JP7458062B2 - How to improve seed productivity in plants - Google Patents
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Description
本発明は、花を観賞用とする植物、すなわち花卉の種子生産性を向上する方法に関する。 The present invention relates to a method for improving seed productivity of ornamental plants, i.e., flowering plants.
種子が食用とされるイネ、コムギ、トウモロコシ、ダイズなどの作物（穀物）では、個体ならびに単位面積あたりの種子の数や重さを増大させ、「子実収量性」を向上させる技術が数多く存在する。例えば、植物の成長を促進する働きを持った微生物（Bacillus pumilus TUAT-1株）の芽胞を含有する農業資材（バイオ肥料「キクイチ」）を育苗期の水稲実生に処理することで、実生の根系発達を促進し（非特許文献１）、水田移植後の植物体の生育を旺盛にして、穂数を増加させることで、水稲の子実収量を約18％増加させることができる技術が報告されている（特許文献１）。 For crops (cereals) whose seeds are edible, such as rice, wheat, corn, and soybeans, there are many techniques to increase the number and weight of seeds per individual and unit area, and to improve ``grain yield.'' do. For example, by applying an agricultural material (biofertilizer "Kikuichi") containing spores of a microorganism (Bacillus pumilus TUAT-1 strain) that promotes plant growth to paddy rice seedlings during the seedling-raising stage, the root system of the seedlings can be improved. A technology has been reported that can increase the grain yield of paddy rice by approximately 18% by promoting development (Non-Patent Document 1), increasing the number of panicles by stimulating the growth of plants after transplanting into paddy fields. (Patent Document 1).
一方、観賞用植物あるいは種子以外の部分を食用とする作物では、品種改良や種苗生産のために採種される種子の数や重さを増大させ、「種子生産性」を向上させることができる技術に関する知見はわずかである。例えば、本発明者等のグループでは、アサガオ品種「紫」において、花弁老化関連遺伝子として単離された14-3-3タンパク質をコードする遺伝子InPSR42の発現をRNAi法で抑制した遺伝子組換え体では、花弁老化が促進され、花持ち性は低下するが、種子の数や重さは増大し、種子生産性は向上することが確認されている。 On the other hand, for ornamental plants or crops whose parts other than seeds are edible, technology can increase the number and weight of seeds collected for breeding purposes and seedling production, thereby improving "seed productivity." There is little knowledge regarding this. For example, the present inventors' group has developed a genetically engineered morning glory cultivar "Murasaki" in which the expression of the gene InPSR42, which encodes the 14-3-3 protein isolated as a petal aging-related gene, was suppressed by RNAi method. It has been confirmed that, although petal senescence is accelerated and flower longevity is reduced, the number and weight of seeds are increased and seed productivity is improved.
また、ペチュニア品種「Mitchell Diploid」では、変異型エチレン受容体をコードする遺伝子etr1-1をCaMV35Sプロモーターで過剰発現させた遺伝子組換え体で、花弁老化が抑制され、花持ち性は向上するが、種子の100粒重が減少し、種子生産性は低下することが報告されている（非特許文献２）。 In addition, the petunia variety "Mitchell Diploid" is a genetically modified plant in which the gene etr1-1, which encodes a mutant ethylene receptor, is overexpressed using the CaMV35S promoter, which suppresses petal senescence and improves flower longevity. It has been reported that the 100 grain weight of seeds decreases and seed productivity decreases (Non-Patent Document 2).
しかし、このような技術では、種子生産性は向上しても、花持ち性が低下してしまうため、花持ち性の向上を求める消費者の需要に応えることができなくなってしまう。また、この技術には遺伝子組換え法が用いられているため、屋外で栽培する作物への適用は、カルタヘナ法の「第一種使用等」に該当し、環境に対する安全性の評価が必須となる。また、アサガオのように、交雑可能な在来野生種が国内に自生している場合、組換え遺伝子の環境拡散防止技術を併用しなければならず、当該技術の適用は極めて困難と判断される。 However, with such technology, even if seed productivity is improved, flower shelf life is reduced, making it impossible to meet consumer demand for improved flower shelf life. Additionally, since this technology uses genetic recombination, its application to crops grown outdoors falls under "Type 1 use, etc." of the Cartagena Act, and an evaluation of environmental safety is essential. Become. In addition, when native wild species that can be hybridized, such as morning glory, grow naturally in Japan, technology to prevent the spread of recombinant genes in the environment must also be used, and the application of such technology is judged to be extremely difficult. .
この知見に基づき、野生型エチレン受容体遺伝子の発現をRNAi法などにより抑制して、花弁老化を促進すれば、種子生産性を向上することができるかもしれない。しかし、その場合、アサガオで報告された技術と同様に、遺伝子組換え法を用いた技術となる。また、花弁老化を促進して種子生産性を向上しても、花持ち性が低下してしまうため、実用化は困難と判断される。 Based on this knowledge, it may be possible to improve seed productivity by suppressing the expression of the wild-type ethylene receptor gene using RNAi methods and promoting petal senescence. However, in that case, the technology would use genetic recombination, similar to the technology reported for morning glory. Furthermore, even if seed productivity is improved by accelerating petal senescence, the flower life will be reduced, so it is considered difficult to put it into practical use.
したがって、遺伝子組換え法などを用いて遺伝的に花弁老化を促進し、種子生産性を向上するのではなく、薬剤処理等により特定の個体で一過的に花弁老化を促進することで種子生産性を向上させることのできる、実用的で簡便な手法を確立する必要がある。そのような手法が確立されれば、消費者に販売する種子や個体は高い花持ち性を示すが、採種用の個体では、薬剤処理により花弁老化を促進して種子生産性を高めることが可能になる。 Therefore, rather than genetically promoting petal senescence and improving seed productivity using recombinant gene techniques, etc., it is necessary to establish a practical and simple method that can improve seed productivity by temporarily promoting petal senescence in specific individuals through chemical treatment, etc. If such a method is established, seeds and individuals sold to consumers will have long flower life, but in individuals for seed production, it will be possible to promote petal senescence through chemical treatment to increase seed productivity.
ペチュニアやアサガオのような観賞用の植物では、花の日持ち性（花持ち性）を向上させ、観賞期間を延長させた品種の開発が進められてきた。しかし、花持ち性の良い品種は、個体あたりの種子の数や重さが減り、種子生産性が低下するため、種苗生産上の問題となっている。また、遺伝子組換え技術により実験的に花持ち性を良くしたペチュニアでは種子生産性が低下し、悪くしたアサガオでは種子生産性が向上することも確認されている。さらに、160種類のアサガオ品種について花持ち性と種子生産性を調査したところ、両者の間に有意な負の相関も認められた。このように、花持ち性を遺伝的に改良した品種では種子生産性が低下することが確認されているが、その詳細なメカニズムや種子生産性を向上するための実用的で簡便な手法については知られていなかった。 For ornamental plants such as petunias and morning glories, efforts have been made to develop varieties with improved flower shelf life (flower retention) and extended ornamental periods. However, varieties with good flower retention have a reduced number and weight of seeds per individual, resulting in lower seed productivity, which poses a problem in seedling production. It has also been confirmed that seed productivity decreases in petunia, which has been experimentally improved in flower longevity using genetic modification technology, and that seed productivity improves in morning glory, which has been improved in flower longevity. Furthermore, when flower retention and seed productivity were investigated for 160 morning glory cultivars, a significant negative correlation was found between the two. In this way, it has been confirmed that seed productivity decreases in varieties genetically improved for flower retention, but the detailed mechanism and practical and simple methods for improving seed productivity are unknown. It wasn't known.
上記のようなこれまでの知見に基づき、本発明者等は、成長促進、発芽抑制、追熟促進等の植物成長調節作用を有することが知られているエチレンを適用することで、花弁老化を特定の花又は花卉個体に対して生じさせ、結果として種子生産性を向上させることを検討し、この方法が非常に有効であることを確認することができた。 Based on the above-mentioned knowledge, the present inventors have attempted to reduce petal aging by applying ethylene, which is known to have plant growth regulating effects such as promoting growth, suppressing germination, and promoting ripening. We investigated the possibility of producing seeds in specific flowers or individual flowering plants to improve seed productivity as a result, and were able to confirm that this method is very effective.
すなわち、本発明は以下を提供するものである。
１． 花卉における種子生産性を向上させる方法であって、
開花した花をエチレンガス又はエチレン発生剤と接触させて花弁老化を促進し、そして
種子を取得する
ことを含む、上記方法。
２． 花卉が、キク科、キキョウ科、キョウチクトウ科、キンポウゲ科、シソ科、スミレ科、ナス科、ナデシコ科、ハゼリソウ科、ハナシノブ科、ヒルガオ科、ムラサキ科、ユリズイセン科、及びリンドウ科から選択される種子植物である、上記１記載の方法。
３． 花卉個体を0.1～10 ppmの範囲の濃度のエチレンガス存在下に0.5～48時間置くことを特徴とする、上記１又は２記載の方法。
４． 開花した花の花弁に対して10～1000 ppmの範囲の濃度のエチレン発生剤を適用することを特徴とする、上記１又は２記載の方法。
５． エチレン発生剤が液剤である、上記４記載の方法。
６． エチレン発生剤が2-クロロエチルホスホン酸である、上記４又は５記載の方法。
７． 個体当たりの種子生産量が、エチレンガス又はエチレン発生剤と接触させていない対照と比較して10％以上増加する、上記１～６のいずれか記載の方法。
８． 果実当たりの平均種子重量が、エチレンガス又はエチレン発生剤と接触させていない対照と比較して10％以上増加する、上記１～６のいずれか記載の方法。
９． エチレン又はエチレン発生剤を有効成分として含む、花卉における種子生産性向上剤。
１０． 花卉個体に対してエチレン濃度0.1～10 ppmの範囲で個体と接触させることを特徴とする、上記９記載の種子生産性向上剤。
１１． 開花した花の花弁に10～1000 ppmの範囲の濃度のエチレン発生剤を適用することを特徴とする、上記９記載の種子生産性向上剤。
That is, the present invention provides the following.
1. A method for improving seed productivity in floriculture, comprising:
The above method comprising contacting the bloomed flowers with ethylene gas or an ethylene generator to promote petal senescence and obtaining seeds.
