Haploid Embryogenesis in Isolated Microspore Culture of Carrots (Daucus carota L.)

The process of embryogenesis in isolated microspore culture was studied in eight carrot accessions of different origin. The ½NLN-13 medium supplemented with 0.2 mg/L 2,4D and 0.2mg/L kinetin was used to induce embryogenesis. The temperature treatment was performed at 5–6 °C for three days, followed by cultivation at 25 °C in darkness. As was shown, the first embryogenesis was only observed in microspores at the late vacuolated stage when the nucleus moved from the center to one pole following the long cell axis. Depending on the nucleus position, the microspore can divide into two equal or two different sized cells. Following divisions occurred either in one of these cells or in two. However, microspores that divided into two unequal cells were morphologically different form bi-cellular pollen grain. Embryogenic divisions in bi-cellular pollen grains were not observed. First divisions began by the third day of cultivation, and continued until the globular embryoid stage that was well-seen after the fourth week of cultivation. The already-formed embryoids can develop the secondary embryoids on their surface. Depending on the genotype, up to 1000 secondary embryoids can be produced from one embryoid in the liquid MSm medium supplemented with 0.1 mg/L of kinetin for regeneration. All carrot accessions studied were split into three groups: responsive genotypes, weakly responsive genotypes, and reluctant genotypes. The highest yield was 53 initial embryoids per a 6 cm diameter petri dish. Thus, the Nantskaya 4 cultivar totally produced 256 initial embryoids, out of which 94 developed into green plantlets and 162 into albino plantlets, whereas 97 initial embryoids with 45 albino plantlets formed from them were obtained from Chantenay cultivar.

[1]  O. V. Romanova,et al.  Production of doubled haplois in Brassica purpuraria , 2019 .

[2]  F. Békés,et al.  Utilization of in Vitro Anther Culture in Spelt Wheat Breeding , 2019, Plants.

[3]  U. Kowalska,et al.  Comparison of methods for obtaining doubled haploids of carrot , 2017 .

[4]  I. Żur,et al.  Identification of QTLs associated with albino plant formation and some new facts concerning green versus albino ratio determinants in triticale (×Triticosecale Wittm.) anther culture , 2015, Euphytica.

[5]  M. Catal,et al.  Haploids in the improvement of Crucifers , 2014 .

[6]  M. Asif.  Progress and Opportunities of Doubled Haploid Production , 2013, SpringerBriefs in Plant Science.

[7]  J. M. Seguí-Simarro,et al.  Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.) , 2013, Acta Physiologiae Plantarum.

[8]  Hong Hu,et al.  Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.) , 2012, Plant Cell, Tissue and Organ Culture (PCTOC).

[9]  C. Möllers,et al.  Haploids and doubled haploids in Brassica spp. for genetic and genomic research , 2011, Plant Cell, Tissue and Organ Culture (PCTOC).

[10]  Z. Zhiwei,et al.  Induction of Microspores-derived Embryos and Calli from Anther Culture in Carrot , 2010 .

[11]  U. Kowalska,et al.  Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures , 2010, Journal of Applied Genetics.

[12]  K. Siddique,et al.  Albinism in Plants: A Major Bottleneck in Wide Hybridization, Androgenesis and Doubled Haploid Culture , 2009 .

[13]  Jinfeng Chen,et al.  Regeneration of doubled haploid plants by androgenesis of cucumber (Cucumis sativus L.) , 2007, Plant Cell, Tissue and Organ Culture.

[14]  E. Jacobsen,et al.  Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.) , 2006, Plant Cell Reports.

[15]  Ó. Vicente,et al.  In situ characterization of the late vacuolate microspore as a convenient stage to induce embryogenesis inCapsicum , 1995, Protoplasma.

[16]  R. Hagemann,et al.  The cytological basis of the plastid inheritance in angiosperms , 1989, Protoplasma.

[17]  K. Górecka,et al.  The influence of several factors on the efficiency of androgenesis in carrot. , 2005, Journal of applied genetics.

[18]  H. N. Murthy,et al.  Effect of Sugars and Amino Acids on Androgenesis of Cucumis sativus , 2004, Plant Cell, Tissue and Organ Culture.

[19]  P. Stamp,et al.  The improvement in regenerated doubled haploids from anther culture of wheat by anther transfer , 2000, Plant Cell, Tissue and Organ Culture.

[20]  R. Jain,et al.  Anther culture of recalcitrant indica × Basmati rice hybrids , 2000, Euphytica.

[21]  E. Heberle‐Bors,et al.  Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies , 1996, Plant Cell Reports.

