domonkos I, Valkai I, Szőllősi R, Ferenc G, Ayaydin F, Fehér A, Gémes K. Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots. Plant Cell Tiss Org, 2020, 140(2): 327-339.

[2] Binte Mostafiz S, Wagiran A. Efficient callus induction and regeneration in selected indica rice. Agron J, 2018 8 5 77

[3] Bradaï F, Pliego-Alfaro F, Sánchez-Romero C. Long-term somatic embryogenesis in olive (Olea europaea L.): influence on regeneration capability and quality of regenerated plants. Sci Horitic, 2016, 199: 23-31.

[4] Chen J, Zhang Y, Li T, Wang P, Wang G, Shi J. Study on origin and development of somatic embryos of Liriodendron hybrids. J Nanjing For Univ (Nat Sci Edition), 2012, 36(1): 16-20. (in Chinese)

[5] Chen TT, Wang PK, Zhang JJ, Shi JS, Cheng TL, Chen JH. Effects of combined ABA and ZT treatment on somatic embryogenesis and development of liriodendron sino-americanum. Sci SilvaeSinicae, 2019, 55(3): 64-71. (in Chinese)

[6] Cong JM, Shen HL, Li YH, Zhang P, Yang L, Huang J. Physiological and biochemical status of different-types of explants in somatic embryogenesis of Fraxinus mandshurica. J South China Agri Univ, 2012, 33(1): 48-52. (in Chinese)

[7] Corredoira E, Ballester A, Ibarra M, Vieitez AM. Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna× E. maideniitrees. Tree physiol, 2015, 35(6): 678-690.

[8] Corredoira E, Valladares S, Martínez MT, Vieitez AM, San José MC. Somatic embryogenesis in Alnus glutinosa (L.) Gaertn. Trees, 2013, 27(6): 1597-1608.

[9] Du N, Pijut PM. Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons. Sci Horitic, 2008, 118(1): 74-79.

[10] Fehér A. Callus, dedifferentiation, totipotency, somatic embryogenesis: what these terms mean in the era of molecular plant biology?. Front plant sci, 2019, 10: 536.

[11] Guan Y, Li SG, Fan XF, Su ZH. Application of somatic embryogenesis in woody plants. Front plant sci, 2016, 7: 938.

[12] He CS, Chen XF, Huang H, Xu L. Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS Geneti, 2012 8 8 e1002911

[13] Horstman A, Bemer M, Boutilier K. A transcriptional view on somatic embryogenesis. Regeneration, 2017, 4: 201-216.

[14] Ibrahim K, Alromaihi KB, Elmeer KMS. The combined role of sucrose with IBA and NAA in rooting of date palm somatic embryos cv. Khanaizi Plant Tiss Cult Biotech, 2009, 19(2): 127-132.

[15] Jiang RC, Peng FR, Tan PP. Somatic embryogenesis and the physiological and biochemical characteristics in Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour. China Forestry Science and Technology, 2014, 1: 7. (in Chinese)

[16] Khan T, Reddy VS, Leelavathi S. High-frequency regeneration via somatic embryogenesis of an elite recalcitrant cotton genotype (Gossypium hirsutum L.) and efficient Agrobacterium-mediated transformation. Plant Cell Tiss Org, 2010, 101(3): 323-330.

[17] Konan KE, Durand-Gasselin T, Kouadio YJ, Flori A, Rival A, Duval Y, Pannetier C. In vitro conservation of oil palm somatic embryos for 20 years on a hormone-free culture medium: characteristics of the embryogenic cultures, derived plantlets and adult palms. Plant cell rep, 2010, 29(1): 1-13.

[18] Lelu-Walter MA, Gautier F, Eliášová K, 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 Tiss Org, 2018, 132(1): 137-155.

[19] 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.

[20] Li M, Wang S, Feng D. The advance of plant somatic embryogenesis and development. Chin Agric Sci Bull, 2011, 27(03): 237-241. (in Chinese)

[21] Liu CP, Yang L, Shen HL. Proteomic analysis of immature Fraxinus mandshurica cotyledon tissues during somatic embryogenesis: effects of explant browning on somatic embryogenesis. j mol sci, 2015, 16(6): 13692-13713.

[22] Liu Y, Dong Q, Kita D, Huang JB, Liu G, Wu X, Zhu X, Cheung AY, Wu HM, Tao LZ. RopGEF1 plays a critical role in polarauxin transport in early development. Plant Phys, 2017, 175(1): 157-171.

[23] Lu D, Wei W, Zhou W, McGuigan LD, Ji FY, Li X, Xing Y, Zhang Q, Fang KF, Cao QQ, Qin L. Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut (Castaneamollissima Blume). Plant Cell Tiss Org, 2017, 130(3): 601-616.

