Abstract
Haploid induction in rocket (Eruca sativa Mill.), a novel and increasingly important vegetable, was studied in microspore culture. A procedure based on a high sucrose NLN medium and heat shock treatment resulted in nuclear divisions and embryo induction. The effect of genotype both among seed lots and among single plants was a major factor influencing embryo formation. The addition of activated charcoal was essential for obtaining reproducible results, 0.2 mg l−1 being superior to 1.0 mg l−1. A 24 h heat shock treatment at 32°C doubled the embryogenic response compared to a 48 h treatment. Embryo conversion was only efficient (23%) for embryos that had been cultured on medium with activated charcoal and subcultured on solid B5 medium; pretreatment of embryos with ABA or desiccation for 1–3 weeks inhibited embryo conversion. Analysis of ploidy level revealed that the majority (65.6%) of 489 regenerated plantlets tested were diploid. Breeding programs and genetic studies of rocket are likely to benefit substantially from the established method.
Similar content being viewed by others
Abbreviations
- ABA:
-
Abscisic acid
- AC:
-
Activated charcoal
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- MTT:
-
3-4,5 Dimethylthiazol-2-yl-2,5 diphenyl-tetrazol bromide
References
Adamus A (2000) Androgenesis in Polish Brassica oleracea L. genotypes. In: COST Action 824-Biotechnological approaches for utilisation of gametic cells. Office for Official Publications of the European Communities, Bled, Slovenia, pp 133–136
Agarwal PK, Agarwal P, Custers JBM, Liu CM, Bhojwani SS (2006) PCIB an antiauxin enhances microspore embryogenesis in microspore culture of Brassica juncea. Plant Cell Tiss Organ Cult 86:201–210
Akeroyd J (2006) Rocket-wild or wall? Plantsman 5(2) 98–99
Bennett RN, Carvalho R, Mellon FA, Eagles J, Rosa EAS (2007) Identification and quantification of glucosinolates in sprouts derived from seeds of wild Eruca sativa L. (salad rocket) and Diplotaxis tenuifolia L. (wild rocket) from diverse geographical locations. J Agric Food Chem 55:67–74
Cegielska-Taras T, Tykarska T, Szala L, Kuras M, Krzymanski J (2002) Direct plant development from microspore-derived embryos of winter oilseed rape Brassica napus L. ssp oleifera (DC.) Metzger. Euphytica 124:341–347
Chanana NP, Dhawan V, Bhojwani SS (2005) Morphogenesis in isolated microspore cultures of Brassica juncea. Plant Cell Tiss Organ Cult 83:169–177
Custers J (2003) Microspore culture in rapeseed (Brassica napus L.). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 185–193
Dias JCD (1999) Effect of activated charcoal on Brassica oleracea microspore culture embryogenesis. Euphytica 108:65–69
Dias JCD (2001) Effect of incubation temperature regimes and culture medium on broccoli microspore culture embryogenesis. Euphytica 119:389–394
Duijs JG, Voorrips RE, Visser DL, Custers JBM (1992) Microspore culture is successful in most crop types of Brassica oleracea L. Euphytica 60:45–55
Ferrie A (2003) Microspore culture of Brassica species. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 205–215
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Germanà MA, Chiancone B, Lain O, Testolin R (2005) Anther culture in Citrus clementina: a way to regenerate tri-haploids. Aust J Agric Res 56:839–845
Gland A, Lichter R, Schweiger HG (1988) Genetic and exogenous factors affecting embryogenesis in isolated microspore cultures of Brassica napus L. J Plant Physiol 132:613–617
Gu HH, Zhou WJ, Hagberg P (2003) High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis. Euphytica 134:239–245
Gu HH, Hagberg P, Zhou WJ (2004) Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.). Plant Growth Regul 42:137–143
Halkjaer M, Ringgaard S (2000) Genetic and exogenous factors affecting the induction of double haploid cabbage (Brassica oleracea var. capitata L.). In: COST Action 824-Biotechnological approaches for utilisation of gametic cells. Office for Official Publications of the European Communities, Bled, Slovenia, pp 147–150
Hansen M (2000) ABA treatment and desiccation of microspore-derived embryos of cabbage (Brassica oleracea ssp capitata L.) improve plant development. J Plant Physiol 156:164–167
Hansen M, Svinnset K (1993) Microspore culture of swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media. Plant Cell Rep 12:496–500
