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Protoplast isolation and culture for somatic hybridization of Lupinus angustifolius and L. subcarnosus

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Abstract

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 105 protoplasts g−1 fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.

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Abbreviations

BAP:

6-Benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

GA3 :

Gibberellic acid

IBA:

Indole-3-butyric acid

IAA:

Indole-3-acetic–acid

MES:

2-N-morpholino ethanesulfonic acid

NAA:

1-Naphthaleneacetic acid

PVP:

Polyvinyl pyrrolidone

References

  • Babaoglu M (2000) Protoplast isolation in lupin (Lupinus mutabilis Sweet): determination of optimum explant sources and isolation conditions. Turk J Bot 23:177–185

    Google Scholar 

  • Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep 16:513–519

    Google Scholar 

  • Binding H, Nehls R, Schieder O, Sopory SK, Wenzel G (1978) Regeneration of mesophyll protoplasts isolated from dihaploid clones of Solanum tuberosum. Physiol Plant 43:52–54. doi:10.1111/j.1399-3054.1978.tb01565.x

    Article  Google Scholar 

  • Boehmer P, Meyer B, Jacobsen HJ (1995) Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus. Plant Cell Rep 15:26–29. doi:10.1007/BF01690247

    Article  CAS  Google Scholar 

  • Dan Y, Reichert NA (1998) Organogenic regeneration of soybean from hypocotyl explants. In Vitro Cell Dev Biol Plant 34:14–21. doi:10.1007/BF02823117

    CAS  Google Scholar 

  • Davey MR, Busch E, Power JB (1974) Cultural stadies of a dividing legume protoplast system. Plant Sci Lett 3:127–133. doi:10.1016/0304-4211(74)90112-6

    Article  Google Scholar 

  • Davey MR, Marchant R, Power JB (2003) Protoplasts of grain and forage legumes: their exploitation in genetic manipulation, physiological investigation and plant-pathogen interactions. In: Jaiwal PK, Singh RP (eds) Improvement strategies for Leguminosae biotechnology, pp 133–153

  • Dorokhov DB, Klocke E (1997) A rapid and economic technique for RAPD analysis of plant genomes. Russ J Genet 33:358–365

    CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158. doi:10.1016/0014-4827(68)90403-5

    Article  PubMed  CAS  Google Scholar 

  • Griesbach RJ (1988) Recent advances in the protoplast biology of flower crops. Sci Hortic (Amsterdam) 37:247–256. doi:10.1016/0304-4238(88)90026-X

    Article  Google Scholar 

  • Kao KN (1977) Chromosomal behavior in somatic hybrids of soybean-Nicotiana glauca. Mol Gen Genet 150:225–230. doi:10.1007/BF00268120

    Article  Google Scholar 

  • Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126:105–110. doi:10.1007/BF00380613

    Article  CAS  Google Scholar 

  • Kasten W, Paradies T, Kunert R, Straka P (1991) Progress in realization of interspecific hybrids in the genus Lupinus by means of an embryo rescue technique. Biol Zent Bl 110:301–309

    Google Scholar 

  • Menczel L, Nagy F, Kiss ZS, Maliga P (1981) Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195. doi:10.1007/BF00264975

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:472–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  Google Scholar 

  • Nelson MN, Phan HTT, Ellwood SR, Moolhuijzen PM, Hane J, Williams A, O’Lone CE, Fosu-Nyarko J, Scobie M, Cakir M, Jones MGK, Bellgard M, KsiSkiewicz M, Wolko B, Barker SJ, Oliver RP, Cowling WA (2006) The first gene-based map of Lupinus angustifolius L.-location of domestication genes and conserved synteny. Theor Appl Genet 113:225–238. doi:10.1007/s00122-006-0288-0

    Article  PubMed  CAS  Google Scholar 

  • Ochatt SJ, Mousset-Declas C, Rancillac M (2000) Fertile pea plants regenerate from protoplasts when calluses have not undergo endoreplication. Plant Sci 156:177–183. doi:10.1016/S0168-9452(00)00250-8

