Skip to main content
SpringerLink
Log in

Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Genetic improvement of sunflower (Helianthus annuus L.) through the use of biotechnological tools requires a reliable in vitro shoot regeneration system. Tissue culture protocols reported to date for sunflower suffer from low efficiency, poor reproducibility, genotype dependence and a tendency for flowering in vitro. The present study describes an efficient protocol system for shoot regeneration via direct adventitious shoot organogenesis from cotyledons of mature seeds of sunflower. About 169 media combinations comprising 12 different growth regulator combinations in various concentrations were assessed for induction of shoots from cotyledons derived from mature seeds and also from seedling tissues of 2–20-day-old seedlings. Appearance of shoots from seedling tissues was sporadic and the frequency of shoot regeneration was low. Cotyledon explants from mature seeds were consistent with regard to frequency of adventitious shoot regeneration and number of shoots per explant. A high frequency (93.86 %) of adventitious shoot regeneration was obtained within 2 weeks of culture initiation on Murashige and Skoog (MS) medium supplemented with 9.84 μM 2-isopentenyladenine (2-iP), 2.85 μM indole-3-acetic acid (IAA) and 0.45 μM thidiazuron (TDZ). Use of 2-iP in the shoot induction and elongation media prevented precocious flowering. Statistical analysis revealed significant effects of explant orientation, age of seedlings, and genotype on adventitious organogenesis. Maximum shoot regeneration was obtained when cotyledons from 0 and 1-day-old seedlings were placed with their adaxial surface in contact with the medium surface. The protocol developed was tested on 42 genotypes and found to be applicable to a wide range of genotypes. Histological studies indicated that the shoots originated predominantly through adventive organogenesis from the sub-epidermal and cortical regions.

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

Similar content being viewed by others

Abbreviations

BA:

N6-Benzyladenine

2,4-D:

2,4-Dichlorophenoxyacetic acid

DMRT:

Duncan’s Multiple Range Test

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

Kn:

Kinetin

2-iP:

N6-[2-Isopentenyl]adenine

NAA:

α-Naphthaleneacetic acid

TDZ:

1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)

References

  • Abdoli M, Moieni A, Dehghani H (2003) Effects of genotype and cotyledon section on organogenesis in sunflower. Iran J Biotechnol 1:234–238

    Google Scholar 

  • Abdoli M, Moieni A, Dehghani H (2007) Effects of cultivar and agar concentration on in vitro shoot organogenesis and hyperhydricity in sunflower (Helianthus annuus L.). Pak J Bot 39:31–35

    Google Scholar 

  • Alibert G, Aslane-Chanabe JC, Burrus M (1994) Sunflower tissue and cell cultures and their use in biotechnology. Plant Physiol Biochem 32:31–44

    CAS  Google Scholar 

  • Alibert B, Lucas D, Le Gall V, Kallerhoff J, Alibert G (1999) Pectolytic enzyme treatment of sunflower explants prior to wounding and cocultivation with Agrobacterium tumefaciens, enhances efficiency of transient β-glucuronidase expression. Physiol Plant 106:232–237

    Article  CAS  Google Scholar 

  • Baker CM, Munoz-Fernandez N, Carter CD (1999) Improved shoot development and rooting from mature cotyledons of sunflower. Plant Cell Tiss Org Cult 58:39–49

    Article  CAS  Google Scholar 

  • Bell RL, Scorza R, Lomberk D (2012) Adventitious shoot regeneration of pear (Pyrus spp.) genotypes. Plant Cell Tiss Organ Cult 108:229–236

    Article  Google Scholar 

  • Bidney D, Scelonge C, Martich J, Burrus M, Sims L, Huffman G (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol Biol 18:301–313

    Article  PubMed  CAS  Google Scholar 

  • Burrus M, Chanabe C, Alibert G, Bidney D (1991) Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.). Plant Cell Rep 10:161–166

    Article  CAS  Google Scholar 

  • Burrus M, Molinier J, Himber C, Hunold R, Bronner R, Rousselin P, Hahne G (1996) Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns. Mol Breed 2:329–338

    Article  CAS  Google Scholar 

  • Ceriani MF, Hopp HE, Hahne G, Escandon AS (1992) Cotyledons: an explant for routine regeneration of sunflower plants. Plant Cell Physiol 33:157–164

    Google Scholar 

  • Charriere F, Hahne G (1998) Induction of embryogenesis versus caulogenesis on in vitro cultured sunflower (Helianthus annuus L.) immature zygotic embryos: role of plant growth regulators. Plant Sci 137:63–71

    Article  CAS  Google Scholar 

  • Chraibi KMB, Latche A, Roustan JP, Fallot J (1991) Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors, silver and cobalt. Plant Cell Rep 10:204–207

