Sperm cells can be isolated from the mature pollen grains of medicinal wild rice (Oryza officinalis) using an osmotic shock method, and the viable egg cells can be isolated by enzymatic digestion and mechanical dissection steps.
Favorable alleles for rice breeding have been identified in natural cultivars and wild rice by association analysis of known functional genes with target trait performance. Transferring these genes from wild rice into cultivated rice varieties is one of the important objectives for rice breeders. The isolation of the sperm and egg cells of wild and cultivated rice is a prerequisite for the in vitro hybridization of distantly related cultivated rice and wild rice lines. Here, we provide a technical approach for isolating the sperm and egg cells of wild rice (Oryza officinalis). In this method, sperm cells were isolated from the mature pollen grains of medicinal wild rice (O. officinalis) according to an osmotic shock method. Additionally, viable O. officinalis egg cells were isolated following enzymatic digestion and mechanical dissection steps. Specifically, ovules were digested in an enzymatic solution containing pectinase and cellulase for 30 min, after which the ovule was cut into two halves. Three egg apparatus cells were released by gently applying pressure to the micropylar end. Generally, six or seven egg cells could be isolated from 20 ovules in 60 min. The same method was used to isolate zygotes from flowers at 24 h after pollination. This technology solved the difficulty of isolating sperm and egg cells in O. officinalis and allowed the isolated sperm and egg cells to be combined by in vitro hybridization of distantly related wild and cultivated rice lines.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Availability of data and materials
All relevant data are provided in the file.
Cao YJ, Russell SD (1997) Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica. Sex Plant Reprod 10:368–373
Guo S, Wei Y, Li X, Gao G, Deng G (2016) Advances in excavation and utilization of elite genes from Oryza minuta. J Plant Genet Resources 17:371–376 (in Chinese with English abstract)
He EM, Wang YY, Liu HH, Zhu XY, Tian HQ (2012) Egg cell isolation in Datura stramonium (Solanaceae). Annales Botanici Fennici. 49:7–12
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
Hoshino Y, Murata N, Shinoda K (2006) Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump. Ann Bot 97:1139–1144
Khalequzzaman M, Haq N (2005) Isolation and in vitro fusion of egg and sperm cells in Oryza sativa. Plant Physiol Biochem 43:9–75
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
Kranz E, Bautor J, Lörz H (1991) In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex Plant Reprod 4:12–16
Li C, Xu H, Russell SD, Sundaresan V (2019) Step-by-step protocols for rice gamete isolation. Plant Reprod 32:5–14
Maryenti T, Kato N, Ichikawa M, Okamoto T (2019) Establishment of an in vitro fertilization system in wheat (Triticum aestivum L.). Plant Cell Physiol 60(4):835–843
Uchiumi T, Komatsu S, Koshiba T, Okamoto T (2006) Isolation of gametes and central cells from Oryza sativa L. Sex Plant Reprod 19:37–45
Uchiumi T, Uemura I, Okamoto T (2007) Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226:581–589
Wang YY, Kuang A, Russell SD, Tian HQ (2006) In vitro fertilization as a tool for investigating sexual reproduction of angiosperms. Sex Plant Reprod 19:103–115
Yang SJ, Wei DM, Tian HQ (2015) Isolation of sperm cells, egg cells, synergids and central cells from Solanum verbascifolium L. J Plant Biochem Biotechnol. https://doi.org/10.1007/s13562-014-0290-6
Zhang H, Liu R, Guo H, Li Y (2009) Advancement on Mining and Utilization of Elite Genes in Oryza officinalis Wall. Chin Agric Sci Bull 25(19):42–45 (in Chinese with English abstract)
Zhang YN, Wei DM, He EM, Miao S, Tian HQ, Russell SD (2010) Isolation of male and female gametes of rice. Crop Sci 50:2457–2463
Zhao J, Zhou C, Yang HY (2000) Isolation and in vitro culture of zygotes and central cells of Oryza sativa L. Plant Cell Rep 19:321–326
Zhong D, Luo L, Ying C (2000) Advances on transferring elite gene from wild rice species into cultivated rice. Chinese Journal Rice Science 14(2):103–106 (in Chinese with English abstract)
This research was financially supported by the National Natural Science Foundation of China (No. 31570297; No. 31100225), the Natural Science Foundation of Ningxia (No. NZ17033).
Conflicts of interest
The authors declare that they have no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Communicated by S. D. Russell.
About this article
Cite this article
Deng, W., Cheng, C., Luo, C. et al. Isolation of the sperm and egg cells in wild rice (Oryza officinalis) as a mechanism for crop improvement. Plant Reprod (2020). https://doi.org/10.1007/s00497-020-00383-z
- Egg cell
- Sperm cell
- Medicinal wild rice (Oryza officinalis)