JP4868812B2 - Cutting method of cuttings - Google Patents
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- JP4868812B2 JP4868812B2 JP2005288358A JP2005288358A JP4868812B2 JP 4868812 B2 JP4868812 B2 JP 4868812B2 JP 2005288358 A JP2005288358 A JP 2005288358A JP 2005288358 A JP2005288358 A JP 2005288358A JP 4868812 B2 JP4868812 B2 JP 4868812B2
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本発明は、挿し穂を培養して植物個体を得るための方法、特に、挿し穂からの発根率を向上させることのできる挿し穂の培養方法であって、発根が難しい植物を発根させるのに適した方法に関する。 The present invention relates to a method for cultivating cuttings to obtain a plant individual, in particular, a method for cultivating cuttings that can improve the rooting rate from cuttings, and rooting plants that are difficult to root. It relates to a suitable method.
植物を産業的に利用する場合において、目的に適った形質を持つ均質な苗を大量に増殖するステップは必ず要求される。これは、育種を行うにしても、植林を行うにしても変わらない。このとき苗の大量増殖手段として有用なのが、伝統的な挿し木法や、近年のバイオテクノロジーの発達により生まれた組織培養法である。これらの方法によれば、単に、苗の大量増殖ができるばかりではなく、同一の遺伝的性質を有する植物個体、即ちクローン苗を大量かつ迅速に増殖することができる。 When a plant is used industrially, a step for multiplying a large amount of a homogeneous seedling having a trait suitable for the purpose is always required. This is the same whether breeding or planting. At this time, the traditional cutting method and the tissue culture method born by the development of biotechnology in recent years are useful as a means for mass propagation of seedlings. According to these methods, not only can a large amount of seedling be grown, but also a plant individual having the same genetic properties, that is, a cloned seedling, can be rapidly and rapidly grown.
挿し木法においては、増殖しようとする植物個体から枝や、場合によっては芽、葉等を切取って挿し穂とし、これを適当な培養土に挿し付けて発根させ、苗を生産する。一方、組織培養法において木本植物を大量増殖しようとする場合には、多芽体や苗条原基を経由することが多い。具体的には、増殖しようとする植物個体から芽や茎頂点等を切取って培養し、多芽体や苗条原基を発生させた後、これらから伸長してくる不定芽を切取り、この不定芽を挿し穂として適当な支持体に挿し付けて発根させる。つまり、いずれの方法を用いてクローン苗を生産するにしても、最終的には挿し穂からの発根という過程を経ることが多い。 In the cutting method, branches, or in some cases, buds, leaves, etc., are cut out from plant individuals to be proliferated to form cuttings, which are inserted into an appropriate culture soil and rooted to produce seedlings. On the other hand, when a large amount of woody plants are to be proliferated in the tissue culture method, they often go through multi-buds or shoot primordia. Specifically, buds and stem vertices are cut from the individual plant to be grown, cultured, and after generating shoots and shoot primordia, adventitious buds extending from these are cut, and this indefinite. The buds are inserted as roots and inserted into an appropriate support to cause rooting. That is, regardless of which method is used to produce a clonal seedling, in the end, it often goes through a process of rooting from the cutting head.
しかし、挿し穂の発根性は、植物の種類により大きく異なっている。一般に、木本植物は、草本植物よりも発根性が劣っている。また、同じ木本植物においても、例えば、ヤナギやヒノキ等は挿し穂からの発根が容易であるが、ユーカリやマツでは挿し穂からの発根が極めて難しい。 However, the rooting of cuttings varies greatly depending on the type of plant. In general, woody plants are less rooted than herbaceous plants. In the same woody plant, for example, willow and cypress are easy to root from cuttings, but eucalyptus and pine are extremely difficult to root from cuttings.
このような難発根性植物の発根性を改良する方法は、そのアプローチを、外的要因に着目するものと内的要因に着目するものとに、大きく分けることができる。この場合において、外的要因に着目したアプローチでは、挿し穂を培養する際の、温度、湿度、酸素、炭酸ガス濃度、光条件、培地等の環境条件を検討することで、発根に最も適する環境条件を見出し、これを実現しようとする。 Such methods for improving the rooting ability of difficult-to-root plants can be broadly divided into approaches that focus on external factors and those that focus on internal factors. In this case, the approach that focuses on external factors is most suitable for rooting by examining environmental conditions such as temperature, humidity, oxygen, carbon dioxide concentration, light conditions, and culture medium when cultivating cuttings. Find environmental conditions and try to achieve this.