2. Seeds in which the flowering plant is selected from the family Asteraceae, Campanulaceae, Apocynaceae, Ranunculaceae, Lamiaceae, Violaceae, Solanaceae, Caryophyllaceae, Capulaceae, Convolvulaceae, Convolvulaceae, Urticaceae, Liliaceae, and Gentianaceae. The method according to 1 above, wherein the method is a plant.
3. 3. The method according to 1 or 2 above, characterized in that the individual flowers are placed in the presence of ethylene gas at a concentration in the range of 0.1 to 10 ppm for 0.5 to 48 hours.
4. 3. The method according to 1 or 2 above, characterized in that the ethylene generator is applied at a concentration in the range of 10 to 1000 ppm to the petals of a blooming flower.
5. 4. The method according to 4 above, wherein the ethylene generator is a liquid agent.
6. 6. The method according to 4 or 5 above, wherein the ethylene generator is 2-chloroethylphosphonic acid.
7. 7. The method according to any one of 1 to 6 above, wherein the seed production per individual is increased by 10% or more compared to a control not contacted with ethylene gas or an ethylene generator.
8. 7. The method according to any one of 1 to 6 above, wherein the average seed weight per fruit is increased by 10% or more compared to a control not contacted with ethylene gas or an ethylene generator.
9. A seed productivity improving agent for flowers, containing ethylene or an ethylene generator as an active ingredient.
10. 9. The seed productivity improving agent as described in 9 above, which is brought into contact with individual flower plants at an ethylene concentration in the range of 0.1 to 10 ppm.
11. 9. The seed productivity improver according to 9 above, characterized in that the ethylene generator is applied to the petals of bloomed flowers at a concentration in the range of 10 to 1000 ppm.
本発明の方法は、遺伝子組換え法などを用いて遺伝的に花弁老化を促進するのではなく、特定の個体で一過的に花弁老化を促進することで、種子生産性を向上させることが可能である。本発明により得られる効果は、花卉個体あるいは果実レベルでの種子生産性の向上である。 The method of the present invention does not genetically promote petal senescence using genetic recombination methods, but it can improve seed productivity by temporarily promoting petal senescence in specific individuals. It is possible. The effect obtained by the present invention is an improvement in seed productivity at the individual flower or fruit level.
本発明の方法で用いるエチレン発生剤は、植物成長調節剤として市販されており、果樹の着色・熟期促進、摘果・落葉促進、開花促進など、多面的に利用されている。従って、本発明は、安全性が確認された実用的な手法である。 The ethylene generator used in the method of the present invention is commercially available as a plant growth regulator, and is used in a variety of ways, such as promoting coloration and ripening of fruit trees, promoting fruit thinning and defoliation, and promoting flowering. Therefore, the present invention is a practical method whose safety has been confirmed.
＜花卉における種子生産性向上方法＞
本発明は、花卉における種子生産性を向上させる方法であって、
開花した花をエチレンガス又はエチレン発生剤と接触させて花弁老化を促進し、そして
種子を取得する
ことを含む、上記方法を提供する。
<Method for improving seed productivity in floriculture>
The present invention is a method for improving seed productivity in floriculture, comprising:
The method comprises contacting the bloomed flower with ethylene gas or an ethylene generator to promote petal senescence and obtaining seeds.
本発明の方法を用いて種子生産性が向上する花卉としては、限定するものではないが、例えばキク科、キキョウ科、キョウチクトウ科、キンポウゲ科、シソ科、スミレ科、ナス科、ナデシコ科、ハゼリソウ科、ハナシノブ科、ヒルガオ科、ムラサキ科、ユリズイセン科、及びリンドウ科から選択される科に属する種子植物を挙げることができる。 Examples of flowers whose seed productivity can be improved using the method of the present invention include, but are not limited to, Asteraceae, Campanulaceae, Apocynaceae, Ranunculaceae, Lamiaceae, Violataceae, Solanaceae, Caryophyllaceae, and Caryophyllaceae. Mention may be made of seed plants belonging to the families selected from the family Aracinaceae, Convolvulaceae, Urinaceae, Urinaceae, and Gentianaceae.
上記の科に属する種子植物としては、例えばガーベラ、キンセンカ、ヒマワリ、マリーゴールド、キキョウ、ニチニチソウ、アネモネ、クレマチス、デルフィニウム（オオヒエンソウ）、サルビア、ラベンダー、パンジー、ビオラ、ペチュニア、ミリオンベル、カーネーション、カスミソウ、シバザクラ、アサガオ、ヒルガオ、ネモフィラ、アルストロメリア、リンドウ、トルコギキョウ等を挙げることができるが、本発明の方法はこれらの植物のみに限定されるものではない。 Seed plants belonging to the above families include, for example, gerberas, calendula, sunflowers, marigolds, bellflowers, periwinkles, anemones, clematis, delphiniums, salvias, lavender, pansies, violas, petunias, million bells, carnations, and gypsophila. , Shibazakura, Morning Glory, Convolvulus, Nemophila, Alstroemeria, Gentiana, Lisianthus, etc., but the method of the present invention is not limited to these plants.
本発明の方法は、上記のステップを含むものである限り、通常の花卉の栽培条件の下で実施することができる。花卉をはじめとする植物が正常に生育し、開花し、種子を形成するためには、温度及び日照条件が対象の植物にとって適したものであることが必要である。 The method of the present invention can be carried out under normal flower cultivation conditions as long as it includes the above steps. In order for plants including flowers to grow normally, bloom, and form seeds, temperature and sunlight conditions need to be suitable for the target plants.
一般的に、植物が生育可能な温度範囲は5～35℃とされており、生育適温は15～25℃とされている。従って、種子生産性を向上させるための本発明の方法において、開花した花をエチレンガス又はエチレン発生剤と接触させるステップも、上記の温度範囲内、すなわち5～35℃、好ましくは15～25℃の温度条件で行うことが適している。 Generally, the temperature range in which plants can grow is 5 to 35°C, and the optimum temperature for growth is 15 to 25°C. Therefore, in the method of the invention for improving seed productivity, the step of contacting the bloomed flowers with ethylene gas or an ethylene generator is also carried out within the above temperature range, i.e. 5-35°C, preferably 15-25°C. It is suitable to carry out under temperature conditions of .
また、栽培に適した光の強さとしては、0.4～100 klx（キロルクス）、あるいは光量子束密度で7～1680 μmol m-2s-1の範囲が挙げられる。日長時間としては、8～24時間の範囲で選択することができる。 In addition, the light intensity suitable for cultivation is in the range of 0.4 to 100 klx (kilolux), or a photon flux density of 7 to 1680 μmol m -2 s -1 . Day length can be selected from 8 to 24 hours.
本発明の方法において、エチレンガス又はエチレン発生剤を適用する時期は、花が開花している時期に行うことを必要とする。従って、エチレンガス又はエチレン発生剤の適用は、開花前に行うものではなく、また花が終わった後（花弁老化が終了した後）、例えば種子の生長段階で行うものでもない。 In the method of the present invention, the ethylene gas or ethylene generator needs to be applied during the flowering period. Therefore, the application of ethylene gas or ethylene generator is not carried out before flowering, nor after flowering (after petal senescence has ended), for example at the seed growth stage.
また、エチレンガス又はエチレン発生剤は、花が開いている時間帯に適用することが特に好ましい。花が開く時間帯は植物種により異なっているため、エチレンガス又はエチレン発生剤の適用は、１日のうち、対象の植物の花が咲いている時間帯に行うこととするのが好適である。 Moreover, it is particularly preferable to apply ethylene gas or an ethylene generator during the time when flowers are open. Since the time when flowers open differs depending on the plant species, it is preferable to apply ethylene gas or an ethylene generator during the time of the day when the flowers of the target plants are blooming. .
エチレンガスを用いて本発明の方法を実施する場合、限定するものではないが、例えば最初に形成された花（第1花）の開花が認められた個体、又は一定数の花（例えば2～30個の花）が開花した個体に、1～7日の間隔で、1日あたり1～5回の処理を行うことが好適であり得る。 When carrying out the method of the present invention using ethylene gas, for example, but not limited to, an individual whose first flower (first flower) is observed to have bloomed, or a certain number of flowers (for example, 2 to 2 flowers) It may be suitable to apply 1 to 5 treatments per day, at intervals of 1 to 7 days, to individuals in bloom (30 flowers).
エチレン発生剤を用いて本発明の方法を実施する場合、限定するものではないが、例えば開花した花に対してそれぞれ1～5回、全ての花に対して1花ずつ処理を行うことが好適であり得る。この場合、一度に処理する花は全ての花であっても、例えば1～5個であっても良く、また、一定数の花（例えば2～30個の花）が開花した後に、新たに開花した花に対して処理を同様に行うものとしても良い。 When carrying out the method of the present invention using an ethylene generator, it is preferable, but not limited to, to treat each blooming flower 1 to 5 times, for example, and to treat all flowers once. It can be. In this case, the flowers to be treated at once may be all flowers or, for example, 1 to 5 flowers, or after a certain number of flowers (for example, 2 to 30 flowers) have bloomed, new flowers may be treated. The same treatment may be applied to bloomed flowers.
本発明の方法の一実施形態は、花卉個体を0.1～10 ppmの範囲の濃度のエチレンガス存在下に0.5～48時間置くことによって開花した花をエチレンガス又はエチレン発生剤と接触させるものであり得る。 One embodiment of the method of the present invention is to contact the bloomed flowers with ethylene gas or an ethylene generator by placing the individual plants in the presence of ethylene gas at a concentration ranging from 0.1 to 10 ppm for 0.5 to 48 hours. obtain.
エチレンガスは気体のため、エチレンガスを用いた処理のためには密閉容器が必要とされる。例えば、密閉容器内のエチレン濃度が上記の範囲となるように、シリンジを用いてエチレンを注入することができる。 Since ethylene gas is a gas, a closed container is required for processing with ethylene gas. For example, ethylene can be injected using a syringe so that the ethylene concentration within the closed container falls within the above range.
