JPH02242622A - Stevia triploid and method for proliferation thereof - Google Patents

Abstract

PURPOSE:To carry out clone proliferation of stevia triploid in a large amount by mating a haploid using an artificial tetraploid as a mother stock to provide stevia triploid seeds and using shoot apexes of grown seedlings after germinating the above-mentioned seeds. CONSTITUTION:Stevia tetraploid and haploid are cultivated to artificially mate a haploid using a tetraploid as a mother stock in a flowering period to afford seeds of stevia triploid. The afore-mentioned seeds are subsequently sown in soil used, germinated and grown to extract shoot apical parts thereof. The resultant shoot apical parts are then rotated and cultivated in a liquid at 2,000-10,000lx illuminance, 18-28 deg.C temperature and 0.5-5 r.p.m. to induce shoot primordia, which are subsequently maintained, proliferated, further transplanted to an immobilizing culture medium, differentiated and grown into a plant body.

Description

[Detailed Description of the Invention] [Industrial Application] The present invention relates to Stevia triploid and its propagation, and relates to breeding and biology by selective breeding.

Agriculture 2 Applied to tissue culture, etc.

[Problems to be solved by conventional techniques and inventions] Stevia (Slcyi & Reb8udii Bertoni
) is a perennial herb native to Paraguay in South America, and its leaves mainly contain highly sweet diterpene glycosides such as stevioside (Slema 1 osidc) and rebaudioside A (Rebsudioside^), and it is currently used as a natural sweetener. It is one of the useful cultivated varieties.

Until now, the only example of breeding for stevia has been to increase the ratio of rebaudioside A, which has good taste, compared to conventional varieties.

Breeding using polyploids that has been put to practical use in major crops, such as seedless watermelon (Yoshibe Watanabe +982.Cytogenetics in breeding, I!!!!!), freesia (Kiyoshi Saito 1961 in flower rebreeding) Research on polyploid areas and their utilization 1. Role of polyploid varieties in Freesia breeding Breeding 11 (1) +1-9
．． ) and other new breeding methods are also desired for stevia.

Furthermore, since triploids are sterile, it is also necessary to develop a method for efficiently propagating them.

[Means to solve the problem]

As a result of various studies on methods for producing triploid plants in Stevia, the present inventors found that the seeds obtained are triploid only when crossing diploid plants with artificial tetraploid mother plants. I found it.

The resulting triploids were able to be propagated in large quantities by subjecting the shoot tips to liquid rotation culture to induce shoot primordia, and then transplanting them to a fixed medium to differentiate and propagate them into plants.

That is, the gist of the present invention is to use an artificial tetraploid as a mother stock,
This method is characterized in that triploid seeds are obtained by crossing diploids, and after germination, the shoot tips of the grown seedlings are used for mass clonal propagation.

[Effect] The present invention will be explained in further detail.

Artificial tetraploids and diploids are cultivated and flowered, and the diploids are artificially crossed using the tetraploid as a mother plant. The seeds obtained by fertilization are triploid.

Artificial tetraploidy is caused by benzyladenine, colchicine, etc.
It can be created by doubling the number of chromosomes by processing a ploid.

Mating with combinations other than those listed above will not result in fertilization.

When the obtained triploid seeds are sown in soil and allowed to germinate and grow, the plant height becomes larger than that of diploid seeds, and
It has excellent resistance such as disease resistance and insect resistance, and the leaves also contain a high content of sweet components. Furthermore, it exhibits characteristics such as a high content rate of rebaudioside A, which has good taste.

After sterilizing the grown stem containing the triploid shoot apex, the shoot apex is removed and placed on an artificial medium containing inorganic salts, plant growth hormones, and a carbon source.

Next, rotational culture under illumination is performed to induce shoot primordia.

Examples of the above-mentioned rotational culture under illumination include conditions such as a lower limit of illumination intensity of 2, On lux, an upper limit of 10,000 lux, a temperature of 18 to 28° C., and a rotation speed of 0.5 to 5/min.

The above-mentioned inorganic salts include Murashige Skoog (Mua
shige-skooO, Gzlborg
), White, etc. can be used.

As the above-mentioned plant growth hormones, indoleacetic acid, α-naphthaleneacetic acid, etc. can be used as auxin, and 6-benzylaminopurine, kinetin, etc. can be used as cytokinin. It is desirable to use seeds together.

For example, one containing 1.5 to 3 nm of 6-benzylaminopurine as cytokinin, 0.2 to 2.0 ppm of α-naphthaleneacetic acid and 1 to 5% of sucrose as auxin is used.

The induced shoot primordia have a pale green morphology with many small protrusions. Thereafter, by periodically dividing and transplanting into a medium of the same composition, it is possible to maintain and proliferate semi-permanently.

The shoot primordium thus obtained is multiplied in large quantities and then transplanted to a fixed medium, where it differentiates into a plant with leaves and stems and grows. After that, after gradual acclimatization, it can be grown outdoors. The obtained plant body is triploid, that is, 2 n = 33 (X = Il
).

〔Example〕

After sterilizing Stevia seeds, they were sown to grow sterile seedlings. Furthermore, cut into pieces between each box and place in Murashige-Skoog medium with 3% sucrose and 1.0 p of 6-benzylaminopurine.
They were immersed in a treatment solution containing p11 for 10 days, and the number of chromosomes was calculated from newly generated axillary buds to select tetraploids, that is, 2n=44.

