Advertisement

Optimising protocol for successful development of haploids in marigold (Tagetes spp.) through in vitro androgenesis

  • K. Ravindra Kumar
  • Kanwar Pal Singh
  • Reeta BhatiaEmail author
  • D. V. S. Raju
  • Sapna Panwar
Original Article
  • 34 Downloads

Abstract

For the first time, we have developed a protocol for rapid and successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. Various factors affecting the in vitro androgenesis response in Tagetes spp. were optimised. At first, a correlation was established between floret size/bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. The floret size of 3.5–4.0 mm (French marigold) and 3.0–3.5 mm (African marigold) contained the highest percent of microspores at early-uninucleate to early-binucleate stage. The frequency of shoot formation from anthers ranged from 0 to 78.6% in T. patula cv. Pusa Arpita and 0 to 63.9% in T. erecta cv. Pusa Basanti Gainda. The French marigold cultivars exhibited higher androgenic response over the African marigold cultivars. The effects of basal media, plant growth regulator combinations and sucrose concentration for direct differentiation of shoot buds and haploid induction were studied. Marigold anthers cultured on newly formulated EMS1 basal media (enriched with coconut water, AgNO3, PVP etc.) exhibited a very high androgenic response over the other commercially available media. We have also optimised the cold temperature pre-treatments and culture condition for high frequency androgenesis in marigold. Ten days cold pre-treated anthers along with 20 days dark incubation of cultures proved highly beneficial for haploid induction. Among a total of 424 anther-regenerants, 56 plants were randomly selected for flow cytometry and cytological analysis. The ploidy analysis revealed 14.3% of anther regenerants as dihaploids, 66.1% as tetraploid (similar to donor mother plant) and 19.6% as polyploid. The determination of ploidy level by counting the number of chloroplast in stomatal guard cells of marigold was also established for rapid screening of haploids. The identified haploids were successfully diploidised and are being utilised in the hybrid breeding programme at our institute. The developed protocol will facilitate doubled haploids based breeding in Tagetes spp.

Key message

This is the first report of successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. We have studied various factors affecting the in vitro androgenesis response in Tagetes spp. A correlation was established between floret size/ bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. Basal media and cultural conditions were optimised for high frequency androgenesis in marigold. Haploids were successfully induced and their ploidy level was assessed through flowcytometry and cytological analysis.

Keywords

African marigold French marigold Anther culture Haploid Flow cytometry Cytology Chloroplast 

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

BAP

Benzylamino purine

EMS

Enriched Murashige and Skoog medium

NAA

1-Naphthalene-acetic acid

TDZ

Thidiazuron

PA

Pusa Arpita

PBG

Pusa Basanti Gainda

Notes

Acknowledgements

Authors sincerely acknowledge the ICAR-Indian Agricultural Research Institute, New Delhi for providing the research facilities and Dr. YSR Horticultural University, Andhra Pradesh for grant of deputation to the first author to carrying out this research work.

Author contributions

KRK: He is responsible for conducting all the tissue culture related experiments and analysing the ploidy levels of anther induced regenerants. KPS: He has planned the experiments and provided the basic research facility for conducting these experiments. RB: She has planned the experiments and assisted in conducting the ploidy analysis of anther induced regenerants. DVSR: He has assisted in planning and data analysis of this study. SP: She has contributed in raising the marigold cultivars and maintenance of anther induced regenerants.

Compliance with ethical standards

Conflict of interest

The consent of all the authors for submission of this manuscript has been taken. There is no conflict of interest among the authors of this manuscript.

Supplementary material

11240_2019_1598_MOESM1_ESM.docx (528 kb)
Online Resource 1 (DOCX 528 KB)