    Google Scholar 

  • Chraibi KMB, Castelle JC, Latche A, Roustan JP, Fallot J (1992) A genotype-independent system of regeneration from cotyledons of sunflower (Helianthus annuus L.). The role of ethylene. Plant Sci 86:215–221

    Article  CAS  Google Scholar 

  • Cueto MA, Dathe W (1986) Effect of auxins on flower formation in coffee (Coffea arabica L). Biol Plant 28:355–360

    Article  CAS  Google Scholar 

  • Dagustu N, Fraser P, Enfissi E, Bramley P (2008) Screening for high callus induction and Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.). Biotechnol Biotechnol 22:933–937

    Google Scholar 

  • Deglene L, Lesignes P, Alibert G, Sarrafi A (1997) Genetic control of organogenesis in cotyledons of sunflower (Helianthus annuus). Plant Cell Tiss Org Cult 48:127–130

    Article  Google Scholar 

  • Dhaka N, Kothari SL (2002) Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L. Plant Cell Rep 21:29–34

    Article  CAS  Google Scholar 

  • Espinasse A, Lay C (1989) Shoot regeneration of callus derived from globular to torpedo embryos from 59 sunflower genotypes. Crop Sci 29:201–205

    Article  Google Scholar 

  • Espinasse A, Lay C, Volin J (1989) Effects of growth regulator concentrations and explant size on shoot organogenesis from callus derived from zygotic embryos of sunflower (Helianthus annuus L.). Plant Cell Tiss Org Cult 17:171–181

    Article  CAS  Google Scholar 

  • Finer JJ (1987) Direct somatic embryogenesis and plant regeneration from immature embryos of hybrid sunflower (Helianthus annuus L.) on a high sucrose-containing medium. Plant Cell Rep 6:372–374

    Article  CAS  Google Scholar 

  • Fiore MC, Trabace T, Sunseri F (1997) High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis. Plant Cell Rep 16:295–298

    CAS  Google Scholar 

  • Flores Berrios E, Gentzbittel L, Serieys H, Alibert G, Sarrafi A (1999) Influence of genotype and gelling agents on in vitro regeneration by organogenesis in sunflower. Plant Cell Tiss Org Cult 59:65–69

    Article  Google Scholar 

  • Freyssinet M, Freyssinet G (1988) Fertile plant regeneration from sunflower (Helianthus annuus L.) immature embryos. Plant Sci 56:177–181

    Article  Google Scholar 

  • Grayburn WS, Vick BA (1995) Transformation of sunflower (Helianthus annuus L.) following wounding with glass beads. Plant Cell Rep 14:285–289

    Article  CAS  Google Scholar 

  • Greco B, Tanzarella OA, Carrozzo G, Blanco A (1984) Callus induction and shoot regeneration in sunflower (Helianthus annuus L.). Plant Sci Lett 36:73–77

    Article  Google Scholar 

  • Hewezi T, Perrault A, Alibert G, Kallerhoff J (2002) Dehydrating immature embryo split apices and rehydrating with Agrobacterium tumefaciens: a new method for genetically transforming recalcitrant sunflower. Plant Mol Biol Rep 20:335–345

    Article  CAS  Google Scholar 

  • Huang XQ, Nabipour A, Gentzbittel L, Sarrafi A (2007) Somatic embryogenesis from thin epidermal layers in sunflower and chromosomal regions controlling the response. Plant Sci 173:247–252

    Article  CAS  Google Scholar 

  • Hunold R, Burrus M, Bronner R, Duret JP, Hahne G (1995) Transient gene expression in sunflower (Helianthus annuus L.) following microprojectile bombardment. Plant Sci 105:95–109

    Article  CAS  Google Scholar 

  • Ivanov P, Encheva J, Ivanova I (1997) A protocol to avoid precocious flowering of sunflower plantlets in vitro. Plant Breed 117:582–584

    Article  Google Scholar 

  • Jain RK, Bhat AI, Varma A (2003) Sunflower necrosis disease-an emerging viral problem. Technical Bulletin -1, Unit of Virology, IARI, New Delhi, 11 pp

  • Jain RK, Vemana K, Bag S (2006) Tobacco streak virus: an emerging virus in vegetable crops. In: Govind P Rao, Lava Kumar P, Holguin-Pena RJ (eds) Characterization, diagnosis & management of plant viruses vol 3: vegetables and pulse crops. Studium Press, Texas, pp 207–216

  • Jan CC (1997) Cytology and interspecific hybridization. In: Sunflower technology and production. Monograph No 35. American Society of Agronomy, Madison, pp 497–558

  • Jeannin G, Hahne G (1991) Donor plant growth conditions and regeneration of fertile plants from somatic embryos induced on immature zygotic embryos of sunflower (Helianthus annuus L.). Plant Breed 107:280–287

