Skip to main content

Advertisement

SpringerLink
Log in

Field Evaluation and Genetic Stability Assessment of Regenerated Plants Produced Via Direct Shoot Organogenesis from Leaf Explant of an Endangered ‘Asthma Plant’ (Tylophora indica) Along with Their In Vitro Conservation

  • Research Article
  • Published:
National Academy Science Letters Aims and scope Submit manuscript

Abstract

Adventitious shoot buds were induced directly without intervening callus phase from the cut edge of the ‘aged leaves’ explants as well as via nodular meristemoids from the cut edge of the ‘young leaves’ of Tylophora indica an endangered medicinal climber of the family Asclepiadaceae. Shoot primordia developed directly from the cut edge of the ‘aged leaf’ explant without any callus phase within 26–30 days and maximum 18.6 ± 0.36 shoot per explant were induced on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 6-benzyl-aminopurine (BAP). Whereas maximum 25.2 ± 0.57 shoots per explant were produced from ‘young leaf’ explant via nodular meristemoids on 2.0 mg/l BAP and 0.2 mg/l indole-3-acetic acid fortified medium within 40–45 days. Multiplied shoots were successfully rooted with maximum 8.3 ± 0.36 root per shoot with 15.5 ± 0.44 cm length on half strength MS medium supplemented with 0.2 mg/l indole-3-butyric acid. Finally, the rooted plants were hardened and transferred to soil with a 93.3 % success rate. After 18 months of field transfer, a 69.0 % of regenerated plants produce flowers and 28.5 % produce fruits. Seeds of these fruits show 78.3 % viability more or less similar with in vivo grown plants with 71.7 % viability on in vitro germination, which ensures the fertility of the regenerants. These regenerated plants were conserved in vitro for up to 21 months without sub-culturing in presence of osmoticums (Mannitol and Sorbitol) and best results found on MS medium supplemented with 3 % sucrose and 2 % mannitol, where 53.3 ± 3.33 % plants survive up to 21 months and 86.7 ± 3.33 % of these survived plants could turn to normal plants after a re-growth for 2 month which showed no obvious difference in morphology. Further, clonal fidelity assessment of the micropropagated plants was carried out using cytological studies as well as using randomly amplified polymorphic DNA (RAPD) markers analysis. All the micropropagated plants have shown normal diploid 2n = 22 chromosomes, same as that of the mother plant. A result of RAPD markers analysis indicates all the regenerants are true-to-type and there were no somaclonal variations among these regenerants.

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

References

  1. Gupta M, Mukhtar HM, Ahmad S (2010) Phyto-pharmacological and plant tissue culture overview of Tylophora indica (burm f.) Merill. J Pharm Sci Res 2:401–411

    Google Scholar 

  2. Gurav S, Devprakash, Senthilkumar GP, Tembare R, Mani T (2011) Tylophora indica: a review on its ethnobotany, phytochemical and pharmacological profile. Asian J Biochem Pharm Res 1:405–414

    Google Scholar 

  3. Faisal M, Ahmad N, Anis M (2007) An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant. Plant Biotechnol Rep 1:155–161

    Article  Google Scholar 

  4. Islam AS (2009) Utilization of indigenous medicinal plants and their conservation in Bangladesh. In: Akerele O, Heywood V, Synge H (eds) The conservation of medicinal plants. Cambridge University Press, New York, p 323

    Google Scholar 

  5. Engelmann F (1991) In vitro conservation of tropical plant germplasm—a review. Euphytica 57:227–243

    Article  Google Scholar 

  6. Bunn E, Turner SR, Panaia M, Dixon KW (2007) The contribution of in vitro technology and cryogenic storage to conservation of indigenous plants. Aust J Bot 55:345–355

    Article  Google Scholar 

  7. Pence VC (2011) Evaluating costs for the in vitro propagation and preservation of endangered plants. In Vitro Cell Dev Biol Plant 47:176–187

    Article  Google Scholar 

  8. Fay MF (1992) Conservation of rare and endangered plants using in vitro methods. In Vitro Cell Dev Biol Plant 28:1–4

    Google Scholar 

  9. Sarasan V, Cripps R, Ramsay MM, Atherton C, McMichen M, Prendergast G, Rowntree JK (2006) Conservation in vitro of threatened plants—progress in the past decade. In Vitro Cell Dev Biol Plant 42:206–214

    Article  Google Scholar 

  10. Al-Mahmood HJ, Shatnawi MA, Shibli RA, Makhadmeh IM, Abubaker SM, Shadiadeh AN (2012) Clonal propagation and medium-term conservation of Capparis spinosa: a medicinal plant. J Med Plants Res 6:3826–3836

    Google Scholar 

  11. Negash A, Krens F, Schaart J, Visser B (2001) In vitro conservation of enset under slow-growth conditions. Plant Cell Tissue Organ Cult 66:107–111

