JP2000004701A - Creation of genetically pure genus eustoma plant by culturing ovule using false fertilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently growing the F1 species of genus Eustoma plant in a short time by culturing ovules under specific conditions using false fertilization on the pistils of male sterility type genus Eustoma plant or the like, to create the genetically pure genus Eustoma plant. SOLUTION: This method comprises steps of (A) pollination of the ovules of a male sterility type or castrated genus Eustoma plant with the pollen irradiated with ionizing radiation of 50 KR or more, preferably 50 to 200 KR to deactivate its cell nuclei, (B) picking up the ovules 2 to 10 weeks, preferably 3 to 4 weeks after the pollination, and (C) culturing and growing the ovules in a medium incorporated with, e.g. coconut milk, to create the genetically pure genus Eustoma plant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a genetically pure (homozygous) Eustoma plant. Genetically pure Eustoma plants are doubled haploid Eustoma plants obtained by doubling haploid Eustoma plants. The pure Eustoma plant thus produced can be used for breeding a first-generation hybrid (F1) variety.

[0002]

2. Description of the Related Art Eustoma (Eustoma grandiflorum or Eustoma taxonomically) introduced into Japan around 1938
oma exaltatum) has been bred in Japan and many varieties have been bred. At the beginning of the introduction, those with excellent traits were selected from among those with variations in growth and traits, repeated selfing and genetically fixed to produce varieties. However, this fixed species lacked uniformity such as uniform flowering, plant shape, and plant vigor. In recent years, a large number of F1 varieties have been cultivated to achieve this uniformity, mainly by Japanese seed and seedling companies. In order to grow this F1 variety, it is essential to cross genetically pure pollen parent lines and seed parent lines, and in order to select better F1 varieties, cross many pure lines. There is a need. In order to produce such a pure line, a process of selfing a genetically unfixed plant for 5 to 8 years is required, and a long term is required at present. On the other hand, the life span of the varieties of the developed varieties is
It is getting shorter than in the past. Therefore, it has become difficult to produce varieties corresponding to changes in consumer preferences in a timely manner by the conventional self-breeding method.

[0003] Methods for producing a pure plant in a short period of time include methods such as anther culture, pseudo-fertilized ovule culture, and unfertilized ovule culture. Anther culture is a technique in which pollen in anthers (stamens) is split to regenerate plants and obtain haploid plants. The case where pollen is taken out from the anther and cultured is called pollen culture, and the case where the anther is cultured as it is and the inside pollen is divided is called anther culture. Pseudofertilized ovule culture is a technique for regenerating plants from ovules to obtain haploid plants. The pollen pollinates the pistil (stigma), extends the pollen tube, and the nucleus in the pollen fertilizes with the egg cell. Pseudofertilized ovule culture is a method of culturing haploid ovules by inactivating pollen at this time by inactivating the function of the pollen by radiation or the like so that the plant senses as if fertilized. The unfertilized ovule culture is a method of culturing a haploid ovule without fertilizing the inactivated pollen in the pseudo-fertilized ovule culture. The regenerated plant is a haploid, and as long as natural doubling does not occur, the fruit does not fertilize as it is. Therefore, the callus or the plant is treated with an agent that doubles the chromosome (colchicine, colcemide, oryzalin, etc.) to double the chromosome.

[0004] Studies on the production of haploids of the genus Eustoma have been conducted mainly by anther culture (Okayama Agricultural Exp., 1991-199).
Five). However, up to now, there has been only one report of the production of Eustoma haploids, but they have died at the seedling stage (Okayama Agricultural Sciences, 1995). And the retest by this experimental system has not been successful. A method of culturing unfertilized ovules and pseudo-fertilized ovules has also been attempted (Okayama Agricultural Research Institute, 1994-1995), but this method has not succeeded in producing haploids. For example, Okayama agricultural test inactivates pollen with an X-ray irradiation dose of 5 to 40 kR,
75 days after pollination, the ovules are taken out and cultured (Okayama Agricultural Research Institute, “Single Year Study Results” (created January 1995) and (created January 1996)). "The absence of haploids in the rooting individuals did not clarify the conditions for producing haploids by irradiating pollen with X-rays."
(Created January 1996) 3. Summary of the results (2)).