Tetraploid and diploid (2n-22) were potted using soil and grown in a greenhouse, and for confirmation, the number of chromosomes was calculated using root tip tissue.

Artificial hybridization was performed during flowering. The combinations were carried out in cases where a haploid female flower was crossed with a 2@ploid male flower, and a case where a diploid female flower was crossed with a tetraploid male flower, and the flowers were wrapped in bags. Although Stevia is amphoteric, it has strong self-incompatibility and hardly self-pollinates.

As a result of crossing, fertilization and seeds were obtained only when a diploid male flower was crossed with a tetraploid female flower.

The obtained seeds are placed in soil mixed with river sand and peat moss.
has sown. The seeds germinated in about one week, and when the number of chromosomes was calculated using the root tissue, they were all triploid with 2n=:13.

After that, cultivation continued, and the growth was vigorous, and the plant was 50 to 100 cm taller than the diploid, resulting in an overall larger size. Moreover, it was also very good in resistance such as disease resistance and insect resistance.

Furthermore, the sweet components contained in the leaves were also compared.

Leaves of both triploid and conventional products (diploid) were harvested and dried in the IQ month. Next, sweet components were extracted with a solvent (acetonitrile 82%), and the content of stevioside and rebaudioside A was determined by high performance liquid chromatography.

The measurement method is 5hodex R5pxk as a column.
Using DC613 (6+ux 15 Qnm), the eluent was 82% acetonitrile, the flow rate was 1+nl/l in 1, the elution temperature was 45°C, and the ultraviolet detection was 214n11, and the stability was determined by the two-point calibration curve method.

As a result, the triploid has 13.08% stevioside.

The total amount was 21.8%, including rebaudioside A 8.72%. Further, the content ratio between stevioside and liver abuocide was 3:2.

The average content of diploids was 7.6% for stevioside and 1.9% for rebaudioside A, totaling 9.5%, and the ratio was 4:1.

To propagate triploids, cut the stem including the shoot apex into approximately 1 cm pieces during the period of active growth, and add 5% to 0.1% benzalkonium chloride solution.
After sterilizing the plants by immersing them in a 1% sodium subzinc oxide solution for 5 minutes, the shoot tips were removed under a stereomicroscope and used as material for planting. The artificial liquid medium was Gambok's medium, with 2% sucrose and 6-benzylaminopurine and α-naphthalene acetic acid as plant growth hormones.
The concentration was adjusted to 2.0.2 and 2.0 ppm.

The pH of the medium was adjusted to 5.7-58.

Add 25 medium to each test tube (27 x 200 mn+)
The solution was then sterilized in an autoclave at 121°C for 15 minutes. Plant the shoot tip in the adjusted medium and light for 24 hours (
Rotational culture (2 rotations per minute) under conditions of illuminance (lower limit: 2,000 to upper limit: 10,000 lux) and temperature of 22 (±2)°C.
I did it. After about 1 month, light green shoot primordia with many small protrusions were obtained using the combination of 2 ppm of 6-benzylaminopurine, o, and 2 ppn of α-naphthalene acetic acid. Other combinations resulted in callus and early branching. Thereafter, proliferation was continued by periodically dividing and transplanting into a medium of the same composition.

The transformation from shoot primordia to plants was carried out by static culture on a fixed medium.

As a fixed medium, inorganic salt composition 1/2 diluted Gambok, 6-benzylaminopurine 0. O2ppm,
Add 1% sucrose and 0.85% agar to pH 5.7-5.
The one adjusted to 8 was used. The culture conditions at this time were light 16
The time was 2,000 lux illumination and the temperature was 22 (±2)°C.

When shoot primordia were planted in the prepared medium, leaf primordia formed at the tip and gradually grew into plantlets with stems and leaves. Once rooting was observed, the plants were acclimatized and then transferred to outdoor cultivation. The period required from planting the shoot primordium to outdoor cultivation was approximately one month.

The number of chromosomes in this plant is 2, which is the same number as the plant from which the shoot apex was removed.
It was triploid with n=33 (X=II).

There was also no difference in external morphology.

〔Effect of the invention〕

By using the method of the present invention, triploids can be created for the first time in Stevia, and variety improvement, that is, breeding, using polyploids, which has not been done until now, can be achieved. In addition, the obtained triploid is superior to the diploid (conventional product) in that it has a larger plant body and an increased sweetness component. Furthermore, even in triploid plants, which are sterile and for which there is no efficient method of propagation, by inducing shoot primordia, propagating them, and then converting them into plants, efficient mass propagation can be achieved, which is extremely effective.

June 23, 1999

Claims (5)

[Claims] (1) Stevia triploid (2) A method for producing a stevia triploid, which is characterized by crossing a stevia tetraploid (female) with a stevia diploid (male) (3) A method for propagating Stevia triploid, which is characterized by inducing shoot primordia by liquid rotary culture of the shoot apex. (4) Liquid rotation culture with illumination intensity of 2,000 to 10,000
lux, temperature of 18 to 28°C, and rotation speed of 0.5 to 5 revolutions/min. (5) The propagation method according to claim 3, wherein the shoot primordium is maintained and propagated, and further transplanted to a fixed medium to differentiate and grow into a plant body.