Ilić-Grubor K, Attree SM, Fowke LC (1998) Induction of microspore-derived embryos of Brassica napus L. with polyethylene glycol (PEG) as osmoticum in low sucrose medium. Plant Cell Rep 17:329–333
Kasha KJ, Maluszynski M (2003) Production of doubled haploids in crop plants. An introduction. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 1–4
Keller WA, Armstrong KC (1979) Stimulation of embryogenesis and haploid production in Brassica campestris anther cultures by elevated temperature treatments. Theor Appl Genet 55: 65–67
Leelavathi S, Reddy VS, Sen SK (1987) Somatic-cell genetic-studies in Brassica species. 2. production of androgenetic haploid plants in Brassica nigra (L.) Koch. Euphytica 36:215–219
Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434
Lichter R (1989) Efficient yield of embryoids by culture of isolated microspores of different Brassicaceae species. Plant Breed 103:119–123
Mathias R (1988) An improved in vitro culture procedure for embryoids derived from isolated microspores of rape (Brassica napus L). Plant Breed 100:320–322
Mathias R, Röbbelen G (1991) Effective diploidization of microspore-derived haploids of rape (Brassica napus L.) by in vitro colchicine treatment. Plant Breed 106:82–84
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 Tiss Organ Cult 32:241–246
Pignone D (1996) Present status of rocket genetic resources and conservation activities. In: Rocket: a Mediterranean crop for the world. Int Plant Genet Resour Inst., Legnaro, Italy, pp 2–12
Prem D, Gupta K, Agnihotri A (2005) Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (Brassica juncea (L.) Czern and Coss). In Vitro Cell Dev Biol Plant 41:266–273
Rudolf K, Bohanec B, Hansen M (1999) Microspore culture of white cabbage, Brassica oleracea var. capitata L.: genetic improvement of non-responsive cultivars and effect of genome doubling agents. Plant Breed 118:237–241
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the re-programming of plant microspores towards in vitro embryogenesis. Physiol Plant 127:519–534
Stipic M, Campion B (1997) An improved protocol for androgenesis in cauliflowers (Brassica oleracea var. botrytis). Plant Breed 116:153–157
Sunderland N, Collins GB, Dunwell JM (1974) Role of nuclear fusion in pollen embryogenesis of Datura innoxia Mill. Planta 117:227–241
Tian H, Yao CY, Sun MX (2004) High frequency conversion of microspore-derived embryos of Brassica napus cv. Topas by supplemental calcium and vitamins. Plant Cell Tiss Organ Cult 76:159–165
Vižintin L, Bohanec B (2004) In vitro manipulation of cucumber (Cucumis sativus L.) pollen and microspores: isolation procedures, viability tests, germination, maturation. Acta Biol Crac Ser Bot 46:177–183
Zhang GQ, Zhou WJ, Gu HH, Song WJ, Momoh EJJ (2003) Plant regeneration from the hybridization of Brassica juncea and B. napus through embryo culture. J Agron Crop Sci 189:347–350
Zhang GQ, He Y, Xu L, Tang GX, Zhou WJ (2006a) Genetic analyses of agronomic and seed quality traits of doubled haploid population in Brassica napus through microspore culture. Euphytica 149:169–177
Zhang GQ, Zhang DQ, Tang GX, He Y, Zhou WJ (2006b) Plant development from microspore-derived embryos in oilseed rape as affected by chilling, desiccation and cotyledon excision. Biol Plant 50:180–186
Zhao JP, Gervais C, Simmonds J, Newcom W, Simmonds DH (2005) Inhibitory effects of filter-sterilized media on microspore development and embryogenesis in Brassica napus and Triticum aestivum. In Vitro Cell Dev Biol Plant 41:281–284
Zhou WJ, Hagberg P, Tang GX (2002) Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus. Euphytica 128:27–34
Acknowledgements
The authors thank Dr. JBM Custers (Plant Research International, Wageningen, NL) for his generous help with initial stages of protocol establishment and critically reading the manuscript. This work was supported by Slovenian Research project P4-0077.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Leskovšek, L., Jakše, M. & Bohanec, B. Doubled haploid production in rocket (Eruca sativa Mill.) through isolated microspore culture. Plant Cell Tiss Organ Cult 93, 181–189 (2008). https://doi.org/10.1007/s11240-008-9359-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-008-9359-z