    Article  Google Scholar 

  12. Montalvo-Peniche MC, Iglesias-Andreu LG, Mijangos-Corte′s JO, Nahuat-Dzib SL, Barahona-Pe′rez F, Canto-Flick A, Santana-Buzzy N (2007) In vitro germplasm conservation of habanero pepper (Capsicum chinense jacq.). Hortic Sci 42:1247–1252

    Google Scholar 

  13. Holobiuc I, Mitoi M, Blindu R, Helepciuc F (2010) The establishment of an in vitro gene bank in Dianthus spiculifolius schur. and D. glacialis ssp. gelidus (schott nym. et kotschy) tutin: II. medium term cultures characterization in minimal growth conditions. Romanian Biotechnol Lett 15:5111–5119

    Google Scholar 

  14. Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 47:5–16

    Article  Google Scholar 

  15. Sharma N, Chandel KPS (1992) Effect of ascorbic acid on axillary shoot induction in Tylophora indica (burm. f.) merrill. Plant Cell Tissue Organ Cult 29:109–113

    Article  Google Scholar 

  16. Faisal M, Anis M (2003) Rapid mass propagation of Tylophora indica via leaf callus culture. Plant Cell Tissue Organ Cult 75:125–129

    Article  Google Scholar 

  17. Faisal M, Anis M (2005) An efficient in vitro method for mass propagation of Tylophora indica. Biol Plant 49:257–260

    Article  Google Scholar 

  18. Chaudhuri KN, Ghosh B, Jha S (2004) The root: a potential new source of competent cells for high-frequency regeneration in Tylophora indica. Plant Cell Rep 22:731–740

    Article  Google Scholar 

  19. Thomas TD, Philip B (2005) Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of a medicinal climber, Tylophora indica (burm. f.) merrill. In Vitro Cell Dev Biol Plant 41:124–128

    Article  Google Scholar 

  20. Thomas TD (2009) Isolation, callus formation and plantlet regeneration from mesophyll protoplasts of Tylophora indica (burm. f.) merrill: an important medicinal plant. In Vitro Cell Dev Biol Plant 45:591–598

    Article  Google Scholar 

  21. Jayanthi M, Mandal PK (2001) Plant regeneration through somatic embryogenesis and RAPD analysis of regenerated plants in Tylophora indica (burm. F.) merrill. In Vitro Cell Dev Biol Plant 37:576–580

    Article  Google Scholar 

  22. Sahai A, Shahzad A, Anis M (2010) High frequency plant production via shoot organogenesis and somatic embryogenesis from callus in Tylophora indica, an endangered plant species. Turk J Bot 34:11–20

    Google Scholar 

  23. Cheruvathur MK, Abraham J, Thomas TD (2013) Plant regeneration through callus organogenesis and true-to-type conformity of plants by RAPD analysis in Desmodium gangeticum (Linn.) DC. Appl Biochem Biotechnol 169:1799–1810

  24. 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  Google Scholar 

  25. Mohanty S, Parida R, Singh S, Joshi RK, Subudhi E, Nayak S (2011) Biochemical and molecular profiling of micropropagated and conventionally grown Kaempferia galangal. Plant Cell Tiss Organ Cult 106:39–46

    Article  Google Scholar 

  26. Roy AR, Sajeev S, Pattanayak A, Deka BC (2012) TDZ induced micropropagation in Cymbidium giganteum Wall. Ex Lindl. and assessment of genetic variation in the regenerated plants. Plant Growth Regul 68:435–445

    Article  Google Scholar 

  27. Kumar A, Prakash K, Sinha RK, Kumar N (2013) In vitro plant propagation of Catharanthus roseus and assessment of genetic fidelity of micropropagated plants by RAPD marker assay. Appl Biochem Biotechnol 169:894–900

    Article  Google Scholar 

  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:437–497

    Article  Google Scholar 

  29. Stewart CN Jr, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Bio Techniques 14:748–749

    Google Scholar 

  30. Neumann KH, Kumar A, Imani J (2009) Plant cell and tissue culture-a tool in biotechnology, principles and practice. Springer-Verlag, Heidelberg, pp 139–143

    Book  Google Scholar 

  31. Sarwar M, Skirvin RM (1997) Effect of thidiazuron and 6-benzylaminopurine on adventitious shoot regeneration from leaves of three strains of ‘McIntosh’ apple (Malus × domestica borkh) in vitro. Sci Hortic 68:95–100

    Article  Google Scholar 

  32. Northmore JA, Zhou V, Chuong SDX (2012) Multiple shoot induction and plant regeneration of the single-cell C4 species Bienertia sinuspersici. Plant Cell Tissue Organ Cult 108:101–109

    Article  Google Scholar 

  33. Ma GH, He CX, Ren H, Zhang QM, Li SJ, Zhang XH, Eric B (2010) Direct somatic embryogenesis and shoot organogenesis from leaf explants of Primulina tabacum. Biol Plant 54:361–365

