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Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm.

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Abstract

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.

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Abbreviations

BAP:

6-Benzylaminopurine

B5:

Gamborg et al.

2,4-D:

2,4-Dichlorophenoxyacetic acid

EFC:

Embryo forming capacity

GA3 :

Gibberellic acid

IZE:

Immature zygotic embryo

MS:

Murashige and Skoog

NAA:

α-Naphthaleneacetic acid

N6:

Chu et al.

PGRs:

Plant growth regulators

SE:

Somatic embryo

SH:

Schenk and Hildebrandt

SSE:

Secondary somatic embryo

ZE:

Zygotic embryo

References

  • Anita W, Agnieszka G, Anna PB, Norikazu T, Sabina Z, Rafal W, Zbigniew P, Stefan M, Marcin F (2012) Identification of genes upregulated during somatic embryogenesis of cucumber. Plant Physiol Biochem 50:54–64

    Article  Google Scholar 

  • Chu CC, Wang CC, Sun CS (1978) The N6 medium and its application to anther culture of cereal crops. Proceedings of symposium plant tissue culture. Science Press, Peking, pp 45–50

    Google Scholar 

  • Ćosić T, Vinterhalter B, Vinterhalter D, Mitić N, Cingel A, Savić J, Bohanec B, Ninković S (2013) In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes). In Vitro Cell Dev Biol Plant 49:294–303

    Article  Google Scholar 

  • Cui J, Chen J, Henny RJ (2009) Regeneration of Aeschynanthus radicans via direct somatic embryogenesis and analysis of regenerants with flow cytometry. In Vitro Cell Dev Biol Plant 45:34–43

    Article  CAS  Google Scholar 

  • Das S, Ray S, Dey S, Dasgupta S (2001) Optimisation of sucrose, inorganic nitrogen and abscisic acid levels for Santalum album L. somatic embryo production in suspension culture. Process Biochem 37:51–56

    Article  CAS  Google Scholar 

  • Deng DW, Lauren DR, Cooney JM, Jensen DJ, Wurms KV, Upritchard JE, Cannon RD, Wang MZ, Li MZ (2008) Antifungal saponins from Paris polyphylla Smith. Planta Med 74:1397–1402

    Article  CAS  PubMed  Google Scholar 

  • Devkota KP, Tareq HKM, Ranjit R, Lannang AM, Samreen, Choudhary MI (2007) Tyrosinase inhibitory and antileishmanial constituents from the rhizomes of Paris polyphylla. Nat Prod Res 21:321–327

    Article  CAS  Google Scholar 

  • Dolezel J, Bartos J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99–110

    Article  CAS  PubMed  Google Scholar 

  • Elkonin AL, Pakhomova NV (2000) Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. Plant Cell Tiss Organ Cult 61:115–123

    Article  Google Scholar 

  • Feher A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tiss Organ Cult 74:201–228

    Article  CAS  Google Scholar 

  • Fisichella M, Silvi E, Morini S (2000) Regeneration of somatic embryos and roots from quince leaves cultured on media with different macroelement composition. Plant Cell Tiss Organ Cult 63:101–107

    Article  CAS  Google Scholar 

  • Fu YL, Yu ZY, Tang XM, Zhao Y, Yuan XL, Wang S, Ma BP, Cong YW (2008) Pennogenin glycosides with a spirostanol structure are strong platelet agonists: structural requirement for activity and mode of platelet agonist synergism. J Thromb Haemost 6:524–533

    Article  CAS  PubMed  Google Scholar 

  • Gaj MD (2001) Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana. Plant Cell Tiss Organ Cult 64:39–46

    Article  Google Scholar 

  • Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 43:27–47

    Article  CAS  Google Scholar 

  • Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues. Science 220:1049–1051

    Article  CAS  PubMed  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • George EF (1993) Plant propagation by tissue culture, Part 1: In theory, 2nd edn. Exegetics Ltd., Edington

    Google Scholar 

  • Gingas VM, Lineberger RD (1989) Asexual embryogenesis and plant regeneration in Quercus. Plant Cell Tiss Organ Cult 17:191–203

    Article  Google Scholar 

  • Hai-xia Y, Ding C, Yang R, Zhang L, Zhou Y, Li Y (2011) Karyomorphology of some taxa of Paris (Melanthiaceae) from Sichuan province, China. Caryologia 64:288–296

    Article  Google Scholar 

  • Hirai G, Kasai N, Harada T (1997) Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis. Plant Cell Tiss Organ Cult 48:175–180

    Article  Google Scholar 

  • Jimenez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regul 47:91–110

    Article  CAS  Google Scholar 

  • Koh WL, Loh CS (2000) Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid-cycling Brassica napus. Plant Cell Rep 19:1177–1183

