High-throughput in vitro culture system targeting genetic transformation in sugarcane

Abstract

Conventional recombination breeding suffers setbacks for genetic improvement of sugarcane owing to its narrow gene pool, large complex genome, rare flowering, low fertility, long breeding cycle, and complex environmental interactions. However, production of transgenic plants can be a better alternative to improve quality traits and resistance to biotic and abiotic stresses. The authors report a ready in hand efficient in vitro culture protocol for genetic transformation in a popular sugarcane cv. Sabita (CoOR 03151) of Odisha. 2, 4-D at 3.0 mg/l resulted in highest callus induction frequency (87.8%) with white, friable, and nodular embryogenic calli suitable for plantlet regeneration. 2 mg/l BAP resulted in moderately higher number of shoots/responsive callus and higher percentage of plant survival. However, addition of TDZ (0.05 m/l) with 2 mg/l BAP in R medium (modified MS) may be considered optimum for regeneration of profuse healthy plantlets. A combination of PBZ (0.05 mg/l) with BAP (2 mg/l) and TDZ (0.05 mg/l) revealed profuse multiple shoots (25 microshoots/responsive callus) and higher percentage of survival during follow-up plant establishment than above hormonal supplementation. Among various hormone recipes in MS medium with 3.0 mg/l NAA resulted in excellent rhizogenesis response (88.0%) with more or less normal rooting within 2 weeks. The above efficient and highly reproducible in vitro culture system can be amenable for genetic transformation in this crop.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

BAP:

6-Benzylaminopurine

2,4-D:

2,4-Dicchlorophenoxyacetic acid

GA3 :

Gibberellic acid

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

Kn:

Kinetin(6-Furfuryladenopurine)

NAA:

α-Naphthalene acetic acid

PBZ:

Paclobutrazol

PVP:

Polyvinylpyrrolidone

RH:

Relative humidity

TDZ:

Thidizuron

References

  1. Abdu SL, Yahaya M, Shehu UI (2012) In vitro regeneration of commercial sugarcane (Saccharum spp.) cultivars in Nigeria. J Life Sci 6:721–725

    CAS  Google Scholar 

  2. Ali S, Iqbal J (2012) Influence of physical factors on callogenesis in sugarcane (Saccharum officinarum L.). Sci Int (Lahore) 24(2):167–170

    CAS  Google Scholar 

  3. Ali A, Shagufta N, Fayyaz AS, Javed I (2008) An efficient protocol for large scale production of sugarcane through micropropagation. Pak J Bot 40:139–149

    CAS  Google Scholar 

  4. Arencibia A, Molina PR, de la Riva G, Selman-Housein G (1995) Production of transgenic sugarcane (Saccharum officinarum L.) plants by intact cell electroporation. Plant Cell Rep 14(5):305–309. https://doi.org/10.1007/bf00232033

    CAS  Article  PubMed  Google Scholar 

  5. Ather A, Khan S, Rehman A, Nazir M (2009) Optimization of the protocols for callus induction, regeneration and acclimatization of sugarcane cv. Thatta-10. Pak J Bot 41(2):815–820

    CAS  Google Scholar 

  6. Baksha R, Alam R, Karim MZ, Mannan SA, Podder BP (2003) Effect of auxin, sucrose pH level on in vitro rooting of callus induced micro shoots of sugarcane (Saccharum officinarum L.). J Biol Sci 3:915–920

    Google Scholar 

  7. Behera KK, Sahoo S (2009) Rapid in vitro micro propagation of sugarcane (Saccharum officinarum L. cv- Nayana) through callus culture. Nat Sci 7:1–10

    Google Scholar 

  8. Blaydes DF (1966) Interaction of kinetin and various inhibitors in the growth of soybean tissue. Physiol Plant 19:748–753

    CAS  Google Scholar 

  9. Chandran K (2011) In vitro regeneration of Saccharum edule from immature inflorescence. Sugar Tech 13:170–173

    CAS  Google Scholar 

  10. D’Hont A, Glaszmann JC (2001) Sugarcane genome analysis with molecular markers—a first decade of research. In: Proceedings of International Society of Sugar Cane Technologists, pp 556–559

