In Vitro Cellular & Developmental Biology - Plant

, Volume 54, Issue 6, pp 689–700 | Cite as

In vitro growth profile and comparative leaf anatomy of the C3–C4 intermediate plant Mollugo nudicaulis Lam.

  • Meena Barupal
  • Vinod KatariaEmail author
  • Narpat S. Shekhawat
Plant Tissue Culture


Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C3–C4 intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel™. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel™ and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots.


Micropropagation Tropical C3–C4 intermediate Phytagel Gibberellic acid Aeroponics 



Authors are thankful to the University Grant Commission (UGC), New Delhi, for providing Special Assistance Program (SAP) in the form of Centre of Advanced Study (CAS) to the Department of Botany, Jai Narain Vyas University, and Jodhpur.


  1. Bhandari MM (1990) Flora of the Indian desert. MPS Repros, Jodhpur, pp 159–160Google Scholar
  2. Chaâbani G, Tabart J, Kevers C, Dommes J, Khan MI, Zaoui S, Chebchoub L, Lachaâl M, Karray BN (2015) Effects of 2,4-Dichlorophenoxyacetic acid combined to 6-Benzylaminopurine on callus induction, total phenolic and ascorbic acid production, and antioxidant activities in leaf tissue cultures of Crataegus azarolus L. var. Aronia. Acta Physiol Plant 37:16CrossRefGoogle Scholar
  3. Christin PA, Osborne CP (2014) The evolutionary ecology of C4 plants. New Phytol 204:765–781CrossRefGoogle Scholar
  4. Christin PA, Sage TL, Edwards EJ, Ogburn RM, Khoshravesh R, Sage RF (2011) Complex evolutionary transitions and the significance of C3–C4 intermediate forms of photosynthesis in Molluginaceae. Evolution 65:643–660CrossRefGoogle Scholar
  5. Compton ME, Mize CW (1999) Statistical considerations for in vitro research: I- birth of an idea to collecting data. In Vitro Cell Dev Biol-Plant 35:115–121CrossRefGoogle Scholar
  6. Duncan DB (1955) Multiple range and multiple F test. Biometrics 11:1–42CrossRefGoogle Scholar
  7. Gomez KA, Gomez AA (1984) Single factor experiments. In: Statistical analysis procedure of agricultural research. John Wiley and sons, New York, pp 7–29Google Scholar
  8. Gowik U, Westhoff P (2011) The path from C3 to C4 photosynthesis. Plant Physiol 155:56–63CrossRefGoogle Scholar
  9. Hazarika BN (2006) Morpho-physiological disorders in in vitro culture of plants. Sci Hortic 108:105–120CrossRefGoogle Scholar
  10. Huang LC, Kohashi C, Vangundy R, Murashige T (1995) Effects of common components on hardness of culture media prepared with gelrite™. In Vitro Cell Dev Biol-Plant 31:84–89CrossRefGoogle Scholar
  11. Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult 33:105–119CrossRefGoogle Scholar
  12. Huh YS, Lee JK, Nam SY (2017) Effect of plant growth regulators and antioxidants on in vitro plant regeneration and callus induction from leaf explants of purple passion fruit (Passiflora edulis Sims). J Plant Biotech 44:335–342CrossRefGoogle Scholar
  13. Ignacimuthu S, Ayyanar M, Sankarasivaraman K (2008) Ethnobotanical study of medicinal plants used by Paliyar tribals in Theni district of Tamil Nadu, India. Fitoterapia 79:562–568CrossRefGoogle Scholar
  14. Jauhar PP (2006) Modern biotechnology as an integral supplement to conventional plant breeding: the prospects and challenges. Crop Sci 46:1841–1859CrossRefGoogle Scholar
  15. Kaur RP (2015) Photoautotrophic micropropagation an emerging new vista in micropropagation- a review. Agri Rev 36:198–207CrossRefGoogle Scholar