一方、内的要因に着目したアプローチでは、挿し穂自体として高い発根能を有するものを得るべく検討を行う。このような挿し穂を得る方法として、例えば、暗黒状態で挿し穂を萌芽させる(黄化処理)、挿し穂を種々のオーキシンで処理する、挿し穂の葉からの蒸散を抑制する、樹齢の若い親木からの挿し穂を用いる、親木をB−ナインやパクロブトラゾールなどのジベレリン生合成阻害剤(特許文献1)で処理する、挿し穂に負電圧を印加する(特許文献2)等の方法が、これまでに報告されている。 On the other hand, in the approach focusing on the internal factors, the cutting head itself is examined to obtain a high rooting ability. As methods for obtaining such cuttings, for example, the cuttings are germinated in a dark state (yellowing treatment), the cuttings are treated with various auxins, the transpiration from the cuttings of the cuttings is suppressed, and the young are young Using cuttings from the parent tree, treating the parent tree with a gibberellin biosynthesis inhibitor such as B-nine or paclobutrazol (Patent Document 1), applying a negative voltage to the cuttings (Patent Document 2), etc. This method has been reported so far.
難発根性植物の発根性を向上させるには、挿し穂の外的要因のみならず、内的要因をも発根に最も適した状態とする必要がある。しかしながら、挿し穂の内的要因に着目した発根性の向上については、まだ十分に検討がなされているとは言えない。 In order to improve the rooting ability of difficult-to-root plants, not only external factors of cuttings but also internal factors need to be in a state most suitable for rooting. However, it cannot be said that sufficient studies have been made on the improvement of rooting, focusing on the internal factors of cuttings.
本発明は、かかる問題点を踏まえ、挿し穂の内的要因に着目し、その発根性を向上させるべくなされたものである。 In consideration of such problems, the present invention has been made in order to improve the rooting property by focusing on the internal factors of cuttings.
本発明者らは鋭意研究の結果、挿し穂を超音波処理すると、挿し穂の内的要因にこの超音波処理が影響を及ぼして、不定根の形成が促進されることを見出し、本発明を完成させた。 As a result of diligent research, the present inventors have found that, when the cuttings are sonicated, this sonication affects the internal factors of the cuttings, and the formation of adventitious roots is promoted, completing the present invention. I let you.
即ち、本発明は、培地及び/又は培養土を用いて挿し穂を培養するにあたり、挿し穂の培地及び/又は培養土と接する部分の少なくとも一部を超音波処理することにより、不定根を形成させることを特徴とする、挿し穂の培養方法に関する。 That is, the present invention forms adventitious roots by sonicating at least a part of the portion of the cutting head in contact with the medium and / or culture soil when cultivating the cutting using the medium and / or the culture soil. The present invention relates to a method for cultivating cuttings.
本発明によれば、挿し穂からの不定根形成が促進される。 According to the present invention, adventitious root formation from cuttings is promoted.
従って、本発明によれば、挿し穂からの発根率が向上し、また、このとき生じる根の本数も増加する。そして、かかる効果は、挿し穂からの発根が難しいとされている、難発根性植物においても発揮される。 Therefore, according to the present invention, the rooting rate from the cutting head is improved, and the number of roots generated at this time is also increased. And such an effect is exhibited also in a hard-to-root plant which is said to be difficult to root from cuttings.
即ち、本発明によれば、ユーカリプタス・グロブラス(Eucalyptus globulus、以下、単にE.グロブラスと略記する。)等、有用な形質を有する難発根性植物のクロ−ン苗の挿し木法又は組織培養法による大量増殖が、産業的に可能となる。 That is, according to the present invention, by the cutting method or the tissue culture method of a clone seedling of a hardly rooted plant having a useful trait such as Eucalyptus globulus (hereinafter simply abbreviated as E. globula). Mass growth is industrially possible.