[24] Machakova I, Zazimalova E, George EF. George EF, Hall MA, De Klerk GJ. Plant growth regulators I introductions auxins their analogous and inhibitors. Plant propagation by tissue culture, 2008 3 Dordrecht: Springer 175 204

[25] Márquez-Martín B, Sesmero R, Quesada MA, Pliego-Alfaro F, Sánchez-Romero C. Water relations in culture media influence maturation of avocado somatic embryos. J plant phys, 2011, 168(17): 2028-2034.

[26] Ming NG, BinteMostafiz S, Johon NS, Zulkifli A, Saliha N, Wagiran A. Combination of plant growth regulators, maltose, and partial desiccation treatment enhance somatic embryogenesis in selected malaysian rice cultivar. Plants, 2019 8 6 144

[27] Natarajan N, Sundararajan S, Ramalingam S, Chellakan PS. Efficient and rapid in-vitro plantlet regeneration via somatic embryogenesis in ornamental bananas (Musa spp.). Biologia, 2020, 75(2): 317-326.

[28] Orłowska A, Kępczyńska E. Oxidative status in Medicago truncatula Gaertn. non-embryogenic and embryogenic tissues with particular reference to somatic embryogenesis. Plant Cell Tiss Org, 2020, 140(1): 35-48.

[29] Ozudogru EA, Capuana M, Kaya E, Panis B, Lambardi M. Cryopreservation of Fraxinus excelsior L. embryogenic callus by one-step freezing and slow cooling techniques. Cryo Lett, 2010, 31(1): 63-75.

[30] Park YS. Fenning T. Conifer somatic embryogenesis and multi-varietal forestry. Challenges and Opportunities for the World's Forests in the twenty-first Century, 2014, Dordrecht: Springer 425 439

[31] Pasternak TP, Prinsen E, Ayaydin F, Miskolczi P, Potters G, Asard H, Onckelen HAV, Dudits D, Fehér A. The role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiol, 2002, 129(4): 1807-1819.

[32] Perez-Perez Y, El-Tantawy AA, Solis MT, Risueno MC, Testillano PS. Stress-induced microspore embryogenesis requires endogenous auxin synthesis and polar transport in barley. Front plant sci, 2019, 10: 1200.

[33] Shin U, Chandra R, Kang H. In vitro and ex vitro propagations of astilboidestabularis (Hemsl.) Engl. as a rare and endangered species. J Hort, 2019, 6(260): 2376-354.

[34] Solórzano-Cascante P, Sánchez-ChiangJiménez NVM. Explant type, culture system, 6-benzyladenine, meta-topolin and encapsulation affect indirect somatic embryogenesis and regeneration in Carica papaya L. Front plant sci, 2018, 9: 1769.

[35] Su YH, Zhao XY, Liu YB, Zhang CL, O’Neill SD, Zhang XS. Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis. Plant J, 2009, 59(3): 448-460.

[36] Sun GJ, Kong DM, Zhang LJ, Shen HL. Effect of collection time and source tree of zygotic embryo explants on somatic embryogenesis of Fraxinus mandshurica. J Northeast For Univ, 2010, 38(1): 28-30. (in Chinese)

[37] Us-Camas R, Rivera-Solís G, Duarte-Aké F, De-la-Pena C. In vitro culture: an epigenetic challenge for plants. Plant Cell Tiss Org, 2014, 118(2): 187-201.

[38] Wang YY, Chen FJ, Wang YB, Li XL, Liang HW. Efficient somatic embryogenesis and plant regeneration from immature embryos of TapisciasinensisOliv., an endemic and endangered species in China. Hort Sci, 2014, 49(12): 1558-1562.

[39] Wójcikowska B, Jaskóła K, Gąsiorek P, Meus M, Nowak K, Gaj MD. LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis. Planta, 2013, 238(3): 425-440.

[40] Yang L, Bian L, Shen HL, Li YH. Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshuricaRupr.). Plant Cell Tiss Org, 2013, 115(2): 115-125.

[41] Yang L, Liu HN, Zhang DY, Wei C, Shen HL. Effect of plant growth regulators and osmoticums on somatic embryogenesis of Fraxinus mandshuricarupr. Bull Botan Res, 2017, 37(5): 682-689. (in Chinese)

[42] Zavattieri MA, Frederico AM, Lima M, Sabino R, Arnholdt-Schmitt B. Induction of somatic embryogenesis as an example of stress-related plant reactions. Electro J Biotech, 2010, 13(1): 12-13.

[43] Zhang LJ, Zhao LM, Lu XJ, Shen HL. Callus Induction and Somatic Embryogenesis from Zygotic Cotyledons and Hypocotyls of Fraxinus mandshurica Rupr. Mol Plant Breed, 2015, 13(7): 1645-1652.

[44] Zhang Y, Shen HL. Control of synchronization for Plant somatic embryogenesis. Plant Phys Commun, 2007, 43(3): 583-587. (in Chinese)