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In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.)

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Abstract

In clinical treatment, Pleione bulbocodioides (Franch.) Rolfe (PB) has been widely used as an anti-tumor drug. However, low productivity impeded its extensive application. The lack of endosperm in the seeds of PB led to a low natural germination rate, causing inefficient production and long reproductive cycle. Improving production and retaining clonal fidelity are the challenges in its efficient micropropagation. This study is to optimize medium and evaluate clonal fidelity on induction and differentiation of Protocorm-like Body (PLB) from PB. The effects of plant growth regulators (PGR) and natural additives on the formation and proliferation of protocorms from seeds and the induction and proliferation of PLBs were investigated. The ultra-structural were performed via a scanning electron microscope (SEM) and transmission electron microscope (TEM) and clonal fidelity assessment was conducted using histochemical assays, DNA electrophoresis analysis and Inter-simple sequence repeat (ISSR) molecular marker analysis. The optimum medium for inducing protocorm was that the Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-D, 2.0 mg/L KT (Kinetin), 2.0 mg/L AgNO3, and 50 mL/L coconut water in the orthogonal experiments and validation experiments, and for inducing PLBs was that MS medium with 0.3 mg/L indole-3-butyric acid (IBA), 0.3 mg/L KT, 100 mg/L potato extract, and 1 g/L activated charcoal. The SEM and TEM showed the ultra-structural of PB had no change from protocorm to PLBs, and the DNA electrophoresis showed the total DNA did not change in different stages of PB by induction and differentiation and ISSR analysis revealed no DNA polymorphisms included bulb, PLBS, and the differentiated tissue of PLBs. In conclusion, the DNA hereditary character and ultra-structural of PB was retained using our optimum medium induction and differentiation of PLBs from PB, which will be useful for the efficient micropropagation popularization for PB.

Key message

The present work reports the DNA hereditary stability character and optimum medium for induction and differentiation of PLBs from Pleione bulbocodioides (Franch.) Rolfe (PB), which will be useful for the efficient micropropagation popularization for PB.

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Abbreviations

PB :

Pleione bulbocodioides (Franch.) Rolfe

MS:

Murashige and Skoog (1962) media

PLB:

Protocorm-like body

KT:

Kinetin

2,4-D:

2,4-Dichlorophenoxyacetic acid

NAA:

1-Naphthaleneacetic acid

IBA:

Indole-3-butyric acid

6-BA:

Amino purine

TDZ:

Thidiazuron

PE:

Potato extract

BE:

Banana extract

CW:

Coconut water

SEM:

Scanning electron microscope

TEM:

Transmission electron microscope

ISSR:

Inter-simple sequence repeat

References

  • Anjum S, Zia M, Chaudhary MF (2006) Investigations of different strategies for high frequency regeneration of Dendrobium malones ‘Victory’. Afr J Biotechnol 5:1738–1743

    CAS  Google Scholar 

  • Babbar SB, Singh DK (2016) Protocols for in vitro mass multiplication and analysis of medicinally important phenolics of a Salep Orchid, Satyrium nepalense D. Don (“Salam Mishri”). Methods Mol Biol 1391:1–11

    CAS  PubMed  Google Scholar 

  • Cardoso JC, Zanello CA, Chen J-T (2020) An overview of Orchid protocorm-like bodies: mass propagation, biotechnology, molecular aspects, and breeding. Int J Mol Sci 21(3):985

    CAS  PubMed Central  Google Scholar 

  • Chen JT, Chang WC (2000) Plant regeneration via embryo and shoot bud formation from flower-stalk explants of Oncidium ‘Sweet Sugar’. Plant Cell, Tissue Organ Cult 62:95–100

    CAS  Google Scholar 

  • Chen JT, Chang WC (2001) Effects of auxins and cytokinins on direct somatic embryogenesis on leaf explants of Oncidium ‘Gower Ramsey’. Plant Growth Regul 34:229–232

    CAS  Google Scholar 

  • Chen JT, Chang WC (2004) Induction of repetitive embryogenesis from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu. Vitro Cell Dev Biol 40:290–293

    CAS  Google Scholar 

  • Chen ND, You T, Li J, Bai LT, Hao JW, Xu XY (2016) A comparative study of three tissue-cultured Dendrobium species and their wild correspondences by headspace gas chromatography-mass spectrometry combined with chemometric methods. J Food Drug Anal 24:839–847

