JPH0695866B2 - Method for producing tetraploid stevia by plant growth hormone - Google Patents
Method for producing tetraploid stevia by plant growth hormoneInfo
- Publication number
- JPH0695866B2 JPH0695866B2 JP1064120A JP6412089A JPH0695866B2 JP H0695866 B2 JPH0695866 B2 JP H0695866B2 JP 1064120 A JP1064120 A JP 1064120A JP 6412089 A JP6412089 A JP 6412089A JP H0695866 B2 JPH0695866 B2 JP H0695866B2
- Authority
- JP
- Japan
- Prior art keywords
- stevia
- artificial
- tetraploid
- producing
- cytokinin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Seasonings (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は植物成長ホルモン処理によって、人為４倍体を
作出する方法に関するものであり、農業、林業における
品種改良、又は組織培養等に利用できる。DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a method for producing an artificial tetraploid by treatment with plant growth hormone, which can be used for breeding in agriculture, forestry, tissue culture, or the like. .
植物の倍数体作出による育種は、種々の作物において利
用され、実用化されている。そしてこれら倍数体を人為
的に誘発させる薬剤としては、コルヒチン、コルセミ
ド、ピンプラスチン、ポドフィロトキシンさらには、笑
気ガス、クマリン等が用いられてきた。Breeding by producing a polyploid plant has been utilized and put to practical use in various crops. As agents that artificially induce these polyploids, colchicine, colcemid, pinplastin, podophyllotoxin, laughing gas, coumarin, etc. have been used.
上記誘発剤は、いずれも、細胞分裂能の高い部位、例え
ば茎頂、えき芽、根端等の細胞に施すことで、人為４倍
体を作出するものである。All of the above-mentioned inducers produce artificial tetraploids by applying them to cells having a high cell division ability, for example, cells such as shoot tips, shoots, root tips and the like.
また最近では、培養組織であるカルス、苗条原基にも同
様の誘発剤で処理することで、人為４倍体を作出する方
法も開発されている。Recently, a method for producing artificial tetraploids has also been developed by treating callus and shoot primordium, which are cultured tissues, with the same inducer.
しかし、これら誘発剤は、植物体または、培養組織に与
える毒性が強いため副作用があり、処理した後の栽培に
悪影響を及ぼしている。さらに、これらの薬剤は、毒物
又は、劇物で取扱いに注意を要するなど効率、安全の面
で問題がある。However, these inducers have side effects because they are highly toxic to plants or cultured tissues, and have an adverse effect on cultivation after treatment. Further, these drugs are poisonous or deleterious substances and require careful handling, which poses a problem in terms of efficiency and safety.
また、特にコルヒチンを誘発剤として使用する場合、４
倍体の作出は容易であるが、得られた４倍体の順化によ
る屋外栽培が困難であるといった問題もあった。他方、
ステビア甘味料の抽出原料として使用される南米原産の
キク科植物であるステビアにおいて、価格の低減のため
甘味成分の増大が求められていた。Moreover, especially when colchicine is used as an inducer, 4
Although it is easy to produce a tetraploid, there is a problem that it is difficult to cultivate the obtained tetraploid for outdoor cultivation. On the other hand,
In Stevia, which is an Asteraceae plant originating in South America and used as a raw material for extracting Stevia sweeteners, it has been required to increase the amount of sweeteners in order to reduce the price.
従って、その対策としてステビア植物への毒性が少なく
取扱の容易な新規な誘発剤による甘味成分含量の多いス
テビア倍数体の効率的な作出と安定した生育が求められ
ている。Therefore, as a countermeasure, there is a demand for efficient production and stable growth of polyploid Stevia having a high content of sweet components by a novel inducer which is less toxic to Stevia plants and is easy to handle.
そこで、本発明者らは、上記問題点を解決すべく安全に
取り扱える薬剤について種々検討した結果、無菌的に育
成したステビア苗を、植物成長ホルモンの一種であるサ
イトカイニンで処理することにより、新たに発生するえ
き芽に変異誘発された人為４倍体が、短期間に出現する
ことを見い出した。Therefore, the present inventors have variously studied the drug that can be safely handled to solve the above problems, aseptically grown Stevia seedlings, by treating with cytokinin, which is a type of plant growth hormone, It was found that artificial tetraploids, which were mutagenized in the developing shoots, appeared in a short period of time.