[22]  W. Keller,et al.  Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line , 1995, Plant Cell Reports.

[23]  G. Hause.  Ultrastructural investigations of mature embryo sacs of Daaucus carota, D. aureus and D. muricatus — possible cytological explanations of paternal plastid inheritance , 1991, Sexual Plant Reproduction.

[24]  E. Heberle‐Bors.  Isolated pollen culture in tobacco: plant reproductive development in a nutshell , 1989, Sexual Plant Reproduction.

[25]  C. Phippen,et al.  Genotype, plant, bud size and media factors affecting anther culture of cauliflowers (Brassica oleracea var. botrytis) , 2004, Theoretical and Applied Genetics.

[26]  J. M. Seguí-Simarro,et al.  Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L. , 2003, Journal of structural biology.

[27]  H. N. Murthy,et al.  Embryogenesis and plant regeneration from anther cultures of Cucumis sativus L. , 2003 .

[28]  P. Devaux,et al.  High frequency regeneration of barley doubled haploid plants from isolated microspore culture , 2003 .

[29]  J. Pauk,et al.  Protocol of triticale (x Triticosecale Wittmack) microspore culture , 2003 .

[30]  S. Iwai,et al.  Effect of Low Temperature Pretreatment of Buds or Inflorescence on Isolated Microspore Culture in Brassica rapa (syn. B. campestris) , 2002 .

[31]  P. Testillano,et al.  The switch of the microspore developmental program in Capsicum involves HSP70 expression and leads to the production of haploid plants , 2001 .

[32]  P. Smýkal,et al.  Androgenesis: Affecting the fate of the male gametophyte , 2001 .

[33]  C. Konzak,et al.  Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.) , 1999, Plant Cell Reports.

[34]  K. Kasha,et al.  A cytological study of pretreatments used to improve isolated microspore cultures of wheat (Triticum aestivum L.) cv. Chris , 1999 .

[35]  E. Heberle‐Bors,et al.  Assessment of various stresses and carbohydrates for their effect on the induction of embryogenesis in isolated wheat microspores , 1999 .

[36]  N. Shmykova,et al.  CYTOLOGICAL STUDY OF EMBRYOGENESIS IN CULTURED CARROT ANTHERS , 1999 .

[37]  C. Clément,et al.  Androgenesis and albinism in Poaceae: influence of genotype and carbohydrates , 1999 .

[38]  J. Cordewener,et al.  A short severe heat shock is required to induce embryogenesis in late bicellular pollen of Brassica napus L. , 1997, Sexual Plant Reproduction.

[39]  V. Gustafson,et al.  Wheat anther culture as affected by various cultural changes and supplements , 1997 .

[40]  中村 幸司,et al.  Varietal Differences in Embryogenic and Regenerative Ability in Microspore Culture of Chinese Cabbage(Brassica rapa L. ssp. pekinensis). , 1997 .

[41]  A. Jähne,et al.  Cereal microspore culture , 1995 .

[42]  K. Murakami,et al.  CALLUS FORMATION AND REGENERATION OF ADVENTITIOUS EMBRYOS FROM CARROT, FENNEL AND MITSUBA MICROSPORES BY ANTHER AND ISOLATED MICROSPORE CULTURES , 1995 .

[43]  K. Murakami,et al.  Haploid Plant Production by Anther Culture in Carrot (Daucus carota L.) , 1993 .

[44]  Mohan B. Singh,et al.  Development of Plastids in Pollen and Tapetum of Rye-grass, Lolium perenne L , 1992 .

[45]  F. J. Zapata,et al.  Plant Regeneration from Isolated Microspore of Indica Rice , 1990 .

[46]  S. Andersen,et al.  Carrot (Daucus carota L.): In Vitro Productionof Haploids and Field Trials , 1990 .

[47]  F. J. Zapata,et al.  Callus formation and plant regeneration in isolated pollen culture of rice (Oryza sativa L. cv. Taipei 309) , 1988 .

[48]  R. Lichter.  Induction of Haploid Plants From Isolated Pollen of Brassica napus , 1982 .

[49]  Y. Kikuta,et al.  A REVISION OF THE MEDIUM FOR SOMATIC EMBRYOGENESIS IN CARROT SUSPENSION CULTURE , 1981 .

[50]  N. Sunderland,et al.  Cold-pretreatment of Excised Flower Buds in Float Culture of Tobacco Anthers , 1979 .

[51]  K. Kasha.  Haploids in higher plants : advances and potential : proceedings of the first international symposium, Guelph, Ontario, Canada June 10 to 14, 1974 , 1974 .