  11. de Alcantara GB, Dibax R, de Oliveira RA, Filho JCB, Daros E (2014) Plant regeneration and histological study of the somatic embryogenesis of sugarcane (Saccharum spp.) cultivars RB855156 and RB72454. Acta Scientiarum Agron 36(1):1–6. https://doi.org/10.4025/actasciagron.v36i1.16342

    Article  Google Scholar 

  12. Dellaporta SL, Wood J, Hick JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21

    CAS  Google Scholar 

  13. Desai NS, Suprasanna P, Bapat A (2004) Simple and reproducible protocol for direct somatic embryogenesis from cultured immature inflorescence segments of sugarcane (Saccharum spp.). Curr Sci 87:764–768

    Google Scholar 

  14. Dewanti P, Widuri LI, Narulita F, Susilo HA, Addy Okviandari P, Sugiharto B (2016) Rapid propagation of virus-free sugarcane (Saccharum officinarum) by somatic embryogenesis. Agric Agric Sci Procedia 9:456–461

    Google Scholar 

  15. Dibax R, de Alcântara GB, Filho JCB, Machado MP, de Oliveira Y, da Silva ALL (2011) Plant regeneration of sugarcane cv. RB931003 and RB98710 from somatic embryos and acclimatization. J Biotech Biodivers 2:32–37

    CAS  Google Scholar 

  16. Donde R, Samal KC, Rout GR (2014) Studies on Agrobacterium mediated genetic transformation of rice (Oryza sativa L.) for drought tolerance using Dreb 1 gene. Int J Agril Sci 4(12):341–350

    Google Scholar 

  17. Duncan DB (1955) Multiple range and multiple F-tests. Biometrics 11:1–42

    Google Scholar 

  18. Efendi, Matsuoka M (2011) An efficient Agrobacterium-mediated transformation method for sugarcane (Saccharum officinarum L.). In: Proceedings of the annual international conference Syiah Kuala University, 2011, Banda Aceh, Indonesia, November 29–30, 2011

  19. Eldessoky DS, Ismail RM, Abdel-Hadi AHA, Abdallah N (2011) Establishment of regeneration and transformation system of sugarcane cultivar GT54-9 (C9). GM Crops 2(2):126–134

    PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  21. Gandonou C, Errabii T, Abrinii J, Idaomar M, Chibi F, Skali Senhaji NS (2005) Effect of genotype on callus induction and plant regeneration from leaf explants of sugarcane. Afr J Biotechnol 4:1250–1255

    Google Scholar 

  22. Godheja J, Shekhar SK, Modi DR (2014) The standardization of protocol for large scale production of sugarcane (Co-86032) through micro propagation. Int J Plant Anim Environ Sci 4:135–143

    CAS  Google Scholar 

  23. Gopitha K, Bhavani LA, Senthilmanickam J (2010) Effect of the different auxins and cytokinins in callus induction, shoot, root regeneration in sugarcane. Int J Pharma Biol Sci 1:1–7

    Google Scholar 

  24. Hapsor D, Febrianie AP, Yusnita (2012) In vitro shoot formation on sugarcane (Saccharum officinarum L.) callus as affected by benzyladenine concentrations. J Agron Indonesia 40(1):56–61

    Google Scholar 

  25. Ijaz S, Rana IA, Khan IA, Saleem M (2012) Establishment of an in vitro regeneration system for genetic transformation of selected sugarcane genotypes. Genet Mol Res 11:512–530

    CAS  PubMed  Google Scholar 

  26. Jahangir GZ, Nasir IA (2010) Various hormonal supplementations activate sugarcane regeneration in vitro. J Agril Sci 2:231–237

    Google Scholar 

  27. Kaur A, Gill MS, Ruma D, Gosal SS (2008) Enhanced in vitro shoot multiplication and elongation in sugarcane using cefotaxime. Sugar Tech 10(1):60–64

    CAS  Google Scholar 

  28. Khaliq A, Ashfaq M, Akram W, Choi JK, Lee J (2005) Effect of plant factors, sugar contents, and control methods on the Top Borer (Scirpophaga nivella F.) Infestation in selected varieties of sugarcane. Entomol Res 35:153–160

