KR20020032467A - Introduction of CMS traits into Mustard by Multi-cell fusion method - Google Patents

Abstract

PURPOSE: Provided is an introduction of CMS to a leaf mustard with a symmetric multi-cell fusion process based which supplements an asymmetric fusion process to obtain CMS objects so that it helps to develop inbred line with high fertility. CONSTITUTION: The introduction of CMS to the leaf mustard with the symmetric multi-cell fusion process based is characterized by fusing cabbages, broccolis and the leaf mustards by cell unit in a symmetric way to produce callus and differentiating again to transplant to a pot wherein the three fused materials are mixed to make one fused cell and CMS objects with various forms and the callus is differentiated again at 3% MS medium with 5mg/l of zeatin and 2mg/l of IAA added as a plant growth adjuster.

Description

Introduction of cytoplasmic infertility characteristics to gat by developing symmetric multicellular fusion method {Introduction of CMS traits into Mustard by Multi-cell fusion method}

In plant breeding, hybrid methods are widely used as a means for improving the traits of cultivated crops. The fundamental reason for the use of this method in crops is the lack of desirable traits in the crop or the lack of diversity in the gene pool. Somatic hybrids and transformation methods, modern biotechnologies, can be used to introduce useful genes in distant or similar crops.

Somatic cell hybridization, a cell or protoplast fusion technique, has several characteristics, unlike conventional breeding methods.

First, it is a combination of two exchanged genomes, which can form heterologous diploids.

Secondly, the genome of a plant to be used as a recipient of partial genome transfer for asymmetric hybrid production is fragmented by radioactive irradiation and fused with the awarded genome.

Third, it is a fusion method of receiving a nucleus from a donor and receiving cytoplasm from a donor.

Many somatic hybrids have been obtained by this symmetric fusion method or asymmetric fusion method, and some species are actually used for production.

The introduction of specific traits through protoplast fusion has been actively carried out and has brought about continuous development, but fusion has been experimented with only two kinds of plants as fusion materials.

The main purpose of plant breeding is to produce new varieties with new genotypes. The easiest way is to find genetic variability within the same species and transfer the useful traits to the desired breed or breeding line through breeding. Therefore, making various breeding materials to be used for breeding can be said to be the most important step in breeding. By varying the genetic and breeding materials available for breeding, new genotypes can be created through cross-breeding or interbreeding. In general, in the case of hybrids between plant species, it is difficult to obtain hybrid plants because of incompatibility with each other. Sometimes, even if the cultivation of plants through pears or baechu culture is likely to be infertile, it is difficult to use as a breeding material. Cruciferous vegetables have been successfully attempted for oily hybrids to transfer necessary traits between dependents over the last few decades. However, in the case of cross-breeding, it is not easy to obtain fertility plants according to the mating combination, so in advanced countries, many studies are being carried out to cultivate new varieties through protoplast fusion.

Since Korea is ahead of other countries in traditional cruciferous vegetable breeding, if it is possible to produce more excellent breeding materials using protoplast fusion technology in other cruciferous vegetables, including Chinese cabbage, which is grown in Korea, there is a high possibility of developing new varieties. In addition, the time taken to foster new varieties can be greatly reduced. Therefore, the present invention suggests the possibility of producing more various breeding materials using the multi-fusion method than the fusion between two plants.

1 is a process in which cells fused by the Multi-Fusion method divide and re-differentiate into plants.

Figure 2 is a test of the CMS characteristics of somatic hybrid plants by PCR analysis method using a CMS specific primer.

Figure 3 confirms the somatic hybrid plants by Southern blot hybridization method and confirm the introduction of cytoplasmic sterility characteristics.

Figure 4 is a chromosome observation picture of the cell fusion.

Figure 5 compares the shape of the cabbage, broccoli, gat (from left) as a model of cell fusion.

6 shows various forms of plants redifferentiated in cells fused to Multi-Fusion.

Figure 7 compares the lobe types of broccoli, gat, cabbage (from left), which are examples of cell fusion.

Figure 8 compares the shape of the various leaves of the somatic hybrids produced.

9 is a comparison between the somatic cell hybrids and broccoli (from left), the sticking and flowering habits.

Inbred line B. campestris (cabbage kw67), B. oleracea (broccoli kw105) and B. juncea (fresh kw68) were used as protoplast separation and symmetric fusion. Seeds sown were soaked in 70% ethanol for 1 minute and then sterilized for 15 minutes in 50% lactose with 2-3 drops of tween 20 after primary surface sterilization and then washed three times with sterile water. Sterilized seeds were sown in solid medium: MS medium (Murashige and Skoog medium), 3% Sucrose, 0.8% agar, pH5.6-5.8 and cultured in 16-hour light and 8-hour dark conditions and 25 ° C ± 1 conditions. Incubated at. Cotyledons and hypocotyls of plants 3 to 7 days old were used as experimental materials.