本発明において挿し穂として用いることのできる植物の種類に特に制限はない。ユーカリ、マツ、アカシア、ヤマモモ、クヌギ、ブドウ、リンゴ、サクラ、バラ、ツバキ、ウメ等の木本植物の他、キクやカーネーション等の草本植物にも本発明を適用することができる。もっとも、本発明は、ユーカリやマツ等の難発根性の植物に適用した場合に、特に効果を発揮する。 There is no restriction | limiting in particular in the kind of plant which can be used as a cutting head in this invention. The present invention can be applied to herbaceous plants such as chrysanthemum and carnations as well as woody plants such as eucalyptus, pine, acacia, bayberry, kunugi, grape, apple, cherry, rose, camellia and ume. However, the present invention is particularly effective when applied to difficult-to-root plants such as eucalyptus and pine.
これらの挿し穂は、温室や屋外に生育している植物個体から得られたものでも、組織培養法により得られたものでもよい。植物個体から挿し穂を得る場合には、枝、茎、芽又は葉を切取り、これらを挿し穂として用いればよい。木本植物の場合は緑枝(当年枝)や熟枝(前年以前に伸びた枝)、草本植物の場合は芽や葉を用いるのが普通である。挿し穂として枝を用いる場合には、その枝についた葉の蒸散作用を抑制して不定根の形成をより促進させるため、葉の一部を切除することも有効である。なお、本発明において、不定根とは、枝、茎、葉など、通常は根が形成されない部分に形成される根のことをいう。 These cuttings may be obtained from individual plants growing in a greenhouse or outdoors, or may be obtained by a tissue culture method. When cuttings are obtained from a plant individual, branches, stems, buds or leaves may be cut out and used as cuttings. In the case of woody plants, green branches (current year branches) and mature branches (branches extending before the previous year) are usually used, and in the case of herbaceous plants, buds and leaves are usually used. When branches are used as cuttings, it is also effective to excise part of the leaves in order to suppress the transpiration of the leaves attached to the branches and promote the formation of adventitious roots. In the present invention, the adventitious root means a root formed in a portion where a root is not normally formed, such as a branch, a stem, or a leaf.
組織培養により挿し穂を得る場合には、多芽体や苗条原基を誘導し、これらの組織から伸長してくる不定芽を、その根元付近から切取って、これを挿し穂として用いればよい。多芽体又は苗条原基は、それぞれの植物において公知の方法を用い、誘導することができる。例えば、前記の木本植物から、多芽体を形成させて本発明で使用する挿し穂を取得するには、概ね次のようにして行う。 When obtaining cuttings by tissue culture, it is possible to induce multi-buds and shoot primordia, cut adventitious buds extending from these tissues from their roots, and use them as cuttings . The multi-bud or shoot primordia can be induced in each plant using a known method. For example, in order to obtain the cuttings used in the present invention by forming multi-buds from the above-mentioned woody plant, it is generally performed as follows.
まず、材料とする植物から頂芽、腋芽等の組織を採取し、採取した組織について、有効塩素量0.5〜4%の次亜塩素酸ナトリウム水溶液又は有効塩素量5〜15%の過酸化水素水溶液に10〜20分間浸漬して表面殺菌を行う。次いで、これを滅菌水で洗浄し、固体培地に挿し付けて芽を開じょさせ、伸長してきたシュートを同じ組成の培地で継代培養することにより、多芽体を形成させる。ユーカリ属やアカシア属の腋芽を用いる場合には、固体培地は、ショ糖1〜5重量%、植物ホルモンとしてベンジルアデニン(以下、BAと略す。)0.02〜1mg/l、ゲランガム0.2〜0.3重量%もしくは寒天0.5〜1重量%を含有するムラシゲスクーグ(以下、MSと略す。)培地又はこのMS培地の硝酸アンモニウム成分と硝酸カリウム成分とを半減させた改変MS培地を用いるのが好ましい。こうして形成された多芽体からは活発に不定芽が分化し、伸長してくるので、本発明においてはこの伸長して来た不定芽を切取り、挿し穂として使用すればよい。多芽体自体は、適当に分割して多芽体形成に用いた培地と同一組成の培地で培養することにより維持し、増殖させることができる。 First, tissues such as apical buds and axillary buds are collected from a plant as a material, and the collected tissues are subjected to sodium hypochlorite aqueous solution having an effective chlorine content of 0.5 to 4% or peroxidation having an effective chlorine content of 5 to 15%. Surface sterilization is performed by immersing in an aqueous hydrogen solution for 10 to 20 minutes. Next, this is washed with sterilized water, inserted into a solid medium to open the buds, and the elongated shoots are subcultured in a medium having the same composition to form multi-buds. When Eucalyptus or Acacia sprouts are used, the solid medium is 1 to 5% by weight of sucrose, benzyladenine (hereinafter abbreviated as BA) as a plant hormone, 0.02 to 1 mg / l, gellan gum 0.2 A Murashige scoog (hereinafter abbreviated as MS) medium containing ~ 0.3 wt% or agar 0.5-1 wt% or a modified MS medium in which the ammonium nitrate component and potassium nitrate component of this MS medium are halved is used. preferable. Since the adventitious buds are actively differentiated from the multi-buds thus formed and extend, in the present invention, the adventitious buds that have been extended may be cut out and used as cuttings. The multiblasts themselves can be maintained and proliferated by appropriately dividing them and culturing them in a medium having the same composition as the medium used for the formation of the multibuds.