一方、エチレン発生剤は液剤が市販されており、植物体へのスプレー噴霧が可能である。エチレン発生剤は、気体であるエチレンガスの操作性向上のために、液剤として利用でき、適用後に植物体内で容易に分解してエチレンを発生する薬剤として開発されている。適用後数日で消失することが確認されているため、意図する効果を発揮する以外に植物に対する影響を考慮する必要はないと考えられている。 On the other hand, ethylene generators are commercially available in liquid form and can be sprayed onto plants. Ethylene generators have been developed as agents that can be used as liquid agents and easily decompose within plants after application to generate ethylene in order to improve the operability of gaseous ethylene gas. Since it has been confirmed that the drug disappears within a few days after application, it is considered that there is no need to consider the effects on plants other than to achieve the intended effect.
エチレン発生剤は、特に限定するものではないが、例えばエテホン（2-クロロエチルホスホン酸）を挙げることができる。エテホンは、日産化学株式会社及び石原バイオサイエンス株式会社からエスレル（登録商標）10という商品名の液剤が販売されている。 Although the ethylene generator is not particularly limited, for example, ethephon (2-chloroethylphosphonic acid) can be mentioned. Ethephon is sold as a liquid formulation under the trade name Esrel (registered trademark) 10 by Nissan Chemical Co., Ltd. and Ishihara Bioscience Co., Ltd.
2-クロロエチルホスホン酸は、水、及びエタノール、アセトン等の有機溶媒に溶解性の吸湿性粉末であり、エスレル（登録商標）10は、この有効成分エテホン（2-クロロエチルホスホン酸）を10.0％含有する黄褐色液体の液剤であり、噴霧して使用することができる。エスレル（登録商標）10は、開花促進、着色・熟期促進、離層形成促進、摘花・摘果、花ぶるい防止、開花抑制等の効果を有するものとして販売されているが、花卉における種子生産性に対する効果はこれまで全く知られていない。 2-Chloroethylphosphonic acid is a hygroscopic powder that is soluble in water and organic solvents such as ethanol and acetone. It is a yellowish-brown liquid containing 1.5% of the total amount of alcohol, and can be used by spraying. Ethrel (registered trademark) 10 is sold as having effects such as promoting flowering, promoting coloration and ripening, promoting abscission, thinning flowers and fruit, preventing blooming, and suppressing flowering. The effect on sex has not been known until now.
本発明の方法の別の実施形態は、開花した花の花弁に対して10～1000 ppmの範囲の濃度のエチレン発生剤を適用することによって開花した花をエチレンガス又はエチレン発生剤と接触させるものであり得る。例えば、エテホン10.0％の液剤を100～10000倍に希釈して、0.3～3 mLの範囲の量で１回噴霧することで良好な花弁老化効果、従って種子生産性向上効果を得ることができる。 Another embodiment of the method of the invention is to contact the bloomed flower with ethylene gas or an ethylene generator by applying the ethylene generator at a concentration ranging from 10 to 1000 ppm to the petals of the bloomed flower. It can be. For example, by diluting a 10.0% ethephon solution 100 to 10,000 times and spraying it once in an amount in the range of 0.3 to 3 mL, it is possible to obtain a good effect on petal aging and, therefore, an effect on improving seed productivity.
本発明の方法における種子を取得するステップは、通常の花卉における種子の取得方法により実施することができる。
本発明の方法を用いることで、花卉個体当たりの種子生産量を、エチレンガス又はエチレン発生剤と接触させていない対照と比較して10％以上、15％以上、又は20％以上増加させることができる。
The step of obtaining seeds in the method of the present invention can be carried out by a conventional method for obtaining seeds in floriculture.
By using the method of the present invention, it is possible to increase the seed production per individual flower by 10% or more, 15% or more, or 20% or more compared to a control that is not contacted with ethylene gas or an ethylene generator. can.
加えて、あるいはまた、本発明の方法を用いることで、花卉の果実当たりの平均種子重量を、エチレンガス又はエチレン発生剤と接触させていない対照と比較して10％以上、15％以上、又は20％以上増加させることができる。 Additionally or alternatively, using the methods of the present invention, the average seed weight per fruit of a flower plant is increased by 10% or more, 15% or more compared to a control that is not contacted with ethylene gas or an ethylene generator, or It can be increased by more than 20%.
本発明の方法は、目的とする花卉個体の種子生産量を向上させるものである一方で、その花卉個体の花持ち性等の特徴に影響を与えないことが最大の利点である。 The greatest advantage of the method of the present invention is that while it improves the seed production of the desired individual flower plant, it does not affect the characteristics of the individual flower plant, such as flower retention.
また、本発明の方法は、花弁老化を促進する一方で、葉の老化は促進させないことが確認されている。葉では光合成によりショ糖が合成されているが、葉が老化すると、若い葉や花、果実、種子等の他の器官に転流可能なショ糖量が減少するため、新たな葉や花の形成が阻害され、種子生産性も低下し得る。従って、本発明の方法は、葉の老化を促進しないことで、種子生産性に対する悪影響を排除することができるという更なる利点も有し得る。 Furthermore, it has been confirmed that the method of the present invention accelerates petal senescence, but does not accelerate leaf senescence. Sucrose is synthesized in leaves through photosynthesis, but as leaves age, the amount of sucrose that can be translocated to other organs such as young leaves, flowers, fruits, and seeds decreases, making it difficult for new leaves and flowers to be produced. formation may be inhibited and seed productivity may also be reduced. Therefore, the method of the invention may also have the additional advantage of not accelerating leaf senescence, thereby eliminating negative effects on seed productivity.
＜花卉における種子生産性向上剤＞
本発明はまた、エチレン又はエチレン発生剤を有効成分として含む、花卉における種子生産性向上剤を提供する。
本発明の種子生産性向上剤は、例えば花卉個体に対してエチレン濃度0.1～10 ppmの範囲で個体と接触させるように適用するものであり得る。
あるいはまた、本発明の種子生産性向上剤は、開花した花の花弁に10～1000 ppmの範囲の濃度のエチレン発生剤を適用するように適用するものであり得る。
<Seed productivity improver in floriculture>
The present invention also provides an agent for improving seed productivity in floriculture, which contains ethylene or an ethylene generator as an active ingredient.
The seed productivity improving agent of the present invention may be applied to individual flower plants by contacting them with an ethylene concentration in the range of 0.1 to 10 ppm, for example.
Alternatively, the seed productivity improver of the present invention may be applied to the petals of a blooming flower with an ethylene generator at a concentration ranging from 10 to 1000 ppm.
本発明の種子生産性向上剤の作用機序は、特に本発明を限定するために記載するものではないが、花卉における花弁老化を促して、花弁中のタンパク質を分解して窒素再転流に効果的なアミノ酸を合成し、種子形成にこれを再利用するものと考えられる。本発明者等は、エチレン又はエチレン発生剤と接触させた花弁において、接触させていない対照と比較して有意なタンパク質量の減少、及びグルタミン及びアスパラギン含量の増大を確認している。 Although the mechanism of action of the seed productivity enhancer of the present invention is not described in order to limit the present invention, it is believed that the agent promotes petal senescence in flowers, decomposes proteins in the petals, synthesizes amino acids that are effective in nitrogen retranslocation, and reuses these for seed formation. The inventors have confirmed a significant decrease in protein amount and an increase in glutamine and asparagine content in petals contacted with ethylene or an ethylene generating agent compared to uncontacted controls.
以下に本発明を実施例によって更に説明するが、本発明はこれらの実施例に限定されるものではない。 The present invention will be further explained below with reference to Examples, but the present invention is not limited to these Examples.
［実施例１ エチレン処理がペチュニア花弁老化に及ぼす影響］
ペチュニア品種「おゆきちゃん」の苗を、培養土（サカタスーパーミックスA，サカタのタネ社）を入れたプラスチック製ポット（直径14 cm）を用いて恒温室内で24℃、蛍光灯下（光量子束密度100μmol m-2 s-1）の16時間日長（明期6:00～22:00）で栽培した。
開花前日の12:00を基点（-1日）とし、24時間おきに0日、1日、2日、3日、4日、5日、6日、7日とした。
Example 1: Effect of ethylene treatment on petunia petal senescence
Seedlings of the petunia cultivar "Oyuki-chan" were grown in plastic pots (14 cm in diameter) filled with culture soil (Sakata Super Mix A, Sakata Seed Co., Ltd.) at 24°C in a thermostatic room under fluorescent lights (photon flux density 100 μmol m -2 s -1 ) with a 16-hour day length (light period 6:00 to 22:00).
The base point (-1 day) was 12:00 on the day before flowering, and each 24-hour interval was designated as day 0, 1, 2, 3, 4, 5, 6, and 7.
未受粉区の花弁は-1日で除雄を行い、受粉区の花弁は除雄せずに0日で他の開花した花の雄しべからピンセットで採取した花粉を用いて人工受粉した。
エチレン処理区は0日で人工受粉を行うとともに、エチレン濃度が1 ppmとなるようにガラスシリンジを用いて外生的にエチレンを封入した40.5 L容チャンバー内に6時間静置した。
図１に、開花前日から24時間毎に観察した未受粉区、受粉区およびエチレン処理区の鉢花の時系列画像を示す。
Petals of the unpollinated area were emasculated on day -1, whereas petals of the pollinated area were not emasculated and artificially pollinated on day 0 using pollen collected with tweezers from the stamens of other blooming flowers.
The ethylene-treated group was artificially pollinated on day 0 and was then placed in a 40.5 L chamber into which ethylene had been exogenously added using a glass syringe so that the ethylene concentration was 1 ppm for 6 hours.
Figure 1 shows a time series of images of unpollinated, pollinated, and ethylene-treated potted flowers observed every 24 hours from the day before flowering.
開花した花弁の直径を3箇所ノギスで測定し、3箇所の平均値をそれぞれ求め、その最大値を1.0とした相対直径として算出した。未受粉、受粉区の花弁は相対直径が0.8以上で「開花」、1.0で「花弁老化の開始」、0.4以下で「花弁老化の完了」とした。エチレン処理区の花弁はエチレン処理開始時点を「開花」、1.0で「花弁老化の開始」、0.4以下で「花弁老化の完了」とした。「開花」から「花弁老化の開始」までの期間を花弁老化の誘導期間として評価および比較した。 The diameter of open petals was measured at three points with a vernier caliper, the average of the three points was calculated, and the maximum value was set to 1.0 to calculate the relative diameter. For petals in unpollinated and pollinated areas, a relative diameter of 0.8 or more was considered to be "open", 1.0 was considered to be "start of petal senescence", and 0.4 or less was considered to be "completion of petal senescence". For petals in the ethylene treatment area, the start of ethylene treatment was considered to be "open", 1.0 was considered to be "start of petal senescence", and 0.4 or less was considered to be "completion of petal senescence". The period from "opening" to "start of petal senescence" was evaluated and compared as the induction period for petal senescence.
