Summary
This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CPW:
-
basic protoplast washing medium according to Power and Chapman
- CPW972 CPW:
-
basic medium supplemented with 9% mannitol and 7.2% sorbitol
- DAPI:
-
4′,6-diamidino-2-phenylindole
- NLN:
-
nutrient medium according to Lichter modified by Pechan and Keller
- NLN13 NLN:
-
medium supplemented with 13% sucrose
- NLNP NLN13:
-
supplemented with 7.2% sorbitol
References
Bajaj YPS (1974) Isolation and culture studies on pollen tetrad and pollen mother cell protoplasts. Plant Sci Lett 3: 93–99
— (1983) Haploid protoplasts. Int Rev Cytol Suppl 16: 113–141
—, Cocking EC (1973) The isolation, culture and fusing of pollen protoplasts. In Vitro 8: 420
—, Davey MR (1974) The isolation and ultrastructure of pollen protoplasts. In: Linskens HF (ed) Fertilization in higher plants. Elsevier, Amsterdam, pp 73–80
Baldi BG, Franceschi VR, Loewus FA (1987) Preparation and properties of pollen sporoplasts. Protoplasma 141: 47–55
Bhojwani SS, Cocking EC (1972) Isolation of protoplast from pollen tetrads. Nat New Biol 239: 29–30
Fu CM, Sun MX, Zhou C, Yang HY (1996) Isolation of fertilized embryo sac and triggering of zygote division in vitro inNicotiana tabacum. Acta Bot Sin 38: 262–267
Hause B, Hause G (1996) Induction of embryogenesis in microspores and pollen ofBrassica napus L. cv. Topas. PhD thesis, Wageningen Agricultural University, Wageningen, The Netherlands
—, van Veenendaal WLH, Hause G, van Lammeren AAM (1994) Expression of polarity during early development of microspore-derived and zygotic embryos ofBrassica napus L. cv. Topas. Bot Acta 107: 407–415
Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Technol 45: 115–120
Holm PB, Knudsen S, Mouritzen P, Nigri D, Olsen FL, Roue C (1994) Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell. Plant Cell 6: 531–543
Hu H, Huang B (1987) Application of pollen-derived plants to crop improvement. Int Rev Cytol 107: 397–420
Imamura J, Potrykus I (1983) Isolation tetrad protoplasts develop to the binucleate stage in tobacco (Nicotiana tabacum cv. Havana). In: Potrykus I, Harms CT, Hinnen A, Hütter R, King PJ, Shillito RD (eds) Protoplasts 1983: Poster proceedings 6th International Protoplast Symposium. Birkhäuser, Basel, pp 48–49
Kao KN, Michayluk MR (1975) Nutritional requirements for growth ofVicia hajastana cells and protoplasts at very low population density in liquid media. Planta 126: 105–110
Knox RB, Williams EG, Dumas C (1986) Pollen, pistil, and reproductive function in crop plants. Plant Breed Rev 4: 9–71
Kranz E, Lörz H (1993) In vitro fertilization with isolated single gametes results in zygotic embryogenesis and fertile maize plants. Plant Cell 5: 739–746
Liang L, Xiu B, Zheng C, Zhou C (1993) Pollen cryopreservation and protoplast isolation inBrassica napus. J Wuhan Univ (Nat Sci Ed) 6: 133–136
Lichter R (1982) Induction of haploid plants from isolated pollen ofBrassica napus. Z Pflanzenphysiol 105: 211–221
Moloney MM, Holbrook LA (1993) Transformation and foreign gene expression. In: Labana KS, Banga SS, Banga SS (eds) Breeding oilseed brassicas. Springer, Berlin Heidelberg New York Tokyo, pp 148–167 (Monographs on theoretical and applied genetics, vol 19)
Morikawa H, Nishihara M (1997) Use of pollen in gene transfer. In: Shivanna KR, Sawhney VK (eds) Pollen biotechnology for crop production and improvement. Cambridge University Press, Cambridge, pp 423–437
Palmer CE, Keller WA (1997) Pollen embryos. In: Shivanna KR, Sawhney VK (eds) Pollen biotechnology for crop production and improvement. Cambridge University Press, Cambridge, pp 392–422
Pechan P, Keller WA (1988) Identification of potentially embryogenic microspores inBrassica napus. Physiol Plant 74: 377–384
Power JB, Chapman JV (1985) Isolation, culture and genetic manipulation of plant protoplasts. In: Dixon RA (ed) Plant cell culture: a practical approach. IRL Press, Oxford, pp 37–66
Rajasekhar EW (1973) Nuclear divisions in protoplasts isolated from pollen tetrads ofDatura metel. Nature 246: 223–224
Southworth D (1974) Solubility of pollen exine. Am J Bot 61: 36–44
Sun M, Yang HY, Zhou C (1995) Single pair fusion of various combinations between female gametoplasts and other protoplasts inNicotiana tabacum. Acta Bot Sin 37: 1–6
—, Kieft H, van Lammeren AAM (1998) Cotyledon derived haploid and diploid protoplast culture and diploid plant regeneration inBrassica napus L cv. Topas. Can J Bot 76: 530–541
Tanaka L, Kitazuma C, Ito M (1987) The isolation and culture of lily pollen protoplasts. Plant Sci 50: 205–211
Wu Y, Zhou C (1992) An ultrastructural study on triggering of cell division in young pollen protoplast culture ofHemerocallis fulva L. Acta Bot Sin 34: 20–25
Xia HJ, Zhou C, Yang HY (1996) Isolation and early in vitro development of young pollen protoplasts inNicotiana tabacum. Acta Bot Sin 38: 113–117
Zhou C (1988) A study on isolation and culture of pollen protoplasts. Plant Sci 59: 101–109
—, (1989) Cell division in pollen protoplast culture ofHemerocallis fulva L. Plant Sci 62: 229–235
—, Wu Y (1990) Two pathways in pollen protoplast culture: cell division and tube growth. In: Nijkamp HJJ, Plas LHW van der, Aartrijk I (eds) Progress in plant cellular and molecular biology: proceedings of the 7th International Congress on Plant Tissue and Cell Culture. Kluwer, Dordrecht, pp 222–227
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sun, M., Kieft, H., Zhou, C. et al. A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas. Protoplasma 208, 265–274 (1999). https://doi.org/10.1007/BF01279098
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01279098