    Article  PubMed  CAS  Google Scholar 

  • Patel MB, Bhardwaj BR, Joshi A (1991) Organogenesis in Vigna radiata (L) Wilzek. Indian J Exp Biol 29:619–622

    Google Scholar 

  • Pigeare A, Abernethy D, Smith PM, Simpson K, Fletcher N, Lu CY, Atkins CA, Cornish E (1997) Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices. Mol Breed 3:341–349. doi:10.1023/A:1009642620907

    Article  Google Scholar 

  • Prem Anand R, Ganapathi A, Anbazhagan VR, Vengadesan G, Kolothungan S, Selvaraj N (2001) Plant regeneration from immature derived cotyledons of Vigna unguiculata (L.). Walp Curr Sci 80:671–674

    Google Scholar 

  • Przyborowski JA, Packa D (1997) Embryo development after interspecific hybridization of Lupinus albus L., L. mutabilis Sweet and L. angustifolius. J Appl Genet 38:131–141

    Google Scholar 

  • Ramana Rao VV, Chary SN, Bhall JK (1992) Somatic embryogenesis from mesophyll protoplasts of wild and cultivated species of pigeonpea. In: Moss JP (ed) Biotechnology and crop improvement in Asia ICRISAT. Patancheru, India

    Google Scholar 

  • Sacristan MD, Gerdemann-Knörck M, Schieder O (1989) Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion. Theor Appl Genet 78:174–200. doi:10.1007/BF00288799

    Article  Google Scholar 

  • Sarangi BK, Kuchuk N, Gleba YY (1992) Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.). Plant Cell Rep 11:462–465. doi:10.1007/BF00232691

    Article  Google Scholar 

  • Sator C (1985) Studies on shoot regeneration of lupins. Plant Cell Rep 4:126–128. doi:10.1007/BF00571297

    Article  Google Scholar 

  • Schäfer-Menuhr A (1987) Isolation und Kultur von Lupinenprotoplasten. I. Protoplasten aus Blättern von Lupinus angustifolius Sorte Kubesa. Landbauforschung Völkenrode 37:117–120

    Google Scholar 

  • Schäfer-Menuhr A (1988) Isolation und Kultur von Lupinenprotoplasten. III. Protoplasten aus Zellsuspensionskulturen von Lupinus polyphyllus. Landbauforschung Völkenrode 38:99–102

    Google Scholar 

  • Schäfer-Menuhr A (1989a) Isolation und Kultur von Lupinenprotoplasten. IV. Regeneration von Pflanzen aus Protoplasten. Landbauforschung Völkenrode 39:133–136

    Google Scholar 

  • Schäfer-Menuhr A (1989b) Biotechnology as a tool for plant breeders. In: Biotechnology of food and feed production. Proceedings of an UNECE symposium. Budapest, Hungary, 13–17 June 1988, pp 115–124

  • Schäfer-Menuhr A, Stürmer S (1987) Isolation und Kultur von Lupinenprotoplasten. II. Modifikation von Nährmedien zur beschleunigten Teilung von Protoplasten aus Blättern von Lupinus angustifolius Sorte Kubesa. Landbauforschung Völkenrode 37:231–234

    Google Scholar 

  • Sen J, Mukherje SG (1998) In vitro induction of multiple shoots and plant regeneration in Vigna. In Vitro Cell Dev Biol Plant 34:276–280. doi:10.1007/BF02822733

    Article  CAS  Google Scholar 

  • Shekhawat NS, Galston AW (1983) Isolation, culture and regeneration of mothbean Vigna acanitifolia leaf protoplasts. Plant Sci Lett 32:43–51. doi:10.1016/0304-4211(83)90097-4

    Article  Google Scholar 

  • Sinha A, Caligari PDS (2004) Aspects of isolation underpinning mitotic behaviour in lupin protoplasts. Aust J Bot 52:669–676. doi:10.1071/BT03153