その結果、花弁老化の誘導期間は、未受粉区、受粉区およびエチレン処理区でそれぞれ5日、2日、および1日であり、受粉区では未受粉区と比較して花弁老化の誘導期間が短くなっていることが示され、エチレン処理区の花弁では、更に短くなっていることが示された。 As a result, the induction period of petal senescence was 5 days, 2 days, and 1 day in the unpollinated, pollinated, and ethylene-treated areas, respectively, and the induction period of petal senescence was longer in the pollinated area than in the unpollinated area. It was shown that the petals in the ethylene-treated area were shorter.
［実施例２］
2018年度及び2019年度において、実施例１と同様の条件でペチュニア6個体をそれぞれ栽培し、対照区の3個体は各花の開花後0日で他の開花した花の雄しべからピンセットで採取した花粉を用いて、人工受粉させた。エチレン処理区の3個体は、人工受粉を行うとともに鉢花を1週間おきにエチレン濃度1 ppmの密閉された容器内に6時間静置し、これを種子採取が完了するまで継続して行った。
[Example 2]
In 2018 and 2019, six petunia plants were cultivated under the same conditions as in Example 1, and the three plants in the control group were artificially pollinated using pollen collected with tweezers from the stamens of other flowers that had bloomed 0 days after each flower bloomed. The three plants in the ethylene treatment group were artificially pollinated and the potted flowers were left in a sealed container with an ethylene concentration of 1 ppm for 6 hours every other week, and this was continued until seed collection was completed.
2018年度の実験では、対照区およびエチレン処理区の各鉢花から50花分の種子を採取した。ペチュニアの種子は極小であり1粒重の測定が困難であったため、5花ごとに種子数および種子重量を計測し、5花あたりの種子重量 / 5花あたりの種子数により1花ごとの種子数および種子重量を算出した。1花あたりの種子生産量は、1花あたりの種子数×種子1粒重により算出し、種子生産性の評価に用いた。結果を図２－１に示す。 In the 2018 experiment, seeds from 50 flowers were collected from each potted flower in the control and ethylene-treated areas. Because petunia seeds are extremely small and it was difficult to measure the weight of each seed, the number and weight of seeds were measured for every five flowers, and the number and weight of seeds per flower were calculated by dividing the seed weight per five flowers by the number of seeds per five flowers. Seed production per flower was calculated by multiplying the number of seeds per flower by the weight of one seed, and used to evaluate seed productivity. The results are shown in Figure 2-1.
2019年度の実験は、恒温室の故障により栽培が中断されたため、採取できた20花分の種子において、1花ごとに種子数および種子重量を測定した。種子1粒重は1花あたりの種子重量 / 1花あたりの種子数により算出した。そこから1花あたりの種子生産量を1花あたりの種子数×種子1粒重により算出し、種子生産性の評価に用いた。結果を図２－２に示す。
下記の表１は、図２－１及び２－２の結果をまとめたものである。
In the 2019 experiment, cultivation was interrupted due to a malfunction in the thermostatic chamber, so the number and weight of seeds were measured for each flower of the 20 flowers that were collected. The weight of one seed was calculated by the weight of seeds per flower/number of seeds per flower. From this, the amount of seeds produced per flower was calculated as the number of seeds per flower x the weight of one seed, and was used to evaluate seed productivity. The results are shown in Figure 2-2.
Table 1 below summarizes the results of Figures 2-1 and 2-2.
図２－１及び２－２、並びに表１に示す通り、2018年度、2019年度ともにエチレン処理区の鉢花の1花あたりの種子数および種子生産量は有意に増加し、いずれも個体当たりの種子生産量の10％を超える増加が認められた。一方、種子1粒重および開花数は対照区と比較して有意な差は見られなかった。 As shown in Figures 2-1 and 2-2 and Table 1, the number of seeds per flower and the amount of seeds produced in potted flowers in the ethylene-treated plots increased significantly in both 2018 and 2019, and both An increase of more than 10% in seed production was observed. On the other hand, no significant difference was observed in the weight of each seed and the number of flowers compared to the control plot.
［実施例３ エチレン処理が種子発芽率に及ぼす影響］
2018年度に得られた種子を、1個体40粒ずつ2.0 mL容量のマイクロチューブに入れ、0.05％ジベレリン溶液で30分間催芽処理を行った後、蒸留水で3回洗浄した。それぞれの種子を蒸留水を染みこませたろ紙を敷いたプラスチックシャーレに播種した。24℃、24時間日長、白色蛍光下（光量子束密度100μmol m-2 s-1）で静置し、1日おきに発芽率を測定した。
[Example 3 Effect of ethylene treatment on seed germination rate]
Seeds obtained in 2018 were placed in a 2.0 mL microtube (40 seeds each), germinated with 0.05% gibberellin solution for 30 minutes, and then washed three times with distilled water. Each seed was sown in a plastic Petri dish lined with filter paper soaked with distilled water. The seeds were allowed to stand at 24°C, 24-hour photoperiod, and under white fluorescence (photon flux density: 100 μmol m -2 s -1 ), and the germination rate was measured every other day.
その結果、図３に示すように、2018年度に得られた種子では、播種後13日目の発芽率が対照区で61.5％、エチレン処理区で62.0％であり、有意な差はなかった。しかし、播種後5日目で比較すると、対照区で18.0％、エチレン処理区で40.5％であり、播種後7日目では対照区で37.5％、エチレン処理区で54.5％となり、エチレン処理区で有意に高い値を示した（図３Ａ）。
一方、2019年度に得られた種子を用いて同様の実験を行った結果、対照区とエチレン処理区で播種後の発芽率に有意な差は認められなかった（図３Ｂ）。
As a result, as shown in Figure 3, for seeds obtained in 2018, the germination rates on the 13th day after sowing were 61.5% in the control group and 62.0% in the ethylene-treated group, showing no significant difference. However, when compared on the 5th day after sowing, the germination rates were 18.0% in the control group and 40.5% in the ethylene-treated group, and on the 7th day after sowing, the germination rates were 37.5% in the control group and 54.5% in the ethylene-treated group, showing a significantly higher value in the ethylene-treated group (Figure 3A).
On the other hand, a similar experiment was conducted using seeds obtained in 2019, and no significant difference was observed in the germination rate after sowing between the control and ethylene-treated areas (Figure 3B).
［実施例４］
光合成活性を測定することにより、本発明の方法によるエチレン処理が葉の老化に及ぼす影響の有無を検討した。
2019年度の種子生産性の調査に使用した対照区および外生エチレン処理区における各植物個体の葉の中央部において、エチレン処理開始後28 日目でJUNIOR-PAM（ナモト貿易株式会社）を用いて葉の老化マーカーとして使用できるクロロフィル蛍光比Fv / Fmを調査した。各個体の茎2本をランダムに選択し、根本から先端まで生えている葉で1個体27枚、3個体合計81枚のクロロフィル蛍光比Fv / Fmを測定した。各区の数値の有意差をt検定により確認した。
[Example 4]
By measuring photosynthetic activity, we examined whether ethylene treatment according to the method of the present invention had an effect on leaf senescence.
28 days after the start of ethylene treatment, JUNIOR-PAM (Namoto Trading Co., Ltd.) was used to test the central part of the leaf of each plant in the control plot and exogenous ethylene treatment plot used in the 2019 seed productivity survey. We investigated the chlorophyll fluorescence ratio Fv/Fm, which can be used as a leaf senescence marker. Two stems from each individual were randomly selected, and the chlorophyll fluorescence ratio Fv/Fm was measured for 27 leaves for each individual, and a total of 81 leaves for three individuals. Significant differences in numerical values for each section were confirmed by t-test.
その結果、図４に示すように、クロロフィル蛍光比Fv / Fmは対照区で0.733、エチレン処理区で0.728となり有意な差はなく、エチレン処理によって葉の老化は促進されていないことが示唆された。 As a result, as shown in Figure 4, the chlorophyll fluorescence ratio Fv/Fm was 0.733 in the control plot and 0.728 in the ethylene-treated plot, with no significant difference, suggesting that ethylene treatment did not accelerate leaf senescence. .
［実施例５ 可溶性タンパク質含量に及ぼす影響］
ペチュニアの未受粉区、受粉区およびエチレン処理区の鉢花のそれぞれについて、開花前日(-1日）、および実施例１に記載した「開花」、「花弁老化の開始」、「花弁老化の完了」の各段階で植物体から花弁を採取した。また、未受粉区の鉢花は開花期間が長いため、「開花」から「花弁老化」の開始の期間に更に2回サンプリングを行った。
エチレン処理区の鉢花は、恒温室の故障により栽培が中断されたため、採取可能であった蕾（開花前日-1日）および「開花」のステージを採取した。
[Example 5 Effect on soluble protein content]
For petunia potted flowers in unpollinated plots, pollinated plots, and ethylene-treated plots, the day before flowering (-1 day), and the "flowering", "beginning of petal senescence", and "completion of petal senescence" described in Example 1, respectively. Petals were collected from the plants at each stage. In addition, since potted flowers in unpollinated plots have a long flowering period, sampling was conducted two more times during the period from ``flowering'' to the start of ``petal senescence.''
Cultivation of potted flowers in the ethylene-treated area was interrupted due to a malfunction in the thermostatic chamber, so buds that could be collected (1 day before flowering) and the "blooming" stage were collected.
採取した花は1花あたりのタンパク質含量を求めるために、採取した花から雌蕊より上部の花弁を切り取って新鮮重を測定した。切り取った花弁から約50 mg採取し、タンパク質の定量に用いるため6 mm ステンレスビーズ（バイオメディカルサイエンス）を入れた2.0 mL 容量のマイクロチューブに入れ、液体窒素で凍結後、Tissue Lyser LT（QIAGEN）により50 Hz, 1分間で破砕する作業を2回行った。 To determine the protein content per flower, the petals above the pistil were cut from the flowers and their fresh weight was measured. Approximately 50 mg was collected from the cut petals, placed in a 2.0 mL microtube containing 6 mm stainless steel beads (Biomedical Science) for protein quantification, frozen in liquid nitrogen, and then frozen using Tissue Lyser LT (QIAGEN). Crushing was performed twice at 50 Hz for 1 minute.
