JPH0636698B2 - How to efficiently obtain rice haploids - Google Patents

Description

TECHNICAL FIELD The present invention is a technique for obtaining a haploid plant from rice anthers or pollen through callus, and is used for efficient breeding of rice.

(Prior Art) A haploid production method by anther culture of rice was first reported in 1968 (Niizeki and Oono (1968) Proc. Jan. Acad 44,554-5.
57) Since then, much research has been done.

Their method, and subsequently the improved method, is basically a method of implanting rice anthers on an agar medium to obtain a callus and differentiating a plant from the callus, and various improvements have been made thereafter.

As an improvement of the medium, N6 suitable for anther culture of gramineous plants
Development of medium (Chu et al. (1975) Scientia Sinica 18,659-66
There is 8). There are also reports on selection of various hormones suitable for inducing callus formation and differentiation, addition of potato extract to the medium for improving the differentiation rate, and the like. There is also a report that the callus rate was improved by pre-treating rice ears at low temperature (5 to 10 ° C).

However, in the conventional method in which anthers are directly placed on an agar medium and cultured, no matter how improved medium is used or pretreatment is applied, the callus formation rate per planted anther is about 10% at most. And was very low. The rate of plant formation from the callus formed is also no more than 10% per planted callus, even with the improved medium.
When the conventional technique is used, the formation rate of plant bodies per anther planted as a whole is about 1% at the highest.

Generally, more than 10,000 individuals are used for testing in the field of rice breeding, but if such individuals are to be produced using conventional techniques, one million or more anthers must be planted. That is, applying the conventional technique to the actual breeding work was inefficient and was not practical at all.

Furthermore, it is reported that most of the plants formed using these methods are diploid and the role of haploid plant individuals is 50% at most. That is, as a whole, this method was far from a practical technology.

(Problems to be Solved by the Invention) The conventional method of culturing rice anthers on an agar medium cannot regenerate plants frequently, and regenerated plants also have high diploid frequencies. , It is impossible to actually use for breeding.

The haploid breeding technology using anther culture is considered to make a large contribution to rice breeding in principle, but the low regeneration frequency of plants from anthers as described above is a bottleneck for practical application. Has become.

The task to be solved is to develop a technique for regenerating plants from anthers at a higher frequency and to open the way for applying this haploid breeding technique to rice breeding.

(Means for Solving Problems) In order to regenerate plants from anthers at a higher frequency, as a first step, it is necessary to form more callus from one anther.

Then, the plant is regenerated next, but it is difficult for the plant to directly differentiate from the callus formed in the first step.
Therefore, in the second step, the callus formed in the first step is once grown in a growth medium. In this case, the problem is that rice callus generally has a property that the rate of differentiation into a plant decreases rapidly as the culture is subcultured, and it is necessary to efficiently grow the callus in a short period of time. . This second step is indispensable in the third step for efficient regeneration of the next plant, but this third step also requires careful attention.

The third stage requires efficient regeneration of plants from the callus grown in the second stage.

The above will be described in more detail.

Regarding the method of forming more callus from one anther at a high rate in the first step, since the anther was cultivated on the agar medium in the conventional technique, even if it is theoretically the most, Only one callus could be regenerated from one anther. On the other hand, one anther contains thousands of pollens, and if all these pollens can be used effectively to form callus, theoretically, one anther can Should be able to form the callus of.

The following two methods can be considered to enable this. The first method is to cultivate anthers on a liquid medium, and the callus formed by the splitting of pollen inside the anther spills from the anthers, and each callus can grow independently in the liquid medium. Such a method, that is, a floating culture method of anthers.

The second method is a pollen culture method, in which pollen is directly taken out from an anther, and the pollen is carefully cultured to obtain a callus formed by splitting each directly from the pollen grain.

In both of these methods, in principle, male gametes are dispersed in a liquid and cultured to form thousands of callus from one anther, and the method itself is not novel.

However, neither the first method nor the second method has been tried so far in rice.

Hereinafter, specific methods of applying the first step to meet the purpose of the present invention using these methods will be described.