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to solve various problems related to a method for producing a genetically pure Eustoma plant which has been conventionally performed. In other words, the object is to provide means capable of producing a pure plant in a short period of time.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on a method for producing a haploid or doubled haploid line of a plant belonging to the genus Eustoma. We have completed a means of efficiently producing genetically pure plants in plants. That is, the present invention pollinates the pistil of a male sterile line Eustoma plant or a emasculated Eustoma plant with pollen in which the cell nucleus is inactivated, and extracts an ovule 3 to 4 weeks after the pollination. A method for producing a genetically pure Eustoma plant characterized by culturing and growing ovules.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The present invention is applicable to all Eustoma plants, but is particularly preferably applied to Eustoma.

[0008] The Eustoma plant of the present invention can be produced as follows. First, the anthers are removed from the buds of the genus Eustoma. The emasculation is performed before flowering, and the emasculated buds are covered with a bag or the like. When using male sterile plants, emasculation is not required, but bagging is performed. Next, when the flower buds bloom and the stigma is opened and pollination is possible, pollen whose nucleus has been inactivated in advance is pollinated to the stigma. Nuclear inactivation is
It can be carried out by irradiating the normal pollen at the flowering time with X-rays. The irradiation amount is preferably 50 to 150 kR, but is not particularly limited as long as it can inactivate pollen nuclei. The pistil after pollination is again covered with a bag or the like, and left until the ovary is sufficiently enlarged. The period until the ovary is sufficiently enlarged depends on the female parent plant used and the cultivation environment, but is usually about 3 to 4 weeks after pollination. The enlarged ovary is collected, sterilized with ethanol or the like, and then aseptically decomposed to remove an ovule. The removed ovule is placed on a predetermined medium. At this time, it is possible to place and culture with the placenta attached, but preferably only the ovule is placed. The basic composition of the medium used here is not particularly limited, and for example, a medium obtained by adding sucrose to the inorganic salt and organic substance composition of the MS medium is used. It is desirable to add coconut milk. Plants appear about 3 to 5 weeks after the start of culture. Usually, natural doubling occurs during the culture process,
A doubled haploid line, that is, a genetically pure plant can be obtained without special treatment, but doubling treatment of the chromosome may be performed if necessary. The doubling treatment can be performed by using a solution containing a reagent used in a usual doubling treatment, such as colchicine, colcemide, orizarin. Alternatively, a haploid plant and a doubled haploid plant may be identified in advance, and the doubling process may be performed only on the haploid plant. As the identification method, chromosome investigation, chloroplast number in guard cells, quantification of DNA content by flow cytometry, and the like can be used.

[0009]

[Example 1] [Example 1] Confirmation of genetic properties of a plant used as a pollen parent (1) Selection of plants that have been confirmed to be genetically fixed with respect to the trait of purple flower color From among breeding materials Plants with a purple flower color were selected and selfing was repeated until the plants were confirmed to be genetically fixed in purple with respect to flower color. (2) Investigation that the trait of purple flower color is genetically dominant Plants that have been genetically confirmed to have a fixed purple flower color in (1) can be used for individuals with other flower colors They were crossed according to the usual method. The flower color of all the F1 generations became purple, and the trait of purple flower color was shown to be genetically dominant.

[Example 2] Production of a plant by pseudo-fertilized ovule culture of Eustoma (1) Removal of buds before opening of anthers Genetic unfixed (not pure) Eustoma strain cultivated in a pipe house Anthers before anther opening were removed from the buds with tweezers, and the emasculated buds were covered with a paraffin paper bag. (2) False fertilization Two to seven days after emasculation, the pistil is removed from the flower bag in a pollinable state, and the plant is confirmed to be genetically fixed for the trait of purple flower color. X-rays were applied to the collected pollen at 0 kR (unirradiated), 50 kR, and 10 kR, respectively.
Irradiation was performed at 0 kR and 150 kR, and these were pollinated to the stigma of the pistil of the flower, which had been emasculated in advance. Thereafter, a bag of paraffin paper was again placed over the flowers to prevent unintentional hybridization with other pollen.