次いで、抽出バッファー［100 mM Tris/HCL（pH 8.0）、5 mM EDTA（pH 8.0）、10 mM NaCl、1%（v/v）Triton X-100、0.2% β-メルカプトエタノール］ 1 mLをマイクロチューブに加えて攪拌後、サンプルを新しい1.5 mL容量のマイクロチューブに移し、氷上に30分間静置した。Centrifuge 5417R（eppendorf）を用いて2,200×g、4℃で5分間遠心し、上清を新しい1.5 mL容量のマイクロチューブに25μL移し、これを可溶性タンパク質とした。 Next, add 1 mL of extraction buffer [100 mM Tris/HCL (pH 8.0), 5 mM EDTA (pH 8.0), 10 mM NaCl, 1% (v/v) Triton X-100, 0.2% β-mercaptoethanol] to a microcentrifuge. After adding the sample to the tube and stirring, the sample was transferred to a new 1.5 mL microtube and left on ice for 30 minutes. Centrifuge 5417R (Eppendorf) was used to centrifuge at 2,200 x g and 4°C for 5 minutes, and 25 μL of the supernatant was transferred to a new 1.5 mL microtube, which was used as a soluble protein.
タンパク質濃度測定はRC DCプロテインアッセイ（Bio Rad）のキットを使用し、手順はマニュアルに従った。分光光度計（U5100、日立）を用いて750 nmで吸光度を測定し、検量線を用いてタンパク質含量を算出した。 Protein concentration was measured using the RC DC Protein Assay (Bio Rad) kit, and the procedure was according to the manual. Absorbance was measured at 750 nm using a spectrophotometer (U5100, Hitachi), and protein content was calculated using a calibration curve.
その結果、図５に示すように、1花あたりの可溶性タンパク質含量は開花前日から開花後1日にかけて、未受粉区および受粉区で増加したが、エチレン処理区では減少していた。開花後1日以降は未受粉区および受粉区ともに減少していた。 As a result, as shown in Figure 5, the soluble protein content per flower increased in unpollinated and pollinated plots from the day before flowering to one day after flowering, but decreased in ethylene-treated plots. After 1 day after flowering, the number decreased in both unpollinated and pollinated plots.
［実施例６ 花弁のグルタミン及びアスパラギン含量に及ぼす影響］
ペチュニアの未受粉区、受粉区およびエチレン処理区の開花前日から開花後の鉢花花弁について、窒素再転流のために行われるアミノ酸合成に関わることが知られているアミノ酸であるグルタミン及びアスパラギン含量の経時的変化を検討した。
[Example 6 Effect on glutamine and asparagine content of petals]
The contents of glutamine and asparagine, which are amino acids known to be involved in amino acid synthesis for nitrogen retranslocation, were measured in the petals of potted flowers from the day before flowering to after flowering in unpollinated, pollinated, and ethylene-treated areas of petunia. Changes over time were examined.
ペチュニアの未受粉区、受粉区およびエチレン処理区の鉢花について、開花前の蕾の段階、および実施例１に記載した「開花」、「花弁老化の開始」および「花弁老化の完了」の各段階で植物体から花を採取した。採取した花は、1花あたりのアミノ酸含量を求めるために、実施例５と同様にして花弁を切り取り、保存した。 Regarding potted petunia flowers in unpollinated plots, pollinated plots, and ethylene-treated plots, the bud stage before flowering, and each of "flowering", "start of petal senescence" and "completion of petal senescence" described in Example 1 Flowers were collected from plants at this stage. The petals of the collected flowers were cut out and stored in the same manner as in Example 5 in order to determine the amino acid content per flower.
直径6.0 mmステンレスビーズと共に2.0 mL容量のマイクロチューブに入れた花弁50 mgを30秒間液体窒素で凍結させ、Tissue Lyser LTにより50 Hz、1分間で破砕する作業を行った。マイクロチューブに10 mM HClを、各サンプルの新鮮重の10倍量入れ（花弁1 mgあたり10 mM HClを10μL）、激しく転倒混和した。10,000×g、4℃で10分間の遠心分離を行い、上清を新しい2 mL容量のマイクロチューブに移し、アミノ酸の測定に用いた。 50 mg of petals were placed in a 2.0 mL microtube together with 6.0 mm diameter stainless steel beads and frozen in liquid nitrogen for 30 seconds, then disrupted using a Tissue Lyser LT at 50 Hz for 1 minute. 10 mM HCl was added to the microtube in an amount 10 times the fresh weight of each sample (10 μL of 10 mM HCl per 1 mg of petals) and mixed vigorously by inversion. Centrifugation was performed at 10,000 × g for 10 minutes at 4°C, and the supernatant was transferred to a new 2 mL microtube and used for amino acid measurements.
アミノ酸の測定は、AccQ-Fluor Reagent Kit（Waters）を使用し、マニュアルに従って、抽出したアミノ酸の誘導体化を行った。定量にはHPLC（LCMS-2020、島津製作所）を用いた。移動相には、キットに含まれるAccQ・Tag Eluent Aと超純水を1 : 10の割合で混合したものをA液、60％アセトニトリルをB液、超純水をC液として、マニュアルに定められた割合および時間で流した。なお、移動相の流量は1.0 mL/分とした。分析カラムにはAccQ・Tag 3.9×150 mm Columnを用い、オーブンの温度を37℃に設定した。また、1回のサンプル注入量は10μLとした。 For the measurement of amino acids, the extracted amino acids were derivatized using AccQ-Fluor Reagent Kit (Waters) according to the manual. HPLC (LCMS-2020, Shimadzu Corporation) was used for quantitative determination. For the mobile phase, use a 1:10 mixture of AccQ/Tag Eluent A and ultrapure water included in the kit as solution A, 60% acetonitrile as solution B, and ultrapure water as solution C, as specified in the manual. flowed at the specified rate and time. Note that the flow rate of the mobile phase was 1.0 mL/min. AccQ・Tag 3.9×150 mm Column was used as the analytical column, and the oven temperature was set at 37°C. In addition, the amount of sample injection per time was 10 μL.
1花あたりのアミノ酸含量は以下の計算式により求めた。
1花あたりのアミノ酸含量（mg/花）
＝アミノ酸濃度（nmol/μL）×抽出に使用したHCl量（μL）
/サンプル重（mg）×1花あたりの新鮮重（fresh weight(FW)、mg）×各アミノ酸の分子量
The amino acid content per flower was calculated using the following formula.
Amino acid content per flower (mg/flower)
= Amino acid concentration (nmol/μL) × HCl volume used for extraction (μL)
/sample weight (mg) x fresh weight (FW) per flower (mg) x molecular weight of each amino acid
その結果、図６Ａに示すように、グルタミン含量は、未受粉区および受粉区の花弁では、開花前日～開花後1日（蕾～開花）で増加し、1日（開花）以降減少し、一方、エチレン処理区の花弁は、開花前日～開花後2日（蕾～花弁老化開始）まで継続して増加し、2日（花弁老化開始）以降減少した。 As a result, as shown in Figure 6A, the glutamine content increased from the day before anthesis to the 1st day after anthesis (buds to anthesis) in the petals of the unpollinated and pollinated areas, and decreased after the 1st day (anthesis); The number of petals in the ethylene-treated plots increased continuously from the day before flowering to 2 days after flowering (bud to start of petal senescence), and decreased after 2 days (start of petal senescence).
また、図６Ｂに示すように、アスパラギン含量も、未受粉区および受粉区の花弁では、開花前日～開花後1日（蕾～開花）で増加し、1日（開花）以降減少し、一方、エチレン処理区の花弁は、開花前日～開花後2日（蕾～花弁老化開始）まで継続して増加し、2日（花弁老化開始）以降減少した。 In addition, as shown in Figure 6B, asparagine content also increased in the petals of unpollinated and pollinated areas from the day before anthesis to the 1st day after anthesis (buds to anthesis), and decreased after the 1st day (anthesis); The number of petals in the ethylene-treated plot continued to increase from the day before flowering to 2 days after flowering (bud to start of petal senescence), and decreased after 2 days (start of petal senescence).
［実施例７ 花弁のグルタミン及びアスパラギン合成酵素遺伝子の転写に及ぼす影響］
７－１．転写産物増幅のためのプライマーの設計
シロイヌナズナ（A. thaliana）のグルタミン合成酵素遺伝子として同定されている5種のGS1遺伝子（GLN1;1、GLN1;2、GLN1;3、GLN1;4、GLN1;5）、およびアスパラギン合成遺伝子として同定されている3種のAS遺伝子（ASN1、ASN2、ASN3）の推定アミノ酸配列を用いて、ペチュニア（Petunia inflata）の遺伝子がコードするタンパク質の推定アミノ酸配列群に対して、Sol Genomicsのtblastnプログラムを用いて相同性検索を行い、高い相同性が示された推定アミノ酸配列をコードする遺伝子をGS1遺伝子およびAS遺伝子のホモログ候補とした。
[Example 7] Effect on transcription of glutamine and asparagine synthetase genes in petals
7-1. Primer design for amplifying transcripts
Using the deduced amino acid sequences of five GS1 genes (GLN1;1, GLN1;2, GLN1;3, GLN1;4, GLN1;5) identified as glutamine synthetase genes in Arabidopsis thaliana, and three AS genes (ASN1, ASN2, ASN3) identified as asparagine synthesis genes, a homology search was performed against the deduced amino acid sequences of proteins encoded by genes in Petunia inflata using the tblastn program from Sol Genomics. Genes encoding deduced amino acid sequences that showed high homology were selected as candidate homologs of the GS1 and AS genes.
続いて、このホモログ候補の塩基配列情報を用いて、A. thalianaのアミノ酸配列群に対してxblastnプログラムを用いて相同性検索を行った。検出されたA. thalianaのアミノ酸配列が最初の相同性検索に用いたA. thalianaのGS1 遺伝子およびAS遺伝子と一致した場合、ホモログ候補の遺伝子をペチュニアにおけるGS1遺伝子およびAS遺伝子のホモログと決定した。 Next, using the nucleotide sequence information of this homolog candidate, a homology search was performed on the amino acid sequence group of A. thaliana using the xblastn program. If the detected A. thaliana amino acid sequence matched the A. thaliana GS1 gene and AS gene used in the initial homology search, the candidate homolog gene was determined to be a homolog of the GS1 gene and AS gene in petunia.