Collect young shoots of rice (including pollen from the mononuclear stage) for 4-10
Hold at a temperature of ℃ for 5-14 days. The whole ear is sterilized with 70% ethyl alcohol, the spikelets are taken out, and the anthers are taken out from the spikelets one by one. In the first method, the anther thus taken out is suspended in a liquid medium as described below and cultured. As the medium to be used, almost any medium (eg MS, B5) that has been used for anther culture of rice can be used. However, the N6 medium developed for rice anther culture gives even better results. Basic medium was prepared by adding 5 to 15% potato extract and 30 g / sucrose to these medium, and auxins such as NAA (naphthalene acetic acid) and 2,4-D (2,4-dichloro) were added to the medium. Phenoxyacetic acid) or IAA (indole acetic acid) 0.1 to 10 ppm, and cytokine kinetin,
A medium to which 0.5 to 10 ppm of zeatin, benzyladenenyl or isopentenyladenine was added was used as a medium.

Each of the plant hormones may be given alone, but depending on the rice cultivar, the combined use may bring about a more reproducible result.

When cultured in this manner, formation of minute cell aggregates (callus) is observed in the liquid medium about 20 days after the start of culture. When the culture is continued, the number of minute callus formed increases for about one month.

Callus formed in this way has a size
Sequentially transfer to the second stage culture within 1 mm.

In the second method, the anthers are cultured for 7 days in the same manner as in the first method, the pollen is isolated from the anthers, and the pollen is cultured in the medium described below.

The medium for cultivating pollen is 2,4-D 0.5-5 ppm, B5 medium containing vitamin and sucrose 3%, N6 or
R2 medium was used. After 2 to 3 weeks of culture, small callus formed by splitting pollen is formed. As in the first method, the formed callus is successively transferred to the second-stage culture before its size exceeds 1 mm in diameter.

In the second step, the callus formed in the first step is cultured on the following agar medium.

Any medium can be used as long as it is a medium commonly used for tissue culture, but N6 or MS medium has produced particularly good results.

1-5 g / casein hydrolyzate, 10-1 in these media
5 g / sucrose and 0.5 to 1.2% agar were added to the basic medium, and auxin NAA, 2,4-D or IAA was 0.1 to 10 ppm, and cytokinins kaitinene, zeatin or benzyladenine were used. 0.5 to 1
A medium supplemented with 0 ppm was used.

After culturing in this medium for 10 to 20 days, callus grown to a diameter of 3 to 5 mm and not exceeding 7 mm at the maximum is transferred to the third stage culture.

In the third step, the callus grown in the second step is cultured on the following agar medium to regenerate the plant body.

The medium used is N6 or MS medium containing 30 g / sucrose and 0.
A medium supplemented with 7 to 1.2% agar was used as a basic medium, and a medium supplemented with 0.1 to 3 ppm of auxin NAA or IAA and 1 to 5 ppm of cytokinin kinetin or benzyladenine was used as a medium.

When culturing in this way, 5 to 10 days after the start of culturing,
Small green spots or green spots form on the surface of the white callus. From here, further differentiation,
Shoot and root differentiation is seen on day 20-30.

By once performing the second-stage culture and then performing the third-stage culture, the differentiation rate of the plant body in the third stage reached 30 to 50% per planted callus.

After softening the root tips of the regenerated plants with pectinase, they were stained by the flame method and Giemsa stain, and the number of chromosomes was measured using a microscope. As a result, about 75% of the regenerated plants were haploid.

After sufficiently growing these redifferentiated plantlets, they are transplanted to a pot containing seedling cultivation soil and grown at room temperature to give healthy plant bodies. The grown plant blooms and bears fruit. At this time, the plant having 2n = 24 chromosomes bears fruit.
On the other hand, haploid plants do not bear fruit and their fruit is poor.

(Function) Compared to the case of using the conventionally used method, by using the method of the present invention, it is possible to efficiently and dramatically form a large number of callus from anther or pollen. Even better, many of the plants formed from callus derived from anthers or pollen using this method are haploid.

Example 1 Ears at the earing stage of rice plants (variety: Nihonbare, Sasanishiki) were collected and subjected to low temperature treatment at 10 ° C. for 10 days, and then the whole leaf sheath was sterilized with 70% ethanol and washed with sterile water. did. The spikes were aseptically removed from the leaf sheath sterilized in this manner, and the anthers inside were removed from the spikelets containing pollen at the mononuclear stage and then planted in a liquid medium for callus induction.

The medium for cultivating anthers was 4 ppm of NAA (naphthalene acetic acid) as a plant hormone and 2 pp of kinetin in a basic medium of N6.
The pH was adjusted to 5.8 by adding m and adding 3% of sucrose and 15% of potato extract.