    Article  Google Scholar 

  • Jeannin G, Bronner R, Hahne G (1995) Somatic embryogenesis and organogenesis induced on the immature zygotic embryos of sunflower (Helianthus annuus L.) cultivated in vitro: role of the sugar. Plant Cell Rep 15:200–204

    Article  CAS  Google Scholar 

  • Johansen DA (1940) Plant microtechnique. McGraw-Hill, New York

    Google Scholar 

  • Joshi M, Nadgauda RS (1997) Cytokinins and in vitro induction of flowering in bamboo: Bambusa arundinacea (Retz.) Willd. Curr Sci 73:523–526

    CAS  Google Scholar 

  • Ju HJ, Eck JV, Gray SM (2012) Factors influencing plant regeneration from seedling explants of Hairy nightshade (Solanum sarrachoides). Plant Cell Tiss Organ Cult 108:121–128

    Article  CAS  Google Scholar 

  • Knittel N, Escandon AS, Hahne G (1991) Plant regeneration at high frequency from mature sunflower cotyledons. Plant Sci 73:219–226

    Article  Google Scholar 

  • Knittel N, Gruber V, Hahne G, Lenee P (1994) Transformation of sunflower (Helianthus annuus L): a reliable protocol. Plant Cell Rep 14:81–86

    Article  CAS  Google Scholar 

  • Konieczny R, Libik M, Tuleja M, Niewiadomska E, Miszalski Z (2008) Oxidative events during in vitro regeneration of sunflower. Acta Physiol Plant 30:71–79

    Article  CAS  Google Scholar 

  • Korell M, Brahm L, Friedt W, Horn R (1996) Interspecific and intergeneric hybridization in sunflower breeding. II. Specific uses of wild germplasm. Plant Breed Abst 66:1081–1091

    Google Scholar 

  • Laparra H, Burrus M, Hunold R, Damm B, Bravo-Angel AM, Bronner R, Hahne G (1995) Expression of foreign genes in sunflower (Helianthus annuus L.): evaluation of three gene transfer methods. Euphytica 85:63–74

    Article  CAS  Google Scholar 

  • Lin CS, Liang CJ, Hsaio HW, Lin MJ, Chang WC (2007) In vitro flowering of green and albino Dendrocalamus latiflorus. New Forest 34:177–186

    Article  Google Scholar 

  • Lupi MC, Bennici A, Locci F, Gennai D (1987) Plantlet formation from callus and shoot tip culture of Helianthus annuus (L). Plant Cell Tiss Org Cult 11:47–55

    Article  Google Scholar 

  • Malone-Schoneberg J, Scelonge CJ, Burrus M, Bidney DL (1994) Stable transformation of sunflower using Agrobacterium and split embryonic axis explants. Plant Sci 103:199–207

    Article  CAS  Google Scholar 

  • Mayor ML, Nestares G, Zorzoli R, Picardi LA (2003) Reduction of hyperhydricity in sunflower tissue culture. Plant Cell Tiss Org Cult 72:99–103

    Article  CAS  Google Scholar 

  • Moyo M, Finnie JF, Staden JV (2009) In vitro morphogenesis of organogenic nodules derived from Sclerocarya birrea subsp. Caffra leaf explants. Plant Cell Tiss Organ Cult 98:273–280

    Article  Google Scholar 

  • Muller A, Iser M, Hess D (2001) Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker. Transgenic Res 10:435–444

    Article  PubMed  CAS  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 

  • Nataraja K, Ganapathi TR (1989) In vitro plantlet regeneration from cotyledons of Helianthus annuus cv. Morden (Sunflower). Indian J Exp Biol 27:777–779

    CAS  Google Scholar 

  • Nestares G, Zorzoli R, Mroginski L, Picardi L (1996) Plant regeneration from cotyledons derived from mature sunflower seeds. Helia 19:107–112

    Google Scholar 

  • Ozyigit II, Gozukirmizi N, Semiz BD (2006) Callus induction and plant regeneration from mature embryos of sunflower. Russ J Plant Physiol 53:556–559

    Article  CAS  Google Scholar 

  • Paterson KE (1984) Shoot tip culture of Helianthus annuus: flowering and development of adventitious and multiple shoots. Am J Bot 71:925–931

    Article  Google Scholar 

  • Paterson KE, Everett NP (1985) Regeneration of Helianthus annuus inbred plants from callus. Plant Sci 42:125–132

    Article  CAS  Google Scholar 

  • Pelissier B, Bouchefra O, Pepin R, Freyssinet G (1990) Production of isolated somatic embryos from sunflower thin cell layers. Plant Cell Rep 9:47–50

    Article  Google Scholar 

  • Power CJ (1987) Organogenesis from Helianthus annuus inbreds and hybrids from the cotyledons of zygotic embryos. Am J Bot 74:497–503

