JP2748141B2 - How to grow Stevia triploid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Use] The present invention relates to a triploid stevia and its proliferation, and is applied to breeding by breeding, biology, agriculture, tissue culture and the like.

[Problems to be solved by conventional technology and invention]

Stevia (Stevia rebaudiana BERTONI) is a perennial herb native to Paraguay in South America, with mainly Stevioside and Rebaudioside A (Rebaudio) leaves.
It contains a highly sweetened diterpene glycoside such as side A), is currently used as a natural sweetener, and is one of useful cultivars.

Stevia breeding is the only example in which the ratio of rebaudioside A with good taste is increased compared to the past. Breeding using polyploids practically used in major crops, such as seedless watermelon (Yoshio Watanabe 1982. Cytogenetics in breeding, Yokendo), Freesia (Kiyoshi Saito)
1961 Research on polyploid territory and its utilization in flower breeding, I. Role of polyploid varieties in freesia breeding New breeding methods such as breeding 11 (1): 1-9,) are also desired in Stevia ing.

Since triploids are sterile, it is also necessary to develop a method for efficiently growing them.

[Means for solving the problem]

Therefore, the present inventors have conducted various studies on a method for producing a triploid in Stevia. As a result, it was found that only when the diploid is crossed using an artificial tetraploid as a mother strain, the seed obtained is a triploid. I found it.

The obtained triploids were able to be mass-produced by inducing shoot primordia by spinning the shoot apical liquid, and then transplanting them into a fixed medium to differentiate and proliferate into plants.

That is, the gist of the present invention is to obtain a triploid seed by crossing a diploid with an artificial tetraploid as a mother strain, and to use a shoot apex of a grown seedling after germination to propagate a large amount of clones. It is a method characterized by making it.

[Action]

 The present invention will be described in further detail.

The artificial tetraploid and the diploid are cultivated and flowered, and the diploid is artificially crossed using the tetraploid as a mother strain. Seeds obtained by fertilization are triploid.

An artificial tetraploid can be produced by treating a diploid with benzyladenine, colchicine, or the like, thereby doubling the number of chromosomes.

 Crosses other than the above will not fertilize.

When the obtained triploid seeds are seeded and germinated and grown in the soil, the plant height becomes larger as compared with the diploid seeds, and they have excellent resistance to diseases and insects. And also
The sweetness component contained in the leaves is also increased. Furthermore, it has the characteristic that the content of rebaudioside A having good taste is also increased.

After sterilization of the stem containing the grown triploid stem apex, the stem apex is excised and placed on an artificial medium containing inorganic salts, plant growth hormone and a carbon source. Next, rotation culture is performed under illumination, and the shoot primordia are induced and printed.

The rotation culture under the above-mentioned illumination is performed at a light intensity lower limit of 2,000 lux, an upper limit of 10,000 lux, a temperature of 18 to 28 ° C. and a rotation speed of 0.
The condition is 5 to 5 / min.

The above inorganic salts include Murashige School (Mura
shige-skoog), Gamborg, White (Wh
medium having a composition such as ite).

Examples of the plant growth hormone include indole acetic acid and α-naphthalene acetic acid as auxins and 6-benzylaminopurine and kinetin as cytokinins. At least one auxin and at least one cytokinin It is desirable to use together.

For example, 1,5 to 3 ppm of 6-benzylaminopurine as cytokinin and 0.2 of α-naphthaleneacetic acid as auxin
Those containing 2.02.0 ppm and sucrose 1-5% are used.

The derived shoot primordium has a form with many light green small projections. Thereafter, it is possible to semi-permanently maintain and proliferate by dividing and transplanting into a medium having the same composition regularly.

When the shoot primordia obtained in this manner is transplanted into a fixed medium after mass-proliferation, the shoot primordia differentiates into a plant having foliage and grows. Then, if it is gradually acclimated, it can be cultivated outdoors. The obtained plant is triploid, that is, 2n = 33 (X = 11).

〔Example〕

After sterilizing Stevia seeds, they were seeded and germ-free seedlings were grown. Furthermore, the cells were cut at each internode, immersed in a treatment solution containing 3% sucrose and 1.0 ppm of 6-benzylaminopurine in Murashige-Skoog medium for 10 days, and the number of chromosomes was calculated from the newly generated shoots. Tetraploid, that is, 2n = 44.

The tetraploids and diploids (2n = 22) were potted using a medium and grown in a greenhouse, and the number of chromosomes was calculated using root tip tissues for confirmation.

Artificial mating was performed at the time of flowering. The combination was performed for a case where a diploid male flower was mated with a tetraploid female flower and a case where a tetraploid female flower was mated with a tetraploid male flower. Stevia is an amphoteric flower, but has strong self-incompatibility and hardly self-pollinates.

As a result of the cross, the seeds obtained by fertilization were 4
Only when a diploid male flower was crossed to a diploid female flower.

The obtained seeds were seeded on a medium in which river sand and peat moss were mixed. Germination occurred in about one week, and the number of chromosomes was calculated using the root tip tissue.

