JP2000342093A - Method for creating pure line genus eustoma plant genetically by using high degree polyploid pollen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To create a pure line (homozygote) of genus Eustoma plant efficiently genetically by growing the ovule or seed contained in the ovary of a pistil of a genus Eustoma plant after the pistil is pollolinated of a pollen obtained from a specific plant. SOLUTION: A pistil of a genus Eustoma plant is pollinated of a pollen obtained from a genus Eustoma high-degree polyploid plant, and subsequently the ovule or seed contained in the ovary of the pistil is grown. Further, a pistil of a genus Eustoma plant is pollinated of the pollen obtained from a genus Eustoma high-degree polyploid plant, subsequently the oval or seed contained in the ovary of the pistil is taken out and cultured on a culture medium. Thus, a pure line of genus Eustoma plant can be created genetically.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a genetically pure (homozygous) Eustoma plant. The genetically pure Eustoma plant is a doubled haploid Eustoma plant obtained by doubling a haploid Eustoma plant, and can be used for producing a first-generation hybrid (F1) variety.

[0002]

[ Prior Art] Turkey Gyokyo ( Eustoma grandiflorum (Raf.)
breeding in Japan has progressed, 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 breed this F1 variety, it is essential to cross the genetically fixed almost pure pollen parent line and the seed parent line, and in order to select a better F1 variety, many pure lines must be crossed. Must be crossed. In order to produce such a pure line, a process of self-breeding 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 becoming shorter than in the past due to the acceleration of changes in consumer trends. 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] As a method of producing a pure plant in a short period of time, anther culture (Sudhir K. Sopory and Meenakshi Munsh
i, 1996, In Vitro Haploid Production in Higher Plan
ts, 1, 145-176), pollen culture (JM Dunwell, 1996, In
Vitro Haploid Productionin Higher Plants, 1, 205-2
16), pseudo-fertilized ovule culture (Sara Sestili and Nadia Ficca
denti, 1996, In Vitro Haploid Production in Higher
Plants, 1, 263-274), unfertilized ovule culture (ER Joachi
m Keller and Larissa Korzun, 1996, In Vitro Haploid
Production in Higher Plants, 1, 217-235). Anther culture is a technique in which pollen in anthers (stamens) is split to regenerate plants and obtain haploid plants. In the anther culture, the case where pollen is taken out from the anther and cultured is particularly called pollen culture. In addition, pseudo-fertilized ovule culture is a technique for regenerating plants from ovules that have undergone pseudo-fertilization to obtain haploid plants. Normally, pollen pollinates pistils (stigmas), extends the pollen tube, and the sperm in the pollen fertilizes with the egg cell. Pseudofertilized ovule culture is a method in which pollen whose fertilization ability has been inactivated by radiation or the like is crossed to promote parthenogenesis of unfertilized eggs (pseudofertilization), and culture ovules containing pseudozygous eggs that have been parthenogenously generated. And regenerate the plant to obtain a haploid plant. Unfertilized ovule culture is a method of culturing ovules including unfertilized eggs without fertilizing the inactivated pollen in the pseudo-fertilized ovule culture. On the other hand, production of haploids by interspecies hybridization is also known. Successful haploids due to interspecific crosses include the barbosum method in barley ( Hordeum vulgare ) (KJ Kasha and KN Kao, 1970, Nat.
ure 225, 874-876) and tobacco ( Nicotiana tabacum )
To produce haploid tobacco by pollinating N.africana pollen (Burk, LG et al. 1979, Science 206, 585),
Example of pollinating a tetraploid potato ( Solanum tuberosum ) with pollen of a diploid wild species S. phureya to obtain a diploid haploid potato (Hougas, RW et al. 1964, Crop Scie)
nce 4, 593-595) are known. As an example of haploid production by intergeneric crossing, a report was obtained in which wheat ( Triticum aestivum ) was crossed with barbosum ( H. bulbosum ) or maize ( Zea mays ) to obtain a wheat haploid (IR Barclay 19
75, Nature 256, 410-411, Ushiyama et al., 1989, Breeding Journal 3
9, Separate volume 2, 146) and a case where barley was crossed with corn or Italian ryegrass to produce a barley haploid (Furusho et al., 1991, Japan J.
Breeding, 41, 41-44).

[0004] Plants regenerated by the above method are haploid unless natural doubling occurs. Then, the callus or the plant is treated with a drug that doubles the number of chromosomes (colchicine, colcemide, oryzalin, etc.) to double the number of chromosomes (PS Rao and P. Suprasanna 199
6, In Vitro Haploid Production in Higher Plants, 1,
317-339), and a doubled haploid (pure plant) can be obtained (except for potato, which is a diploid haploid).

