阅读说明：本技术 一种猕猴桃离体花粉活力测定方法 (Actinidia chinensis in-vitro pollen activity determination method ) 是由 姚春潮 刘占德 蒋宏勤 李建军 杨开宝 白雪 钱峥 王潇 仇长斌 于 2020-05-30 设计创作，主要内容包括：本发明涉及一种花粉活力测定方法,尤其涉及一种猕猴桃离体花粉活力测定方法,属于植物技术领域。本发明公开了一种猕猴桃离体花粉活力测定方法,可有效的测定猕猴桃花粉活力。所用的花粉离体萌发液体培养基,以纯净水为溶剂,组分为10％蔗糖及浓度为100mg·L<Sup>-1</Sup>的硼酸。在10mL试管内装上6-7mL花粉培养液,然后加入0.1％～0.2％的花粉,试管封口,置于摇床之中,在25℃、100rpm/min的条件下培养2-3h,然后将花粉培养液混均,加一滴于载玻片,盖上盖玻片,在电子显微镜下观察花粉萌发和花粉管生长情况。培养液制备方便,操作简单,耗时短,成本低,效果好。本发明用于猕猴桃花粉萌发能力的测定领域。(The invention relates to a pollen activity determination method, in particular to a kiwi fruit in-vitro pollen activity determination method, and belongs to the technical field of plants. The invention discloses a method for determining the vitality of kiwi fruit in-vitro pollen, which can effectively determine the vitality of kiwi fruit pollen. The pollen in vitro germination liquid culture medium comprises purified water as solvent, 10% sucrose and 100 mg.L ‑1 Boric acid of (a). Putting 6-7mL of pollen culture solution in a 10mL test tube, adding 0.1% -0.2% of pollen, sealing the test tube, placing the test tube in a shaking table, culturing for 2-3h at 25 ℃ and 100rpm/min, uniformly mixing the pollen culture solution, adding a drop of the pollen culture solution to a glass slide, covering the glass slide, and observing the pollen germination and pollen tube growth conditions under an electron microscope. The culture solution is convenient to prepare, simple to operate, short in time consumption, low in cost and good in effect. The method is used for the field of determination of pollen germination capacity of kiwi fruits.)

1. The method for determining the vitality of the in vitro pollen of the kiwi fruit is characterized by comprising the following steps:

1) preparation of culture solution

2) Pollen preparation

3) Pollen culture

Putting pollen culture solution into a test tube, then adding kiwi fruit in-vitro pollen accounting for 0.1-0.2% of the weight of the culture solution, sealing the test tube, and putting the test tube in a shaking table for culturing for 2-3 h;

4) observe pollen germination condition

Dropping a drop of pollen culture solution on a glass slide, covering the glass slide, observing the germination condition of pollen on the glass slide under an electron microscope, taking the condition that the length of a pollen tube is greater than the diameter of the pollen as pollen germination, randomly observing 5 visual fields for recording, and repeating the experiment for 3 times;

5) calculating the germination rate of pollen

And (3) calculating the pollen germination rate of 15 fields, and averaging to obtain the vitality of the kiwi in vitro pollen, wherein the pollen germination rate is the number of the pollen germinated in each field/the total number of the pollen grains in the field.

2. The method for determining the viability of the isolated pollen of kiwi fruit according to claim 1, wherein the culture solution is purified water as a solvent, and purified water is addedAdding sucrose and boric acid, wherein the mass fraction of sucrose in the culture solution is 10%, and the concentration of boric acid is 100 mg.L^-1。

3. The method for determining the vitality of the in-vitro kiwi pollen according to claim 1, wherein the pollen is fresh pollen or frozen pollen, the fresh pollen is collected from the male kiwi plant flowers which shed pollen on the day at 9: 00-10: 00 am, the pollen sacs which shed pollen are lightly brushed by a brush, and the pollen is collected; the frozen pollen is taken out from a freezing storage environment, is placed in the environment at 4-5 ℃ for 10-12 hours, is opened, is filled in a pollen bottle or is poured in a glass vessel or a plastic vessel, is placed in the environment at 15-22 ℃ and humidity of 70-80% and is rewarmed and hygroscopic for 2-3 hours under the environment of preventing sunlight from directly shining, and the pre-wetted frozen stored pollen is obtained.

4. The method for determining the vitality of the isolated pollen of the kiwi fruit of claim 1, wherein the pollen culture is performed by loading 6-7mL of pollen culture solution into a 10mL test tube, adding the isolated pollen of the kiwi fruit, and culturing for 2-3h at 25 ℃ and 100 rpm/min.

Technical Field

The invention belongs to the technical field of plants, relates to pollen viability determination of plants, and particularly relates to a method for determining in-vitro pollen viability of kiwi fruits.