After cultivation, it grew vigorously.
The overall size was larger, such as the height of the plant was 50 to 100 cm higher than that of the haploid. Further, it was very excellent in resistance to diseases such as disease resistance and insect resistance.

Furthermore, the sweetness components contained in the leaves were also compared. Both triploid and conventional (diploid) leaves were harvested and dried in October. Next, the sweet component was extracted with a solvent (acetonitrile 82%), and the contents of stevioside and rebaudioside A were determined by high performance liquid chromatography.

The measurement method is as follows: Shodex RS pak DC-613 (6m
eluent 82% acetonitrile, flow rate 1m
l / min, elution temperature 45 ° C., ultraviolet detection 214 nm, and quantified by the two-point calibration method.

As a result, triploid was 13.08% stevioside and A8.72% rebaudioside, totaling 21.8%. The content ratio of stevioside to rebaudioside A was 3: 2.

The average content of diploid was 9.5% in total of 7.6% stevioside and 1.9% rebaudioside, and the ratio was 4: 1.

Triploid multiplication is carried out in a vigorous growth period by cutting a stem including an apex into approximately 1 cm and immersing in a benzalkonium chloride solution 0.1% for 5 minutes and further in a sodium hypochlorite solution 1% for 5 minutes to perform a sterilization treatment. After this, the shoot apex was excised under a stereoscopic microscope to obtain a planting material. The artificial liquid medium used was a gamboque medium, and 2% of sucrose and 6-benzylaminopurine and α-naphthaleneacetic acid were used as plant growth hormones at 0, 0.02, 0.2, and 2.0, respectively.
It was adjusted by adding to a concentration of ppm.

 The pH of the medium was adjusted to 5.7 to 5.8.

Dispense 25 ml of each medium into test tubes (27 x 200 mm),
Then, it was sterilized in a high-pressure sterilizer at 121 ° C. for 15 minutes. The shoot apex was inoculated on the adjusted medium, and rotation culture (two rotations per minute) was performed under the condition of illumination for 24 hours (illuminance lower limit 2,000 to upper limit 10,000 lux) and temperature of 22 (± 2) ° C. After about one month, 6
-Benzylaminopurine 2 ppm, α-naphthaleneacetic acid 0.02
In the combination of ppm, a light green shoot primordium with many small protrusions was obtained. The other combinations resulted in castle and premature branching. Thereafter, the growth was continued by periodically dividing and transplanting into a medium having the same composition.

Conversion from shoot primordia to plants was performed by static culture in a fixed medium.

As a fixed medium, a medium adjusted to PH 5.7 to 5.8 by adding 6-benzylaminopurine 0.02 ppm, sucrose 1%, and agar 0.85% to a 1/2 diluted inorganic salt composition gamborg was used.
The culture conditions were 2,000 lux light for 16 hours and a temperature of 22.
(± 2) ° C.

When shoot primordium was inoculated on the adjusted medium, leaf primordium was formed at the tip and gradually grew into a small plant having foliage. After rooting was observed, the plants were acclimated and then transferred to field cultivation. The time required from planting the shoot primordia to field cultivation was about one month.

The number of chromosomes in this plant is 2n, the same number as the strain from which the shoot apex was removed.
= 33 (X = 11). There was no difference in the external form.

[Effect of the Invention] Using the method of the present invention, the first time in Stevia is 3
Diploids are produced, and breeding, or breeding, using polyploids, which had not been performed before, can be achieved. Moreover, the obtained triploids are extremely superior to diploids (conventional products), such as an enlarged plant and an increase in sweetness components. Furthermore, even in a triploid, which is sterile and has no efficient propagation method, by inducing and growing shoot primordia and then converting it to a plant, the effect is enormous, such as efficient mass growth.

 ──────────────────────────────────────────────────続 き Continued on the front page (56) References “Breeding Science” by Takao Matsuo, published by Yokendo Co., Ltd. (March 1, 1978) P. 217-218 “Practical technology for mass production of cloned plants” By Takaso Tanaka, published by CMC Corporation (December 25, 1985) 4 to 12

Claims (4)

(57) [Claims] 1. The production of a stevia triploid as a starting material for a stevia diploid enables the production of a stevia triploid having a higher content of sweet components, especially rebaudioside A, than the diploid of the starting material. How to grow. 2. Steploid tetraploid (male) obtained by artificially treating stevia diploid is crossed with stevia diploid (male) to produce stevia triploid. Item 4. The method for growing a stevia triploid according to Item 1. 3. The method of growing stevia triploids, wherein the shoot apex of the stevia triploid grown by the growing method according to claim 1 is subjected to liquid rotation culture to induce shoot primordia, wherein the liquid rotation culture is performed with illumination intensity. Stevia 3 characterized by a 2,000 to 10,000 lux, a temperature of 18 to 28 ° C. and a rotation speed of 0.5 to 5 rotations
Diploid multiplication method. 4. The method according to claim 3, wherein the shoot primordia are maintained and grown, and further transplanted into a fixed medium to differentiate and grow into a plant.