In Vitro Pollination and Fertilization

Abstract

In angiosperms, the female gamete (egg) is formed and remains fixed at the micropylar end of the embryo sac deeply embedded in the sporophytic tissues of the ovule, which is enclosed in the ovary well removed from the stigma. The male gametes (sperms) are enclosed in the pollen grain. To effect fertilization, the pollen germinate on the stigma to form a pollen tube that transports the two non-motile sperms to the embryo sac and delivers them in the vicinity of the egg. Whereas one of the sperms fertilizes the egg (syngamy), the other fertilizes the central cell (triple fusion). The fertilized egg (zygote) develops into an embryo, the progenitor of the next generation, and the fertilized central cell forms the endosperm tissue, the main source of nutrition for the developing and germinating embryo. Thus, in the angiosperms the gametes, the process of double fertilization, zygote, early stages of embryo and endosperm development are not readily accessible to study the cellular and molecular aspects of fertilization and embryogenesis. Therefore, for almost 100 years since the discovery of double fertilization in angiosperms, by Nawaschin (1898), not much progress could be made in this area. Whatever little information is known is based mainly on mutant analysis in Arabidopsis. Sexual incompatibility is a serious handicap in developing desirable hybrids. In this the pollen fails to germinate on the stigma or the pollen tube gets arrested or bursts before reaching the ovary. Two in vitro techniques developed to overcome this problem are in vivo pollination (IVP) and In vitro fertilization (IVF). IVF, developed in nineties involved isolation of male and female gametes and their in vitro fusion (fertilization) culture of the in vitro zygote to regenerate full plants. Complete IVF technique has been developed in only two plants, namely maize and rice. This technique is proving to be an invaluable aid to directly observe and analyse fertilization and post-fertilization process in flowering plants which is not possible under in vivo conditions.

Appendix

• 1. IVF Protocol for Zea mays (after Kranz 1999, 2001)

• i) Isolation of Egg and Central Cell

  • a) Collect ears after silk emergence and sterilize the outer leaves with 70 % ethanol.

  • b) Dissect out 20–30 nucellar tissue pieces from the ovules under a dissecting microscope. The embryo sac should be visible in the nucellar tissue.

  • c) Collect the nucellar tissue pieces in one ml of mannitol solution (750 mosmol kg⁻¹ H₂O) in 3-cm plastic Petri dishes and add 0.5 ml of enzyme mixture, containing 1.5 % pectinase, 0.5 % pectolyase Y23, 1 % cellulase Onozuka RS, 1 % hemicellulase and 530 mM mannitol (osmolality 570 mosmol kg⁻¹ H₂O) and pH adjusted to 5. Incubate the plates at room temperature without shaking.

  • d) After 30 min, store the dishes in a refrigerator at 6 °C, or dissect out manually the embryo sac cells in the incubation dish with glass needles under microscopic observation. Using a micropump, transfer the cells to microdroplets (2 μl of 600 mosmol kg⁻¹ H₂O mannitol droplet overlaid with 300 μl of autoclaved mineral oil) by microcapillary (tip opening of 100–200 μm for egg and 300 μm for central cell) on a siliconized and UV-sterilized cover glass.

• (ii) Isolation of Sperm Cells

  • (e) Overlay about 1000 pollen grains, in a 3.5-cm Petri dish, with 1.5 ml of mannitol solution (600 mosmol kg⁻¹ H₂O). After the bursting of the pollen, pick the sperm cells with a capillary (tip opening of 20 μm) and transfer them into the microdroplet containing egg or central cells using a micropump.

• (iii) Gametic Fusion

  • (f) Fix two electrodes (50-μm-diameter platinum wire) to an electrode support under the condenser of the microscope. Adjust the electrodes to a crosshair position and lower these onto the cover slip and into one of the microdroplets. Sterilize the electrodes in light flame before use.

  • (g) Align and fix the two gametes at one electrode. Prepare and adjust the electrodes carefully. By moving the microscope stage, first move an egg cell towards one of the electrodes. Finally, fix the egg cell to the electrode by dielectrophoresis (1 MHz, 70 V cm⁻¹). Using the same procedure, fix the sperm cell to the egg cell. Add 0.5–1.0 μl of mannitol solution of 520 mosmol kg⁻¹ H₂O. Now, the final distance between the two electrodes is adjusted to about double the sum of the diameters of the two gametes.

  • (h) Induce egg–sperm fusion by applying a single or a maximum of three negative DC pulses (50 μs, 0.9–1.0 kVcm⁻¹). With well-prepared electrodes, nearly 100 % fusion occurs. When no fusion occurs, lower the distance between the electrodes. Low fusion rate could also be due to very low turgor pressure of the gametes. In that case, reduce the osmolality of the fusion mixture. The central cell–sperm fusion is induced by a single or 2–3 negative DC pulses (50 μs, 0.4–0.5 kV cm⁻¹).

• (iv) Culture of In Vitro Zygote and Primary Endosperm Cells

  • (i) Gently moving the stage, remove the fusion product from the electrode. Move the electrode out of the droplet and transfer the fertilized egg (in vitro zygote) or central cell (in vitro primary endosperm cell), using microcappillary, to a 12-mm Millicell-CM insert plate with 100 μl of liquid ZMS medium (for composition see Table 13.2). Insert the plate into a 3.5-cm plastic petri dish containing 1.5 ml of a feeder suspension.

