Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice

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Plant Physiology
Mar 2016



Electro-fusion system with isolated gametes has been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. In this protocol, procedures for isolation of rice gametes, electro-fusion of gametes, and culture of the produced zygotes are described.

Keywords: Egg cell (卵细胞), Embryogenesis (胚胎发育), Fertilization (受精), in vitro fertilization (体外受精), Sperm cell (精细胞), Rice (水稻), Zygote (合子)


Fertilization and subsequent events in angiosperms, such as embryogenesis and endosperm development, occur in the embryo sac deeply embedded in ovular tissue (Nawaschin, 1898; Guignard, 1899; Russell, 1992; Raghavan, 2003). Therefore, isolated gametes have been used for in vitro fertilization (IVF) system to observe and analyze fertilization and postfertilization processes (reviewed in Wang et al., 2006). The IVF system used for angiosperms includes a combination of three basic micro-techniques: (i) the isolation and selection of male and female gametes; (ii) the fusion of pairs of gametes and (iii) single cell culture (Kranz, 1999). Procedures for isolating viable gametes have been established in a wide range of plant species, including monocotyledonous and dicotyledonous plants (reviewed in Kranz, 1999 and in Okamoto, 2011). The isolated gametes can be fused electrically (Kranz et al., 1991; Uchiumi et al., 2006 and 2007) or chemically using calcium (Faure et al., 1994; Kranz and Lörz, 1994; Khalequzzaman and Haq, 2005), polyethyleneglycol (Sun et al., 1995; Tian and Russell, 1997) or bovine serum albumin (Peng et al., 2005), as the gametes are generally protoplasts. Although gametes can be fused using these different procedures, only zygotes produced by electro-fusion are only known to divide and develop into embryo-like structures and plantlets. A complete IVF system was developed by Kranz and Lörz (1993) using maize gametes and electrical fusion, and, to take advantage of the abundant resources stemming from rice research, a rice IVF system was also established by Uchiumi et al. (2007). By the use of these electro-fusion based IVF systems, post-fertilization events, such as karyogamy (Faure et al., 1993; Ohnishi et al., 2014), egg activation and zygotic development (Kranz et al., 1995; Nakajima et al., 2010; Sato et al., 2010), paternal chromatin decondensation in zygote nucleus (Scholten et al., 2002), the microtubular architecture in egg cells and zygotes (Hoshino et al., 2004), fertilization-induced/suppressed gene expression (Okamoto et al., 2005), epigenetic resetting in early embryos (Jahnke and Scholten, 2009), have been successfully observed and investigated. Moreover, polyspermic triploid zygotes were produced by the modification of the rice IVF system and the triploid zygotes were grown into triploid mature plants (Toda et al., 2016). The rice IVF system described here might become an important technique for generating new cultivars with desirable characters as well as for investigating post fertilization events.