本発明においては、こうして得られた挿し穂を培地や培養土で培養する。このとき用いる培地及び/又は培養土は、それぞれの植物の発根用に適したものを用いるとよい。例えば、培地としては、ムラシゲ・スクーグ(MS)培地、リンスマイヤー・スクーグ培地、ガンボーグのB5培地、ホワイトの培地、ニッチ・ニッチの培地等、植物の組織培養用培地として一般的に良く知られた基本培地又はこれを希釈したものに、必要に応じ、植物ホルモンとして1種類以上のオーキシン類、及び/又は、炭素源としてショ糖5〜30g/lを添加して用いることができる。オーキシン類も特に限定されるものではないが、インドール酪酸(IBA)やナフタレン酢酸(NAA)等が入手も容易であり使いやすい。本発明において、培地は液体培地であっても固体培地であっても構わない。固体培地として使用する場合には、上記成分に寒天又はゲランガムを更に加え、固化させて使用する。なお、炭素源として、微生物の炭素源でもあるショ糖等を添加した培地を使用する場合には、無菌環境下で挿し穂を培養する。炭素源としてショ糖等の炭水化物を用いる代わりに、炭酸ガスを培養環境中に濃度300〜1500ppm程度付与して培養することもでき、この場合には、挿し穂を無菌環境下で培養する必要がない。 In the present invention, the cuttings thus obtained are cultured in a medium or culture soil. The medium and / or culture soil used at this time may be those suitable for rooting each plant. For example, as a culture medium, a Murashige-Skoog (MS) medium, a Rinsmeier-Skoog medium, a Gamborg B5 medium, a white medium, a niche niche medium, or the like is generally well known as a tissue culture medium for plants. If necessary, one or more auxins as plant hormones and / or 5 to 30 g / l of sucrose as a carbon source can be added to the basic medium or a diluted one thereof. Auxins are not particularly limited, but indole butyric acid (IBA), naphthalene acetic acid (NAA) and the like are easily available and easy to use. In the present invention, the medium may be a liquid medium or a solid medium. When used as a solid medium, agar or gellan gum is further added to the above ingredients and solidified for use. In addition, when using the culture medium which added sucrose etc. which are also a carbon source of microorganisms as a carbon source, cuttings are cultured in a sterile environment. Instead of using carbohydrates such as sucrose as a carbon source, carbon dioxide can be cultured in a culture environment with a concentration of about 300 to 1500 ppm. In this case, it is necessary to cultivate the cuttings in a sterile environment. Absent.
培養土としては、赤土(赤玉土)、川砂、山砂、鹿沼土、バーミキュライト、パーライト、ピートモス、水ごけ等、挿し木に用いられる一般的な培養土を使用することができる。その他の発根用資材として、スミザーオアシス社製「オアシス(登録商標)」、日清紡績(株)製「フロリアライト(登録商標)」等も用いることができる。 As the culture soil, common culture soil used for cuttings such as red soil (red bean soil), river sand, mountain sand, kanuma soil, vermiculite, perlite, peat moss, and water can be used. As other rooting materials, “Oasis (registered trademark)” manufactured by Smither Oasis, “Florialite (registered trademark)” manufactured by Nisshinbo Industries, Ltd., and the like can be used.