    Google Scholar 

  29. Khamrit R, Jaisil P, Bunnag S (2012) Callus induction, regeneration and transformation of sugarcane (Saccharum officinarum L.) with chitinase gene using particle bombardment. Afr J Biotech 11:6612–6618

    CAS  Google Scholar 

  30. Khan MR, Rashid H (2003) Studies on the rapid clonal propagation of Saccharum officinarum. Pak J Biol Sci 6:1876–1879

    Google Scholar 

  31. Khan IA, Khatri A, Nizamani GS, Siddiqui MA, Khanzada MH, Dahar NA, Seema N, Naqvi MH (2004) In vitro culture studies in sugarcane. Pak J Biotech 1:6–10

    Google Scholar 

  32. Khan SA, Hanif Z, Irshad U, Rashid H (2013) Genetic transformation of sugarcane Variety HSF-240 with marker gene GUS. Int J Agril Biol 15:1258–1264

    CAS  Google Scholar 

  33. Kumar T, Uzma Khan MR, Abbas Z, Ali GM (2014) Genetic improvement of sugarcane for drought and salinity stress tolerance using Arabidopsis Vacuolar Pyrophosphatase (AVP1) Gene. Mol Biotech 56(3):199–209

    CAS  Google Scholar 

  34. Linsmaier EH, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Plant Physiol Plant 18:100–127

    CAS  Google Scholar 

  35. Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhakar B, Selvaraj N, Vasudevan A, Kasthurirengan S (2004) Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Rep 23(3):134–143. https://doi.org/10.1007/s00299-004-0794-y

    CAS  Article  PubMed  Google Scholar 

  36. Mayavan S, Subramanyam K, Arun M, Rajesh M, Kapil Dev G, Sivanandhan G, Jaganath B, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane. Plant Cell Rep 32(10):1557–1574. https://doi.org/10.1007/s00299-013-1467-5

    CAS  Article  PubMed  Google Scholar 

  37. Michael PS (2007) Micropropagation of elite sugarcane planting materials from callus culture in vitro. J Proc R Soc NSW 140:79–86

    Google Scholar 

  38. Mittal P, Devi R, Gosal SS (2016) Effect of genotypes and activated charcoal on high frequency in vitro plant regeneration in sugarcane. Indian J Biotech 15:261–265

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  40. Patel VS, Mehta R, Naik KH, Singh D, Patel DU, Mali SC (2015) Callus induction & whole plant regeneration in sugarcane (Saccharum spp. complex) variety Co 86032. Green Farm 6(5):935–939

    Google Scholar 

  41. Pierik RLM (1997) In vitro culture of higher plants. Kluwar Academic Publishers, Netherlands, pp 95–196

    Google Scholar 

  42. Pillay E (2013) In vitro culture and genetic transformation of selected ancestral and commercial sugarcane germplasm. A dissertation submitted for the degree of Master of Science in the School of Life Science, University of KwaZulu-Natal, Durban, South Africa

  43. Rashid AHA, Lateef DD (2016) Novel techniques for gene delivery into plants and its applications for disease resistance in crops. Am J Plant Sci 7:181–193

    CAS  Google Scholar 

  44. Rashid H, Khan SA, Zia M, Chaudhary MF, Hanif Z, Chaudary Z (2009) Callus induction and regeneration in elite sugarcane cultivar HSF-240. Pak J Bot 41(4):1645–1649

    Google Scholar 

  45. Satpal SB, Routray AK, Mishra R (2011) Rapid in vitro propagation technique for sugarcane variety 018. Int J Pharma Biol Sci 2:B242–B279

    Google Scholar 

  46. Singh BD (2012) Transgenic Plants:I. Gene construct, vectors and transformation methods. In: Biotechnology expanding horizons, Fourth edition, pp 372–405

  47. Snyman S, Meyer G, Richards J, Haricharan N, Ramgareeb S, Huchett B (2006) Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transferon transformation efficiency. Plant Cell Rep 25:1016–1023

