Skip to main content
SpringerLink
Log in

An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop

  • Research Article
  • Published:
Physiology and Molecular Biology of Plants Aims and scope Submit manuscript

Abstract

Momordica dioica Roxb. ex Willd., is a perennial and dioecious (2n = 28) plant of family Cucurbitaceae. Conventional methods of propagation through seeds, stem cuttings and rhizomatous/tuberous roots are inadequate for its mass cultivation as a vegetable crop. This paper reports an improved and efficient micropropagation method for wild female M. dioica using nodal explants. Shoot amplification was achieved using subculturing of in vitro raised shoots on MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid (IAA). The maximum number of shoots (45.30 ± 3.83) with an average length 6.52 ± 0.89 cm were differentiated on MS medium containing 0.5 mg L−1 BAP, 0.1 mg L−1 IAA and additives (50 mg L−1 ascorbic acid, 25 mg L−1 each of adenine sulphate, citric acid and l-arginine). The cloned shoots were rooted ex vitro. Each shoot treated with 250 mg L−1 IBA for 5 min produced 12.3 ± 1.33 with a mean length 5.4 ± 0.73 cm. More than 85% (46 plants) of ex vitro rooted plantlets were successfully hardened in a greenhouse with normal growth characteristics. In order to evaluate the genetic stability of micropropagated plants, the two PCR-based techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used. The amplification patterns of the micropropagated and mother plant were monomorphic thus depicting genetic stability of the micropropagation system. This protocol could be effectively employed for the mass multiplication of wild female M. dioica, a popular summer vegetable crop.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

BAP:

6-benzylaminopurine

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

ISSR:

Inter Simple Sequence Repeat

Kin:

Kinetin

MS:

Murashige and Skoog (1962)

NOA:

Naphthoxyacetic acid

PCR:

Polymerase chain reaction

PFD:

Photon flux density

PGRs:

Plant growth regulators

RAPD:

Random Amplified Polymorphic DNA

RH:

Relative humidity

References

  • Agarwal M, Kamal R (2004) In vitro clonal propagation of Momordica charantia. Indian J Biotechnol 3:426–430

    CAS  Google Scholar 

  • Agarwal T, Gupta AK, Patel AK, Shekhawat NS (2015) Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers. Plant Cell Tissue Organ Cult 120:313–323

    Article  CAS  Google Scholar 

  • Ali M, Okubo H, Fujii T, Fujieda K (1991) Techniques for propagation and breeding of kakrol (Momordica dioica Roxb.). Sci Hortic 47:335–343

    Article  Google Scholar 

  • Asthana P, Jaiswal VS, Jaiswal U (2011) Micropropagation of Sapindus trifoliatus L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis. Acta Physiol Plant 33:1821–1829

    Article  CAS  Google Scholar 

  • Bairu MW, Aremu AO, Staden JV (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173

    Article  CAS  Google Scholar 

  • Bandyopadhyay S, Mukherjee SK (2009) Wild edible plants of Koch Bihar district. Nat Prod Radiance 8:64–72

    Google Scholar 

  • Bawara B, Dixit M, Chauhan NS, Dixit VK, Saraf DK (2010) Phyto-pharmacology of Momordica dioica Roxb. ex. Willd: a review. Int J Phytomed 2:1–9

    CAS  Google Scholar 

  • Bennici A, Anzidei M, Vendramin GG (2004) Genetic stability and uniformity of Foeniculum vulgare Mill. regenerated plants through organogenesis and somatic embryogenesis. Plant Sci 166:221–227

    Article  CAS  Google Scholar 

  • Bharathi LK, Naik G, Singh HS, Dora DK (2007) Spine gourd. In: Peter KV (ed) Underutilized and underexploited horticultural crops. New India Publishing, New Delhi, pp 289–295

    Google Scholar 

  • Bohra P, Waman AA, Sathyanarayana BN, Umesha K (2016) Concurrent ex vitro rooting and hardening in Ney Poovan Banana (Musa AB): effect of carbon sources and their concentrations. Erwerbs-Obstbau 58:193–198

    Article  Google Scholar 

  • Catalogue of Life (2016) http://www.catalogueoflife.org/col/details/species/id/f2baec8 be6488a79329413ec8d0db88f. Accessed 16 July 2016

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Ganasan K, Huyop F (2010) In vitro regeneration of Citrullus lanatus cv. Round Dragon. J Biol Sci 10:131–137

    Article  CAS  Google Scholar 

  • Hatzilazarou SP, Syros TD, Yupsanis TA, Bosabalidis AM, Economou AS (2006) Peroxidases, ligin and anatomy during in vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots. J Plant Physiol 163:827–836

