Hostname: page-component-78c5997874-s2hrs Total loading time: 0 Render date: 2024-11-10T20:50:57.506Z Has data issue: false hasContentIssue false
  • English
  • Français

Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb)

Published online by Cambridge University Press:  03 April 2020

Yi Hua Lin ,
Mei Zhen Lin ,
Yu Qing Chen  and
Hui Qiao Tian Open the ORCID record for Hui Qiao Tian [Opens in a new window]
Yi Hua Lin
Affiliation:
Zhangzhou Health Vocational College, Zhangzhou363000, China
Mei Zhen Lin
Affiliation:
Zhangzhou Health Vocational College, Zhangzhou363000, China
Yu Qing Chen
Affiliation:
Zhangzhou Health Vocational College, Zhangzhou363000, China
Hui Qiao Tian*
Affiliation:
School of Life Sciences, Xiamen University, Xiamen361102, China
*
Address for correspondence: Hui Qiao Tian. School of Life Sciences. Xiamen University, Xiamen, Fujian361102, China. Tel: +11 86 592 2186486. E-mail: hqtian@xmu.edu.cn
Get access Rights & Permissions [Opens in a new window]

Summary

The isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6–8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

Keywords

Allium tuberosum RoxbEggProembryoSpermZygote
Type
Research Article
Information
Zygote , Volume 28 , Issue 4 , August 2020 , pp. 278 - 285
Copyright
© Cambridge University Press 2020

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abiko, M, Maeda, H, Tamura, K, Hara-Nishimura, I and Okamoto, T (2013) Gene expression profiles in rice gametes and zygotes: identification of gamete-enriched genes and up-or down-regulated genes in zygotes after fertilization. J Exp Bot 64, 1927–40.CrossRefGoogle ScholarPubMed
Bayer, M, Nawy, T, Giglione, C, Galli, M, Meinnel, T and Lukowitz, W (2009) Paternal control of embryonic patterning in Arabidopsis thaliana. Science 323, 1485–8.CrossRefGoogle ScholarPubMed
Berger, F (2008) Double-fertilization, from myths to reality. Sex Plant Reprod 21, 3–5.CrossRefGoogle Scholar
Deng, W, Xie, YL and Qiu, YL (2018) Isolation of sperm and egg cells from pepper. J Amer Soc Hort Sci 143, 310–5.CrossRefGoogle Scholar
Frank, AC and Johnson, MA (2009) Expressing the diphtheria toxin a subunit from the HAP2 (GCS1) promoter blocks sperm maturation and produces single sperm-like cells capable of fertilization. Plant Physiol 151, 1390–400.CrossRefGoogle ScholarPubMed
Gou, XP, Yuan, T, Wei, XP and Russell, SD (2009) Gene expression in the dimorphic sperm cells of Plumbago zeylanica: transcript profiling, diversity, and relationship to cell type. Plant J 60, 33–47.CrossRefGoogle ScholarPubMed
He, EM, Wang, YY, Liu, HH, Zhu, XY and Tian, HQ (2012) Egg cell isolation in Datura stramonium L. Ann Bot Finnici 49, 7–12.CrossRefGoogle Scholar
Heslop-Harrison, J and Heslop-Harrison, Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Tech 45, 115–20.CrossRefGoogle ScholarPubMed
Imre, K and Kristof, Z (1999) Isolation and osmotic relations of developing megagametophytes of Torenia fournieri. Sex Plant Reprod 12, 152–7.CrossRefGoogle Scholar
Kranz, E, Bautor, J and Lörz, H (1991) In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex Plant Reprod 4, 12–6.CrossRefGoogle Scholar
Kranz, E and Lörz, H (1993) In vitro fertilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plants. Plant Cell 5, 739–46.CrossRefGoogle ScholarPubMed
Leljak-Levanić, D, Juranić, M and Sprunck, S (2013) De novo zygotic transcription in wheat (Triticum aestivum L.) includes genes encoding small putative secreted peptides and a protein involved in proteasomal degradation. Plant Reprod 26, 267–85.CrossRefGoogle Scholar
Lin, MZ, Chen, L, Zhu, XY, Tian, HQ and Teixeira, SJA (2012) Isolation of eggs and synergids in Ceiba speciosa. Ann Bot Fennici 49, 229–33.CrossRefGoogle Scholar
Okamoto, T, Scholten, S, Lörz, H and Kranz, E (2005) Identification of genes that are up- or down-regulated in the apical or basal cell of maize two-celled embryos and monitoring their expression during zygote development by a cell manipulation- and PCR-based approach. Plant Cell Physiol 46, 332–8.CrossRefGoogle ScholarPubMed
Read, SM, Clark, AE and Bacic, A (1993) Requirement for division of generative nucleus in cultured pollen tubes of Nicotiana. Protoplasma 174, 101–5.CrossRefGoogle Scholar
Russell, SD (1984) Ultrastructure of the sperm of Plumbago zeylanica. II. Quantitative cytology and three-dimensional organization. Planta 162: 385–91.CrossRefGoogle ScholarPubMed
Russell, SD, Gou, X, Wei, X and Yuan, T (2010) Male gamete biology in flowering plants. Biochem Soc Trans 38, 598–603.CrossRefGoogle ScholarPubMed
Russell, SD, Gou, X, Wong, CE, Wang, X, Yuan, T, Wei, X, Bhalla, PL and Singh, MB (2012) Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements in the flowering plant male germ lineage. New Phytol 195, 560–73.CrossRefGoogle ScholarPubMed
Sauter, M, von Wiegen, P, Lörz, H and Kranz, E (1998) Cell cycle regulatory genes from maize are differentially controlled during fertilization and first embryonic cell division. Sex Plant Reprod 11, 41–8.CrossRefGoogle Scholar
Shivanna, KR, Xu, H, Taylor, P and Knox, RB (1988) Isolation of sperms from the pollen tubes of flowering plants during fertilization. Plant Physiol 87, 647–50.CrossRefGoogle ScholarPubMed
Sprunck, S, Baumann, U, Edwards, K, Langridge, P and Dresselhaus, T (2005) The transcript composition of egg cells changes significantly following fertilization in wheat (Triticum aestivum L.). Plant J 41, 660–72.CrossRefGoogle Scholar
Uchiumi, T, Uemura, I and Okamoto, T (2007) Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226, 581–9.CrossRefGoogle Scholar
von Besser, K, Frank, AC, Johnson, MA and Preuss, D (2006) Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization. Development 133, 4761–9.CrossRefGoogle ScholarPubMed
Wang, YY, Kuang, A, Russell, SD and Tian, HQ (2006) In vitro fertilization as a tool for investigating sexual reproduction of angiosperms. Sex Plant Reprod 19, 103–115.CrossRefGoogle Scholar
Yang, H, Kaur, N, Kiriakopolos, S and McCormick, S (2006) EST generation and analyses towards identifying female gametophyte-specific genes in Zea mays L. Planta 224, 1004–14.CrossRefGoogle ScholarPubMed
Yang, SJ, Wei, DM and Tian, HQ (2015) Isolation of sperm cells, egg cells, synergids and central cells from Solanum verbascifolium L. J Plant Biochem Biotech 24, 400–7.CrossRefGoogle Scholar
Yang, YH, Qiu, YL, Xie, CT and Tian, HQ (2005) Isolation of two populations of sperm cells and micro-electrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum). Sex Plant Reprod 18, 47–53.CrossRefGoogle Scholar