    CAS  PubMed  Google Scholar 

  • Cheng W, Li H, Zhou F, Zhu B, Yu J, Ding Z (2020) Cryopreservation of Pleione bulbocodioides (Franch.) Rolfe protocorm-like bodies by vitrification. Acta Physiol Plant 42:1–11

    Google Scholar 

  • Chevala NP, Chevala NT, Dhanakodi K, Nadendla RR, Nagarathna CK (2016) In vitro accumulation of polyphenols in tea callus derived from anther. Pharmacogn Mag 12:400–406

    Google Scholar 

  • Chugh S, Guha S, Rao IU (2009) Micropropagation of orchids: a review on the potential of different explants. Sci Hortic 122(4):507–520

    CAS  Google Scholar 

  • Fang SC, Chen JC, Wei MJ (2016) Protocorms and protocorm-like bodys are molecularly distinct from zygotic embryonic tissues in Phalaenopsis aphrodite. Plant Physiol 171:2682–2700

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fengyi L, Lijuan F, Haijing F, Yujia L, Ling W (2021) An efficient in vitro regeneration system via callus from hypocotyl and root of Iris laevigata. Res Sq. https://doi.org/10.21203/rs.3.rs-46031/v1

    Article  Google Scholar 

  • Gantait S, Bustam S, Sinniah UR (2012) Alginate-encapsulation, short-term storage and plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.(Orchidaceae). Plant Growth Regul 68(2):303–311

    CAS  Google Scholar 

  • Ghosh A, Hossain MM, Sharma M (2014) Mass propagation of Cymbidium giganteum Wall. ex Lindl. using in vitro seedlings. Indian J Exp Biol 52:905–911

    PubMed  Google Scholar 

  • Gnasekaran P, Mahmood M, Subramaniam S (2016) Ultrastructure study of Vanda Kasem’s Delight orchid’s protocorm-like body. Horticultura Brasileira 34(3):333–339

    Google Scholar 

  • Hong PI, Chen JT, Chang WC (2008) Plant regeneration via protocorm-like body formation and shoot multiplication from seed-derived callus of maudiae type slipper orchid. Acta Physiol Plant 30:755–759

    CAS  Google Scholar 

  • Huang X, Lu Y (2016) Callus induction and plant regeneration of Oncidium ‘Gower Ramsey’ by means of protocorm-like body culture. Guihaia 36(9):1082–1086

    Google Scholar 

  • Huang YW, Tsai YJ, Cheng TC, Chen JJ, Chen FC (2014) Physical wounding and ethylene stimulated embryogenic stem cell proliferation and plantlet regeneration in protocorm-like bodys of Phalaenopsis orchids. Genet Mol Res 13:9543–9557

    CAS  PubMed  Google Scholar 

  • Huang H, Zi XM, Lin H, Gao JY (2018) Host-specificity of symbiotic mycorrhizal fungi for enhancing seed germination, protocorm formation and seedling development of over-collected medicinal orchid, Dendrobium devonianum. J Microbiol 56:42–48

    PubMed  Google Scholar 

  • Jaime A, da Silva Teixeira (2013) Cryopreservation of hybrid Cymbidium protocorm-like bodys by encapsulation-dehydration and vitrification. In Vitro Cell Dev Biol Plant 49(6):690–698

    Google Scholar 

  • Kalimuthu K, Senthilkumar R, Vijayakumar S (2007) In vitro micropropagation of orchid, Oncidium sp (Dancing Dolls). Afr J Biotechnol 6:1171–1174

    CAS  Google Scholar 

  • Kosir P, Skof S, Luthar Z (2004) Direct shoot regenerated from nodes of Phalaenopsis orchids. Acta Agric Slovenica 83:233–242

    CAS  Google Scholar 

  • Lee YI, Lee N (2003) Plant regeneration from protocorm-derived callus of Cypripedium formosanum. In Vitro Cell Dev Biol Plant 39:475–479

    Google Scholar 

  • Li Q, Zhang S, Wang J (2015) Transcriptomic and proteomic analyses of embryogenic tissues in Picea balfouriana treated with 6-benzylaminopurine. Physiol Plant 154:95–113

    CAS  PubMed  Google Scholar 

  • Lin YH, Chang C, Chang WC (2000) Plant regeneration from callus culture of a Paphiopedilum hybrid. Plant Cell, Tissue Organ Cult 62:21–25

    CAS  Google Scholar 

  • Liu XQ, Guo YQ, Gao WY, Zhang TJ, Yan LL (2008) Two new phenanthrofurans from Pleione bulbocodioides. J Asian Nat Prod Res 10:453–457