    Article  CAS  Google Scholar 

  • Pugliesi C, Cecconi F, Mandolfo A, Baroncelli S (1991) Plant regeneration and genetic variability from tissue cultures of sunflower (Helianthus annuus L.). Plant Breed 106:114–121

    Article  CAS  Google Scholar 

  • Rao KS, Rohini VK (1999) Agrobacterium–mediated transformation of sunflower (Helianthus annuus L.): a simple protocol. Ann Bot 83:347–354

    Article  CAS  Google Scholar 

  • Saravitz CH, Blazich FA, Amerson HV (1993) Histology of in vitro adventitious bud development on cotyledons and hypocotyls of Fraser fir. J Am Soc Hortic Sci 118:163–167

    Google Scholar 

  • Sarrafi A, Bolandi AR, Serieys H, Berville A, Alibert G (1996) Analysis of cotyledon culture to measure genetic variability for organogenesis parameters in sunflower (Helianthus annuus L.). Plant Sci 121:213–219

    Article  CAS  Google Scholar 

  • Schrammeijer B, Sijmons PC, Van den Elzen PJM, Hoekema A (1990) Meristem transformation of sunflower via Agrobacterium. Plant Cell Rep 9:55–60

    Article  CAS  Google Scholar 

  • Shin DH, Kim JS, Kim IJ, Yang J, Oh SK, Chung GC, Han KH (2000) A shoot regeneration protocol effective on diverse genotypes of sunflower (Helianthus annuus L.). In Vitro Cell Dev Biol Plant 36:273–278

    Article  CAS  Google Scholar 

  • Song JY, Mattson NS, Jeong BR (2011) Efficiency of shoot regeneration from leaf, stem, petiole and petal explants of six cultivars of Chrysanthemum morifolium. Plant Cell Tiss Organ Cult 107:295–304

    Article  Google Scholar 

  • Sujatha M, Vijay S, Vasavi S, Veera Reddy P, Chander Rao S (2012) Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.). Plant Cell Tiss Organ Cult. doi:10.1007/s11240-012-0149-2

  • Vega TA, Nestares GM, Zorzoli R, Picardi L (2006) Responsive regions for direct organogenesis in sunflower cotyledons. Acta Physiol Plant 28:427–432

    Article  CAS  Google Scholar 

  • Vega TA, Nestares GM, Pratta G, Zorzoli R, Gattuso S, Picardi L (2007) Biochemical and histological changes associated with in vitro responses in sunflower cotyledonary explants. In Vitro Cell Dev Biol Plant 43:415–422

    Article  CAS  Google Scholar 

  • Von Arnold S, Gronroos R (1986) Anatomical changes and peroxidase activity after cytokinin treatments inducing adventitious bud formation on embryos of Picea abies. Bot Gaz 147:425–431

    Article  Google Scholar 

  • Weber S, Friedt W, Landes N, Molinier J, Himber C, Rousselin P, Hahne G, Horn R (2003) Improved Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): assessment of macerating enzymes and sonication. Plant Cell Rep 21:475–482

    PubMed  CAS  Google Scholar 

  • Witrzens B, Scowcroft WR, Downes RW, Larkin PJ (1988) Tissue culture and plant regeneration from sunflower (Helianthus annuus) and interspecific hybrids (H. tuberosus x H. annuus). Plant Cell Tiss Org Cult 13:61–76

    Article  Google Scholar 

Download references

Acknowledgments

We thank the Project Director, Directorate of Oilseeds Research for providing the necessary facilities for carrying out the work; Dr. Gerald Seiler, Research Botanist, USDA-ARS, Fargo, USA for careful editing of the manuscript and Dr. Laura Marek, Sunflower Curator, USDA-ARS, Ames, USA for supplying the seeds of sunflower core germplasm. The Financial support from the Department of Biotechnology, Government of India for the project (BT/PR9572/AGR/02/432/2007) is duly acknowledged.

Author information

Authors and Affiliations

  1. Directorate of Oilseeds Research, Rajendranagar, Hyderabad, 500 030, India

    Mulpuri Sujatha, Sheri Vijay, Singareddy Vasavi & Sankaraneni Chander Rao

  2. NBPGR Regional Station, Rajendranagar, Hyderabad, 500 030, India

    Natarajan Sivaraj

Authors
  1. Mulpuri Sujatha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sheri Vijay
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Singareddy Vasavi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Natarajan Sivaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sankaraneni Chander Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mulpuri Sujatha.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sujatha, M., Vijay, S., Vasavi, S. et al. Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds. Plant Cell Tiss Organ Cult 111, 359–372 (2012). https://doi.org/10.1007/s11240-012-0202-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-012-0202-1

Keywords

Search

Navigation