Materials and Reagents

  1. Coverslips (24 x 40 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 125485J ) (siliconized at the edges with 5% dichlorodimethylsilane in 1,1,1-trichloroethane, see Note 3)
  2. Glass capillaries made from 50 μl aspirator tubes (Drummond Scientific, catalog number: 2-000-050 ) (Figures 1A and 1B, Video 1; see Note 4)
  3. Manual handling injector (ST Science, type UJB)
  4. Non-treated plastic dishes with diameter of 3.5 cm (Iwaki, catalog number: 1000-035 )
  5. Razor blade
  6. Millicell-CM inserts, diameter 12 mm (EMD Millipore, catalog number: PICM01250 )
  7. Glass needles (2 mm diameter) with fine tips
  8. Rice plants (Oryza sativa L. cv. Nipponbare) (see Note 1)
  9. Feeder cells: rice suspension cell culture (Line Oc, provided by RIKEN Bio-Resource Center, Tsukuba, Japan) (see Note 2)
  10. Mineral oil (Sigma-Aldrich, catalog number: M8410-100ML )
  11. Mannitol (Wako Pure Chemicals Industries, catalog number: 133-00845 )
  12. Absolute ethanol (Wako Pure Chemicals Industries, catalog number: 057-00451 )
  13. 370 mosmol/kg H2O (330 mM) mannitol solution (autoclaved)
  14. 450 mosmol/kg H2O (385 mM) mannitol solution (autoclaved)
  15. 520 mosmol/kg H2O (430 mM) mannitol solution (autoclaved)
  16. CHU (N6) basal salt mixture (Sigma-Aldrich, catalog number: C1416 )
  17. Na2MoO4.2H2O (Wako Pure Chemicals Industries, catalog number: 514-30001 )
  18. CoCl2·6H2O (Wako Pure Chemicals Industries, catalog number: 036-03682 )
  19. CuSO4·5H2O (Wako Pure Chemicals Industries, catalog number: 039-19385 )
  20. Retinol (Sigma-Aldrich, catalog number: R7632 )
  21. Calciferol (Wako Pure Chemicals Industries, catalog number: 039-00291 )
  22. Biotin (Wako Pure Chemicals Industries, catalog number: 023-08711 )
  23. Thiamin·HCl (Nacalai Tesque, catalog number: 36319-82 )
  24. Nicotinic acid (Sigma-Aldrich, catalog number: N4126 )
  25. Pyridoxine·HCl (Wako Pure Chemicals Industries, catalog number: 163-05402 )
  26. Choline chloride (Sigma-Aldrich, catalog number: C7527 )
  27. Ca-pantothenate (Wako Pure Chemicals Industries, catalog number: 031-14161 )
  28. Riboflavin (Wako Pure Chemicals Industries, catalog number: 180-00171 )
  29. 2,4-D (Wako Pure Chemicals Industries, catalog number: 040-18532 )
  30. Cobalamine (Sigma-Aldrich, catalog number: V2876 )
  31. p-aminobenzoic acid (Sigma-Aldrich, catalog number: 100536 )
  32. Folic acid (Wako Pure Chemicals Industries, catalog number: 062-01801 )
  33. Ascorbic acid (Wako Pure Chemicals Industries, catalog number: 012-04802 )
  34. Malic acid (Sigma-Aldrich, catalog number: M1000 )
  35. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  36. Fumaric acid (Wako Pure Chemicals Industries, catalog number: 069-00652 )
  37. Na-pyruvate (Tokyo Chemical Industry, catalog number: P0582 )
  38. Glutamine (Wako Pure Chemicals Industries, catalog number: 078-00525 )
  39. Casein hydrolysate (BD, catalog number: 22 3050 )
  40. Myo-inositol (Wako Pure Chemicals Industries, catalog number: 094-00281 )
  41. Glucose (Wako Pure Chemicals Industries, catalog number: 049-31165 )
  42. MS salt (Wako Pure Chemicals Industries, catalog number: 392-00591 )
  43. MS vitamin (Sigma-Aldrich, catalog number: M7150 )
  44. Sucrose (Wako Pure Chemicals Industries, catalog number: 196-00015 )
  45. Sorbitol (Wako Pure Chemicals Industries, catalog number: 198-03755 )
  46. 1-naphthaleneacetic acid (Nacalai Tesque, catalog number: 23628-32 )
  47. Kinetin (Sigma-Aldrich, catalog number: K3378 )
  48. Gelrite (Wako Pure Chemicals Industries, catalog number: 075-05655 )
  49. Dichlorodimethylsilane (Tokyo Chemical Industry, catalog number: D0358 )
  50. 1,1,1-trichloroethane (Tokyo Chemical Industry, catalog number: T0380 )
  51. Zygote culture medium (see Recipes)
  52. Regeneration media (see Recipes)
  53. Rooting media (see Recipes)


  1. Stereo microscope (OLYMPUS, model: SZ61 )
  2. Inverted microscope (OLYMPUS, model: IX-71 or IX-73 )
  3. Forceps
  4. Electrofusion apparatus (Nepa Gene, model: ECFG21 )
  5. Manipulator (NARISHIGE, model: M-152 ) with a double pipette holder (NARISHIGE, model: HD-21 )
  6. Electrodes (Nepa Gene, model: CUY5100Ti100 ) fixed to the pipette holder
  7. Sliding stage for the insertion of a coverslip and a plastic dish
  8. Environmental chambers
  9. Charge-coupled device camera (Pixcera, model: Penguin 600CL )


  1. InStudio software (Pixcera)


  1. Isolation of gametes
    1. Overlay the siliconized coverslip with 0.3 ml mineral oil and make several 1-2 μl mannitol droplets (370 mosmol/kg H2O) inside the mineral oil on the coverslip (Figure 1C) using a glass capillary connected to a handling injector. Take care that the droplets have no access to the air (Figure 1D).

      Figure 1. Glass capillary (A, B) and mannitol droplets on coverslip (C, D). A. A glass capillary drawn by hand. B. Tip opening of the glass capillary in panel A. C and D. A photo (C) and an image (D) of mannitol droplets inside the mineral oil over the siliconized coverslip. Bars = 1 cm in A and C, 200 μm in B.

      Video 1. Making glass capillary

    2. Collect panicles in which some flowers have already opened and others remain unflowered. Pick up the unflowered ones from the panicles and dissect them under a stereo microscope. Isolate ovaries, and transfer them into 3.5 cm plastic dishes filled with 3 ml of mannitol solution (370 mosmol/kg H2O) (Figure 2A).
    3. For egg cell isolation, remove the stigmas from ovaries and transfer them into new 3.5 cm plastic dishes filled with 3 ml of the mannitol solution (see Note 5). Allow the ovaries to sink to the bottom of the dishes and cut them transversely with a razor blade at the middle (Figures 2A and 2B). Under an inverted microscope, identify the egg cells which are released from the lower parts of the cut ovaries (Figure 2C; see Note 6). By using a glass capillary connected to a handling injector, collect egg cells and transfer them into mannitol droplets prepared in step A1 (Figure 4A; see Note 7).

      Figure 2. Isolation of rice egg cells from ovaries. A. Ovary harvested from a rice flower before flowering. The red line indicates the incision line for egg isolation. B. Cut ovary in mannitol solution. C. Rice egg cell (arrowhead) being released from basal portion of the dissected ovary.