挿し穂から不定根を形成させるにあたっては、通常、上記培地や培養土に挿し穂を挿しつけて培養する。即ち、挿し穂として枝、茎、芽又は不定芽を用いる場合には、もとの個体や組織から切出されたこれらの挿し穂を、上記固体培地、又は、水、市販の液肥もしくは上記液体培地で湿潤させた培養土に挿しつけ、挿し穂として葉を用いる場合には、もとの個体や組織から葉柄をつけて葉を切出し、この葉柄を同様の固体培地や上記培養土に挿しつけて培養すればよい。なお、培養土は、挿し穂の挿しつけ後に水等で湿潤させても構わない。 In order to form adventitious roots from cuttings, usually the cuttings are inserted into the medium or culture soil and cultured. That is, when branches, stems, buds or adventitious buds are used as cuttings, these cuttings cut from the original individual or tissue are used as the solid medium, water, commercially available liquid fertilizer or the liquid. When using leaves as culture heads moistened with culture medium and using leaves as cutting ears, leaves are cut out from the original individuals or tissues, and the leaves are cut into the same solid medium or the above culture soil. Culture. The culture soil may be moistened with water or the like after the insertion of the cutting head.
本発明においては、このようにして不定根を形成させる挿し穂の、培地及び/又は培養土と接する部分の少なくとも一部に超音波処理を行う。このとき用いる超音波の周波数は、10〜100khzの範囲であることが好ましく、特に20〜30khzの範囲であることが好ましい。また、超音波処理の時間は、30〜300秒であることが好ましく、特に60〜180秒であることが好ましい。用いる超音波の周波数が10khz未満の場合、又は、超音波処理の時間が30秒未満の場合は、不定根の形成促進に対する効果があまり期待できない。一方、超音波の周波数が100khzを超える場合、又は、超音波処理の時間が300秒を超える場合は、挿し穂を挿しつけている固体培地や培養土中の水分が発熱して挿し穂の細胞に間接的に障害を与えるおそれがある他、挿し穂の細胞が超音波処理により直接的に障害を受けるおそれも生じる。 In the present invention, ultrasonic treatment is performed on at least a part of the portion of the cutting head that forms the adventitious root in this way and in contact with the medium and / or the culture soil. The frequency of the ultrasonic wave used at this time is preferably in the range of 10 to 100 khz, and particularly preferably in the range of 20 to 30 khz. The ultrasonic treatment time is preferably 30 to 300 seconds, particularly preferably 60 to 180 seconds. When the ultrasonic frequency used is less than 10 khz, or when the ultrasonic treatment time is less than 30 seconds, the effect of promoting the formation of adventitious roots cannot be expected. On the other hand, when the ultrasonic frequency exceeds 100 khz, or when the sonication time exceeds 300 seconds, the moisture in the solid medium or the culture soil in which the cuttings are inserted generates heat and the cells of the cuttings In addition to the possibility of indirect damage, the cells in the cuttings may be directly damaged by the ultrasonic treatment.
具体的に、挿し穂の培地及び/又は培養土と接する部分の少なくとも一部に超音波処理を行うには、例えば、適当な容器内に、固体培地、又は、水、市販の液肥もしくは前記液体培地で湿潤させた培養土を用意し、これに挿し穂を挿しつけた後、水を張っておいた超音波洗浄機の超音波処理槽に、この容器を、容器内に水が入らないように置いて、超音波洗浄機を動作させればよい。このとき超音波は、超音波洗浄槽に張った水、そして培地及び/又は培養土を介して、固体培地や培養土に挿しつけられた挿し穂の組織に達することから、挿し穂の培地及び/又は培養土と接する部分の少なくとも一部に超音波処理を行うことができる。超音波洗浄機としては、出力10〜100w程度の市販の超音波洗浄機を用いることができる。もっとも、本発明においては、固体培地や培養土に挿しつける前の挿し穂に超音波処理を行っても構わない。この場合においても、挿し穂からの不定根形成は促進される。 Specifically, in order to perform ultrasonic treatment on at least a part of the cutting head medium and / or the portion in contact with the culture soil, for example, in a suitable container, solid medium, water, commercially available liquid fertilizer or the liquid Prepare the culture soil moistened with the medium, insert the ears into this soil, and then put this container in the ultrasonic treatment tank of the ultrasonic cleaning machine that is filled with water so that water does not enter the container. It is only necessary to operate the ultrasonic cleaner. At this time, the ultrasonic wave reaches the tissue of the cutting head inserted in the solid medium or the culture soil through the water stretched in the ultrasonic cleaning tank and the medium and / or the culture soil. Sonication can be performed on at least a portion of the portion in contact with the culture soil. As the ultrasonic cleaner, a commercially available ultrasonic cleaner with an output of about 10 to 100 w can be used. However, in the present invention, ultrasonic treatment may be applied to the cutting head before being inserted into a solid medium or culture soil. Even in this case, adventitious root formation from the cuttings is promoted.