    Article  CAS  Google Scholar 

  • Kumar S (2002) The medicinal plants of North-East India. Scientific Publishers, Jodhpur

    Google Scholar 

  • Laxmi DV, Sharma HC, Kirti PB, Mohan ML (1999) Somatic embryogenesis in mango (Mangifera indica L.) cv. Amrapali. Curr Sci 77:1355–1358

    CAS  Google Scholar 

  • Lee MS, Yuet-Wa JC, Kong SK, Yu B, Eng-Choon VO, Nai-Ching HW, Chung-Wai TM, Fung KP (2005) Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft. Cancer Biol Ther 4:1248–1254

    Article  CAS  PubMed  Google Scholar 

  • Li YC (1986) Vegetative propagation of Paris polyphylla var. yunnanensis. Acta Bot Yunnan 8:429–435 (in Chinese)

    Google Scholar 

  • Li H (1998) The genus Paris (Trilliaceae), vol 7. Science Press, Beijing, pp 182–184

    Google Scholar 

  • Lodhi M, Reisch B (1995) Nuclear DNA content of Vitis species, cultivars, and other genera of the Vitaceae. Theor Appl Genet 90:11–16

    Article  CAS  PubMed  Google Scholar 

  • Loureiro J, Rodriguez E, Doležel J, Santos C (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100:875–888

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Man S, Gao W, Zhang Y, Yan L, Ma C, Liuc C, Huang L (2009) Antitumor and antimetastatic activities of Rhizoma Paridis saponins. Steroids 74:1051–1056

    Article  CAS  PubMed  Google Scholar 

  • Mao AA, Hynniewta TM, Sanjappa M (2009) Plant wealth of northeast India with reference to ethnobotany. Indian J Tradit Know 8:96–103

    Google Scholar 

  • Merkle SA (1997) Somatic embryogenesis in ornamentals. In: Geneve RL, Preece JE, Merkle SA (eds) Biotechnology of ornamental plants. CAB International, Wallingford, pp 13–33

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nair RR, Gupta SD (2006) High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.). Plant Cell Rep 24:699–707

    Article  CAS  PubMed  Google Scholar 

  • Pathi KM, Tula S, Tuteja N (2013) High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium-mediated genetic transformation of tobacco. Plant Signal Behav 8(6):e24354

    Article  PubMed Central  PubMed  Google Scholar 

  • Pavlovic S, Vinterhalter B, Zdravkovic-Korac S, Vinterhalter D, Zdravkovic J, Cvikic D, Mitic N (2013) Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis). Plant Cell Tiss Organ Cult 113:397–406

    Article  CAS  Google Scholar 

  • Pence VC, Soukup VG (1986) Plant regeneration from trillium spp. in vitro. HortScience 21:1211–1213

    Google Scholar 

  • Pence VC, Soukup VG (1993) Factors affecting the initiation of mini-rhizomes from Trillium erectum and T. grandiflorum tissues in vitro. Plant Cell Tiss Organ Cult 35:229–235

    Article  Google Scholar 

  • Plata E, Viéitez AM (1990) In vitro regeneration of Camellia reticulata by somatic embryogenesis. J Hort Sci 65:707–714

    Google Scholar 

  • Pullman GS, Johnson S, Peter G, Cairney J, Xu N (2003) Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination and gene expression. Plant Cell Rep 21:747–758

    CAS  PubMed  Google Scholar 

  • Raemakers CJJM, Jacobsen E, Visser RGF (1995) Secondary somatic embryogenesis and applications in plant breeding. Euphytica 81:93–107

    Article  Google Scholar 

  • Rai MK, Akhtar N, Jaiswal VS (2007) Somatic embryogenesis and plant regeneration in Psidium guajava L. cv. Banarasi local. Sci Hortic 113:129–133

    Article  CAS  Google Scholar 

  • Rey HY, Faloci M, Medina R, Dolce N, Engelmann F, Mroginski L (2013) Cryopreservation of Arachis pintoi (Leguminosae) Somatic Embryos. Cryoletters 34:571–582

    CAS  PubMed  Google Scholar 

  • Rout GR, Debata BK, Das P (1991) Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora. Plant Cell Tiss Organ Cult 27:65–69

    Article  CAS  Google Scholar 

  • Samson NP, Campa C, Le Gal L, Noirot M, Thomas G, Lokeswari TS, de Kochko A (2006) Effect of primary culture medium composition on high frequency somatic embryogenesis in different Coffea species. Plant Cell Tiss Organ Cult 86:37–45

    Article  Google Scholar 

  • Schenk RU, Hildebrandt A (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204

    Article  CAS  Google Scholar 

  • Sharp WR, Sondahl MR, Caldas LS, Maraffa SB (1980) The physiology of in vitro asexual embryogenesis. Hortl Rev 2:268–310