    Article  Google Scholar 

  • Sinha A, Caligari PDS (2005) Enhanced protoplast division by encapsulation in droplets: an advances towards somatic hybridization in recalcitrant white lupin. Ann Appl Biol 146:441–448. doi:10.1111/j.1744-7348.2005.040097.x

    Article  Google Scholar 

  • Sinha A, Wetten AC, Caligari PDS (2003) Effect of biotic factors on the isolation of Lupinus albus L. protoplasts. Aust J Bot 51:103–109. doi:10.1071/BT01104

    Article  Google Scholar 

  • Sinha A, Wetten AC, Caligari PDS (2004) Optimisation of protoplast production in white lupin. Biol Plant 47:21–25. doi:10.1023/A:1027316511865

    Article  Google Scholar 

  • Sonntag K, Rudloff E, Ruge-Wehling B (2006) Wide crosses to improve pH-tolerance in the narrow-leafed lupin (Lupinus angustifolius). In: van Santen E, Hill GD (eds) Mexico, where old and new world lupins meet. Proceedings of the 11th international lupin conference, 04–09 May 2005, Guadalajara, pp 17–19

  • Tegeder M, Koehn H, Nibbe M, Schieder O, Pickardt T (1996) Plant regeneration of protoplasts of Vicia narbonensis via somatic embryogenesis and shoot organogenesis. Plant Cell Rep 16:22–25. doi:10.1007/BF01275442

    Article  CAS  Google Scholar 

  • Thomas MR, Johnson LB, White FF (1990) Selection of interspecific somatic hybrids of Medicago by using Agrobacterium-transformed tissues. Plant Sci 69:189–198. doi:10.1016/0168-9452(90)90117-7

    Article  Google Scholar 

  • Warren G (1991) Protoplast isolation and fusion. In: Stafford A, Warren G (eds) Plant cell and tissue culture. Milton Keynes, UK, pp 50–81

    Google Scholar 

  • Webb JK, Woodcock S, Chamberlain DA (1987) Plant regeneration from protoplasts of Trifolium repens and Lotus corniculatus. Plant Breed 98:111–118. doi:10.1111/j.1439-0523.1987.tb01102.x

    Article  Google Scholar 

  • Wetten A, Bese N, Masumbuko L, Caligari PDS (1994) Isolation of protoplasts from Lupinus spp., for the production of interspecific hybrids. Proceedings of 1st Australian lupin technical symposium, Western Australia, Perth, October 1994, p 321

  • Wetten A, Sinha A, Caligari PDS (1999) Electrofusion of lupin protoplasts for the production of interspecific hybrids. In: Hill GD (ed) Proceedings of the 8th international lupin conference. Asilomar, California, USA, 11–16 May 1996, pp 270–272

  • Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining the viability of cultured cells. Stain Technol 47:189–194

    PubMed  CAS  Google Scholar 

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Acknowledgment

The authors thank Irmgard Müller, Susan Dreischhoff and Hildegard Dreier for their excellent technical assistance.

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Authors and Affiliations

  1. Julius Kühn Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Agricultural Crops, Erwin-Baur-Straße 27, 06484, Quedlinburg, Germany

    Karin Sonntag, Brigitte Ruge-Wehling & Peter Wehling

  2. Rudolf-Schick-Platz 3a, 18190, OT Groß Lüsewitz, Sanitz, Germany

    Karin Sonntag

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  1. Karin Sonntag
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  2. Brigitte Ruge-Wehling
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  3. Peter Wehling
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Correspondence to Karin Sonntag.

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Sonntag, K., Ruge-Wehling, B. & Wehling, P. Protoplast isolation and culture for somatic hybridization of Lupinus angustifolius and L. subcarnosus . Plant Cell Tiss Organ Cult 96, 297–305 (2009). https://doi.org/10.1007/s11240-008-9487-5

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