その結果、ペチュニアのScf00947g08014.1、Scf0003g01011.1、Scf1671g03035.1、Scf00576g06002.1、Scf05252g00002.1およびScf01633g06046.1がいずれもA. thalianaのGS1 遺伝子がコードするアミノ酸配列に対してアミノ酸レベルで約60％以上の相同性を示し、これらをペチュニアのGS1遺伝子ホモログとした。 As a result, Scf00947g08014.1, Scf0003g01011.1, Scf1671g03035.1, Scf00576g06002.1, Scf05252g00002.1 and Scf01633g06046.1 of petunia are all about 6 amino acids at the amino acid level with respect to the amino acid sequence encoded by the GS1 gene of A. thaliana. 0 % or more homology, and these were designated as petunia GS1 gene homologs.
また、ペチュニアのScf01313g02018.1、Scf00230g00014.1、およびScf13451180g00004.1がいずれもA. thalianaのAS遺伝子がコードするアミノ酸配列に対してアミノ酸レベルで約60％以上の相同性を示し、これらをペチュニアのAS遺伝子ホモログとした。 In addition, Scf01313g02018.1, Scf00230g00014.1, and Scf13451180g00004.1 of petunia all show approximately 60% or more homology at the amino acid level to the amino acid sequence encoded by the AS gene of A. thaliana. It was designated as an AS gene homolog.
qRT-PCRにより転写産物量を調査するため、上記の各GS1遺伝子およびAS遺伝子のホモログを標的としたプライマーをPrimer3Plus（http://primer3plus.com/cgi-bin/dev/primer3plus.cgi）、NetPrimer（PREMIER Biosoft International software ; http://www.PremierBiosoft.com）およびPrimer-BLAST（Sol Genomics ; https://solgenomics.net/tools/blast/）を用いて設計・評価した。なお、Primer3PlusのパラメータはProduct Size Ranges ; 80-150 bp, Primer Size ; 17-25 b, Primer Tm ; 60-65℃とし、候補として得られたプライマーペアは、NetPrimerおよびPrimer-BLASTによる評価に基づき、ヘアピン、ダイマー、クロスダイマーが形成されにくいものを選択した。 To investigate the amount of transcripts by qRT-PCR, primers targeting the homologs of each GS1 gene and AS gene mentioned above were used with Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi), NetPrimer (PREMIER Biosoft International software; http://www.PremierBiosoft.com) and Primer-BLAST (Sol Genomics; https://solgenomics.net/tools/blast/). The parameters of Primer3Plus are Product Size Ranges; 80-150 bp, Primer Size; 17-25 b, Primer Tm; 60-65℃, and the primer pairs obtained as candidates are based on the evaluation by NetPrimer and Primer-BLAST. , hairpins, dimers, and cross-dimers were selected.
その結果、上記の6種のGS1遺伝子ホモログ及び3種のAS遺伝子ホモログについて、cDNA配列を標的としたqRT-PCRに用いるプライマーを設計することができた（表２）。 As a result, we were able to design primers for use in qRT-PCR targeting cDNA sequences for the six types of GS1 gene homologs and three types of AS gene homologs mentioned above (Table 2).
表２に示すプライマーの特異性の確認のために、KAPA SYBR FAST qPCR Kits（Kapa Biosystems）、Eco Real-Time PCR System（illumina）を用いて、qRT-PCRを行った。5μLのKAPA SYBR FAST Universal 2X qPCR Master Mix、0.2 μLの10μMフォワードプライマー、0.2μLの10μM リバースプライマー、3.6μLのRNase-Free水、1μLのcDNA溶液（10 ng/μL）を混和した反応液（10μL）を0.2 mL容量のPCRチューブに調製した。qRT-PCRの条件は、95℃、5分間の初期変性処理後、｛95℃、10秒間 / 60℃、30秒間｝を40サイクル行い、最後に60℃から95℃への融解曲線分析を加え、4℃で保持し、標的配列の特異的な増幅の有無を調査した。その結果、この予備実験において、標的の配列が特異的に増幅されることを確認した（データは示さない）。 To confirm the specificity of the primers shown in Table 2, qRT-PCR was performed using KAPA SYBR FAST qPCR Kits (Kapa Biosystems) and Eco Real-Time PCR System (illumina). Reaction mixture (10 μL) containing 5 μL of KAPA SYBR FAST Universal 2X qPCR Master Mix, 0.2 μL of 10 μM forward primer, 0.2 μL of 10 μM reverse primer, 3.6 μL of RNase-Free water, and 1 μL of cDNA solution (10 ng/μL). ) was prepared in a 0.2 mL PCR tube. The qRT-PCR conditions were as follows: initial denaturation at 95℃ for 5 minutes, followed by 40 cycles of {95℃, 10 seconds / 60℃, 30 seconds}, and finally melting curve analysis from 60℃ to 95℃. , and the presence or absence of specific amplification of the target sequence was investigated. As a result, it was confirmed that the target sequence was specifically amplified in this preliminary experiment (data not shown).
７－２．花弁からのRNA抽出
未受粉区、受粉区およびエチレン処理区の鉢花を、開花後1日の時点で植物体から採取した。採取した花の雌蕊より上部の花弁から100 mgを切り取り、6 mmステンレスビーズを入れた2.0 mL 容量のマイクロチューブに入れ、30秒間液体窒素で凍結させ、Tissue Lyser LTにより50 Hz、1分間で破砕する作業を2回行った。これに1 mLのRNAiso plus（Takara）を加えて30秒間攪拌し、室温にて5分間静置後、使用時まで-80℃の冷凍庫で保存した。
7-2. RNA Extraction from Petals Potted flowers from unpollinated, pollinated, and ethylene-treated plants were collected from plants one day after flowering. Cut 100 mg from the petal above the pistil of the collected flower, place it in a 2.0 mL microtube containing 6 mm stainless steel beads, freeze it in liquid nitrogen for 30 seconds, and crush it with Tissue Lyser LT at 50 Hz for 1 minute. I did this twice. 1 mL of RNAiso plus (Takara) was added to this, stirred for 30 seconds, left to stand at room temperature for 5 minutes, and then stored in a -80°C freezer until use.
保存した各サンプルを氷上で融解し、4℃、12,000×g、10分間の遠心分離を行い、上清を新しい2.0 mL容量のマイクロチューブに移した。その後、200μLのクロロホルムを加え、転倒混和し、室温にて5分間静置した。次に、4℃、12,000×g、15分間の遠心分離を行い、上清500μLを1.5 mL容量のマイクロチューブに移した。この上清に500μLのイソプロパノールを加えて混和し、室温で10分間静置した。4℃、13,000×g、15分間の遠心分離後、上清を除き、沈殿に冷75％エタノールを1 mL加え転倒混和した。更に4℃、7,500×g、5分間で遠心分離を行った後、上清を除き、沈殿を卓上型微量用遠心濃縮機で10分間乾燥させた。 Each stored sample was thawed on ice, centrifuged at 12,000 x g for 10 minutes at 4°C, and the supernatant was transferred to a new 2.0 mL microtube. Then, 200 μL of chloroform was added, mixed by inversion, and left at room temperature for 5 minutes. Next, centrifugation was performed at 4° C. and 12,000×g for 15 minutes, and 500 μL of the supernatant was transferred to a 1.5 mL microtube. 500 μL of isopropanol was added to this supernatant, mixed, and allowed to stand at room temperature for 10 minutes. After centrifugation at 13,000×g at 4° C. for 15 minutes, the supernatant was removed, and 1 mL of cold 75% ethanol was added to the precipitate and mixed by inversion. After further centrifugation at 4° C. and 7,500×g for 5 minutes, the supernatant was removed and the precipitate was dried for 10 minutes using a tabletop micro centrifugal concentrator.
これに22μLの10 mM Tris-HCl Buffer（pH 8.0）を加え、RNAを溶解させた。抽出したRNAの濃度をQubit 2.0 Fluorometer（Life technologies）で測定し、RNA溶液の濃度を10 mM Tris-HCl Buffer（pH 8.0）を用いて100 ng/μLに調製した。 22 μL of 10 mM Tris-HCl Buffer (pH 8.0) was added to this to dissolve the RNA. The concentration of the extracted RNA was measured using a Qubit 2.0 Fluorometer (Life technologies), and the concentration of the RNA solution was adjusted to 100 ng/μL using 10 mM Tris-HCl Buffer (pH 8.0).
得られたRNAから、PrimeScript RT reagent Kit with gDNA Eraser（TAKARA）およびApplied Biosystems 2720 Thermal Cycler（Life technologies）を用いてcDNAを合成した。具体的には、(1)ゲノムDNA除去反応のため、1μLの5×gDNA Eraser Buffer、0.5μLのgDNA Eraser、1μLのRNase-Free水、調整した2.5μLのRNA溶液（100 ng/μL）を混和した反応液（5μL）を0.2 mL容量のPCRチューブに調製し、穏やかに混和し、室温で5分間インキュベートして4℃で保持した。続いて、(2)逆転写反応のため、(1)の反応液5μLと、2μLの5×gDNA Eraser Buffer 2（for Real Time）、0.5μLの PrimeScript RT Enzyme Mix 1、0.5μLのRT Primer Mix、2μLのRNase-Free水を混和した反応液（10μL）を0.2 mL容量のPCRチューブに調製し、穏やかに混和した。続いて、Applied Biosystems 2720 Thermal Cyclerを用いて37℃、15分間 / 85℃、5秒間 / 4℃保持条件でインキュベートした。その後、40μLのTE Bufferを加え、cDNA濃度を10 ng/μLに調製した。調整したcDNA溶液は使用時まで-80℃で保存した。 cDNA was synthesized from the obtained RNA using PrimeScript RT reagent Kit with gDNA Eraser (TAKARA) and Applied Biosystems 2720 Thermal Cycler (Life technologies). Specifically, (1) for the genomic DNA removal reaction, add 1 μL of 5× gDNA Eraser Buffer, 0.5 μL of gDNA Eraser, 1 μL of RNase-Free water, and 2.5 μL of the prepared RNA solution (100 ng/μL). The mixed reaction solution (5 μL) was prepared in a 0.2 mL PCR tube, mixed gently, incubated at room temperature for 5 minutes, and kept at 4°C. Next, for (2) reverse transcription reaction, add 5 μL of the reaction solution from (1), 2 μL of 5×gDNA Eraser Buffer 2 (for Real Time), 0.5 μL of PrimeScript RT Enzyme Mix 1, and 0.5 μL of RT Primer Mix. A reaction solution (10 μL) mixed with 2 μL of RNase-free water was prepared in a 0.2 mL PCR tube and mixed gently. Subsequently, it was incubated at 37°C for 15 minutes/85°C for 5 seconds/4°C holding conditions using an Applied Biosystems 2720 Thermal Cycler. Then, 40 μL of TE Buffer was added to adjust the cDNA concentration to 10 ng/μL. The prepared cDNA solution was stored at -80°C until use.