    CAS  Google Scholar 

  • Shohael AM, Ali MB, Yu KW, Hahn EJ, Paek KY (2013) Effects of Murashige and Skoog medium strength on germination and secondary metabolites production of Eleutherococcus senticosus’s somatic embryos in bioreactor. Int J Biosci 3:155–163

    Article  CAS  Google Scholar 

  • Sun J, Liu BR, Hu WJ, Yu LX, Qian XP (2007) In vitro anticancer activity of aqueous extracts and ethanol extracts of fifteen traditional Chinese medicines on human digestive tumor cell lines. Phytother Res 21:1102–1104

    Article  CAS  PubMed  Google Scholar 

  • Taniguchi T, Kurita M, Itahana N, Kondo T (2004) Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtuse Sieb. et Zucc.). Plant Cell Rep 23:26–31

    Article  CAS  PubMed  Google Scholar 

  • Tao L, Yang Y, Wang Q, You X (2012) Callose deposition is required for somatic embryogenesis in plasmolyzed Eleutherococcus senticosus zygotic Embryos. Int J Mol Sci 13:14115–14126

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vasic D, Alibert G, Skoric D (2001) Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader). Plant Cell Rep 20:121–125

    Article  CAS  Google Scholar 

  • Wang GX, Han J, Zhao LW, Jiang DX, Liu YT, Liu XL (2010) Antihelmintic activity of steroidal saponins from Paris polyphylla. Phytomedicine 17:1102–1105

    Article  CAS  PubMed  Google Scholar 

  • Wetherall DF, Dougall DK (1976) Sources of nitrogen supporting growth and embryogenesis in cultured wild carrot tissue. Plant Physiol 37:97–103

    Article  Google Scholar 

  • Wu SS, Gao WY, Duan HQ, Jia W (2004) Advances in studies on chemical constituents and pharmacological activities of Rhizoma paridis. Chin Trad Herb Drugs 35:344–347

    CAS  Google Scholar 

  • Wu HC, du Toit ES, Reinhardt CF (2007) A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues. Plant Cell Tiss Organ Cult 89:217–224

    Article  Google Scholar 

  • Yang L, Li Y, Shen H (2012) Somatic embryogenesis and plant regeneration from immature zygotic embryo cultures of mountain ash (Sorbus pohuashanensis). Plant Cell Tiss Organ Cult 109:547–556

    Article  CAS  Google Scholar 

  • You XL, Yi JS, Choi YE (2006) Cellular changes and callose accumulation of zygotic embryos of Eleutherococcus senticosus by plasmolyzing pretreatment result in high frequency single cell-derived somatic embryogenesis. Protoplasma 227:105–112

    Article  Google Scholar 

  • You CR, Fan TJ, Gong XQ, Bian FH, Liang LK, Qu FN (2011) A high-frequency cyclic secondary somatic embryogenesis system for Cyclamen persicum Mill. Plant Cell Tiss Organ Cult 107:233–242

    Article  Google Scholar 

  • Zdravković-Korać S, Ćalić-Dragosavac D, Uzelac B, Janošević D, Budimir S, Vinterhalter B, Vinterhalter D (2008) Secondary somatic embryogenesis versus caulogenesis from somatic embryos of Aesculus carnea Hayne.: developmental stage impact. Plant Cell Tiss Organ Cult 94:225–231

    Article  Google Scholar 

  • Zhao J, Cui J, Liu J, Liao F, Henny RJ, Chen J (2012) Direct somatic embryogenesis from leaf and petiole explants of Spathiphyllum ‘Supreme’and analysis of regenerants using flow cytometry. Plant Cell Tiss Organ Cult 110:239–249

    Article  Google Scholar 

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Acknowledgments

The authors acknowledge Prof. N. Venugopal, Head, Department of Botany, North-Eastern Hill University (NEHU), Shillong for permission to use microtome and staff of Sophisticated Analytical Instrumentation Facility (SAIF) for technical assistance in SEM. The authors would like to thank Prof. A. Chatterjee and D. Tripathi from Department of Biotechnology and Bioinformatics, NEHU for access to the FACS instrumentation facility. SR is thankful to University Grant Commission, India for awarding her Rajiv Gandhi National Fellowship for SC/ST candidate.

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  1. Plant Biotechnology Laboratory, Department of Botany, Centre for Advanced Studies, North-Eastern Hill University, Shillong, 793022, India

    Shiveirou Raomai, Suman Kumaria & Pramod Tandon

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  1. Shiveirou Raomai
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  2. Suman Kumaria
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  3. Pramod Tandon
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Correspondence to Suman Kumaria.

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Raomai, S., Kumaria, S. & Tandon, P. Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm.. Plant Cell Tiss Organ Cult 118, 445–455 (2014). https://doi.org/10.1007/s11240-014-0496-2

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