  16. Kennedy RA, Eastburn JL, Jensen KG (1980) C3-C4 photosynthesis in the genus Mollugo: structure, physiology and evolution of intermediate characteristics. Am J Bot 67:1207–1217CrossRefGoogle Scholar
  17. Khan EU, Wang FXJ, Fan QJ, Huang XS, Zhang GN, Shi J, Liu JH (2009) Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars. Sci Hortic 120:70–76CrossRefGoogle Scholar
  18. Kumar A, Aggarwal D, Gupta P, Reddy MS (2010) Factors affecting in vitro propagation and field establishment of Chlorophytum borivilianum. Biol Plant 54:601–606CrossRefGoogle Scholar
  19. Ludwig-Müller J (2000) Indole-3-butyric acid in plant growth and development. Plant Growth Regul 32:219–230CrossRefGoogle Scholar
  20. Mehandru P, Shekhawat NS, Rai MK, Kataria V, Gehlot HS (2014) Evaluation of aeroponics for clonal propagation of Caralluma edulis, Leptadenia reticulata and Tylophora indica–three threatened medicinal asclepiads. Physiol Mol Biol Plants 20:365–373CrossRefGoogle Scholar
  21. Mendiburu F (2016) Agricolae: statistical procedures for agricultural research. R package version 1:2–4 Google Scholar
  22. Mohlakola EM, Cheng C, Lin Y, Guo R, Min KT, Chen Y, Lai Z (2017) Effects of 2, 4-Dichlorophenoxy acetic acid and light on growth of Gerbera (Gerbera jamesonii cv. Daxueju) callus. J Agr Sci Tech 18:385–393Google Scholar
  23. Morini S, D'onofrio C, Bellocchi G, Fisichella M (2000) Effect of 2, 4-D and light quality on callus production and differentiation from in vitro cultured quince leaves. Plant Cell Tissue Organ Cult 63:47–55CrossRefGoogle Scholar
  24. Mosaleeyanon K, Chan-Um S, Kirmane C (2004) Enhanced growth and photosynthesis of rain tree (Samanea saman Merr.) plantlets in vitro under a CO2-enriched condition with decreased sucrose concentrations in the medium. Sci Hortic 103:51–63CrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Nagesh KS, Shanthamma C (2011) Micropropagation and antioxidant activity of Mollugo nudicaulis lam. J Med Plants Res 5:895–902Google Scholar
  27. Nicholson SE (2011) Dryland climatology. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  28. 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 52:10–19CrossRefGoogle Scholar
  29. Rameshkumar A, Sivasudha T (2012) In vitro antioxidant and antibacterial activity of aqueous and methanolic extract of Mollugo nudicaulis lam. Leaves. Asian Pac J Trop Biomed 2:895–900CrossRefGoogle Scholar
  30. Rajamanikandan S, Sindhu T, Durgapriya D, Sophia D, Ragavendran P, Gopalakrishnan VK (2012) Protective effect of Mollugo nudicaulis lam. On acute liver injury induced by perchloroethylene in experimental rats. Asian Pac J Trop Med 5:862–867CrossRefGoogle Scholar
  31. Rathore MS, Rathore MS, Shekhawat NS (2013) Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn.—an endangered plant. Environ Exper Bot 86:86–93CrossRefGoogle Scholar
  32. Rawsthorne S (1992) C3–C4 intermediate photosynthesis: linking physiology to gene expression. Plant J 2:267–274CrossRefGoogle Scholar
  33. Rufai S, Hanafi MM, Rafii MY, Mohidin H, Omar SRS (2016) Growth and development of Moringa (Moringa oleifera L.) stem cuttings as affected by diameter magnitude, growth media and indole-3-butyric acid. Ann For Res 59:209–218CrossRefGoogle Scholar
  34. Sage RF, Christin PA, Edwards EJ (2011) The C4 lineages of planet earth. J Exp Bot 62:3155–3169CrossRefGoogle Scholar