    Article  CAS  PubMed  Google Scholar 

  • Hoque A, Islam R, Joarder OI (1995) In vitro plantlets differentiation in kakrol (Momordica dioica Roxb.). Plant Tissue Cult 5:119–124

    Google Scholar 

  • Hoque A, Islam R, Arima S (2000) High frequency plant regeneration from cotyledon-derived callus of Momordica dioica (Roxb.) Willd. Phytomorphology 50:267–272

    Google Scholar 

  • Kiba T, Krapp A (2016) Plant nitrogen acquisition under low availability: regulation of uptake and root architecture. Plant Cell Physiol 57:707–714

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kitto S (2015) Micropropagation of Spigelia marilandica L. In Vitro Cell Dev Biol Plant 51:205–213

    Article  Google Scholar 

  • Lodha D, Patel AK, Shekhawat NS (2015) A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub. Physiol Mol Biol Plant 21:407–415

    Article  CAS  Google Scholar 

  • Luo L, Li Z, Zhang Y, Huang R (1998) Triterpenes and steroidal compounds from Momordica dioica. Acta Pharm Sin 33:839–842

    CAS  Google Scholar 

  • Masondo NA, Aremu AO, Finnie JF, Van Staden J (2015) Growth and phytochemical levels in micropropagated Eucomis autumnalis subspecies autumnalis using different gelling agents, explant source, and plant growth regulators. In Vitro Cell Dev Biol Plant 51:102–110

    Article  CAS  Google Scholar 

  • Mendel Soares DM, Sattler MC, Silva Ferreira MF, Praça-Fontes MM (2016) Assessment of genetic stability in three generations of in vitro propagated Jatropha curcas L. plantlets using ISSR markers. Trop Plant Biol 9:229–238

    Article  Google Scholar 

  • Mohanty JN, Nayak S, Jha S, Joshi RK (2016) A sequence tagged site (STS) marker encoding Copia-like retrotransposable element is associated with male specific sex expression in Momordica dioica Roxb. Sci Hortic 201:265–270

    Article  CAS  Google Scholar 

  • Mondal A, Ghosh GP, Zuberi MI (2006) Phylogenetic relationship in different kakrol collections of Bangladesh. Pak J Biol Sci 9:1516–1524

    Article  Google Scholar 

  • Moon JG, Choo BK, Hs D, Kwon TH, Yang MS, Ryu JH (2000) Effects of growth regulators on plant regeneration from the cotyledon explant in oriental melon (Cucumis melo L.). Korean J Plant Tissue Cult 27:1–6

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nabi SA, Rashid MM, Al-Amin M, Rasul MG (2002) Organogenesis in teasle gourd (Momordica dioica Roxb.). Plant Tissue Cult 12:173–180

    Google Scholar 

  • Nagarani G, Abirami A, Siddhuraju P (2014) Food prospects and nutraceutical attributes of Momordica species: a potential tropical bioresources—a review. Food Sci Hum Wellness 3:117–126

    Article  Google Scholar 

  • Ntui VO, Thirukkumaran G, Lioka S, Mii M (2009) Efficient plant regeneration via organogenesis in “Egos” melon (Colocynthis citrullus L.). Sci Hortic 119:397–402

    Article  CAS  Google Scholar 

  • Patel MG, Ishnava KB (2015) Momordica dioica Roxb. (spine gourd): multiple shoot induction from nodal cultures and its antidiabetic activity. J Med Plant Stud 3:82–88

    Google Scholar 

  • Patel AK, Lodha D, Ram K, Shekhawat S, Shekhawat NS (2016) Evaluation of physiochemical factors affecting high frequency plant regeneration of Blyttia spiralis (Forssk.) D.V. Field & J.R.I. Wood [Synonym: Pentatropis spiralis (Forssk.) Decne.], a threatened climber of medicinal values. In Vitro Cell Dev Biol Plant 52:10–19

    Article  Google Scholar 

  • Patil CG, Baratakke RC, Sandigwad AM (2012) Development of a RAPD based SCAR marker for sex identification in Momordica dioica Roxb. Isr J Plant Sci 60:457–465

    Google Scholar 

  • Perveen S, Anis M, Aref IM (2013) Lipid peroxidation, H2O2 content and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions. Biol Plant 57:417–424

    Article  CAS  Google Scholar 

  • Phulwaria M, Ram K, Gahlot P, Shekhawat NS (2011) Micropropagation of Salvadora persica—a tree of arid horticulture and forestry. New For 42:317–327