References

  1. Achar PN (2002) A study of factors affecting embryo yields from anther culture of cabbage. Plant Cell Tissue Organ Cult 69:183–188CrossRefGoogle Scholar
  2. Agarwal PK, Agawam P, Custers JBM, Liu C, Bhojwani SS (2006) PCIB an anti auxin enhances microspore embryogenesis in microspore culture of Brassica juncea. Plant Cell Tissue Organ Cult 86:201–210CrossRefGoogle Scholar
  3. Bal U, Touraev A (2009) Microspore embryogenesis in selected medicinal and ornamental species of the Asteraceae. In: Touraev A (ed) Advances in Haploid Production in Higher Plants, Chap. 19. Springer, Dordrescht, pp 219–229CrossRefGoogle Scholar
  4. Ball ST, Zhou HP, Konzak CF (1993) Influence of 2,4-D, IAA and duration of callus induction in anther culture of spring wheat. Plant Sci 90:195–200CrossRefGoogle Scholar
  5. Bhatia R, Dey SS, Shritika S, Sharma K, Sharma VK, Parkash C, Kumar R (2016) Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B. oleracea var. botrytis L.) in India. Euphytica 212:439–454CrossRefGoogle Scholar
  6. Bhatia R, Dey SS, Shritika S, Sharma K, Parkash C, Kumar R (2017) Efficient microspore embryogenesis in cauliflower (Brassica oleracea var. botrytis L.) for development of plants with different ploidy level and their use in breeding programme. Sci Hort 216:83–92CrossRefGoogle Scholar
  7. Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FI (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci Sin 18:659–668Google Scholar
  8. Croes AF, Bosveld M, Wullems GJ (1988) Control of thiophene accumulation in Tagetes. In: Breteler JH, Arnason T, Hansen L (eds) chemistry and biology of naturally occurring acetylenes and related compound (NOARC). Elsevier publishers, Amesterdam, pp 255–265Google Scholar
  9. Eyster WH (1941) The induction of fertility in genetically self-sterile plants. Science 94:144–145CrossRefGoogle Scholar
  10. Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104:301–309CrossRefGoogle Scholar
  11. Gamborg OL, Miller RA, Rancillac M (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefGoogle Scholar
  12. Ganga M, Chezhiyan N, Kumar N, Soorianathasundaram K (2002) Stomatal and chloroplast traits as ploidy assessment techniques for ploidy screening of in vitro induced tetraploids of banana. Phytomorphology 52(2–3):113–120Google Scholar
  13. Gao Y, Chen B, Zhang J (2011) Anther culture of garden Chrysanthemum. Proceedings of XXVIIIth IHC-IS on micro and macro technologies for plant propagation. Acta Hort 923:103–110CrossRefGoogle Scholar
  14. Germana MA (2011) Anther culture for haploid and double haploid production. Plant Cell Tissue Organ Cult 104:283–300CrossRefGoogle Scholar
  15. Godoy-Hernandez G, Miranda-Ham ML (2007) Marigold biotechnology: Tissue culture and genetic transformation. Transgenic Plant J 1(1):169–174Google Scholar
  16. Gopikrishna V, Baweja PS, Venkateshbabu N, Thomas T, Kandaswamy D (2008) Comparison of coconut water, propolis, HBSS, and milk on PDL cell survival. J Endod 34:587–589CrossRefGoogle Scholar
  17. Guerin M, Huntley ME, Olaizola M (2003) Haematococcusastaxanthin: applications for human health and nutrition. Trends Biotechnol 21:210–216CrossRefGoogle Scholar
  18. Gupta V, Rahman L (2015) An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta. Protoplasma 252:1061–1070CrossRefGoogle Scholar
  19. Hendriks T, Gonthier L, Blassiau C, Legrand S, Gagneul D, Huss B, Bergs H, Quillet M (2014) Unravelling self-incompatibility in the Asteraceae: cloning of the S Locus in Chicory (Cichorium intybus L). International Plant and Animal Genome Conference XXIIGoogle Scholar
  20. Hermsen JGT, Ramanna MS (1981) Haploidy and plant breeding. Philos Trans R Soc Lond B 292:499–507CrossRefGoogle Scholar
  21. Hoveida ZS, Abdollahi MR, Mirzaie-Asl A, Moosavi SS, Seguí-Simarro JM (2017) Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress. Plant Cell Tissue Organ Cult 130:369–378CrossRefGoogle Scholar
  22. Jackson JC, Gordon A, Wizzard G, McCook K, Rolle R (2004) Changes in chemical composition of coconut. J Sci Food Agric 84:1049–1052CrossRefGoogle Scholar
  23. Jacquard C, Nolin F, He´cart C, Grauda D, Rashal I, Dhondt-Cordelier S, Sangwan R, Devaux P, Mazeyrat-Gourbeyre F, Cle´ment C (2009) Microspore embryogenesis and programmed cell death in barley: effects of copper on albinism in recalcitrant cultivars. Plant Cell Rep 28:1329–1339CrossRefGoogle Scholar
  24. Ketel DH, Breteler H, De Groot B (1987) Distribution and accumulation of thiophenes in plants and calli of different Tagetes species. J Exp Bot 38:322–330CrossRefGoogle Scholar
  25. Khandakar RK, Jie YU, Sun-Kyung MIN, Mi-Kyoung WON, Choi HG, Ha-Seung PARK, Tae-Sung KIM (2014) Regeneration of haploid plantlet through anther culture of Chrysanthemum. Not Bot Horti Agrobot Cluj-Na 42(2):482Google Scholar
  26. Kim KM, Baenziger PS, Rybczynski JJ, Arumuganathan K (2003) Characterization of ploidy levels of wheat microspore-derived plants using laser flow cytometry. In vitro Cell Dev Biol Plant 39:663–668CrossRefGoogle Scholar