    CAS  PubMed  Google Scholar 

  48. Soomro F, Lochi S, Lochi R, Lochi F, Ansari TS, Ansari P, Ansari UA, Rajper AA (2014) Callus induction in sugarcane. J Agric Food Tech 4(3):8–11

    Google Scholar 

  49. Souza GM, Berges H, Bocs S, Casu R, D’Hon A, Ferreira JE, Henry RJ, Ming R, Potier B, Sluya MAV, Vincentz M, Paterson A (2011) The sugarcane genome challenge: strategies for sequencing a highly complex genome. Trop Plant Biol 4:145–156. https://doi.org/10.1007/s12042-011-9079-0

    CAS  Article  Google Scholar 

  50. Srivong T, Zhu YJ, Pongdontri P, Pliansinchai U, Sakuanrungsirikul S, Borthakur D, Nagai C, Kosittrakun M (2015) Optimization of callus induction and plant regeneration in sugarcane (Saccharum spp.) for a study of sucrose accumulation in relation to soluble acid invertase expression. Chiang Mai J Sci 42:797–805

    CAS  Google Scholar 

  51. Suprasanna P (2010) Biotechnological interventions in sugarcane improvement: strategies, methods and progress. Nucl Agril Biotech Div BARC Newsl 316:47–53

    CAS  Google Scholar 

  52. Taparia Y, Fouad WM, Gallo M, Altpeter F (2012a) Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis. Vitro Cell Dev Biol Plant 48:15–22

    CAS  Google Scholar 

  53. Taparia Y, Gallo M, Altpeter F (2012b) Comparison of direct and indirect embryogenesis protocols, biolistic gene transfer and selection parameters for efficient genetic transformation of sugarcane. Plant Cell Tiss Organ Cult 111:131–141. https://doi.org/10.1007/s11240-012-0177-y

    CAS  Article  Google Scholar 

  54. Ullah M, Khan H, Khan MS, Jan A, Ahmad K, Khan AW (2016) In vitro plant regeneration of sugarcane (Saccharum officinarum L.); the influence of variety, explant, explant position and growth regulators. ARPN J Agril Biol Sci 11:267–273

    CAS  Google Scholar 

  55. Vinayak V, Dhawan AK, Gupta VK (2009) Efficiency of nonpurine and purine cytokinins on shoot regeneration of sugarcane. Indian J Biotech 8:227–231

    CAS  Google Scholar 

  56. Warakagoda PS, Subasinghe S, Kumari DLC, Neththikumara TS (2007) Micro propagation of sugarcane (Saccharum officinarum L.) through auxiliary buds. In: Proc. Fourth Acad. Sessions 2007, University of Ruhuna, Mapalana, Kamburupitiya, pp 55–60

  57. Yadav S, Ahmad A, Lal M (2012) Effect of different auxins and cytokinins on in vitro multiplication and rooting of shoot cultures in Sugarcane. Int J Biol Pharma Res 3:814–818

    Google Scholar 

  58. Zamir R, Khalil SA, Shah ST, Khan MS, Ahmad K, Shahenshah Ahmad N (2014) Efficient in vitro regeneration of sugarcane (Saccharum officinarum L.) from bud explants. Biotech Biotechnol Equip 26:3094–3099. https://doi.org/10.5504/BBEQ.2012.0049

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We sincerely acknowledge and thank all researchers for their valuable contributions included in the text as references. Besides, special thanks to Breeder, AICRP (Sugarcane), Nayagarh, Odisha (OUAT) for providing the plant material for the study.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Affiliations

Authors

Contributions

SKT conceived the idea, performed the study and data analysis, and wrote the paper. DMI assisted in the in vitro culture work.

Corresponding author

Correspondence to Swapan K. Tripathy.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tripathy, S.K., Ithape, D.M. High-throughput in vitro culture system targeting genetic transformation in sugarcane. J. Crop Sci. Biotechnol. 23, 325–335 (2020). https://doi.org/10.1007/s12892-020-00040-x

Download citation

Keywords

  • Somatic embryogenesis
  • High-throughput plantlet regeneration
  • Genetic
  • Transformation
  • Saccharum officinarum L.