  • (j) Next day, place the dish on a rotary shaker (50–70 rpm) and incubate the cultures at 26 ± 1 °C under 16-h photoperiod and a light intensity of about 50 μmol m⁻²s⁻¹.

  • (k) After 10–14 days, transfer the embryos (0.4 mm long), using a Pasteur pipette or a small blade, to 1.5 ml hormone-free RMS 1 regeneration medium (MS medium with 6 % sucrose and solidified with 0.4 % agarose) in a 3.5-cm plastic petri dish.

  • (l) After about 2 weeks, when a coleoptile and roots are formed, transfer the structures to 1.5 ml of RMS 2 medium (RMS 1 medium with 4 % sucrose).

  • (m) After another 1–2 weeks, transfer plantlet into a glass jar containing 50 ml of RMS 3 medium (RMS 1 medium with 1 % sucrose and macro and micro salts reduced to half concentration).

  • (n) After about 2 weeks, transfer the plants, with 15–20-cm-long leaves, to soil.

• 2. IVF Protocol for Oryza sativa (after Uchiumi et al. 2006, 2007)

• (i) Isolation of Egg and Central Cell

  • (a) The experimental plants of rice (Oryza sativa cv. Nipponbare) are grown in green house under controlled conditions of light (13-h photoperiod with 150-μmol photons m⁻²s⁻¹) and temperature (25 °C).

  • (b) Ovaries are collected from spikes before anthesis, and ovules are dissected out in 0.3 M mannitol solution using sharp forceps and 30G short-needle syringe under a dissecting microscope.

  • (c) The ovules are transferred to 3.5-cm Petri plates containing 3 ml of mannitol solution. The peripheral region of the ovule along the antipodal end of the ovary is gently cut with the tip of a 30G needle, and 0.5 ml of the mannitol solution (650 mosmol kg^-1 H₂O) containing cell wall-degrading enzymes (0.5 % pectolyase Y-23, 1.5 % pectinase, 1 % cellulase Onozuka RS and 1 % hemicellulase) was added to the dish.

  • (d) After 10–15 min of incubation at room temperature, the egg cell and central cell become visible. At this stage, the egg cell could be manually isolated from the micropylar end of the ovule using glass needles under an inverted microscope. Longer incubation in the enzyme solution is damaging to the egg cell.

  • (e) The central cell is isolated from the enzyme-treated ovules by careful micromanipulation of the tissue with glass needle.

• (ii) Isolation of Sperm Cells

  • (f) The anthers isolated from the flower buds just before anthesis are transferred to 0.3 M mannitol solution in 3.5 cm plastic Petri plates, and the anther tissue is torn with the help of a forceps to release the pollen grains. The pollen grains burst after 3–5 min releasing their contents including the two sperms (Fig. 13.3b).

• (iii) Gametic Fusion

  • (g) An isolated egg cell and an isolated sperm cell are transferred to a 0.5–1.0 μl fusion droplet of mannitol solution (370 mosmol kg⁻¹ H₂O) overlaid with mineral oil on a cover slip and electrofused.

  • (h) After aligning the egg cell and the sperm cell on one of the electrodes under a dielectrophoretic AC field of 1 MHz and 0.4 kV cm⁻¹, 0.5 μl of mannitol solution (520 mosmol kg⁻¹ H₂O) containing 2 mM CaCl₂ is added to the fusion droplet. Addition of mannitol solution changes the shape of sperm to oblong and makes the attachment of the egg cell to the electrode more stable.

• (iv) Culture of In Vitro Zygote

  • (i) The fusion product is washed twice by transferring it to fresh mannitol droplets (450 mosmol kg⁻¹ H2O).

  • (j) The cleaned zygote is transferred onto the membrane of 12-mm Millicell-CM dishes, and the dish is placed in the centre of a 3.5 cm plastic dish filled with 3 ml of N₆Z medium (Cullen et al. 1998), modified by using commercial N₆ medium, and molality was adjusted to 450 mosmol kg⁻¹ H₂O with glucose. To the outer dish, 50 μl of rice suspension culture is added as feeder cells. The composition of the N₆Z medium is given in Table 13.2.

  • (k) After overnight culture at 26 °C in dark without shaking, the cultures are continued with gentle shaking (40 rpm).

  • (l) After 110–124 h of the fusion, the Millicell-CM dish with the developing embryo is transferred to a clean 3.5 cm dish filled with 3 ml of fresh N₆Z medium without feeder cells. The cultures are maintained at 26 °C in dark with shaking (40 rpm.)

  • (m) After 18–19 days in culture, the cell colonies (Fig. 13.8b) derived from the in vitro zygote are transferred for plant regeneration to a medium containing MS salts and vitamins, 100 mg L⁻¹ myo-inositol, 2 g L⁻¹ casamino acid, 30 g L⁻¹ sucrose, 30 g L⁻¹ sorbitol, 0.2 mg l⁻¹ NAA and 1 mg L⁻¹ kinetin and gelled with 0.3 % gelrite. The cultures are incubated under light (80-μmol photons m⁻² s⁻¹) at 30 °C.

  • (n) After 12–30 days, the differentiated shoots (Fig. 13.8c, d) are transferred to hormone-free medium containing MS salts, MS vitamins, 100 mg myo-inositol, 30 g L⁻¹ sucrose and 0.3 % gelrite. The cultures are maintained under 13-h photoperiod with photon flux density of 55-μmol photons m⁻² s⁻¹, at 28 °C.

  • (o) After 11–13 days, the plantlets (Fig. 13.8e) are transferred to soil.