    Article  Google Scholar 

  34. Gaspar TH, Kevers C, Penel C, Greppin H, Reid DM, Thorpe TA (1996) Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Dev Biol Plant 32:272–289

    Article  Google Scholar 

  35. Gaspar TH, Kevers C, Faivre-rampant O, Crevecoeur M, Penel C, Greppin H, Dommes J (2003) Changing concepts in plant hormone action. In Vitro Cell Dev Biol Plant 39:85–106

    Article  Google Scholar 

  36. Melara MV, Arias AMG (2009) Effect of BAP and IAA on shoot regeneration in cotyledonary explants of Costa Rican melon genotypes. Agronomía Costarricense 33:125–131

    Google Scholar 

  37. Majumder S, Rahman MM, Bhadra SK (2011) Micropropagation of Scoparia dulcis linn. through induction of indirect organogenesis. Asia Pac J Mol Biol Biotechnol 19:11–17

    Google Scholar 

  38. Rajasekharan PE, Ambika SR, Ganeshan S (2009) In-vitro conservation of Tylophora indica: a threatened medicinal plant. IUP J Genet Evol 2:26–35

    Google Scholar 

  39. Dube P, Gangopadhyay M, Dewanjee S, Ali MN (2011) Establishment of a rapid multiplication protocol of Coleus forskohlii briq. and in vitro conservation by reduced growth. Indian J Biotechnol 10:228–231

    Google Scholar 

  40. Golmirzaie A, Toledo J (1998) Non-cryogenic, long-term germplasm storage. In: Hall R (ed) Methods in molecular biology, vol 111., Plant cell culture protocols, Humana Press, Netherlands, pp 95–101

  41. Dodds JH, Roberts LW (1985) Experiments in plant tissue culture, 2nd edn. Cambridge University press, Cambridge

    Google Scholar 

  42. Tahtamouni RW, Shibli RA, Ajlouni MM (2001) Growth responses and physiological disorders in wild pear (Pyrus syriaca Boiss.) during slow-growth in vitro preservation on osmostressing media. Plant Tissue Cult 11:15–23

    Google Scholar 

  43. Thompson MR, Douglas TJ, Sasamoto HO, Thorpe TA (1986) Mannitol metabolism in cultured plant cells. Physiol Plant 67:365–369

    Article  Google Scholar 

  44. Harb A, Krishnan A, Ambavaram MMR, Pereira A (2010) Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth. Plant Physiol 154:1254–1271

    Article  Google Scholar 

  45. Feng X, Zhao P, Hao J, Hu J, Kang D, Wang H (2011) Effects of sorbitol on expression of genes involved in regeneration of upland rice (Oryza sativa L.). Plant Cell Tissue Organ Cult 106:455–463

    Article  Google Scholar 

  46. Haque SM, Ghosh B (2013) Micropropagation, in vitro flowering and cytological studies of Bacopa chamaedryoides, an ethno-medicinal plant. Environ Exp Biol 11:59–68

    Google Scholar 

  47. Kaur H, Anand M, Goyal D (2011) Optimization of potting mixture for hardening of in vitro raised plants of Tylophora indica to ensure high survival percentage. Int J Med Arom Plants 1:83–88

    Google Scholar 

  48. Samaddar T, Nath S, Halder M, Sil B, Roychowdhury D, Sen S, Jha S (2012) Karyotype analysis of three important traditional Indian medicinal plants, Bacopa monnieri, Tylophora indica and Withania somnifera. Nucleus 55:17–20

    Article  Google Scholar 

  49. Saker MM, Bekheer SA, Taha HS, Fahmy AS, Moursy HA (2000) Detection of somaclonal variations in tissue cultured-derived date palm plants using iso-enzyme analysis and RAPD fingerprints. Biol Plant 43:347–351

    Article  Google Scholar 

  50. Chaturvedi P, Chowdhary A, Kawalkar H (2012) Molecular characterization of Tylophora indica regenerated plants in vitro by RAPD and ISSR analysis. Int J Res Phytochem Pharmacol 2:175–179

    Google Scholar 

Download references

Acknowledgments

We thank Swami Kamalasthananda, Principal of our college for facilities provided in this work. The Financial assistance from West Bengal Biodiversity Board (WBBB), Govt of West Bengal [Sanction No. 52/3k(Bio)-3/2008] is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Plant Biotechnology Laboratory, Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, 700118, India

    Sk. Moquammel Haque & Biswajit Ghosh

Authors
  1. Sk. Moquammel Haque
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Biswajit Ghosh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Biswajit Ghosh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Haque, S.M., Ghosh, B. Field Evaluation and Genetic Stability Assessment of Regenerated Plants Produced Via Direct Shoot Organogenesis from Leaf Explant of an Endangered ‘Asthma Plant’ (Tylophora indica) Along with Their In Vitro Conservation. Natl. Acad. Sci. Lett. 36, 551–562 (2013). https://doi.org/10.1007/s40009-013-0161-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40009-013-0161-z

Keywords

Search

Navigation