    4. For sperm cell isolation, dissect an unopened flower under a stereo microscope to obtain anthers, and transfer anthers into a 3.5 cm plastic dish filled with 3 ml of mannitol solution (370 mosmol/kg H2O). Break anthers in mannitol solution with forceps to free pollen grains (Figure 3A). Pollen grains burst due to the osmotic shock and release sperm cells. Identify the sperm cells under an inverted microscope (Figure 3B). By using a glass capillary connected to a handling injector, collect sperm cells and transfer into mannitol droplets prepared in step A1. (Figure 4B; see Note 8).

      Figure 3. Isolation of rice sperm cells from pollen grains. A. A broken anther (arrow) and released pollen grains (arrowheads) in mannitol solution. B. A pollen grain (arrow) released its content in mannitol solution. Two sperm cells were enclosed with the square. Bars = 500 µm in A and 20 μm in B.

  2. Electro-fusion of isolated gametes
    1. Set up fusion apparatus and adjust the position of electrodes (Figures 4C and 5).
    2. Transfer one egg cell to each of the six mannitol droplets (Figure 4C), then transfer 1 or 2 sperm cells to each droplet.
    3. Lower the position of electrodes into a fusion droplet using manipulator (Videos 2 and 3), and align and fix the two gametes at one electrode under an alternating current (AC) field (1 MHz, 5 V rms). Under the AC field, gametes move toward electrode. By moving the electrode connected with manipulator, first fix an egg cell to the electrode. Using the same procedure, fix a sperm cell to the female gamete (Figure 4D). Adjust the final distance of the electrodes to approximately twice the sum of the diameters of the cells.
    4. Add 0.5-1.0 μl of mannitol solution (520 mosmol/kg H2O) gently to the fusion droplet using a thin glass capillary (Figure 4E, see Note 9).
    5. Induce cell fusion by applying a single negative direct current (DC) pulse (50 μsec, 12-15 V) (Figures 4F and 4G, Video 4, see Note 10).
    6. Remove the fusion products from the electrode by gently moving the electrode. Move the electrodes out of the droplet with manipulator and conduct the next gamete fusion at step B3 (see Notes 11 and 12).

      Figure 4. Isolated rice gametes (A and B) and electro-fusion of gametes (C–G). A. An isolated rice egg cell. B. Rice sperm cells released from pollen grain. C. An illustration of the fusion droplets on a coverslip covered with mineral oil. The gray bar and thin triangle indicate electrodes. D. Alignment of an egg cell with a sperm cell (arrowhead) on one of the electrodes under an alternating current (AC) field in a fusion droplet. E. Aligned egg and sperm cells after the addition of mannitol solution with a higher osmolality to the fusion drop. The sperm cell becomes oblong (arrowhead). F. Fusion of gametes following a negative direct current (DC) pulse. An arrowhead indicates fusion point. G. A zygote 10 sec after fusion. The arrowhead indicates the fusion point. Bars = 50 μm in A, D and 5 μm in B.

      Figure 5. Fusion system consisting of inverted microscope, electrofusion apparatus, manipulator and electrodes

      Video 2. Adjustment of electrodes into mannitol droplet in which an egg cell and a sperm cell have been transferred

      Video 3. Electrodes in mannitol droplet

      Video 4. Electrofusion of rice gametes

  3. Culture and development of zygotes
    1. Place 0.2 ml zygote culture medium in a Millicell-CM insert and put it into a 3.5 cm plastic dish containing 2 ml of the medium. Add 40-60 µl of a rice suspension cell culture into the dish as feeder cells.
    2. After sterilization of the microcapillary by washing with absolute ethanol and sterilized water, transfer IVF-produced zygotes into fresh mannitol droplets (450 mosmol/kg H2O) twice and then transfer them onto the membranes of a Millicell-CM insert (Figure 5A).

      Figure 6. Culture and development of zygotes. A. An illustration of zygote culture; A white circle in the Millicell insert indicates a zygote. Light-blue oblong circles represent aggregates of feeder cells. B. A zygote 1 h after fusion; C. A zygote 4 h after fusion; Two nucleoli are indicated by arrowheads. D. An asymmetric two-celled embryo 18 h after fusion; E and F. Nuclear staining of an embryo 48 h after fusion, visualized by brightfield and fluorescence microscopy, respectively; G. A cell mass 5 days after fusion, which developed from the globular-like embryo; H. A white cell colony 18 days after fusion; I. A developed cell colony 4 days after transferring the white cell colony (panel H) into regeneration medium (22 days after fusion). Green spots are visible in/on the cell colony. J. Regenerated shoots; Generation of shoots can be observed after 8 days of subculturing the white cell colony (26 days after fusion). K. A plantlet after 12 days of subculturing a regenerated shoot in hormone-free medium (43 days after fusion); L. A regenerated plant with seed sets (100 days after fusion). Bars =  50 μm in B-E and G, 1 mm in H-J and 1 cm in K.

