Directional growth of plant pollen tubes to ovules

Summary

Since the sperm cells of angiosperms are unable to move, the completion of sexual reproduction in angiosperms requires the use of pollen tubes to deliver the sperm cells to the ovule's embryo sac. How do pollen tubes sense signals to grow directionally toward the ovule? Early observations revealed that the auxiliary cells in the embryo sac contain abundant endoplasmic reticulum structures, suggesting that the auxiliary cells are active in protein synthesis, and it was hypothesized that proteins secreted by the auxiliary cells are involved in the directional growth of pollen tubes.

In recent years, it has been shown that when the helper cells were burst by laser, the pollen tubes stopped growing toward the ovules. This directly confirms the involvement of helper cells in inducing the directional growth of pollen tubes. At present, the genes expressed by helper cells involved in inducing the directional growth of pollen tubes have been cloned. In vitro, wrapping the protein encoded by this gene with agar spheres also guided the directional growth of pollen tubes toward the agar spheres. In this experiment, Arabidopsis thaliana was chosen as the experimental material to observe the directional growth of pollen tubes to ovules.

Operation method

Directional growth of plant pollen tubes to ovules

Materials and Instruments

Equipment:
Arabidopsis flowers open on the same day.
Apparatus:
① Tweezers
② Dissecting needle
① Dissecting needle ② Dissecting glass
Reagents:
① Boric acid (H
3
BO
3
Boric acid (H 3 BO 3)
② Calcium chloride (CaCl
2
)
③ Potassium chloride (KCl)
④ Magnesium sulfate (MgSO
4
④ Magnesium sulfate (MgSO 4)
⑤ Sucrose
⑥ Sodium hydroxide (NaOH)
⑦ Low melting point agarose

Move

The basic process of directional growth of plant pollen tubes to ovules can be divided into the following steps:

(1) Prepare the medium according to the requirements, and add the final concentration of 1.5% low melting point agarose, and melt it in a water bath at 60 ℃ (medium composition: _H3BO3 0.01%; _{CaCl2 5} mmol/L; KC1 5 mmol/L; _MgSO4 1 mmol/L; Sucrose 15%; and adjust the pH to 7.5 with about 30 μL of 0.1 mol/L NaOH).

(2) Spread the medium in a plastic petri dish of about 3 cm (the thickness of the medium was about 2 mm) and let it solidify in a refrigerator at 4 ℃ (about 20 min).

(3) During this period, freshly opened Arabidopsis flowers were collected (pollen viability was the highest).

(4) Under a dissecting microscope, remove the petals and calyx of the flowers with pointed forceps, leaving the stamens and pistil. The anterior portion of the pistil containing the style is cut off with a sharp, one-sided blade and placed in solidified medium (Fig. 35-1).

(5) Remove the stamens with forceps, hold the filaments, and hand-pollinate the separated style stigmas. Under a high-powered dissecting microscope, the pollen can be seen adhering to the anterior end of the style between the finger-like protruding cells.

(6) The unfertilized ovule is then placed within about 100 μm of the anterior end of the style section (Fig. 35-1). The ovules are separated by fixing the pistil on a slide with double-sided adhesive and gently scratching the ovary along the ventral suture line with a sharp, thin dissecting needle, so that the ovules are visible. After gently scratching the bead stalk of the ovule with the dissecting needle, the ovule can be brought out with the side of the needle tip (be careful not to damage the ovule).

(7) Seal the petri dish and incubate at 21℃. To prevent evaporation of liquid, a circle of moistened cotton can be placed around the inside of the petri dish to maintain the humidity of the dish. To facilitate the growth of pollen tubes from the styles, care should be taken to check that the cut end of the styles is placed on top of the medium rather than buried in the medium.

(8) After 3~4 h, check the pollen tubes growing out from the cut side of the styles under an inverted microscope (the time varies with the length of the cut styles). When the pollen tube grows out, it can be seen under the inverted microscope intermittently that the pollen tube enters into the bead pore by directional growth induced by the ovule (Fig. 35-1).

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/