    Article  Google Scholar 

  • Phulwaria M, Patel AK, Rathore JS, Ram K, Shekhawat NS (2014) An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers. Acta Physiol Plant 36:1115–1122

    Article  CAS  Google Scholar 

  • Rai GK, Singh M, Rai NP, Bhardwaj DR, Kumar S (2012a) In vitro propagation of spine gourd (Momordica dioica Roxb.) and assessment of genetic fidelity of micropropagated plants using RAPD analysis. Physiol Mol Biol Plant 18:273–280

    Article  CAS  Google Scholar 

  • Rai MK, Phulwaria M, Harish, Gupta AK, Shekhawat NS, Jaiswal U (2012b) Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers. Plant Cell Tissue Organ Cult 111:259–264

    Article  CAS  Google Scholar 

  • Rakh MS, Chaudhari SR (2010) Evaluation of analgesic activity of Momordica dioica roxb. Willd fruit pulp. Int J Pharm Sci Res 1:53–56

    Google Scholar 

  • Ram D, Banerjee MK, Pandey S, Srivastava U (2001) Collection and evaluation of Kartoli (Momordica dioica Roxb. Ex. Willd.). Indian J Plant Genet Resour 14:114–116

    Google Scholar 

  • Rana S, Das AB (2016) Assessment of genetic diversity in 48 landraces of Momordica dioica Roxb. ex Willd. from Odisha, India using RAPD and ISSR markers. Nucleus 59:107–114

    Article  Google Scholar 

  • Rao PS, Mohan GK (2016) In vitro alpha-amylase inhibition and in vivo antioxidant potential of Momordica dioica seeds in streptozotocin-induced oxidative stress in diabetic rats. Saud J Biol Sci. doi:10.1016/j.sjbs.2016.01.010

    Google Scholar 

  • Rasul MG, Hiramatsu M, Okubo H (2007) Genetic relatedness (diversity) and cultivar identification by randomly amplified polymorphic DNA (RAPD) markers in teasle gourd (Momordica dioica Roxb.). Sci Hortic 111:271–279

    Article  CAS  Google Scholar 

  • Rathore MS, Yadav P, Mastan SG, Prakash ChR, Singh A, Agarwal PK (2014a) Evaluation of genetic homogeneity in tissue culture regenerates of Jatropha curcas L. using flow cytometer and DNA-based molecular markers. Appl Biochem Biotechnol 172:298–310

    Article  CAS  PubMed  Google Scholar 

  • Rathore NS, Rai MK, Phulwaria M, Rathore N, Shekhawat NS (2014b) Genetic stability in micropropagated Cleome gynandra revealedby SCoT analysis. Acta Physiol Plant 36:555–559

    Article  CAS  Google Scholar 

  • Saha S, Sengupta C, Ghosh P (2015) Encapsulation, short-term storage, conservation and molecular analysis to assess genetic stability in alginate-encapsulated microshoots of Ocimum kilimandscharicum Guerke. Plant Cell Tissue Organ Cult 120:519–530

    Article  CAS  Google Scholar 

  • Salvi J, Katewa SS (2015) Nutritional composition of Momordica dioica fruits: as a wild vegetable. J Food Pharm Sci 3:18–22

    Google Scholar 

  • Sebastiani MS, Ficcadenti N (2016) In vitro plant regeneration from cotyledonary explants of Cucumis melo L. var. cantalupensis and genetic stability evaluation using RAPD analysis. Plant Cell Tissue Organ Cult 124:69–79

    Article  CAS  Google Scholar 

  • Shekhawat MS, Shekhawat NS, Harish, Ram K, Phulwaria M, Gupta AK (2011) High frequency plantlet regeneration from nodal segment culture of female Momordica dioica (Roxb.). J Crop Sci Biotechnol 14:133–137

    Article  Google Scholar 

  • Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin–cytokinin interactions in the control of shoot branching. Plant Mol Biol 69:429–435

    Article  CAS  PubMed  Google Scholar 

  • Singh R, Kashyap SP, Kumari N, Singh M (2016) Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers. Physiol Mol Biol Plant 22:381–389

    Article  CAS  Google Scholar 

  • Sinha SK (1997) Global change scenario: current and future with reference to land cover changes and sustainable agriculture—South and South-East Asian context. Curr Sci 72:846–854

    Google Scholar 

  • Su YH, Zhang XS (2014) The hormonal control of regeneration in plants. Curr Top Dev Biol 108:35–69