    3. After overnight culture of zygotes at 26 °C in the dark without shaking, continue culture with gentle shaking (40 rpm) (Figures 6B-6F; see Notes 13 and 14).
    4. Five days after fusion, remove feeder cells by transferring the Millicell dishes containing the embryos into new 35-mm diameter dishes filled with 2 ml of fresh zygote culture medium (Figure 6G; see Note 15). Continue culturing as above.
    5. After 18 days in culture, subculture cell colonies developed from the IVF-produced zygotes in Millicell-CM inserts onto a regeneration medium in plastic dishes by use of a sterilized Pasteur pipette (Note 16). Incubate under continuous light at 30 °C for 12-30 days (Figure 6H; see Note 17).
    6. Transfer the differentiated shoots into a rooting medium in plastic dishes and culture them under a 13/11 h light/dark cycle at 28 °C for 11-13 days (Figures 6I and 6J).
    7. Transfer the resulting plantlets to soil pods (diameter 20-28 cm, depth 30-35 cm) and grow in environmental chambers as described in Note 1 (Figure 6K). If needed, harvest seeds from the regenerated plants and germinate them (Figure 6L).

Data analysis

Digital images of gametes, zygotes, and their resulting embryos were obtained using a cooled charge-coupled device camera (Penguin 600CL; Pixcera, Los Gatos, CA, USA) and InStudio software (Pixcera).


  1. Rice plants are grown in environmental chamber at 26 °C in a 13/11 h light/dark cycle with a photosynthetic photon flux density of 150-300 μmol photons m-2 sec-1. Under these growth conditions, flowers can be obtained throughout all seasons.
  2. Rice suspension cell, Line Oc, were subcultured once weekly according to instructions from RIKEN Bio-Resource Center. No difference in feeder effects between freshly subcultured cells and one week-cultured cells has been observed.
  3. Coverslips should be non-coated, as using coated coverslips will result in attachment of the cells to the surface of the coverslip.
  4. Tip openings were drawn by hand to 150-250 μm.
  5. Without removing the stigmas, ovaries always float on the mannitol solution. To isolate egg cells, sinking ovaries into the mannitol solution is essential. Usually, 15-25 ovaries are put into a dish.
  6. Usually, 3-8 egg cells are automatically released from approximately 20 cut ovaries. Gentle pushing of the basal portion of the lower part of the cut ovary with a glass needle will produce additional egg cells.
  7. Egg cells can be kept in the mannitol droplet until 6 h after isolation to conduct IVF without decreasing fusion efficiency. Alternatively, egg cells can be stored at 4 °C overnight for next day use.
  8. Sperm cells should be used for IVF within 1 h after isolation. Otherwise, sperm cells appear to degenerate and cannot be fused with egg cells.
  9. The addition of mannitol solution with a higher osmolarity changes the shape of the sperm cell to oblong and makes the attachment of the egg cell to the electrode more stable (Figures 4D and 4E). Without this treatment, egg cells are often released from the electrode upon fusion induced by a DC pulse and fusion efficiency is greatly reduced.
  10. If no cell fusion occurs, reduce the distance between the two electrodes and pulse again.
  11. At each round of fusion procedures, 5-6 sets of gamete fusions are recommended (Figure 4C), since conducting many sets of gamete fusion takes time and sperm cell will be degenerate during the course of the experiments.
  12. The efficiency of successful electrofusion is approximately 85% under optimal conditions. 20 to 40 egg cells can be isolated from one hundred processed ovaries, and 15-30 egg cells can be fused with sperm cells by one experimenter in a day.
  13. The rice zygotes produced by IVF start to form cell walls (Figure 6B) and two nucleoli can be observed in a zygote at least 4 h after fusion (Figure 6C). At around 12 h after fusion, well-developed granular organelles, probably starch granules, are visible in the zygotes and the first asymmetric cell division of the zygotes is observed at 17-22 h after fusion (Figure 6D). After the first division, the two-celled embryos continue to develop into early embryos at 40-50 h after fusion (Figures 6E and 6F).
  14. Approximately 90% IVF-produced zygotes divide into two-celled embryo, and 90% IVF-produced two-celled embryos develop into globular embryos.
  15. After five days culture of the IVF-produced zygotes, co-cultivation with feeder cells is not needed.
  16. Diameter of cell colonies are approximate 1-2 mm, and can be transferred with Pasteur pipette.
  17. Normally, after four days of subculture of the cell colony on a solidified-regeneration medium (22 days after fusion), green spots become visible and the emergence of multiple shoots is observed after eight days of subculturing (26 days after fusion). Most subcultured cell colonies form green spots and multiple shoots.


  1. Zygote culture medium (N6Z-medium [Kumlehn et al., 1998] with modifications)
    2 g/L CHU (N6) basal salt mixture
    0.025 mg/L Na2MoO4·2H2O
    0.025 mg/L CoCl2·6H2O
    0.025 mg/L CuSO4·5H2O
    0.01 mg/L retinol
    0.01 mg/L calciferol
    0.01 mg/L biotin
    1 mg/L thiamin·HCl
    1 mg/L nicotinic acid
    1 mg/L pyridoxine·HCl
    1 mg/L cholin chloride
    1 mg/L Ca-pantothene
    0.2 mg/L riboflavin
    0.2 mg/L 2,4-D
    0.02 mg/L cobalamine
    0.02 mg/L p-aminobenzoic acid
    0.4 mg/L folic acid
    2 mg/L ascorbic acid
    40 mg/L malic acid
    40 mg/L citric acid
    40 mg/L fumaric acid
    20 mg/L Na-pyruvate
    1000 mg/L glutamine
    250 mg/L casein hydrolysate
    100 mg/L myo-inositol
    Osmolality, 450 mosmol kg-1 H2O adjusted with glucose
    Adjust to pH 5.7 and filter sterilized
  2. Regeneration media (Solidified MS medium with some modifications [Hiei et al., 1994])
    MS salt
    MS vitamin
    100 mg/L myo-inositol
    2 g/L casamino acid
    30 g/L sucrose
    30 g/L sorbitol
    0.2 mg/L 1-naphthaleneacetic acid (NAA)
    1 mg/L kinetin
    0.3% gelrite
  3. Rooting media
    The same as the regeneration media but omitting sorbitol, kinetin and NAA