Background

The Actinidia is a plant of Actinidia of Actinidiaceae, native to China. The kiwi fruit is rich in vitamin C, mineral substances and soluble dietary fibers, is one of the most comprehensive fruits in the fruit, and is known as the king of the fruit. Pollen as a male gametophyte of a plant plays an important role in sexual reproduction, in order to solve the problems of inconsistent flowering phases of parents, distant hybridization and artificial supplementary pollination, pollen is generally collected and stored at an early stage, and before the pollen is used, the pollen needs to be identified in viability to evaluate whether the pollen has insemination capability so as to determine the pollen dosage and pollination effect of the artificial supplementary pollination. In the prior art for measuring the vitality of the kiwi fruit pollen, the operation of culturing by adopting a solid culture medium is relatively complex, and the required time is long. Patent CN201610136320.9 describes a method for detecting pollen storage and in-vitro culture activity of actinidia arguta, collecting buds of actinidia arguta in the bud period and taking out anthers in the bud period at 9: 00-10: 30 in sunny morning, placing the anthers in a parchment paper bag, placing the parchment paper bag in a dryer containing allochroic silica gel, drying for 20-28 hours at the temperature of 20-25 ℃ until the water content of the anthers reaches 4.5-6%, sieving with a 60-80-mesh sieve, sealing the collected pollen and storing at the low temperature of-85-75 ℃. Taking out the pollen stored at low temperature, placing the pollen at room temperature for 5-10 min, uniformly spreading the pollen on a solid culture medium containing 250-350 mg/L boric acid, 45-55 g/L sucrose and 9.5-10.5 g/L agar for culture (the spreading density of the pollen is 50-100 grains under 10 x10 times of microscope under each visual field), carrying out dark culture at 19-21 ℃ for 5.5-6.5 h, and then carrying out microscopic examination under a microscope and counting the pollen germination rate. However, the solid culture medium can be used only by boiling and cooling, which is relatively complicated and time-consuming, and the pollen culture time also needs 5.5-6.5 h. The dyeing method is fast and short in time, but the result is extremely unstable (red poplar, yunhe ming, plum blossom, and von glittering & translucent. Actinidia chinensis pollen viability determination method and anther treatment method research [ J ]. northern horticulture, 2015(08): 36-39).

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the method for determining the vitality of the pollen in vitro of the kiwi fruit, which has the advantages of convenient preparation of a culture solution, simple operation, short time consumption, low cost, good effect and capability of quickly and accurately determining the vitality of the pollen in vitro of the kiwi fruit.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for determining the vitality of the in vitro pollen of kiwi fruit comprises the following steps:

1) preparation of culture solution

2) Pollen preparation

3) Pollen culture

Putting pollen culture solution into a test tube, then adding kiwi fruit in-vitro pollen accounting for 0.1-0.2% of the weight of the culture solution, sealing the test tube, and putting the test tube in a shaking table for culturing for 2-3 h;

4) observe pollen germination condition

Dropping a drop of pollen culture solution on a glass slide, covering the glass slide, observing the germination condition of pollen on the glass slide under an electron microscope, taking the condition that the length of a pollen tube is greater than the diameter of the pollen as pollen germination, randomly observing 5 visual fields for recording, and repeating the experiment for 3 times;

5) calculating the germination rate of pollen

And (3) calculating the pollen germination rate of 15 fields, and averaging to obtain the vitality of the kiwi in vitro pollen, wherein the pollen germination rate is the number of the pollen germinated in each field/the total number of the pollen grains in the field.

The culture solution takes purified water as a solvent, sucrose and boric acid are added, the mass fraction of the sucrose in the culture solution is 10%, and the concentration of the boric acid is 100 mg.L^-1。

The pollen is fresh pollen or frozen pollen, the fresh pollen is collected from the male kiwi plant flowers which are scattered on the same day at 9: 00-10: 00 am, the pollen sacs which are scattered are lightly brushed by a brush, and the pollen is collected; the frozen pollen is taken out from a freezing storage environment, is placed in the environment at 4-5 ℃ for 10-12 hours, is opened, is filled in a pollen bottle or is poured in a glass vessel or a plastic vessel, is placed in the environment at 15-22 ℃ and humidity of 70-80% and is rewarmed and hygroscopic for 2-3 hours under the environment of preventing sunlight from directly shining, and the pre-wetted frozen stored pollen is obtained.

The pollen culture is to put 6-7mL pollen culture solution into a 10mL test tube, then add the kiwi fruit isolated pollen, and culture for 2-3h at 25 ℃ and 100 rpm/min.

Compared with the prior art, the invention has the beneficial effects that:

compared with the conventional pollen viability solid culture method, the method for determining the in vitro pollen viability of the kiwi fruit is simple and convenient to operate, and the pollen culture only needs 2-3 hours which is far lower than 5.5-6.5 hours of the solid culture method;

at present, methods for determining vitality of kiwi fruit pollen by using a dyeing method comprise 3 methods of triphenyltetrazolium chloride (TTC) dyeing, iodine-potassium iodide (I2-KI) and methylene blue dyeing, and although dyeing is fast, errors are large. Compared with a dyeing method, the method has the advantages that the kiwi fruit in-vitro pollen is dispersed in the culture solution, the kiwi fruit in-vitro pollen is cultured at a proper temperature by adopting a sucrose-boric acid solution with a proper concentration, sufficient oxygen is provided for kiwi fruit pollen germination by continuously shaking the shaking table, so that the normal germination of pollen with viability is ensured, the statistical method is clear, the pollen viability is determined by applying the method, the result is easy to observe, the statistics is convenient, the repeatability is high, and the obtained data is accurate and reliable.

Drawings

FIG. 1 is the germination rate of pollen at different sucrose concentrations.

FIG. 2 shows the germination rate of pollen at different boric acid concentrations.

FIG. 3 shows the germination rates of pollen at different culture temperatures.

FIG. 4 shows the germination rates of pollen at different cultivation times.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the drawings and examples.