    Article  CAS  PubMed  Google Scholar 

  • Su YH, Liu YB, Zhang XS (2011) Auxin–cytokinin interaction regulates meristem development. Mol Plant 4:616–625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sukumar G, Divyalakshmi D, Mithula S, Rupachandra S (2016) In vitro anticancer effects of methanolic seed coat extract of Momordica dioica against human carcinoma cell lines. Int J ChemTech Res 9:284–289

    CAS  Google Scholar 

  • Talukdar SN, Hossain MN (2014) Phytochemical, phytotherapeutical and pharmacological study of Momordica dioica. Evid Based Complement Altern Med. doi:10.1155/2014/806082

    Google Scholar 

  • The Plant List (2016) http://www.theplantlist.org/tpl1.1/record/tro-9200027. Accessed 15 July 2016

  • Thiruvengadam M, Jayabalan N (2001) In vitro shoot multiplication and field establishment of kakrol (Momordica dioica Roxb.). J Indian Bot Soc 80:31–33

    Google Scholar 

  • Thiruvengadam M, Rekha KT, Jayabalan N (2006) An efficient in vitro propagation of Momordica dioica Roxb. ex. Willd. Philipp Agric Sci 89:165–171

    Google Scholar 

  • Thiruvengadam M, Praveen N, Ye-Ji Lee, Ill-Min Chung (2012) An efficient regeneration from petiole derived callus of male and female spine gourd (Momordica dioica Roxb. ex. Willd.). J Med Plant Res 6:3330–3337

    CAS  Google Scholar 

  • Thiruvengadam M, Rekha KT, Jayabalan N, Praveen N, Kim EH (2013) Effect of exogenous polyamines enhances somatic embryogenesis via suspension cultures of spine gourd (Momordica dioica Roxb. ex. Willd.). Aust J Crop Sci 7:446–453

    CAS  Google Scholar 

  • Thiruvengadam M, Rekha K, Ill-Min Chung (2016) Induction of hairy roots by Agrobacterium rhizogenes mediated transformation of spine gourd (Momordica dioica Roxb. ex. willd) for the assessment of phenolic compounds and biological activities. Sci Hortic 198:132–141

    Article  CAS  Google Scholar 

  • Velivela Y, Narra M, Ellendula R, Kota S, Abbagani S (2016) Establishment of in vitro regeneration from petiole explants and assessment of clonal fidelity by ISSR markers in Luffa acutangula L. Roxb. J Appl Biol Biotechnol 4:41–45

    Google Scholar 

  • Venkatachalam L, Sreedhar RV, Bhagyalakshmi N (2007) Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers. Plant Growth Regul 51:193–205

    Article  CAS  Google Scholar 

  • Vinoth A, Ravindhran R (2016) Efficient plant regeneration of watermelon (Citrullus lanatus Thunb.) via somatic embryogenesis and assessment of genetic fidelity using ISSR markers. In Vitro Cell Dev Biol Plant 52:107–115

    Article  CAS  Google Scholar 

  • Xiao Y, Kozai T (2004) Commercial application of a photoautotrophic micropropagation system using large vessels with forced ventilation: system configuration, plantlet growth and production cost. Hortic Sci 39:1387–1391

    Google Scholar 

Download references

Acknowledgements

SKC thank the University Grant Commission (UGC) for providing financial support in the form of start-up grant [F.-30-16/2014(BSR)]. Also, authors (SKC and AKP) are grateful to UGC for providing the Special Assistance Program (SAP) in the form of Centre of Advanced Study (CAS) to the Department of Botany, Jai Narain Vyas University, Jodhpur, Rajasthan. The authors expressed their gratitude to Dr. Rajshree Ranawat, Assistant Professor of English, Jai Narain Vyas University, Jodhpur for improving English language of this paper.

Author information

Authors and Affiliations

  1. Biotechnology Unit, Department of Botany, UGC – Centre of Advanced Study (CAS), Jai Narain Vyas University, Jodhpur, 342 001, India

    Sumitra Kumari Choudhary, Ashok Kumar Patel & Narpat S. Shekhawat

  2. Department of Botany, Mohanlal Sukhadia University, Udaipur, 313 001, India

    Harish

  3. Department of Botany, University of Delhi, Delhi, 110 007, India

    Smita Shekhawat

Authors
  1. Sumitra Kumari Choudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashok Kumar Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Harish
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Smita Shekhawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Narpat S. Shekhawat
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Narpat S. Shekhawat.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Choudhary, S.K., Patel, A.K., Harish et al. An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop. Physiol Mol Biol Plants 23, 713–722 (2017). https://doi.org/10.1007/s12298-017-0441-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12298-017-0441-z

Keywords

Search

Navigation