つまりサイトカイニンで処理することで細胞分裂が促進
され、染色体の複製過程が細胞分裂と同調しなくなり、
結果的に染色体数の倍加が生じると考えられる。In other words, treatment with cytokinin promotes cell division, and the replication process of chromosomes is not synchronized with cell division.
It is thought that the number of chromosomes doubles as a result.
すなわち、本発明の要旨は、植物成長ホルモンの一種で
あり、現在、組織培養等で広く用いられているサイトカ
イニンで処理することによりステビア人為４倍体を効率
よく得ることを、特徴とする方法である。That is, the gist of the present invention is a kind of plant growth hormone, and is a method characterized in that Stevia artificial tetraploid is efficiently obtained by treating with cytokinin which is widely used at present in tissue culture and the like. is there.
本発明法でサイトカイニンにより誘発されたステビア人
為４倍体植物は、薬剤による害がなく、以後、安定して
生育させることができる。The artificial tetraploid plant of Stevia induced by cytokinin according to the method of the present invention is not damaged by the drug and can be stably grown thereafter.
本発明をさらに、詳しく説明する。 The present invention will be described in more detail.
ステビア無菌苗を節間ごとに切断した供試材料は、サイ
トカイニンを含む処理液（人工培地）に10日から30日間
浸漬して処理する。The test material obtained by cutting the sterile Stevia seedling at each internode is immersed in a treatment solution (artificial medium) containing cytokinin for 10 to 30 days for treatment.
上記、サイトカイニンとしては、カイネチン、６−ベン
ジルアミノプリン、ゼアチン、２−イソペンテニルアデ
ニンを用いることができる。人工培地は、ムラシゲ・ス
クーグ（Murashigeskoog）、ガンボーグ（Gamborg）、
ホワイト（White）等の組成を有する培地を用いること
が出来る。As the above-mentioned cytokinin, kinetin, 6-benzylaminopurine, zeatin, and 2-isopentenyladenine can be used. Artificial medium is Murashigeskoog, Gamborg,
A medium having a composition such as White can be used.
浸漬処理中に、新たに発生したえき芽を切りとり、滅菌
水で洗浄後、無機塩類及び炭素源を含む人工固定培地
に、いわゆるさし木の要領で移植後、１〜２週間で根を
形成させる。根端組織を用いて、染色体数を算定するこ
とで、４倍体を選抜することができる。このようにして
得られた４倍体は、以後、何ら問題なく順調に生育し、
最終的に屋外栽培できる。During the dipping treatment, newly-grown buds are cut off, washed with sterilized water, and then transplanted to an artificial fixed medium containing inorganic salts and a carbon source in the manner of so-called cuttings to form roots in 1 to 2 weeks. The tetraploid can be selected by calculating the number of chromosomes using the root tip tissue. The tetraploid thus obtained grows smoothly without any problems thereafter,
Finally, it can be grown outdoors.
ステビア（Stevia rebaudiana BERTONI）の種子を殺菌
した後、ショ糖２％を含むムラシゲ・スクーグの固定培
地上には種して、無菌苗を生育させた。さらに節間ごと
に切断して実験に供試した。Stevia (Stevia rebaudiana BERTONI) seeds were sterilized, and then seeded on a Murashige-Skoog fixed medium containing 2% sucrose to grow a sterile seedling. Furthermore, it cut for each internode and used for the experiment.
尚、実験に供試した苗の染色体数は、すべて2n＝22の２
倍体であった。The number of chromosomes of the seedlings tested in the experiment was 2n = 22, which is 2
It was a diploid.
処理液は、ムラシゲ・スクーグ培地を用い、ショ糖３％
を加え、６−ベンジルアミノプリンを0,0.5,1.0,2.0ppm
の各濃度になるように添加して調整した。As the treatment liquid, Murashige-Skoog medium was used, and sucrose 3%
And 6-benzylaminopurine at 0, 0.5, 1.0, 2.0 ppm
Was adjusted to each concentration.