[0005] Studies on the production of haploids of the genus Eustoma have been first performed by anther culture (Okayama Noriken, 1991-1995). Although there is only one reported case of the production of Eustoma haploid by this anther culture, it died at the seedling stage (Okayama Agricultural Sciences, 199
Five). And the retest by this experimental system has not been successful.
The same group has also attempted to culture unfertilized ovules and pseudo-fertilized ovules, but they have not succeeded (Okayama Agriculture, 19
94-1995). On the other hand, with regard to interspecies crosses, there has been a report on the production of plants estimated to be haploids or doubled haploids of Eustoma plants using intergeneric crosses with gentian (Shonai Nippo 98, 1998 Dated March 18). In addition,
Although not yet known at the time of filing the present application,
Regarding the pseudo-fertilized ovule culture method, the present inventors have established a system for producing a haploid doubled line by improving the radiation dose and culture conditions (Japanese Patent Application No. 10-177103).

[0006]

As described above, as a method for producing a genetically pure Eustoma plant in a short period of time, a method utilizing pseudofertilized ovule culture and interspecies hybridization is effective. However, in order to carry out pseudo-fertilized ovule culture, inactivation of pollen by radiation or the like is essential, and at that time, a radiation source such as an X-ray generator is required. Therefore, without these machines or equipment, it may be difficult to carry out pseudo-fertilized ovule culture. In addition, the method using intergeneric crossing with Gentian described above does not have a very high doubling haploid production rate (40-50 seeds per sheath), and it is necessary to match the flowering time of Eustoma plants with Gentian. There is a disadvantage that there is. An object of the present invention is 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.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventor has pollinated pollen obtained from a higher-doubled Eustoma genus plant to the pistil of the Eustoma genus plant to receive the pollen. The present inventors have found that parthenogenesis is induced in Eustoma plants. That is, the present invention is a genetically characterized in that after pollinating a pistil of a Eustoma plant with pollen obtained from a higher-doubled Eustoma plant, the ovules or seeds contained in the ovary of the pistil are grown. This is a method for producing pure Eustoma plants. Further, the present invention, after pollinating the pistil of a genus Eustoma, pollen obtained from a higher-doubled Eustoma genus plant, extracting ovules or seeds contained in the ovary of the pistil, and culturing this in a medium. A method for producing a genetically pure Eustoma plant characterized by the following characteristics.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The invention is applicable to all species of the genus Eustoma, but good results are obtained, especially for Eustoma grandiflorum. The Eustoma plant of the present invention can be produced as follows. First, the chromosome of the Eustoma plant is doubled to create a higher-order doubled Eustoma plant. Chromosome doubling can be carried out by treating with a solution containing a reagent used in a usual doubling treatment such as colchicine, colcemide, orizarin. As the higher-order doubled Eustoma plant, a tetraploid plant is preferable.
Diploid or octaploid plants may be used.

Next, pollen obtained from the higher-doubled Eustoma plant is pollinated to the pistil of the Eustoma plant. The pollination time is not particularly limited as long as the flower buds of the genus Eustoma bloom and the stigmas are opened to enable pollination.
Unless the plant that causes parthenogenesis is a male sterile plant, it is preferable to remove the anthers of the plant before flowering and remove males before flowering. Furthermore, after the emasculation, the buds are preferably covered with a bag in order to prevent unintended hybridization. In the case of a male sterile plant, it is not necessary to remove males, but it is preferable to perform bagging. After pollination, leave until the ovary is sufficiently enlarged. The period until the ovary is sufficiently enlarged depends on the type of plant and the cultivation environment, but is usually about 3 to 4 weeks after pollination. The enlarged ovary can be left as it is to collect seeds and grow it to produce a plant.However, after sterilizing with ethanol etc., it is aseptically decomposed and the ovule is extracted. Can also be produced by culturing E. coli in a predetermined medium. The extracted ovule can be placed and cultured 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. Normally, natural doubling occurs during the growth or culturing process.
That is, a genetically pure plant can be obtained, but if necessary, a process for doubling the chromosome may be performed. The doubling treatment can be carried out using a solution containing a reagent used in a usual doubling treatment such as colchicine, colcemide, orizarin. Further, 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 a discrimination method, a method of investigating the number of chromosomes, counting the number of chloroplasts in guard cells, and quantifying the DNA content per cell by flow cytometry can be used.

[0010]

Examples [1] Production of pollen-donating plants and confirmation of their genetic properties (1) Eradication Eustoma plant line "P94IS3B; single, purple flower color" is cultivated in a pipe house, and anthers are obtained from its buds. The previous anthers were removed with forceps and the emasculated buds were covered with a bag of paraffin paper. (2) Pollination Two to seven days after the emasculation, the flower pouch of "P94IS3B" in a state where the pistil can be pollinated is removed, and the Eustoma plant line (variety name: Pear clear pink; occult, light pink F1 cultivar, which has been grown by crossing a single Eustoma plant fixed line and an octuple Eustoma plant fixed line), and pollen collected from P94IS3B Pollinated on the stigma of the pistil of the flower. After that, a bag of paraffin paper was put on the flower of "P94IS3B" again to prevent unintentional hybridization with other pollen.