This protocol was adapted from Uchiumi et al. (2007) and Okamoto (2010). This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid No. 26113715 to T.O.) and the Japan Society for the Promotion of Science (Grant-in-Aid No. 16K14742 to T.O.). I thank Ms. Tomoko Mochizuki (Tokyo Metropolitan University) for technical assistance.


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  28. Uchiumi, T., Uemura, I. and Okamoto, T. (2007). Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226(3): 581-589.
  29. Wang, Y. Y., Kuang, A., Russell, S. D. and Tian, H. Q. (2006). In vitro fertilization as a tool for investigating sexual reproduction of angiosperms. Sexual Plant Reproduction 19(3): 103-115.



背景 被子植物的受精和后续事件,如胚胎发生和胚乳发育,发生在深入嵌入卵母细胞的胚囊中(Nawaschin,1898; Guignard,1899; Russell,1992; Raghavan,2003)。因此,分离的配子已被用于体外受精(IVF)系统,以观察和分析受精和后处理过程(Wang等人,2006年)。用于被子植物的IVF系统包括三种基本微技术的组合:(i)男性和女性配子的分离和选择; (ii)配对对和(iii)单细胞培养物的融合(Kranz,1999)。已经在广泛的植物物种中建立了分离活的配子的程序,包括单子叶植物和双子叶植物(综述于Kranz,1999和Okamoto,2011)。分离的配子可以电融合(Kranz等人,1991; Uchiumi等人,2006和2007)或化学地使用钙(Faure等人,1994; Kranz和Lörz,1994; Khalequzzaman和Haq,2005),聚乙二醇(Sun等,1995; Tian和Russell,1997)或牛血清白蛋白等,2005),因为配子通常是原生质体。虽然配子可以使用这些不同的程序进行融合,但只有通过电融合产生的受精卵才知道分裂成胚状结构和小植株。 Kranz和Lörz(1993)利用玉米配子和电融合开发了一个完整的IVF系统,为了利用水稻研究丰富的资源,Uchiumi等人也建立了一个水稻IVF系统。 (2007)。通过使用这些基于电融合的IVF系统,受精后事件(例如,kureogamy(Faure等人,1993; Ohnishi等人,2014)),卵激活和合子发育(Kranz等人,1995; Nakajima等人,2010; Sato等人,2010),父系受精卵诱导/抑制的受精卵中的染色质解链(Scholten等人,2002),卵细胞和受精卵中的微管结构(Hoshino等,2004)基因表达(Okamoto等人,2005),早期胚胎中的表观遗传重建(Jahnke和Scholten,2009)已被成功地观察和研究。此外,通过水稻IVF系统的修饰产生多核三倍体合子,并将三倍体合子生长成三倍体成熟植物(Toda等人,2016)。这里描述的水稻IVF系统可能成为产生具有理想特征的新品种以及调查后施肥事件的重要技术。