本発明において、挿し穂は、その基部を固体培地、又は、水、市販の液肥もしくは前記液体培地で湿潤させた培養土に挿しつけて、培養することが好ましい。植物は、本来的に頂部と基部とを認識し、不定根は、通常、その基部から形成されるため、このようにして挿し穂を挿しつけることにより、形成された不定根はそのまま、必要な栄養素を含有する固体培地や培養土中に伸長することができるからである。 In the present invention, it is preferable to cultivate the cutting spike by inserting its base into a solid medium, or water, a commercially available liquid fertilizer, or a culture soil moistened with the liquid medium. Plants inherently recognize the top and base, and adventitious roots are usually formed from the base, so by inserting the spikes in this way, the formed adventitious roots remain as they are and the necessary nutrients are added. It is because it can extend into the contained solid medium or culture soil.
また、挿し穂が、その培地及び/又は培養土と接する部分に、切断面又は切込み部を有していれば、その挿し穂からの不定根形成は促進される。これは、挿し穂の切込みや切断により、その部位の細胞が傷つけられたことが生理的刺激となって、その近辺の部位からの不定根形成を促進させるためであると考えられる。このため、人為的に、挿し穂の培地等への挿しつけ部に、カッター等で1以上の小さな傷をつけてもよいが、通常、挿し穂は、もとの個体や組織から枝、芽、葉又は不定芽等を切出すことによって得られ、その基部には切断面を有しているので、普通は、単に挿し穂の基部を培地等に挿しつけるだけで、この効果は得ることができる。このとき、挿し穂を斜めに切出せば、細胞の傷害による刺激が大きくなると共に、その切断面と培地及び/又は培養土との接触面積も大きくなるので、不定根の形成は一層促進される。 Moreover, if the cutting spike has a cut surface or a cut portion at a portion in contact with the medium and / or culture soil, adventitious root formation from the cutting spike is promoted. This is thought to be due to the fact that the cells at the site were damaged by the cutting and cutting of the cutting head, which caused physiological stimulation and promoted adventitious root formation from the nearby site. For this reason, artificially, one or more small scratches may be made on the insertion part of the cutting head to the medium or the like with a cutter or the like. It is obtained by cutting leaves or adventitious buds, etc., and has a cut surface at the base thereof. Usually, this effect can be obtained simply by inserting the base of the cutting head into a medium or the like. it can. At this time, if the cutting head is cut obliquely, the stimulation due to cell damage increases, and the contact area between the cut surface and the medium and / or culture soil also increases, so that adventitious root formation is further promoted.
さらに、培養前の挿し穂に対し、予めオーキシン処理を行っておくことで、挿し穂からの不定根形成を、促進することもできる。オーキシン処理は、挿し穂を、濃度5〜100ppmのオーキシン溶液に浸漬したり、タルク1gあたり1〜20mgのオーキシンを混合した粉末を挿し穂の切断面に塗布することによって行えばよい。 Furthermore, adventitious root formation from cuttings can be promoted by preliminarily treating the cuttings before culturing with auxin. The auxin treatment may be carried out by immersing the cuttings in an auxin solution having a concentration of 5 to 100 ppm, or by applying a powder mixed with 1 to 20 mg of auxin per 1 g of talc and applying it to the cut surface of the cuttings.
なお、固体培地、又は、水、市販の液肥もしくは前記液体培地で湿潤させた培養土に挿しつけた後の挿し穂は、好ましくは、温度23〜28℃、650〜670nmの波長成分と450〜470nmの波長成分とを、9:1〜7:3の割合で含む光照射下で培養することが好ましい。不定根は、こうして培養している挿し穂の基部より、通常、2〜5週間で形成される。 It should be noted that the cutting head after being inserted into a solid medium, or water, commercially available liquid fertilizer or culture soil moistened with the liquid medium, preferably has a temperature of 23 to 28 ° C., a wavelength component of 650 to 670 nm and 450 to It is preferable to culture under a light irradiation containing a 470 nm wavelength component in a ratio of 9: 1 to 7: 3. Adventitious roots are usually formed in 2 to 5 weeks from the base of the cuttings thus cultured.
