Study on seedling development of cotyledon embryo in microspore culture of Brassica napus L.

doi:10.14088/j.cnki.issn0439-8114.2021.16.003

Abstract

Abstract: The embryos induced by microspore cells in Brassica napus L. were cotyledon type and non-cotyledon type such as globular embryo, heart-shaped embryo and unregular embryos structure. Cotyledon embryos can grow into plantlets quickly on seedling medium, but non-cotyledon embryos are easy to browning and die. Therefore, reducing non-cotyledon embryos and obtaining cotyledon embryos is the key point to improve the efficiency of microspore regeneration.In this research, we isolated microspore cells from three varieties and transferred them to different media and culture methods. In order to optimize the experiment, the microspores isolated from three varieties were cultured in the dark at 25 ℃ for 2 weeks. They were transferred from the solid-liquid double-layer culture medium and treated with different agitation period, temperature, concentrations of activated carbon and 6-BA. The results showed that the number of cotyledon embryos grown on the solid-liquid two-layer medium was significantly increased than that in the liquid medium. The number of cotyledon embryos obtained from the combination of solid-liquid double-layer medium and agitation for 1 week was evidently increased and the number of non-cotyledon embryos was significantly reduced. When microspores were cultured at 23 ℃, there was no significant difference between the total number of embryos and the control, but the number of cotyledon embryos increased significantly. The result on the effect of various concentrations of activated charcoal and 6-BA on sublayer medium indicated the combination of 0.1% and 0.25 mg/L 6-BA was very promising, as it outperformed the control treatment in the yield of normal looking embryos. In this study, we developed a two-step （liquid culture and solid-liquid double-layer culture） microspore culture protocol. It can effectively obtain high-quality cotyledon embryos with ability to regenerate seedlings, which may greatly accelerate the application of microspore-derived DH plants as breeding tool.

Key words: Brassica napus L, microspore embryogenesis, cotyledon embryos, solid-liquid double layer culture medium, regenerated plants

CLC Number:

S565.4

WAN Li-li, WANG Zhuan-rong, XU Yi, KANG Lei, YANG Guang-sheng, XIN Qiang, HONG Deng-feng, SUN Yu-hong. Study on seedling development of cotyledon embryo in microspore culture of Brassica napus L.[J]. HUBEI AGRICULTURAL SCIENCES, 2021, 60(16): 15-20.

References

[1] LICHTER R.Induction of haploid plants from isolated pollen of Brassica napus[J]. Z Pflanzenphysiol,1982, 105(5):427-434.
[2] FERRIE A M R, CASWELL K L. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production[J]. Plant cell,tissue and organ culture,2011,104(3):301-309.
[3] FERRIE A M R, MÖLLERS C. Haploid and doubled haploids in Brassica spp. for genetic and genomic research[J]. Plant cell,tissue and organ culture,2011, 104(3):375-386.
[4] GU H H, HAGBERG P, ZHOU W J.Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)[J]. Plant growth regulation,2004,42(2):137-143.
[5] KISZCZAK W, KOWALSKA U, KAPUŚCIŃSKA A, et al. Effect of low temperature on in vitro androgenesis of carrot (Daucus carota L.)[J]. In vitro cellular & developmental biology-plant, 2015, 51(2):135-142.
[6] ZHOU W J, HAGBERG P, TANG G X.Increasing embryogenesis and doubling efciency by immediate colchicine treatment of isolated microaspores in spring Brasscia napus[J]. Euphytica, 2002,128(1):27-34.
[7] LESKOVŠEK L, JAKŠE M, BOHANEC B. Doubled haploid production in rocket (Eruca sativa Mill.) through isolated microspore culture[J]. Plant cell,tissue and organ culture, 2008, 93:181-189.
[8] KIM M, JANG I C, KIM J A, et al.Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture[J]. Plant cell reports,2008, 27(3):425-434.
[9] LANTOS C, JUHÁSZ A G, SOMOGYI G, et al. Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat[J]. Plant cell,tissue and organ culture, 2009, 97(3):285-293.
[10] SUPENA E D J, SUHARSONO S, JACOBSEN E, et al. Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)[J]. Plant cell reports, 2006,25(1):1-10.
[11] SUPENA E D J, MUSWITA W, SUHARSONO S, et al. Evaluation of crucial factors for implementing shed-microspore culture of Indonesian hot pepper (Capsicum annuum L.) cultivars[J]. Scientia horticulturae, 2006,107(3):226-232.
[12] GYULAI G, GÉMESNÉ J A, SÁGI Z S, et al. Doubled haploid development and PCR-analysis of F₁ hybrid derived DH-R 2 paprika (Capsicum annuum L.) lines[J]. Journal of plant physiology, 2000, 156(2):168-174.
[13] NOWAKZYK P, KISIALA A.Effect of selected factors on the effectiveness of (Capsicum annuum L.) anther culture[J]. Journal of applied genetics, 2006, 47(2):113-117.
[14] IRIKOVA T, GROZEVA S, RODEVA V.Anther culture in pepper (Capsicum annuum L.) in vitro[J]. Acta physiologiae plantarum, 2011, 33(5):1559-1570.
[15] STASOLLA C, YEUNG E C.Recent advances in conifer somatic embryogenesis: Improving somatic embryo quality[J]. Plant cell,tissue and organ culture, 2003, 74(1):15-35.
[16] STEINITZ B, KÜSEK M, TABIB Y, et al. Pepper (Capsicum annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot[J]. In vitro cellular & developmental biology-plant,2003, 39(3) : 296-303.
[17] HU T, KASHA K J.Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture[J]. Plant cell reports,1997,16(8):520-525.
[18] PEDROSO M C, PAIS M S.Induction of microspore embryogenesis in (Camellia japonica cv. Elegans)[J]. Plant cell,tissue and organ culture,1994, 37(2):129-136.
[19] LI H, DEVAUX P.High frequency regeneration of barley doubled haploid plants from isolated microspore culture[J]. Plant science,2003, 164(3):379-386.
[20] AGARWAL P K, AGARWAL P, CUSTERS J B M, et al. PCIB an antiauxin enhances microspore embryogenesis in microspore culture of Brassica juncea[J]. Plant cell,tissue and organ culture,2006, 86(2):201-210.
[21] ZENG A, SONG L, CUI Y, et al.Reduced ascorbate and reduced glutathione improve embryogenesis in broccoli microspore culture[J]. South African journal of botany, 2017, 109:275-280.
[22] VERÓNICA P V, RENAU-MORATA B, SIFRES A, et al. Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.)[J]. Plant cell,tissue and organ culture, 2013, 112(3):353-360.
[23] KIM M, PARK E J, AN D, et al.High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.)[J]. Plant cell,tissue and organ culture, 2013, 112(2):191-201.