処理液のpHは5.7〜5.8に調整した。The pH of the treatment liquid was adjusted to 5.7 to 5.8.
それぞれの処理液を200ml三角フラスコに50ml分注し、
次いで高圧滅菌器で121℃15分間滅菌した。調整した処
理液に、節間ごとに切断した植物片を温度24℃、照度20
00ルクス（明期16時間、暗期８時間）の条件下で、それ
ぞれにつき無菌的に10日間、20日間、30日間浸漬処理し
た。Dispense 50 ml of each treatment solution into a 200 ml Erlenmeyer flask,
Then, it was sterilized in a high-pressure sterilizer at 121 ° C. for 15 minutes. The adjusted treatment liquid was used to cut plant pieces cut into internodes at a temperature of 24 ° C and an illuminance of 20.
Under the conditions of 00 lux (light period 16 hours, dark period 8 hours), each was aseptically immersed for 10 days, 20 days, and 30 days.
処理中に新たに発生するえき芽をメスで切りとり、滅菌
水で充分に洗浄後、無機塩類、炭素源に寒天を加えた人
工固定培地にいわゆるさし木の要領で移植した。各濃
度、各処理時間ごとに50本ずつのえき芽を得た。During the treatment, the new shoots were cut with a scalpel, washed thoroughly with sterilized water, and then transplanted to an artificial fixed medium containing inorganic salts and agar as a carbon source in the manner of so-called cuttings. 50 shoots were obtained at each concentration and each treatment time.
固定培地としては、無機塩類組成1/2希釈のムラシゲ・
スクーグにショ糖１％、寒天0.85％を加え、pH5.7〜5.8
に調整して用いた。As the fixed medium, Murashige with 1/2 dilution of inorganic salt composition
Add 1% sucrose and 0.85% agar to the scoog, pH 5.7-5.8
It was used after adjusting to.
２〜３週間すると、根が形成され、それらの根端組織を
用いて染色体数を算定した。After 2-3 weeks, roots were formed, and their root tip tissues were used to count the number of chromosomes.
その結果、６−ベンジルアミノプリン1.0ppmで10日間、
及び20日間浸漬処理して、得たえき芽から生育させた苗
のなかに、それぞれ５本ずつの４倍体（2n＝44）を確認
した。さらに４倍体は、茎頂を用いても染色体数を確認
した。As a result, 6-benzylaminopurine 1.0 ppm for 10 days,
And 20 days of immersion treatment, and 5 tetraploids (2n = 44) were confirmed in each seedling grown from the obtained sprouts. Furthermore, in the tetraploid, the number of chromosomes was confirmed even by using the shoot apex.
他の処理区では、外部形態などの異常は見られたが染色
体数は、いずれも２倍体（2n＝22）であった。In the other treated plots, abnormalities such as external morphology were observed, but the chromosome numbers were all diploid (2n = 22).
得られた４倍体は、その後培養器より取り出し、順化を
へて、屋外栽培に移した。２倍体に比較して、草丈が高
く、葉面積も大きくなる傾向を示した。The tetraploid thus obtained was then taken out of the incubator, acclimated and transferred to outdoor cultivation. Compared with the diploid, the plant height was higher and the leaf area was larger.
さらに、葉に含有される甘味成分についても比較した。
４倍体及び従来品（２倍体）とも10月に葉を収穫して乾
燥させた。次いで溶媒（アセトニトリル82％）で甘味成
分を抽出して高速液体クロマトグラフィーでステビオサ
イドとレバウディオサイドＡの含有率を求めた。Furthermore, the sweetness components contained in the leaves were also compared.
The leaves of both the tetraploid and the conventional product (diploid) were harvested and dried in October. Then, 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.