Protoplast separation was carried out using incubated cabbage, broccoli, fresh cotyledons and hypocotyls. Cotyledon and hypocotyls were cut to 0.5mm in all directions from the pretreatment solution before protoplast separation. After culturing the cotyled cotyledons and hypocotyls in the pretreatment solution, the pretreatment solution was removed and treated with enzyme solution (1% cellulycin (w / v), 0.5% macerozyme (w / v), 0.4M mannitol, pH5.6 to 5.8). It was. After the enzyme solution treatment, the sieve was filtered to 50 µm, and the protoplasts were centrifuged at 1000 rpm for 10 minutes to separate the protoplasts. The separated protoplasts were placed in a new centrifuge tube, and the washing solution W5 was added and washed again at 1000 rpm for 10 minutes and twice, and then the protoplast density was adjusted with a hemocytometer.

The protoplasts of the subjects to be used in the three fusion experiments were diluted with W5 solution so that the final concentration was 1 × 10 ⁶ / ml, the fusion parents were well mixed in a 1: 1: 1 ratio, and the protoplasts were placed in a 6 cm diameter Petri dish. 5 minutes after dropping 7 drops were treated with the same amount of 35% polyethylene glycol (K8P medium, 35% PEG w / v, mol.wt 4000, 0.3M glucose, 50mM CaCl ₂ · H ₂ O). After 5 minutes the PEG solution is removed and washed twice with culture medium.

PEG-treated protoplasts were treated with k8p medium (0.4M glucose, 1mg / l 2.4-D, 1mg / l NAA, 0.5mg / l BAP, 0.2mg / l kinetin) containing 0.4M glucose and hormones at 25 ° C. The incubator was incubated. When cleavage started actively from the fused protoplasts, the plant growth regulator was reduced to 1/4 in 0.1% agarose semi-solid medium. When the callus was about 1 to 2 mm in diameter, it was transferred to hormone-added redistribution medium (3% MS medium, 5 mg / l zeatin, 2 mg / l IAA), and the callus was subcultured every two weeks. 20% of plants were regenerated from callus. Callus cultivation conditions were the same, but plants were re-differentiated. In some callus, green shoots were formed, but in some callus, purple shoots were formed. In most cases, multiple shoots were formed in callus. Replanted plants were transferred to MS 3% medium without plant growth regulators. Plants were normally rooted in this medium. Regenerated plants showed a normal appearance in appearance (FIG. 1). Subdivided stems were subcultured in hormonal-free medium, and then roots were induced and purified in the greenhouse.

The regenerated plants were analyzed by PCR and Southern blot hybridization method for molecular biology. As a result, cytoplasmic male infertility was assayed. In addition, the re-differentiated plants were purified in a greenhouse to compare the morphological characteristics and to confirm the fusion.

Ogura CMS specific primers were used for the PCR method. Specific primers (KNU-3-A, KNU-3-B) prepared from mitochondrial OrfB locus of radish were used. The sequence of KNU-3-A is 5'-GTCGTTATCGACCTCGCAAGG-3 'and the sequence of KNU-3-B is 5'-GTCAAAGCAATTGGGTTCAC-3'. For PCR reaction, add 1 μl of 150ng DNA, 1.5 μl of 25mM MgCl _2, 2.5 μl of 10 × reaction buffer, 0.5 μl of 10mM dNTP, 0.125 μl of 5u / μl Taq polymerase, 0.1 μl of 100pM / μl primer, and 19.275 μl of distilled water. The amount was adjusted to 25 μl and the same amount of mineral oil was added. DNA amplification conditions were performed 30 cycles at 94 ℃ 40 seconds, 55 ℃ 1 minutes, 72 ℃ 1 minutes and finally maintained at 72 ℃ 10 minutes. As a result of amplification of 96 plants by PCR method, only 4 plants did not show specific bands with cytoplasmic male infertility, but the remaining individuals showed specific bands at 500bp position. It could be confirmed (Fig. 2).