关键字:卵细胞, 胚胎发育, 受精, 体外受精, 精细胞, 水稻, 合子


  1. (24 x 40 mm)(Thermo Fisher Scientific,Fisher Scientific,目录号:125485J)(在5%二氯二甲基硅烷在1,1,1-三氯乙烷中边缘硅化,见注3)
  2. 玻璃毛细管由50μl抽吸管(Drummond Scientific,目录号:2-000-050)制成(图1A和1B,视频1;见注4)
  3. 手动注射器(ST科学,UJB型)
  4. 直径3.5厘米的未经处理的塑料盘(岩壁,目录号:1000-035)
  5. 剃刀刀片
  6. Millicell-CM插入件,直径为12mm(EMD Millipore,目录号:PICM01250)
  7. 玻璃针(2毫米直径)与细小的尖端
  8. 水稻植株(日本晴天)(见注1)
  9. 饲养细胞:水稻悬浮细胞培养物(Line Oc,由RIKEN Bio-Resource Center,Tsukuba,Japan提供)(参见注2)
  10. 矿物油(Sigma-Aldrich,目录号:M8410-100ML)
  11. 甘露醇(Wako Pure Chemicals Industries,目录号:133-00845)
  12. 绝对乙醇(Wako Pure Chemicals Industries,目录号:057-00451)
  13. 370 mosmol/kg H 2 O(330mM)甘露醇溶液(高压灭菌)
  14. 450 mosmol/kg H 2 O(385mM)甘露醇溶液(高压灭菌)
  15. 520 mosmol/kg H 2 O(430mM)甘露醇溶液(高压灭菌)
  16. CHU(N6)基础盐混合物(Sigma-Aldrich,目录号:C1416)
  17. Na 2 O 2 O 2 O 2(和光纯药工业公司,目录号:514-30001)
  18. CoCl 2·6H 2 O(Wako Pure Chemicals Industries,目录号:036-03682)
  19. CuSO 4·5H 2 O(Wako Pure Chemicals Industries,目录号:039-19385)
  20. 视黄醇(Sigma-Aldrich,目录号:R7632)
  21. Calciferol(Wako Pure Chemicals Industries,目录号:039-00291)
  22. 生物素(Wako Pure Chemicals Industries,目录号:023-08711)
  23. Thiamin·HCl(Nacalai Tesque,目录号:36319-82)
  24. 烟酸(Sigma-Aldrich,目录号:N4126)
  25. 吡哆醇·HCl(和光纯药工业公司,目录号:163-05402)
  26. 氯化胆碱(Sigma-Aldrich,目录号:C7527)
  27. 泛酸钙(Wako Pure Chemicals Industries,目录号:031-14161)
  28. 核黄素(Wako Pure Chemicals Industries,目录号:180-00171)
  29. 2,4-D(Wako Pure Chemicals Industries,目录号:040-18532)
  30. 钴胺(Sigma-Aldrich,目录号:V2876)
  31. 对氨基苯甲酸(Sigma-Aldrich,目录号:100536)
  32. 叶酸(Wako Pure Chemicals Industries,目录号:062-01801)
  33. 抗坏血酸(和光纯药工业公司,目录号:012-04802)
  34. 苹果酸(Sigma-Aldrich,目录号:M1000)
  35. 柠檬酸(Sigma-Aldrich,目录号:251275)
  36. 富马酸(Wako Pure Chemicals Industries,目录号:069-00652)
  37. 丙酮酸钠(Tokyo Chemical Industry,目录号:P0582)
  38. 谷氨酰胺(Wako Pure Chemicals Industries,目录号:078-00525)
  39. 酪蛋白水解物(BD,目录号:22 3050)
  40. 肌醇(和光纯药工业公司,目录号:094-00281)
  41. 葡萄糖(Wako Pure Chemicals Industries,目录号:049-31165)
  42. MS盐(和光纯药工业公司,目录号:392-00591)
  43. MS维生素(Sigma-Aldrich,目录号:M7150)
  44. 蔗糖(Wako Pure Chemicals Industries,目录号:196-00015)
  45. 山梨醇(和光纯药工业公司,目录号:198-03755)
  46. 1-萘乙酸(Nacalai Tesque,目录号:23628-32)
  47. 激动素(Sigma-Aldrich,目录号:K3378)
  48. Gelrite(Wako Pure Chemicals Industries,目录号:075-05655)
  49. 二氯二甲基硅烷(Tokyo Chemical Industry,目录号:D0358)
  50. 1,1,1-三氯乙烷(Tokyo Chemical Industry,目录号:T0380)
  51. 合子培养基(见食谱)
  52. 再生媒体(见食谱)
  53. 生根媒体(见食谱)


  1. 立体显微镜(OLYMPUS,型号:SZ61)
  2. 倒置显微镜(OLYMPUS,型号:IX-71或IX-73)
  3. 镊子
  4. 电融器(Nepa Gene,型号:ECFG21)
  5. 机械手(NARISHIGE,型号:M-152),带双吸液管支架(NARISHIGE,型号:HD-21)
  6. 电极(Nepa Gene,型号:CUY5100Ti100)固定在移液器支架上
  7. 用于插入盖玻片和塑料盘的滑动台
  8. 环保厅
  9. 电荷耦合器件相机(Pixcera,型号:企鹅600CL)


  1. InStudio软件(Pixcera)


  1. 配子的分离
    1. 用0.3ml矿物油覆盖硅化盖玻片,并使用玻璃毛细管连接在盖玻片上的矿物油内部制备数个1-2μl甘露醇液滴(370 mosmol/kg H 2 O 2)(图1C)到处理注射器。请注意,液滴无法接近空气(图1D)。

      图1.盖玻片上的玻璃毛细管(A,B)和甘露醇液滴(C,D)。 A.用手绘的玻璃毛细管。 B.面板A中的玻璃毛细管的尖端开口。C和D.矿物油中的甘露醇液滴的照片(C)和图像(D))在硅化盖玻片上。酒吧= A,C为1厘米,B为200厘米
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    2. 收集一些鲜花已经开放的圆锥花序,其他花朵仍未开花。从圆锥花序中取出未花粉的,并在立体显微镜下剖析。隔离卵巢,并将其转移到装有3ml甘露醇溶液(370 mosmol/kg H 2 O)的3.5cm塑料盘中(图2A)。
    3. 对于卵细胞分离,从卵巢中取出柱头并将其转移到装有3ml甘露醇溶液的新的3.5cm塑料盘中(参见附注5)。允许卵巢下沉到碗的底部,并用中间的剃刀刀片横向切割(图2A和2B)。在倒置显微镜下,确定从切割卵巢下部释放的卵细胞(图2C;见附注6)。通过使用连接到处理注射器的玻璃毛细管,收集卵细胞并将其转移到步骤A1中制备的甘露醇液滴中(图4A;参见附注7)。

      图2.从卵巢中分离卵卵细胞 A.开花前从米花中收获的卵巢。红线表示卵隔离的切割线。 B.在甘露醇溶液中切割卵巢。 C.从解剖卵巢的基底部分释放的稻蛋细胞(箭头)。