  35. Sage RF, Khoshravesh R, Sage TL (2014) From proto-Kranz to C4 Kranz: building the bridge to C4 photosynthesis. J Exp Bot 65:3341–3356CrossRefGoogle Scholar
  36. Schlüter U, Weber AP (2016) The road to C4 photosynthesis: evolution of a complex trait via intermediary states. Plant Cell Physiol 57:881–889CrossRefGoogle Scholar
  37. Sharma U, Kataria V, Shekhawat NS (2017) In vitro propagation, ex vitro rooting and leaf micromorphology of Bauhinia racemosa lam.: a leguminous tree with medicinal values. Physiol Mol Biol Plants 23:969–977CrossRefGoogle Scholar
  38. Stata M, Sage TL, Rennie TD, Khoshravesh R, Sultmanis S, Khaikin Y, Ludwig M, Sage RF (2014) Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants. Plant Cell Environ 37:2587–2600CrossRefGoogle Scholar
  39. Stobbe H, Schmitt U, Eckstein D, Dujesiefken D (2002) Developmental stages and fine structure of surface callus formed after debarking of living lime trees (Tilia sp.). Ann Bot 89:773–782CrossRefGoogle Scholar
  40. Sukhorukov AP, Kushunina M (2016) Taxonomic revision and distribution of herbaceous Paramollugo (Molluginaceae) in the eastern hemisphere. PhytoKeys 73:93–116CrossRefGoogle Scholar
  41. Terashima I, Hanba YT, Tholen D, Niinemets Ü (2011) Leaf functional anatomy in relation to photosynthesis. Plant Physiol 155:108–116CrossRefGoogle Scholar
  42. The International Plant Names Index (2012) names: 363731–1. Cited on 15 Jan 2018
  43. Thole V, Rawsthorne S (2003) Efficient regeneration systems for two closely related Moricandia species possessing a C3 or C3–C4 intermediate photosynthetic character. Plant Cell Rep 21:707–712PubMedGoogle Scholar
  44. Tholen D, Boom C, Zhu XG (2012) Opinion: prospects for improving photosynthesis by altering leaf anatomy. Plant Sci 197:92–101CrossRefGoogle Scholar
  45. Thulin M, Moore AJ, El-Seedi H, Larsson A, Christin PA, Edwards EJ (2016) Phylogeny and generic delimitation in Molluginaceae, new pigment data in Caryophyllales, and the new family Corbichoniaceae. Taxon 65:775–793CrossRefGoogle Scholar
  46. Vincent MA (2003) Molluginaceae Rafinesque: Flora of North America Editorial Committee. Flora of North America, volume 4: Magnoliophyta: Caryophyllidae, Part 1, Oxford Univ. Press, Oxford, UKGoogle Scholar
  47. Voznesenskaya EV, Koteyeva NK, Edwards GE, Ocampo G (2010) Revealing diversity in structural and biochemical forms of C4 photosynthesis and a C3–C4 intermediate in genus Portulaca L. (Portulacaceae). J Exp Bot 61:3647–3662CrossRefGoogle Scholar
  48. Xiao Y, Niu G, Kozai T (2011) Development and application of photoautotrophic micropropagation plant system. Plant Cell Tissue Organ Cult 105:149–158CrossRefGoogle Scholar
  49. Yancheva SD, Golubowicz S, Fisher E, Lev-Yadun S, Flaishman MA (2003) Auxin type and timing of application determine the activation of the developmental program during in vitro organogenesis in apple. Plant Sci 165:299–309CrossRefGoogle Scholar
  50. Ziv M, Altman A (2003) TISSUE CULTURE: General Principles. In: Thomas B, Murphy DJ, Murray BG (eds) Encyclopedia of applied plant sciences. Elsevier, Oxford, pp 1341–1353CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Meena Barupal
    • 1
  • Vinod Kataria
    • 1
    Email author
  • Narpat S. Shekhawat
    • 1
  1. 1.Biotechnology Unit, Department of Botany (UGC-Centre of Advanced Study)Jai Narain Vyas UniversityJodhpurIndia

Personalised recommendations