７－３．PCR
上記表２に示すプライマーを合成により取得し、下記の条件でPCRを実施し、転写産物量を算出した。
先ず、各ホモログについて既知濃度のcDNA溶液を調製するために、cDNA合成で調製した全てのステージのcDNA溶液を混合したものをテンプレートとして使用し、KAPA Taq Ready Mix PCR KitおよびApplied Biosystems 2720 Thermal Cyclerを用いてPCRを実施した。各サンプルに25μLの2x ReadyMix with Mg2＋、2μLのフォワードプライマー（10 μM）、2μLのリバースプライマー（10μM）、2μLの鋳型DNA（10 ng/μL）、19μLのRNase-Free水を混和した反応液（50μL）を0.2 mL容量のPCRチューブに調製した。PCR条件は、95℃、2分間の初期変性処理後、｛95℃、30秒間 / 55℃、30秒間 / 72℃、30秒間｝を40サイクル行った後、72℃で30秒間処理し、4℃で保持した。
7-3. PCR
The primers shown in Table 2 above were obtained by synthesis, PCR was performed under the following conditions, and the amount of transcript was calculated.
First, in order to prepare cDNA solutions of known concentrations for each homolog, a mixture of cDNA solutions of all stages prepared by cDNA synthesis was used as a template, and the KAPA Taq Ready Mix PCR Kit and Applied Biosystems 2720 Thermal Cycler were used. PCR was performed using For each sample, mix 25 μL of 2x ReadyMix with Mg 2+ , 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM), 2 μL of template DNA (10 ng/μL), and 19 μL of RNase-free water. (50 μL) was prepared in a 0.2 mL PCR tube. PCR conditions were as follows: initial denaturation at 95℃ for 2 minutes, followed by 40 cycles of {95℃, 30 seconds / 55℃, 30 seconds / 72℃, 30 seconds}, followed by treatment at 72℃ for 30 seconds, It was kept at ℃.
次いで、QIAquick Gel Extraction Kit（QIAGEN）を用いて、PCR産物の精製を行った。全ての遠心分離操作は、室温、17,900×gで行った。上記で増幅した50μLのPCR反応液を1％アガロースゲルで電気泳動した後、UV Transilluminator 2000（BioRad）上でカミソリを用いて目的断片を切り出し、2.0 mL容量のマイクロチューブに入れて重量を測定した。以降はQIAquick Gel Extraction Kitのマニュアルに従ってPCR産物を精製し、30μLのBuffer EBで溶出し、NanoDrop Lite（ThremoScientific）で濃度を測定して、既知濃度のcDNA溶液を調製した。 Next, the PCR product was purified using QIAquick Gel Extraction Kit (QIAGEN). All centrifugation operations were performed at room temperature and 17,900×g. After electrophoresing 50 μL of the PCR reaction solution amplified above on a 1% agarose gel, the target fragment was cut out using a razor on a UV Transilluminator 2000 (BioRad), placed in a 2.0 mL microtube, and its weight was measured. . Thereafter, the PCR product was purified according to the QIAquick Gel Extraction Kit manual, eluted with 30 μL of Buffer EB, and the concentration was measured with NanoDrop Lite (ThremoScientific) to prepare a cDNA solution with a known concentration.
KAPA SYBR FAST qPCR KitsおよびEco Real-Time PCR Systemを用いて、調製した全てのサンプルのcDNA溶液を用いてqRT-PCRを行った。GS1遺伝子ホモログおよびAS遺伝子ホモログの転写産物量は、予め調製した既知濃度のcDNA溶液を用いて検量線を作製し、各サンプルの全RNA 10 ngに含まれるmRNAのコピー数を求め、Chapin and Jones（2009）に準じ、内部標準遺伝子アクチン（遺伝子識別番号：CV299322）のmRNAのコピー数に対する数値を求め、相対転写産物量とした。 qRT-PCR was performed using the cDNA solutions of all prepared samples using KAPA SYBR FAST qPCR Kits and Eco Real-Time PCR System. To determine the amount of transcripts of the GS1 gene homolog and AS gene homolog, prepare a standard curve using a cDNA solution of known concentration prepared in advance, calculate the copy number of mRNA contained in 10 ng of total RNA of each sample, and calculate the number of copies of mRNA contained in 10 ng of total RNA of each sample. (2009), the numerical value for the mRNA copy number of the internal standard gene actin (gene identification number: CV299322) was determined and used as the relative transcript amount.
７－４．結果
GS1遺伝子のペチュニアホモログ（Scf00947g08014.1、Scf0003g01011.1、Scf1671g03035.1、Scf00576g06002.1、Scf05252g00002.1およびScf01633g06046.1）について、未受粉区、受粉区およびエチレン処理区のそれぞれの鉢花花弁における6種のGS1遺伝子ホモログの総転写産物量を図７Ａに示す。開花後1日（開花）におけるGS1遺伝子ホモログの総転写産物量は、エチレン処理区の花弁において最も高かった。
7-4. result
The petunia homologues of the GS1 gene (Scf00947g08014.1, Scf0003g01011.1, Scf1671g03035.1, Scf00576g06002.1, Scf05252g00002.1 and Scf01633g06046.1) were analyzed in 6 samples of unpollinated, pollinated and ethylene-treated potted flowers. The total transcript amount of the GS1 gene homologues of the species is shown in Figure 7 A. The total transcript amount of the GS1 gene homologues 1 day after anthesis (anthesis) was highest in the petals of the ethylene-treated group.
AS遺伝子のペチュニアホモログ（Scf01313g02018.1、Scf00230g00014.1、 およびScf13451180g00004.1）について、未受粉区、受粉区およびエチレン処理区のそれぞれの鉢花花弁における3種のAS1遺伝子ホモログの総転写産物量を図７Ｂに示す。開花後1日（開花）におけるAS遺伝子ホモログの総転写産物量は、エチレン処理区の花弁において最も高かった。 Regarding the petunia homologs of the AS gene (Scf01313g02018.1, Scf00230g00014.1, and Scf13451180g00004.1), the total transcript amount of the three AS1 gene homologs in the petals of potted flowers in unpollinated, pollinated, and ethylene-treated areas was calculated. Shown in FIG. 7B. The total transcript amount of AS gene homologs one day after flowering (flowering) was highest in petals of ethylene-treated plants.
［実施例８ アサガオにおけるエチレン処理の効果］
アサガオ品種「ムラサキ」の種子をプラスチックシャーレに置き、キムワイプ（日本製紙クレシア株式会社）をかぶせた上から種子が十分に浸るように蒸留水を加えた状態で、室温で3時間程度吸水させた。種子のへそと反対側の種皮をかみそりで少し削り、へそが上向きになるよう、培養土（スーパーミックスA；サカタのタネ）を入れた4号のポリポットに播種し、恒明条件、24℃に設定した恒温室内の白色蛍光灯下（光量子束密度100μmol m-2 s-1）で、約1か月間、一定の大きさになるまで生長させた。
[Example 8 Effect of ethylene treatment on morning glory]
Seeds of the morning glory cultivar ``Murasaki'' were placed in a plastic petri dish, covered with Kimwipe (Nippon Paper Crecia Co., Ltd.), and distilled water was added to the top so that the seeds were fully immersed, and allowed to absorb water at room temperature for about 3 hours. Slightly scrape the seed coat on the opposite side of the seed with a razor, sow the seeds in a No. 4 polypot filled with culture soil (Super Mix A; Sakata Seed) so that the navel faces upward, and set at 24℃ under constant light conditions. The seeds were grown under white fluorescent lights (photon flux density 100 μmol m -2 s -1 ) in a thermostatic chamber for about one month until they reached a certain size.
その後、12時間日長（明期 9:00～21:00）、24℃に設定した恒温室内の白色蛍光灯下（光量子束密度100μmol m-2 s-1）に移し、花芽形成を誘導させた。育成中は、水で希釈した液体肥料（花工場；住友化学園芸）を週に1回与え、適宜、農薬の散布を行った。種子生産量の計測の実験に用いた植物個体は播種時からグロースチャンバー（MLR-352H；Panasonic）内で育成し、グロースチャンバー内の位置による照度や温度などのわずかな違いを考慮し、植物個体を置く位置は随時入れ替えた。 Thereafter, the plants were placed under white fluorescent lights (photon flux density 100 μmol m -2 s -1 ) in a thermostatic chamber set at 24°C with a 12-hour photoperiod (light period 9:00-21:00) to induce flower bud formation. Ta. During growth, liquid fertilizer (Flower Factory; Sumitomo Chemical Gardening) diluted with water was applied once a week, and pesticides were sprayed as appropriate. The individual plants used in the experiment to measure seed production were grown in a growth chamber (MLR-352H; Panasonic) from the time of sowing. The position of the was changed from time to time.