3 m of the medium thus prepared was dispensed in a plastic petri dish having a diameter of 35 mm. Fifty anthers were suspended on the liquid medium and cultured under the conditions of 26 ° C and about 1001ux.

Pollen-derived callus was formed 3 to 4 weeks after the culture. When the diameter of each callus has grown to 0.5-1 mm,
The callus was transplanted to a growth medium. The number of callus per petri dish formed is 25 with a small number and 22 with a large number.
There were five.

When these calli were cultured by the method as described in Examples 3 and 4, many haploid plants could be obtained.

Example 2 Ears at the earing stage of a rice plant (variety: Nihonbare, Sasanishiki) were collected and subjected to low temperature treatment at 10 ° C. for 10 days, and then the whole leaf sheath was sterilized with 70% ethanol and washed with sterile water. did.
The spikelets were aseptically taken out from the leaf sheath sterilized in this manner, and the anthers inside the spikelets containing the mononuclear stage pollen were extracted and planted in a liquid medium for callus induction.

The medium for cultivating anthers was the addition of 4 ppm of NAA and 2 ppm of kinetin as plant hormones to the basal medium of N6 and sucrose to 3
% And potato extract were added to adjust the pH to 5.8.

After culturing anthers on this medium for 7 days, the anthers were taken out and lightly homogenized with a mortar and pestle to take out pollen.

This pollen was suspended at a concentration of 1 to 2 × 10 ⁵ / m and cultured in a pollen culture medium. 2,4-D 2p as a medium for pollen culture
N6 or R2 medium containing pm, B5 vitamins and 3% sucrose was used. Two to three weeks after the start of culture, pollen was divided and many small calli were formed. This callus has a diameter of 0.5-1 mm
At the time point when it grew up to 1), it was transplanted into a medium for callus growth.

When these calli were cultured by the method as described in Examples 3 and 4, many haploid plants were formed.

Example 3 Callus formed by culturing pollen and anthers was transferred to a callus growth medium to grow the callus. The callus growth medium used was one obtained by removing the potato extract from the callus induction medium and adding 3 g of casein hydrolyzate and 1% of agar. 20 m of this sterilized medium was dispensed into a petri dish having a diameter of 9 cm and solidified, and then 100 callus formed in Examples 1 and 2 were planted per petri dish. When the implanted callus had grown to a diameter of 3 to 5 mm, each callus was transplanted to a medium for inducing differentiation.

When the callus thus carefully grown was transferred to a medium for inducing differentiation, it easily differentiated to form a plant.

Example 4 Callus formed by suspension culture and pollen culture of anthers was further grown on a callus growth medium, and then the grown callus was transplanted to the differentiation medium to induce plant differentiation. The medium consists of N6 medium components containing 1 ppm of NAA, 2 ppm of kaitinene, 3% of sucrose and 1% of agar and adjusted to pH 5.8. The formation of green spots was observed about 1 week after transplantation, and the plants were differentiated into plantlets in about 1 month. The regeneration rate of plantlets per planted callus reached 30-50%.

The plantlets were transplanted to a magenta box containing an agar medium, allowed to grow sufficiently, and then transplanted to a pot containing seedling-raising soil to grow in a greenhouse.

When the number of mature rice chromosomes and the degree of fertility were examined, 25% were normal diploids. The remaining 75% were haploid.

(Effects of the Invention) When anther culture is performed using the method according to the present invention, plants are efficiently formed. The number of plants formed per 100 anthers planted is 25 to 200. This is a very high value as compared with the conventional method. Moreover, reproducibility is very good. Furthermore, what is characteristic of the population of plants regenerated by this method is that the haploid ratio is extremely high (75%) as compared with the conventional method (50%).

That is, using the method devised by the present invention,
From anthers or pollen, a haploid plant individual can be obtained very efficiently. In other words, by using this method, the haploid breeding technology, which had been thought to be a very attractive technology in principle in the past but was not practical, can actually be used for rice. This has enabled efficient breeding of rice.

Claims (1)

[Claims] 1. An anther or pollen of rice is dispersed and cultured in a liquid medium to form callus having a diameter of not more than 1 mm, and then this callus is formed into a callus having a size of not more than 7 mm on a solid medium. A method of efficiently obtaining rice haploids, which comprises growing the callus and growing the callus into a differentiation medium to form a plant.