不定根が形成された挿し穂は、これをある程度の期間、そのまま培養を続けて根を充実させた後、発根苗として育苗容器又は苗畑等に移植して育成することにより、植林等の所定の目的に使用可能な苗とすることができる。苗を育成する際の用土や、温度・光強度等の条件は、その植物に適するように適宜設定すればよい。なお、多芽体や苗条原基等、培養組織由来の不定芽を挿し穂とした場合には、通常、育苗容器等への移植の前に、順化の過程を経る必要がある。 The cuttings in which adventitious roots are formed are continuously cultured for a certain period of time to enrich the roots, and then transplanted to a seedling container or a nursery field as rooting seedlings to grow them. It can be set as the seedling which can be used for the purpose. What is necessary is just to set suitably the conditions at the time of growing a seedling, conditions, such as temperature and light intensity, so that it may be suitable for the plant. When adventitious buds derived from cultured tissues such as multi-buds or shoot primordia are used as cuttings, it is usually necessary to undergo an acclimatization process before transplanting to a seedling container or the like.
[作用]
挿し穂を適当な培地及び/又は培養土で培養すると、培地等に接している部分から、培地等の成分が挿し穂に吸収され、不定根が形成される。このとき、挿し穂のこの部分が超音波を受けると、細胞が活性化し、細胞***も促進されて、不定根形成が促進されると考えられる。特に、難発根性植物は、発根に要する期間が長く、不定根が形成されるまでに挿し穂が枯死してしまうことが多いため、かかる超音波処理による不定根形成の促進効果は、難発根性植物において非常に有利に作用する。
[Action]
When the cuttings are cultured in a suitable medium and / or culture soil, components such as the medium are absorbed into the cuttings from the portion in contact with the medium and so on, and adventitious roots are formed. At this time, if this part of the cutting head is subjected to ultrasonic waves, cells are activated, cell division is promoted, and adventitious root formation is promoted. In particular, difficult rooting plants take a long time to root, and the ears often die before the adventitious roots are formed. Acts very advantageously in plants.
また、超音波処理は、培地や培養土が含有する水のイオン化と水クラスターの低分子化も起こすと考えられる。従って、培地及び/又は培養土を介して挿し穂に超音波処理を行った場合には、水はもちろん、水に溶解している培地及び/又は培養土の成分が、挿し穂の細胞膜やその細胞内に存在するオルガネラの膜を浸透しやすくなって、養分や植物ホルモン等が細胞に有効に利用されるようになるので、不定根の形成が一層促進される。 In addition, sonication is thought to cause ionization of water contained in the culture medium and culture soil and reduction of the molecular weight of the water cluster. Therefore, when sonication is performed on the cuttings through the medium and / or the culture soil, not only water but also the components of the culture medium and / or the culture soil dissolved in the water, the cell membrane of the cuttings and its Since it becomes easy to permeate the organelle membrane present in the cells and nutrients, plant hormones, and the like are effectively utilized by the cells, the formation of adventitious roots is further promoted.
以下、実施例により本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
[実施例1]
E.グロブラス2系統の当年生枝よりそれぞれ芽を取得し、これらの芽を材料として、特開平8−228621に示す方法を用い、これら2系統に由来する多芽体2系統(系統1及び系統2)を誘導した。多芽体の誘導を開始してから約1ヵ月後、得られた多芽体2系統から、2〜5cmの長さに伸長してきた不定芽を切取り、これを挿し穂として用いた。なお、不定芽についた比較的大きな葉は、半分程度に切断して蒸散作用を抑制すると共に、挿し穂を密植した場合に、隣り合った挿し穂の葉と葉が重なり合わないようにした上で、挿し穂として用いた。
[Example 1]
E. Buds are obtained from the current year branches of two globulae strains, and using these buds as materials, the method described in JP-A-8-228621 is used to obtain two multi-buds derived from these two lines (line 1 and line 2). Induced. About one month after the start of induction of multi-buds, adventitious buds extending to a length of 2 to 5 cm were cut out from the two obtained multi-bud lines and used as cuttings. In addition, relatively large leaves attached to adventitious buds were cut in half to suppress transpiration, and when cuttings were densely planted, the leaves of adjacent cuttings were not overlapped. And used as cuttings.