    4. 对于精子细胞分离,在立体显微镜下切开未开封的花,以获得花药,并将花药转移到装有3ml甘露醇溶液(370mosmol/kg H 2 O 2)的3.5cm塑料盘中。用镊子打破甘露醇溶液中的花药以释放花粉颗粒(图3A)。由于渗透冲击和释放精子细胞,花粉粒爆裂。在倒置显微镜下鉴定精子细胞(图3B)。通过使用连接到处理注射器的玻璃毛细管,收集精子细胞并转移到步骤A1中制备的甘露醇液滴中。 (图4B;见注8)

      图3.从花粉粒中分离水稻精子细胞A.在甘露醇溶液中破碎的花药(箭头)和释放的花粉粒(箭头)。 B.花粉粒(箭头)释放其甘露醇溶液中的含量。两个精子细胞用正方形封闭。棒=500μm,A为20μm,B为20μm
  2. 孤立配子的融合
    1. 设置融合装置并调整电极的位置(图4C和5)
    2. 将一个卵细胞转移到六个甘露醇液滴中的每一个(图4C),然后将1或2个精子细胞转移到每个液滴中。
    3. 使用操纵器(视频2和3)将电极的位置降低到融合液滴中,并且在交流(AC)场(1MHz,5V rms)下在一个电极处对准和固定两个配子。在AC场下,配子向电极移动。通过移动与机械手连接的电极,首先将鸡蛋固定在电极上。使用相同的程序,将精子细胞固定在雌配子上(图4D)。将电极的最终距离调整到单元直径之和的大约两倍。
    4. 使用薄玻璃毛细管将0.5-1.0μl甘露醇溶液(520 mosmol/kg H 2 O)轻轻加入熔融液滴(图4E,见附注9)。
    5. 通过施加单负负直流(DC)脉冲(50微秒,12-15伏)诱导细胞融合(图4F和4G,视频4,参见附注10)。
    6. 通过轻轻移动电极,从电极中取出融合产品。使用机械手将电极移出液滴,并在步骤B3进行下一个配子融合(见注11和12)。

      图4.分离的稻配子(A和B)和配子的电融合(C-G)。A.分离的稻卵细胞。 B.从花粉粒释放的水稻精子细胞。 C.用覆盖有矿物油的盖玻片上的融合液滴的说明。灰色条和薄三角形表示电极。 D.在融合液滴中的交流(AC)场下,在电极之一上将卵细胞与精子细胞(箭头)对准。 E.加入甘露醇溶液后,对准卵和精子细胞,渗透压高于融合液滴。精子细胞变成长圆形(箭头)。 F.在直流(DC)脉冲之后,配子融合。箭头表示融合点。 G.融合后10秒的合子。箭头表示融合点。棒在A,D中为50μm,在B中为5μm。


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  3. 合作伙伴的文化与发展
    1. 将0.2ml受精卵培养基置于Millicell-CM插入物中,并放入含有2ml培养基的3.5cm塑料皿中。将40-60μl的悬浮细胞培养物加入到作为饲养细胞的培养皿中
    2. 通过用无水乙醇和无菌水洗涤将微毛细管灭菌后,将IVF生产的合适转移到新鲜的甘露醇液滴(450 mosmol/kg H 2 O 2)中,然后转移到Millicell的膜上-CM插入(图5A)。

      图6.合子的文化与发展。 A.合子文化的例证; Millicell插入中的一个白色圆圈表示一个合子。浅蓝色长圆形代表饲养细胞的聚集体。 B.融合后1 h的合子; C.融合后4 h的合子;两个核仁由箭头指示。 D.融合18小时后的不对称双细胞胚胎; E和F.融合后48 h胚胎核染色,分别通过明场和荧光显微镜观察; G.融合后5天的细胞团,其从球状样胚发育; H.融合18天后的白细胞集落; I.将白细胞集落(图H)转移到再生培养基(融合后22天)4天后开发的细胞集落。绿色斑点在细胞集落中/上可见。再生枝条在白细胞集落传代培养8天后(融合后26天)可以观察到芽的产生。 K.在无激素培养基(融合后43天)中再生培养12天后的一个小植株; L.具有种子组的再生植物(融合后100天)。酒吧= B-E和G为50μm,H-J为1毫米,K为1厘米。

    3. 在26°C的黑暗条件下接受合子过夜培养而不摇动后,继续用轻轻摇动(40 rpm)进行培养(图6B-6F;见注释13和14)。
    4. 融合后5天,通过将含有胚胎的Millicell培养皿转移到装有2ml新鲜的合子培养基的新的35mm直径的培养皿中(图6G;参见附注15),去除饲养细胞。继续上述培养。
    5. 培养18天后,通过使用灭菌的巴斯德吸管(注16),将在Millicell-CM中的IVF产生的合子开发的传代培养细胞集落插入塑料盘中的再生培养基。在连续光照30℃下孵育12-30天(图6H;见附注17)
    6. 将分化的芽转移到塑料盘中的生根培养基中,并在28℃的13/11小时/黑暗循环下培养它们11-13天(图6I和6J)。
    7. 将所得的小植株转移到土壤荚(直径20-28厘米,深度30-35厘米),并在环境室中生长,如注1(图6K)所述。如果需要,从再生植物收获种子并发芽(图6L)