(3) Ovule culture The ovary 3 to 4 weeks after pollination of the pollen irradiated with X-rays was collected, and after sterilizing the surface, the ovary was dissected and the ovule was excised and placed on a medium. (4) Medium used for culture The medium used was an MS medium generally used in tissue culture of plants. However, 3% of sucrose, 50 ml / l of coconut milk and 0.8% of gellan gum were added, and the pH was 5.8.
Was adjusted.

[Example 3] Cultivation test and examination of ploidy of a plant obtained by pseudo-fertilized ovule culture (1) Appearance of plant 3 to 5 weeks after the start of culture after pseudo-fertilization induction treatment A plant has appeared. (2) Plant Flowering Assay Plants obtained by the pseudo-fertilization induction treatment were cultivated and flowered. In the 50 kR, 100 kR, and 150 kR irradiation groups, 88% (114 out of 129 individuals) are not purple flowers that can be an indicator of genetic involvement of inactivated pollen.
Blossomed flowers of other colors. Also, the flower color of these plants was not the same as the flower color of the plants that had been subjected to pseudo-fertilization, but exhibited the color of a separated flower. These results suggest that the plants obtained by the pseudo-fertilization induction treatment are not derived from somatic cells of normal fertilized embryos or plants subjected to pseudo-fertilization, but from embryos in which pseudo-fertilization was induced. . On the other hand, 0kR irradiation (not irradiated)
In the plot, 100% (30 out of 30 individuals) showed purple flower color in which the pollinated pollen was genetically involved.

(3) Investigation of ploidy The ploidy of 200 individuals selected from the obtained plants was examined using DNA analysis by flow cytometry, the number of chloroplasts in guard cells and the size of pollen as indices. As a result, all the individuals examined were found to be diploid.
It is presumed that natural doubling occurred during the culture process.

[Example 4] Cultivation test of self-propagated progeny of plants obtained by pseudo-fertilized ovule culture (1) Cultivation of self-propagated progeny One plant of Example 2 was self-pollinated and self-pollinated. Fifty individuals were cultivated. (2) Investigation of the degree of fixation As a result of investigating the morphological characteristics of the plant, particularly the flower color, of (1), no clear difference was observed between individuals in the same line. Therefore, these lines could be confirmed to be genetically pure. It was confirmed that a plant body was obtained by performing pseudofertilized ovule culture on Eustoma plants according to Example 1, and that the plants obtained by pseudofertilized ovule culture were doubled haploid lines according to Examples 2 and 3. . From the above results, it was found that a doubled haploid line could be created in a genus Eustoma by pseudofertilized ovule culture.

[0015]

Industrial Applicability The present invention provides a method for efficiently producing a genetically pure Eustoma plant. By utilizing such a genetically pure Eustoma plant, it is possible to efficiently grow an F1 variety of Eustoma plant in a short period of time.

 ────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Mamiko Yui 1900 Irai, Oyama, Toyama, Japan Tobacco Industry Co., Ltd. Plant Development Laboratory F-term (reference) 2B030 AA02 AB03 AD12 CB02 CD03 CD07 CD09 CD14 CG01 HA01

Claims (5)

[Claims] 1. A pollen having a cell nucleus inactivated is pollinated to a pistil of a male sterile line Eustoma plant or a emasculated Eustoma plant, and an ovule is extracted 2 to 10 weeks after the pollination. A method for producing a genetically pure Eustoma plant characterized by cultivating and growing Eustoma. 2. The method for producing a genetically pure Eustoma plant according to claim 1, wherein the ovule is removed 3 to 4 weeks after pollination. 3. The pollen in which the cell nucleus has been inactivated is 50 kR.
The method for producing a genetically pure Eustoma plant according to claim 1 or 2, wherein the method is obtained by irradiating the pollen with the above ionizing radiation. 4. The method for producing a genetically pure Eustoma plant according to claim 3, wherein the ionizing radiation is 50 to 200 kR. 5. The method for producing a genetically pure Eustoma plant according to claim 1, wherein a medium to which coconut milk is added is used as a medium for culturing the extracted ovules.