    CAS  PubMed  Google Scholar 

  • Liu XQ, Yuan QY, Guo YQ (2009) Two new stilbenoids from Pleione bulbocodioides. J Asian Nat Prod Res 11:116–121

    CAS  PubMed  Google Scholar 

  • Liu SS, Chen J, Li SC, Zeng X, Meng ZX, Guo SX (2015) Comparative transcriptome analysis of genes involved in GA-GID1-DELLA regulatory module in symbiotic and asymbiotic seed germination of Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae). Int J Mol Sci 16:30190–30203

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liu F, Zhu WL, Huang MZ, Ding ZT, Tang YQ (2017) Inducement of philippine ground orchid (Spathoglottis plicata) protocorm like bodies and plant regeneration. Genom Appl Biol 36(5):2082–2087

    Google Scholar 

  • Malabadi RB, Mulgund GS, Nataraja K (2004) Efficient regeneration of Vanda coerulea, an endangered orchid using thidiazuron. Plant Cell, Tissue Organ Cult 76(3):289–293

    CAS  Google Scholar 

  • Medina RD, Flachsland EA, Gonzalez AM, Terada G, Faloci MM, Mroginski LA (2009) In vitro tuberization and plant regeneration from multimodal segment culture of Habenaria ractescens Lindl., an Argentinean wetland orchid. Plant Cell, Tissue Organ Cult 97:91–101

    Google Scholar 

  • Mohanty P, Nongkling P, Das MC, Kumaria S, Tandon P (2013) Short-term storage of alginate-encapsulated protocorm-like bodys of Dendrobium nobile Lindl.: an endangered medicinal orchid from North-east India. 3 Biotech 3(3):235–239

    PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:495–497

    Google Scholar 

  • Nie J, Liu LF, Ren HH, Liu ZJ, Wang JX (2016) Preliminary study on multiplication and regeneration system of protocorm-like bodys in Phalaenopsi. J Shanxi Univ 39(2):318–324

    CAS  Google Scholar 

  • Park HY, Kang KW, Kim DH, Sivanesan I (2018) In vitro propagation of Cymbidium goeringii Reichenbach fil. through direct adventitious shoot regeneration. Physiol Mol Biol Plants 24:307–313

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rafter M, Yokoya K, Schofield EJ, Zettler LW, Sarasan V (2016) Non-specific symbiotic germination of Cynorkis purpurea (Thouars) Kraezl., a habitat-specific terrestrial orchid from the Central Highlands of Madagascar. Mycorrhiza 26:541–552

    CAS  PubMed  Google Scholar 

  • Rahmah S, Ahmad Mubbarakh S, Soo Ping K, Subramaniam S (2015) Effects of droplet-vitrification cryopreservation based on physiological and antioxidant enzyme activities of Brassidium shooting star orchid. Sci World J 2015:961793. https://doi.org/10.1155/2015/961793

  • Ren HH, Wang JX, Nie J (2017) Protocorm proliferation and regeneration of Dendrobium officinale. Chin Tradit Herb Drugs 48(19):4057–4061

    Google Scholar 

  • Rout G, Mohapatra A, Jain SM (2006) Tissue culture of ornamental pot plant: a critical review on present scenario and future prospects. Biotechnol Adv 24(6):531–560

    CAS  PubMed  Google Scholar 

  • Roy J, Banerjee N (2002) Optimization of in vitro seed germination, protocorm growth and seedling proliferation of Vanda Tessellata (Roxb.) Hook. Ex G. Don. Phytomorphology 52(2–3):167–178

    Google Scholar 

  • Roy J, Naha S, Majumdar M, Banerjee N (2007) Direct and callus mediated protocorm-like body induction from shoot-tips of Dendrobium chrysotoxum Lindl. Plant Cell, Tissue Organ Cult 90:31–33

    CAS  Google Scholar 

  • Shriram V, Nanekar V, Kumar V, Kavi Kishor PB (2014) In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl. Indian J Exp Biol 52:1112–1121

    PubMed  Google Scholar 

  • Tamari F, Hinkley CS, Ramprashad N (2013) A comparison of DNA extraction methods using Petunia hybrida tissues. J Biomol Tech 24:113–118. https://doi.org/10.7171/jbt.13-2403-001

  • Teixeira da Silva JA, Zeng S, Galdiano RF, Jr., Dobranszki J, Cardoso JC, Vendrame WA (2014) In vitro conservation of Dendrobium germplasm. Plant cell rep 33:1413–1423. https://doi.org/10.1007/s00299-014-1631-6