開花12時間前に採取した花蕾を、蒸留水（対照区）または0.2 mM STS溶液を入れた2.0 mLマイクロチューブに挿し、24℃、相対湿度70％、12時間日長に設定したグロースチャンバー内で開花させた。開花後0時間にエテホンまたは蒸留水を噴霧してから、グロースチャンバー内に静置し、固定したデジタルカメラで上方から10分おきに、切り花の花弁老化の様子をインターバル撮影した。各時刻の画像内の花冠領域のピクセル値を画像解析ソフトウェア「Flower Shape Analysis System Version 1.5（Tanabata、http://www.kazusa.or.jp/picasos/）」を用いて計測し、花冠面積を評価した。最大のピクセル値を示した花冠面積を100％として相対的な花冠面積が90％を下回り始める時刻を花弁の老化の開始とし、開花から花弁老化の開始までの時間を開花後時間として計測した。 Flower buds collected 12 hours before flowering were placed in a 2.0 mL microtube containing distilled water (control) or 0.2 mM STS solution, and placed in a growth chamber set at 24°C, relative humidity 70%, and a 12-hour photoperiod. Made it bloom. After spraying ethephon or distilled water 0 hours after flowering, the cut flowers were placed in a growth chamber, and the state of petal aging of the cut flowers was photographed from above every 10 minutes using a fixed digital camera. The pixel values of the corolla area in the image at each time were measured using the image analysis software "Flower Shape Analysis System Version 1.5 (Tanabata, http://www.kazusa.or.jp/picasos/)" to calculate the corolla area. evaluated. The corolla area that showed the maximum pixel value was taken as 100%, and the time when the relative corolla area began to fall below 90% was defined as the start of petal senescence, and the time from flowering to the start of petal senescence was measured as post-anthesis time.
その結果、図８に示すように、蒸留水に挿した切り花では、エテホン噴霧により、開花から花弁老化の開始までの時間が蒸留水噴霧区よりも有意に短くなった。一方、エチレン生成阻害剤であるSTS溶液に挿した切り花へのエテホン噴霧区では、開花から花弁老化の開始までの時間が、蒸留水に挿した切り花への蒸留水噴霧区およびSTS溶液に挿した切り花への蒸留水噴霧区と有意な差異はみられなかった。 As a result, as shown in FIG. 8, for cut flowers placed in distilled water, spraying with ethephon significantly shortened the time from flowering to the start of petal senescence than in the group sprayed with distilled water. On the other hand, in the group where ethephon was sprayed on cut flowers placed in STS solution, which is an ethylene production inhibitor, the time from flowering to the start of petal senescence was significantly longer than that in the group where cut flowers were sprayed with distilled water and placed in STS solution. No significant difference was observed between the cut flowers and the distilled water spraying group.
［実施例９ アサガオにおけるエチレン処理の効果］
実施例８と同様にして生長させたアサガオ品種「ムラサキ」について、開花した花を開花後0時間にポリプロピレン素材のフィルムで隔離し、花以外にかからないように100 ppmエテホン液剤（日産エスレル10；日産化学株式会社）を花弁の向軸面に向けて花から5 cm程度距離を取ってスプレーで1回噴霧した。対照区では、同様のやり方で蒸留水を噴霧した。エテホンまたは蒸留水の噴霧は、第1花または第6花の開花日から2週後までに咲いた全ての花に対して行った。
[Example 9 Effect of ethylene treatment on morning glory]
Regarding the morning glory cultivar "Murasaki" grown in the same manner as in Example 8, the bloomed flowers were isolated with a polypropylene film at 0 hours after flowering, and a 100 ppm ethephon solution (Nissan Esler 10; Nissan Kagaku Co., Ltd.) was sprayed once toward the adaxial surface of the petal at a distance of about 5 cm from the flower. In the control plot, distilled water was sprayed in the same manner. Spraying of ethephon or distilled water was performed on all flowers that bloomed up to 2 weeks after the first or sixth flower bloom date.
本実施例では、対照区として蒸留水を第1花から噴霧する個体を5個体、エテホンを第1花から噴霧する個体（エテホン処理区１）を5個体、第1花から第5花まで蒸留水を噴霧し、第6花からエテホンを噴霧する個体（エテホン処理区２）を4個体用意した。それぞれの処理個体で、第1花の開花日から80日後まで、果皮に包まれていた種子数を記録し、1個体で合計の種子数を出して、処理群ごとに果実あたりおよび個体あたりの平均種子数を求めた。 In this example, five control plants were sprayed with distilled water from the first flower, five plants were sprayed with ethephon from the first flower (ethephon treatment 1), and four plants were sprayed with distilled water from the first to fifth flowers and then with ethephon from the sixth flower (ethephon treatment 2). For each treatment plant, the number of seeds contained within the skin was recorded from the day the first flower bloomed until 80 days later, and the total number of seeds per plant was calculated to determine the average number of seeds per fruit and per plant for each treatment group.
第1花の開花日から80日後までに採取した成熟種子は、封筒に入れ、シリカゲルを入れたジップロック内で4℃で保存し、1週間後、1粒ずつ重さを測定し、個体ごとの平均1粒重を求め、1粒ずつの重さを合計し、果実あたりの平均種子重量を求めた。そして、個体あたりの平均種子数と平均1粒重の積を求め、その値を個体あたりの種子生産量とした。
表３に、果実あたりの平均種子数および平均種子重量、並びに個体あたりの平均種子数、平均1粒重および種子生産量の計測結果を示す。
Mature seeds collected up to 80 days after the first flowering date are placed in an envelope and stored at 4℃ in a Ziploc bag containing silica gel.After one week, each seed is weighed and each individual seed is The average seed weight per fruit was determined, and the weights of each individual seed were totaled to determine the average seed weight per fruit. Then, the product of the average number of seeds per individual and the average grain weight was calculated, and this value was taken as the seed production amount per individual.
Table 3 shows the measurement results of the average number of seeds and average seed weight per fruit, as well as the average number of seeds per individual, average grain weight, and seed production.
表３に示す通り、果実あたりの平均種子数は、対照区と比較してエテホン処理区１で有意ではないが増加傾向を示し、エテホン処理区２で有意に増加していた。一方、個体あたりの平均種子数は、対照区と比較してエテホン処理区で減少する傾向があったが、これは、エテホン処理により、開花前の花（蕾）や開花しても結実に到らない花の脱離が観察されたことと一致する。 As shown in Table 3, the average number of seeds per fruit showed a non-significantly increasing trend in ethephon treatment 1 compared to the control, and was significantly increased in ethephon treatment 2. On the other hand, the average number of seeds per individual tended to decrease in the ethephon treatment compared to the control, which is consistent with the observation that ethephon treatment caused the abscission of flowers (buds) before flowering and flowers that flowered but did not produce fruit.
また、個体あたりの成熟種子の平均1粒重は、エテホン処理区で対照区より増加する傾向が観察された。これより、個体あたりの平均種子生産量は、エテホン処理区で、対照区に比べ有意ではないが低くなった。一方、果実あたりの平均種子重量は、エテホン処理区で対照区より有意に増加した。 In addition, the average weight of mature seeds per individual tended to be higher in the ethephon-treated area than in the control area. As a result, the average seed production per individual was lower in the ethephon-treated area than in the control area, although this was not significant. On the other hand, the average seed weight per fruit was significantly higher in the ethephon-treated area than in the control area.
本発明により、消費者に販売する種子や個体の花持ち性は高く維持したまま、採種用の個体でのみ、花弁老化を促進することで、種子生産性を高めることができる。 According to the present invention, seed productivity can be increased by promoting petal senescence only in individuals for seed collection, while maintaining high flower longevity in seeds and individuals sold to consumers.
本発明の方法によって達成される個体レベルでの種子生産性の向上は、品種あたりの栽培面積が縮小され、同じ場所で同時により多くの品種を栽培して採種することが可能になるため、温室等の採種施設の利用効率の向上につながる。一方、果実レベルでの種子生産性の向上は、アサガオのように1果実に形成される種子数が少ない植物の場合、より少ない果実の収穫により十分な種子を得ることが可能になるため、収穫作業時間の短縮による採種効率の改善につながり得る。 The improvement in seed productivity at the individual level achieved by the method of the present invention is due to the fact that the cultivation area per variety is reduced and it is possible to grow and collect more varieties at the same time in the same location. This will lead to improved utilization efficiency of seed collection facilities such as On the other hand, improvement in seed productivity at the fruit level means that for plants like morning glory that form a small number of seeds per fruit, it is possible to obtain sufficient seeds by harvesting fewer fruits, so This can lead to improved seed collection efficiency by reducing work time.
本発明は、品種改良により花持ち性を向上させた花卉品種における種子生産性の低下を改善することでき、また、通常の花持ち性の花卉品種における種子生産性の向上にも応用することが可能と判断される。 The present invention can improve the decline in seed productivity in flower varieties whose flower longevity has been improved through breeding, and can also be applied to improve seed productivity in flower varieties with normal flower longevity. It is considered possible.
また、本発明は、花持ち性の品種改良にも応用が可能である。例えば、花持ち性が非常に良くても、種子生産性が極端に悪いことで、品種として選抜されてこなかった育成系統であっても、本発明によって種子生産性の向上を図りながら品種改良を進めることで、より花持ち性の良い品種の開発が可能になることが期待される。 The present invention can also be applied to the improvement of varieties with better flower longevity. For example, even if a breeding line has very good flower longevity but extremely poor seed productivity, it is expected that it will be possible to develop varieties with better flower longevity by improving seed productivity while improving the line using the present invention.
更に、本発明の方法を利用して、将来的には例えば品種ごとに展開花弁や肥大果実を自動認識する機能を備えた超小型ドローンを用い、エチレン発生剤を必要に応じて自動散布することも可能となる。このような技術により、品種ごとの種子の生産調整をより効率的に行うことができる。 Furthermore, using the method of the present invention, in the future, for example, it is possible to automatically spray ethylene generators as needed using an ultra-compact drone equipped with a function to automatically recognize unfolded petals and swollen fruits for each variety. is also possible. With such technology, it is possible to more efficiently adjust the production of seeds for each variety.
Claims (6)
開花した花の花弁に対して10～1000 ppmの範囲の濃度のエチレン発生剤を適用することにより花弁老化を促進し、そして
種子を取得する
ことを含み、種子生産性は、花若しくは果実当たりの種子数、花卉個体当たりの種子生産量、及び／又は果実当たりの平均種子重量であり、花卉がペチュニア又はアサガオである、上記方法。 A method for improving seed productivity in floriculture, comprising:
Seed productivity is determined by applying an ethylene generator at a concentration ranging from 10 to 1000 ppm to petals of flowering flowers to accelerate petal senescence and obtaining seeds. The above method, wherein the number of seeds, the amount of seed produced per individual flower plant , and/or the average seed weight per fruit , and the flower plant is a petunia or a morning glory .
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