使用冷却的电荷耦合器件相机(Penguin 600CL; Pixcera,Los Gatos,CA,USA)和InStudio软件(Pixcera)获得配子,合子及其产生的胚胎的数字图像。


  1. 水稻植物在13/11小时的光/暗循环中在26℃的环境室中生长,光合子光子通量密度为150-300μmol光子,其中p <! - SIPO 。在这些生长条件下,可以在整个季节获得花
  2. 根据RIKEN生物资源中心的指示,每周将水稻悬浮液细胞系Oc进行传代培养。新近传代培养的细胞与一周培养细胞之间的饲喂效应没有差异
  3. 盖片应该是非涂层的,因为使用涂覆的盖玻片将导致细胞附着到盖玻片的表面。
  4. 用手将尖端开口拉伸至150-250μm
  5. 不排除柱头,卵巢总是漂浮在甘露醇溶液上。为了分离卵细胞,将卵巢沉入甘露醇溶液是至关重要的。通常,将15-25个卵巢放入盘中。
  6. 通常,3-8个卵细胞从大约20个切割的卵巢中自动释放。用玻璃针轻轻按下切割卵巢下部的基部,会产生额外的卵细胞。
  7. 卵细胞可以保持在甘露醇液滴中,直到分离6小时才能进行IVF,而不会降低融合效率。或者,卵细胞可以在4℃下储存过夜,以备第二天使用。
  8. 分离后1 h内精子细胞应用于体外受精。否则,精子细胞似乎退化,不能与卵细胞融合
  9. 添加具有较高渗透压的甘露醇溶液将精子细胞的形状改变为长圆形,并使卵细胞与电极的连接更稳定(图4D和4E)。没有这种处理,通过DC脉冲诱导的融合,卵细胞通常从电极释放,并且融合效率大大降低
  10. 如果没有发生细胞融合,则减小两个电极之间的距离并再次脉冲。
  11. 在每轮融合程序中,推荐使用5-6组配子融合(图4C),因为进行多组配子融合需要时间,精子细胞在实验过程中会退化。
  12. 在最佳条件下,成功电融合的效率约为85%。可以从一百个加工的卵巢中分离20至40个卵细胞,一天一个实验者可以将15-30个卵细胞与精子细胞融合。
  13. 由IVF产生的稻米合子开始形成细胞壁(图6B),融合后至少4小时可以在受精卵中观察到两个核仁(图6C)。在融合后约12小时,合子中可见的发育良好的颗粒细胞器(可能是淀粉颗粒)可见,并且融合后17-22小时观察到受精卵的第一不对称细胞分裂(图6D)。第一次分裂后,双细胞胚胎在融合后40-50小时继续发育成早期胚胎(图6E和6F)。
  14. 约90%的IVF产生的合子分为双细胞胚胎,90%的IVF产生的双细胞胚胎发育成球状胚胎。
  15. 培养IVF生产的受精卵五天后,不需要与饲养细胞共同培养
  16. 细胞集落直径约为1-2毫米,可用巴斯德吸管转移。
  17. 通常,在固化再生培养基(融合后22天)传代细胞集落四天后,绿斑变得可见,并且在传代培养8天(融合后26天)时观察到多个芽的出现。大多数传代培养的细胞集落形成绿色斑点和多个芽。


  1. 合子培养基(N6Z-培养基[Kumlehn等人,1998],修改)
    2g/L CHU(N6)基础盐混合物
    0.025mg/L Na 2 MoO 4·2H 2 O 0.025mg/L CoCl 2·6H 2 O
    0.025mg/L CuSO 4·5H 2 O
    0.01 mg/L视黄醇
    0.01mg/L calciferol
    0.01 mg/L生物素
    1 mg/L氯化胆碱
    1 mg/L Ca-pantothene
    0.2 mg/L核黄素
    0.2 mg/L 2,4-D
    0.02 mg/L钴胺
    0.4 mg/L叶酸
    40 mg/L柠檬酸
    20毫克/升的丙酮酸钠 1000 mg/L谷氨酰胺
    250 mg/L酪蛋白水解产物
    重量份浓度,450 mosmol kg -1 H 2 O调整葡萄糖
    调整至pH 5.7并过滤消毒的
  2. 再生培养基(具有一些修饰的固化MS培养基[Hiei等人,1994])
    30 g/L山梨醇
    0.2mg/L 1-萘乙酸(NAA)
    1 mg/L激动素
  3. 生根媒体


该协议由Uchiumi等人改编。 (2007)和冈本(2010)。这项工作得到了日本的教育,文化,体育,科技部(TO-26113715 TO TO)和日本的科学促进会(赠款援助号)的部分支持。 16K14742到TO)。感谢Tomoko Mochizuki女士(东京都大学)提供技术援助。


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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Toda, E., Ohnishi, Y. and Okamoto, T. (2016). Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice. Bio-protocol 6(24): e2074. DOI: 10.21769/BioProtoc.2074.
  2. Toda, E., Ohnishi, Y. and Okamoto, T. (2016). Development of polyspermic rice zygotes. Plant Physiol 171(1): 206-214.