  27. Kothari SL, Chandra N (1984) In vitro propagation of African marigold. HortScience 19:703–705Google Scholar
  28. Li Fu (2007) Identification of the microspore development stages and study of anther vitro culture on Tagetes erecta. Thesis submitted to Qinghai University, China (Abstract)Google Scholar
  29. Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434CrossRefGoogle Scholar
  30. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  31. Murovec J, Bohanec B (2012) Haploids and doubled haploids in plant breeding. In: Abdurakhmonov I (ed) Plant breeding. In Tech, ZagrebGoogle Scholar
  32. Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87CrossRefGoogle Scholar
  33. Nitsch C, Norreel B (1973) Effect d’un choc the rmiquesurIepouvoir embryogenedu pollen de Datura culture dansI’ anthereouisole de l’ anthere. Comptes Rendus Acad Sci 276:303–306Google Scholar
  34. Osolnik B, Bohanec B, Jelaska S (1993) Stimulation of androgenesis in white cabbage (Brassica oleracea var. capitata) anthers by low temperature and anther dissection. Plant Cell Tissue Organ Cult 32:241–246CrossRefGoogle Scholar
  35. Pablo E, Vanegas PE, Cruz-Hernandez A, Valverde ME, Paredes-Lopez O (2002) Plant regeneration via organogenesis in marigold. Plant Cell Tissue Organ Cult 69:279–283CrossRefGoogle Scholar
  36. Qi Y, Ye Y, Manzhu B (2011) Establishment of plant regeneration system from anther culture of Tagetes patula. Afr J Biotechnol 10(75):17332–17338Google Scholar
  37. Raghavan V (1990) From microspore to embryo: faces of the angiosperm pollen grain. In: Nijkamp HJJ, van der Plas LH, van Hartrigik J (eds) Progress in plant cellular and molecular biology. I.A.P.T.C. Kluwer, Dordrecht, pp 213–221CrossRefGoogle Scholar
  38. Ravindra Kumar K, Singh KP, Jain PK, Raju DVS, Kumar P, Bhatia R, Sapna Panwar (2018) Influence of growth regulators on callus induction and plant regeneration from anthers of Tagetes spp. Indian J Agr Sci 88(6):970–977Google Scholar
  39. Touraev A, Pfosser M, Heberle-Bors E (2001) The microspore: a haploid multipurpose cell. Adv Bot Res 35:53–109CrossRefGoogle Scholar
  40. Vanegas-Espinoza PE, Ramos-Viveros V, Rios-Salome LB, Orbe-Rogel JC, Parredes-Lopez O, Cruz-Hernandez A, Villar-Martineza AAD (2012) Analysis of callus development and transient expression in Tagetes erecta. Acta Hort 937:963–968CrossRefGoogle Scholar
  41. Vasil IK, Nitsch C (1975) Experimental production of pollen haploids and their uses. Z Pfianzenphysiol 76:191–212CrossRefGoogle Scholar
  42. Veilleux RE (1994) Development of new cultivars via anther culture. Hortscience 29(11):1238–1241CrossRefGoogle Scholar
  43. Wang G, Qin H, Sun D, Fan S, Yang Y, Wang Z, Xu P, Zhao Y, Liu Y, Ai J (2018) Haploid plant regeneration from hardy kiwifruit (Actinidia arguta Planch.) anther culture. Plant Cell Tissue Organ Cult 134:15–28CrossRefGoogle Scholar
  44. Wenzel G, Hoffmann F, Thomas E (1977) Increased induction and chromosome doubling of androgenetic haploid rye. Theor Appl Genet 51:81–86CrossRefGoogle Scholar
  45. Wijnker E, Vogelaar A, Dirks R, van Dun K, de Snoo B, van den Berg M, Lelivelt C, de Jong H, Chunting L (2007) Reverse breeding: Reproduction of F1 hybrids by RNAi-induced asynaptic meiosis. Chromosome Res 15(2):87–88Google Scholar
  46. Winarto B, Mattjik NA, Teixeira da Silva JA, Purwito A, Marwoto B (2010) Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture. Sci Hort 127:86–90CrossRefGoogle Scholar
  47. Xu ZH (1990) Barley (Hordeum vulgare L.) anther culture and the production of haploids. In: Bajaj YPS (ed) Haploids in crop improvement biotechnology in agriculture and forestry. Springer, Berlin, pp 125–175CrossRefGoogle Scholar
  48. Xue-jiao S, TAO CG, WANG P (2010) Interrelation of cytological development period of marigold microspore the morphology of flower organ. Hubei Agri Sci 9:039Google Scholar
  49. Zhang XQ, Yang JS, Zheng ZX (2009) Study on induction of embroid through in vitro anther of Cyclamen persicum. Jiang Su Agri Sci 108:57–58Google Scholar
  50. Zhang P, Zeng L, Su Y, Gong X, Wang X (2011) Karyotype studies on Tagetes erecta L. and Tagetes patula L. Afr J Biotechnol 10(72):16138–16144Google Scholar
  51. Zhong D, Michaux-Ferriere N, Coumans M (1995) Assay for double haploid sunflower (Helianthus annus) plant production by androgenesis: fact or artifact? Part 1. In vitro anther culture. Plant Cell Tissue Organ Cult 41:91–97CrossRefGoogle Scholar
  52. Zhou H, Zeng W, Yan H (2017) In vitro induction of tetraploids in cassava variety ‘Xinxuan 048’ using colchicine. Plant Cell Tissue Organ Cult 128:723–729CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Horticultural Research StationDr. Y.S.R. Horticultural UniversityWest GodavariIndia
  2. 2.Division of Floriculture and Landscape ArchitectureICAR-Indian Agricultural Research InstituteNew DelhiIndia
  3. 3.ICAR-Directorate of Floriculture ResearchPuneIndia

Personalised recommendations