測定方法はカラムとして、Shodex RS pak DC-613（6mm
×150mm）を用い、溶離液82％アセトニトリル、流速1ml
/min、溶離温度45℃、紫外検出214nmの条件で行い、二
点検量線法により定量した。The measuring method is Shodex RS pak DC-613 (6 mm
X150mm), eluent 82% acetonitrile, flow rate 1ml
/ min, elution temperature 45 ° C, UV detection at 214 nm, and quantification was carried out by the two-check curve method.
その結果、４倍体はステビオサイド10.3％、レバウディ
オサイドA10.3％の合計20.6％であった。２倍体の平均
含有率は、ステビオサイド7.6％、レバウディオサイドA
1.9％であった。As a result, tetraploid was 10.3% for stevioside and 10.3% for rebaudioside A, and the total was 20.6%. The average diploid content is Stevioside 7.6%, Rebaudioside A
It was 1.9%.
本発明の方法によれば、取り扱いが容易でかつ安全なサ
イトカイニンを使用することにより、甘味成分含量の多
いステビア人為４倍体を短期間に効率良く、しかも後の
生育に悪影響を与えることなく作出することができる。
さらに、他の倍数体を用いた育種にも応用できその効果
は大である。According to the method of the present invention, by using cytokinin that is easy to handle and safe, a stevia artificial tetraploid with a high sweetening ingredient content can be produced efficiently in a short period of time without adversely affecting the subsequent growth. can do.
Furthermore, it can be applied to breeding using other polyploids, and its effect is great.
Claims (5)
ることを特徴とするステビア人為４倍体の作出方法。1. A method for producing an artificial tetraploid of Stevia, which comprises treating a sterile Stevia seedling with cytokinin.
液で処理することを特徴とするステビア人為４倍体の作
出方法。2. A method for producing an artificial tetraploid of Stevia, which comprises treating a sterile Stevia seedling with a solution containing cytokinin.
ルアミノプテリン、ゼアチン、２−イソペンテニルアデ
ニンである請求項１又は請求項２記載のステビア人為４
倍体の作出方法。3. The Stevia-artificial artificial protein according to claim 1 or 2, wherein the cytokinin is kinetin, 6-benzylaminopterin, zeatin or 2-isopentenyladenine.
How to make a double.
地である請求項２又は請求項３記載のステビア人為４倍
体の作出方法。4. The method for producing a Stevia artificial tetraploid according to claim 2 or 3, wherein the solution is a liquid artificial medium containing an inorganic salt and a carbon source.
液に浸せき処理し、新たに発生するえき芽を固定培地に
移植するステビア人為４倍体の作出方法。5. A method for producing a Stevia artificial tetraploid in which a sterile Stevia seedling is soaked in a solution containing cytokinin and the newly-grown sprouts are transplanted to a fixed medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1064120A JPH0695866B2 (en) | 1989-03-16 | 1989-03-16 | Method for producing tetraploid stevia by plant growth hormone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1064120A JPH0695866B2 (en) | 1989-03-16 | 1989-03-16 | Method for producing tetraploid stevia by plant growth hormone |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02242623A JPH02242623A (en) | 1990-09-27 |
| JPH0695866B2 true JPH0695866B2 (en) | 1994-11-30 |
Family
ID=13248890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1064120A Expired - Fee Related JPH0695866B2 (en) | 1989-03-16 | 1989-03-16 | Method for producing tetraploid stevia by plant growth hormone |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0695866B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4741722B2 (en) * | 2000-10-11 | 2011-08-10 | 耕三 丹羽 | Planting method |
| CN102893860A (en) * | 2011-10-19 | 2013-01-30 | 崔广荣 | Stevia rebaudiana bertoni polyploid in vitro induction technology |
| CN102613085A (en) * | 2012-04-09 | 2012-08-01 | 南京农业大学 | Method for cultivating autotetraploid plants of stevia rebaudiana |
| CN115067209B (en) * | 2022-07-21 | 2023-06-27 | 北京市农林科学院 | Method for improving doubling efficiency of corn haploid by using zeatin |
-
1989
- 1989-03-16 JP JP1064120A patent/JPH0695866B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02242623A (en) | 1990-09-27 |
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| LAPS | Cancellation because of no payment of annual fees |