(3) Seeding 5 to 6 weeks after pollination, the ovary of “P94IS3B” is collected,
The seeds were collected according to a standard method. (4) Confirmation of Genetic Properties of Pollen Donor Plant The seed obtained in (3) was sown according to a standard method, and 70 plants were cultivated and flowered. Of these plants, 30 individuals are octuple and 40 individuals are single, with a separation ratio of approximately 1: 1.
(Χ ² = 1.4286, significant 5% above). If it is assumed that the trait of octuple with `` pair clear pink '' is dominant over the trait of singleness, and that `` P94IS3B '' is homozygous for the trait of singleness,
“P94IS” with “Pear Clear Pink” with heterogeneity
In the progeny obtained by crossing to "3B", the separation ratio between octuple and single was 1: 1, which is consistent with the above results. Therefore, this assumption is correct, and it is inferred that the octuple trait of "Pear clear pink" in Eustoma plants is genetically dominant.

[2] Production of doubled Eustoma plants (1) Doubled treatment “Pear clear pink” seeds were sown by a conventional method. Since cotyledons developed 2 to 3 weeks after sowing, the shoot meristem of this seedling was treated with a 0.1% aqueous colchicine solution. (2) Selection of doubled individuals Cultivated the treated "Pear Clear Pink", examined the number of chloroplasts and the size of pollen grains in guard cells, and quantified the DNA content per cell by flow cytometry did. From the results, individuals with doubled chromosomes were selected. This plant was designated as “doubling pair clear pink”.

[3] Pollination and ovule culture (1) Eliminating [P94IS3B] was emasculated in the same manner as in [1]. (2) Pollination The pollen of "Double Pair Clear Pink" was pollinated to the pistil of "P94IS3B" in the same manner as in [1]. In addition, as in [1], a flower made of pollinated "P94IS3B" was covered with a bag made of paraffin paper. (3) Ovule culture Two to four weeks after pollination, the ovary of "P94IS3B" is collected,
After surface sterilization, the ovary was dissected, the ovule was excised and placed on a medium. The medium used is M, which is generally used in plant tissue culture.
S medium was used. However, 3% of sucrose, 50 ml / L of coconut milk and 0.6% of gellan gum were added, and the pH was adjusted to 5.8.

[4] Pollination and Seeding (1) Eliminating [P94IS3B] was emasculated in the same manner as in [1]. (2) Mating (2) Pollination The pollen of "Double Pair Clear Pink" was pollinated to the pistil of "P94IS3B" in the same manner as in [1]. In addition, as in [1], a flower made of pollinated "P94IS3B" was covered with a bag made of paraffin paper. (3) Seeding 5 to 6 weeks after pollination, the ovary of “P94IS3B” is collected,
The seeds were collected according to a standard method.

[5] Cultivation test and examination of ploidy of plants obtained as a result of pollination (1) Appearance of plants 3 to 5 from the ovule of “P94IS3B” whose culture was started in [3]
Plants appeared in a week. When the seeds collected in [4] were sown as usual, germination occurred in 1 to 2 weeks.

(2) Assay of flowering of plants Plants obtained as a result of pollination were cultivated and flowered. As a result, a plant showing octuple that could be an indicator of genetic involvement of “doubling pair clear pink” used as pollen was 300
None of the individuals were found. In addition, not all of the plants showed the same flower color (purple) as “P94IS3B”, and eight plants with white flower color were observed. That is, the flower color trait was separated. From these results, it was inferred that the plants obtained as a result of pollination were not derived from somatic cells of “P94IS3B”, but were derived from egg cells of “P94IS3B”. In other words, it was presumed that pollen obtained from "doubling pair clear pink" induced parthenogenesis in "P94IS3B".

(3) Investigation of ploidy About the ploidy of 50 individuals selected from the obtained plants,
DNA analysis by flow cytometry revealed that all of the individuals examined were diploid Eustoma plants. From these results, it was found that “P94IS3
It was presumed that the haploid plant derived from the egg cell of "B" spontaneously doubled in the course of culture or in the course of growth from seed formation.

[0018]

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

Claims (2)

[Claims] 1. A genetically pure strain characterized by pollinating a pistil of a Eustoma plant with pollen obtained from a higher-doubled Eustoma plant and then growing an ovule or a seed contained in the ovary of the pistil. Method of producing Eustoma plants. 2. After pollinating a pistil of a Eustoma plant with pollen obtained from a higher-doubled Eustoma plant, extracting an ovule or a seed contained in the ovary of the pistil and culturing it in a medium. A method for producing a genetically pure Eustoma plant which is a feature of the present invention.