Southern blot hybridization analysis was performed using PCR reactions using Ogura specific primers. The product obtained in the PCR reaction was electrophoresed by 1% agarose gel and then transferred to a nylon membrane, and then assayed by hybridization and detection. Samples were randomly selected from 13 of 96 individuals. Among the 16 fusion materials, including the fusion material, no DNA bands were observed at kw68, but not at CMS, but the other groups showed cytoplasmic male sterility specific bands. As well as PCR analysis, CMS characteristics were confirmed by Southern blot hybridization method, so that even though the morphologically similar to Gad among individuals, it was confirmed that they were somatic hybrid plants and CMS characteristics were introduced (Fig. 3).

For chromosome observation, the roots of the purified plants were cut and treated with mono-bromonaphthalene for 1 hour as a pretreatment at room temperature and then fixed in a fixed solution (acetic acid: EtOH = 1: 3) for at least 24 hours. After washing the root end fixed in distilled water and treated with enzyme solution (5% cellulysin, 1% pectolase, 10mM EDTA, pH4.5) and placed in a 37 ℃ constant temperature water bath for 30 minutes. The roots of the enzyme-treated roots were washed in distilled water, placed on a slide glass, and a drop of Acetic acid: EtOH = 1: 3 solution was added to the well, followed by air tweezers, followed by air drying. The dried slides were again stained with 5% Geimsa dye solution for 10 minutes and observed. The chromosome of Chinese cabbage has 2n = 20, broccoli has 2n = 18 and freshly has 2n = 36. When the three plants are fused, the probability of recombination of the chromosomes will be more complex than when the two cells are fused, and the number of chromosomes will vary.

From the root tip chromosome observation, the chromosome of somatic hybrids was not constant and the number of chromosomes of 36 to 46 was observed, but the shape of the plant in the pot was very similar to broccoli (Fig. 4).

For morphological comparison, the regenerated plants were rooted with 0.2 mg / l NAA and transferred to horticultural soils. The leaf morphology, foliar morphology, and flowering period of the plants grown in the greenhouse were compared and tested for convergence. The fusions between gad, cabbage and broccoli using multi-fusion showed a wide variety of variations in morphology. Plants appeared in three distinct forms: ones that were close to the fresh, the middle of the cabbage and broccoli, and the whole form of broccoli.

Chinese cabbage and gat are cabbage-shaped, and broccoli is a long stem without branch with a firearm, but it is non- cabbage-type when observing the pattern of the re-differentiated plant. It was foreskin and most of the fusion showed a non-frozen state (Figures 5 and 6). The lobes varied widely from those with broccoli-like properties to those with gat. The leaf disease, unlike the broad cabbage and gat, was tricircular or medium in shape, like broccoli, with etched lobes, and many leafy and wrinkled shapes. The leaf color was dark green like the lampshade, and the color was broccoli-like and medium. In addition to the lateral shape, there are many variations in hair usage, so that individuals with various changes in density, from many hair-derived hairs to hairless ones, such as broccoli, also exhibit broccoli's traits in terms of intercalar kidney and stem diameters. Judgment was made (Figs. 7 and 8).

Somatic hybrids were purified in pots on September 6 and grown in glass greenhouses. Under natural conditions, the plants began to climb for two months. Among the fusion models of Chinese cabbage, broccoli and gat, broccoli was similar to the fusions, but the cabbage and gat were not bolstered. However, the flower stalk rises differently from the broccoli, showing the shape of the cabbage or the freshly stalk. The branching form of the broccoli's apical tip is a dense firearm with irregular clusters of flowers, but most of the re-differentiated plants are branched stems with gunshot inflorescences (Fig. 9).

The production of somatic hybrid plants using the multi-cell fusion method is expected to be of great significance in creating new gene sources and plants as the probability of nuclear and cytoplasmic genetic recombination is higher than when two cells are fused. In addition, it is possible to develop a variety of high-fertility lines by enabling a variety of different fusion combinations that can compensate for the deficiencies of asymmetric fusion to obtain a cytoplasmic sterile individual of the model.

Claims (5)

Method of producing somatic hybrid plants in which cabbage, broccoli, and gat are formed by symmetrical cell fusion rather than asymmetrical fusion to remove one nucleus, forming callus in fused cells, and regenerating plants from the formed callus The method of claim 1, unlike the conventional methods used to fuse the three fusion materials into a single fusion cell, cell fusion method of making somatic hybrid plants and cytoplasmic male sterile individuals having various morphological characteristics The method of producing a somatic hybrid plant according to claim 1, wherein the formed callus is regenerated in 3% MS medium to which 5 mg / l zeatin and 2 mg / l IAA which are plant growth regulators are added. 4. The method of claim 3, wherein the formed callus is subcultured at intervals of two weeks in regeneration medium to produce somatic hybrid plants. Somatic hybrid plants produced by claim 1