  • Vergara Martínez VM, Estrada-Soto SE, Arellano-García JJ, Rivera-Leyva JC, Castillo-España P, Flores AF, Cardoso-Taketa AT, Perea-Arango I (2018) Methyl jasmonate and salicylic acid enhanced the production of ursolic and oleanolic acid in callus cultures of Lepechinia caulescens. Pharmacogn Mag 13(Suppl 4):S886–S889

    PubMed  PubMed Central  Google Scholar 

  • Wang Y, Guan SH, Feng RH, Zhang JX, Li Q, Chen XH, Bi KS, Guo DA (2013) Elution-extrusion counter-current chromatography separation of two new benzyl ester glucosides and three other high-polarity compounds from the tubers of Pleione bulbocodioides. Phytochem Anal 24:671–676

    CAS  PubMed  Google Scholar 

  • Wang ZQ, Meng JL, Cui YY (2017) Tissue culture of soot apices for induction of protocorm-like bodys of Dendrobium nobile ‘Huoniao’. J Zhejiang For Sci Technol 37(2):48–54

    Google Scholar 

  • Xu XB, Ma XY, Lei HH, Song HM, Ying QC, Xu MJ, Liu SB, Wang HZ (2015) Proteomic analysis reveals the mechanisms of Mycena dendrobii promoting transplantation survival and growth of tissue culture seedlings of Dendrobium officinale. J Appl Microbiol 118:1444–1455

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We appreciate the great help/technical support/experimental support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.

Funding

This research was supported by Zhejiang Traditional Chinese Medicine Administration (CN) (Project No. 2019ZQ009).

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Authors and Affiliations

  1. College of Medical Technology, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China

    Fangmei Zhou, Weipeng Huang, Wen Cheng, Jingjing Lu, Bingqi Zhu & Zhishan Ding

  2. Puer Kunhong Biotechnology Company, Group C of Chamagu Town A, Simao District, Puer, 665000, Yunnan, People’s Republic of China

    Jie Yu

  3. The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou, 310006, Zhejiang, China

    Qianxing Jin

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  1. Fangmei Zhou
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Contributions

FZ contributed the conception, design, drafting part of the manuscript, and final revision. WH contributed to design and drafting the manuscript. WC and JL analyzed and interpreted the data. JY and BZ designed and critical revision of the manuscript. ZD and QJ contributed to supervise and revise the manuscript. All authors read and approved the final manuscript.

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Correspondence to Qianxing Jin.

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Communicated by Ming-Tsair Chan.

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Supplementary Information

Below is the link to the electronic supplementary material.

11240_2021_2033_MOESM1_ESM.tif

Electronic supplementary material 1 (TIF 1003 kb) Fig S1. The reproductive process of seed to plantlets. The seeds are directly scattered on the medium 50 days (a) and then become seedlings for 70 days (b), plantlets for 88 days (c), and plantlets for 112 days (d).

11240_2021_2033_MOESM2_ESM.tif

Electronic supplementary material 2 (TIF 1086 kb) Fig S2. Proliferation of PLBs on MS with the addition of 1.0mg/L 6-BA+0.5mg/L KT+0.2mg/L NAA+0.5g/L AC culture for (a) 1 day. (b) 10 days (c) 30 days.

11240_2021_2033_MOESM3_ESM.tif

Electronic supplementary material 3 (TIF 614 kb) Fig S3. (a) Differentiation of PLBs on MS with the addition of 0.2 mg/L NAA, 2.0–2.5 mg/L 6-BA, and 1.0 g/L activated charcoal after 3 weeks of culture. (b) PLB-derived plantlets ready for potting after 4 weeks of culture. (c) Clusters of PLBs after 5 weeks of culture. (d) PLBs developed into shoot buds after 7 weeks of culture.

11240_2021_2033_MOESM4_ESM.tif

Electronic supplementary material 4 (TIF 758 kb) Fig S4. The transplanting process of the seedlings put into rooting medium, with long leaves and long seedlings, culturing for (A) 7 days, (B) 14 days (C) 28 days, (D) longer seedling were taken out and transplanted to ex-bottle culturing (E) 30 days

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Zhou, F., Huang, W., Cheng, W. et al. In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.). Plant Cell Tiss Organ Cult 145, 625–639 (2021). https://doi.org/10.1007/s11240-021-02033-2

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