一方、植物ホルモンとしてIBA2mg/l、チオ硫酸銀(STS)2μMを添加した4倍希釈MS液体培地にて湿潤させた、スミザーオアシス社製「オアシス(登録商標)」(縦1cm×横1cm×深さ3cm)を、頂面に孔径0.45μmのポリテトラフルオロエチレン製膜(ミリポア社製『ミリシール』)を貼り付けた直径1cmの円形開口部1個が設けられ、胴部がやや張出した立方体形状をしたポリカーボネート製の培養容器(最大寸法:縦8.4cm×横8.4cm×高さ5.4cm)内に、培養容器1ケースあたり16個用意し、上記のようにして得られた挿し穂を、その1個につき1本づつ挿しつけた。なお、このとき、液体培地中のSTSは、エチレンの発生を抑制して、挿し穂の枯死を防止するために添加されている。 On the other hand, “Oasis (registered trademark)” manufactured by Smither Oasis (vertical 1 cm × width 1 cm × depth) wetted in a 4-fold diluted MS liquid medium supplemented with 2 mg / l of IBA as a plant hormone and 2 μM of silver thiosulfate (STS). 3 cm), a cube with a 1 cm diameter circular opening with a polytetrafluoroethylene membrane (Millipore's “Milliseal”) with a pore diameter of 0.45 μm attached to the top surface and a slightly protruding body 16 pieces are prepared per case in a culture vessel made of polycarbonate (maximum dimension: 8.4 cm long x 8.4 cm wide x 5.4 cm high) and inserted as described above. One ear was inserted for each one. At this time, STS in the liquid medium is added to suppress the generation of ethylene and prevent the cutting heads from dying.
挿しつけ後の挿し穂は、ポリカーボネート製の培養容器ごと、水を張った超音波洗浄機(本多電子製卓上型超音波洗浄機『W−113MKII』)の超音波洗浄槽に、培養容器内に水が入らないように置いて、周波数31khz、出力100wにて、60、180又は300秒間超音波処理を行ってから、炭酸ガス濃度1000ppm、温度25℃、湿度60%に調節した培養室内で、650〜670nmの波長成分と450〜470nmの波長成分とを、8:2の割合で含む光照射下で培養した。 After insertion, the cutting head is placed in the culture vessel in the ultrasonic washing tank of the ultrasonic washing machine (Honda Desktop Electronics W-113MKII) filled with water. In a culture chamber adjusted to a carbon dioxide concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60% after sonication for 60, 180 or 300 seconds at a frequency of 31 kHz and an output of 100 w. The culturing was performed under light irradiation containing a wavelength component of 650 to 670 nm and a wavelength component of 450 to 470 nm at a ratio of 8: 2.
挿しつけから3週間後、目視にて発根状況を観察し、発根した挿し穂の数と、そのときの挿し穂一本当りに生じた根の数を調査した。結果を表1に示す。 Three weeks after the insertion, the state of rooting was visually observed, and the number of rooting cuttings and the number of roots generated per cutting head at that time were investigated. The results are shown in Table 1.
[比較例1]
超音波処理を行わなかった以外は、実施例1と同様にして、2系統のE.グロブラスに由来する多芽体2系統より得られた挿し穂を、「オアシス(登録商標)」に挿しつけて培養し、その発根状況を観察し、発根した挿し穂の数と、そのときの挿し穂一本当りに生じた根の数を調査した。結果を表1に示す。
[Comparative Example 1]
Two systems of E. coli were performed in the same manner as in Example 1 except that the ultrasonic treatment was not performed. The cuttings obtained from two buds derived from globulas were inserted into “Oasis (registered trademark)” and cultured, the rooting situation was observed, the number of rooting cuttings, and then The number of roots produced per cutting head was investigated. The results are shown in Table 1.
表1より明らかなように、E.グロブラス2系統の挿し穂はいずれも、超音波処理を行うことにより不定根形成が促進され、超音波処理を行わなかった場合と比べ、発根率が向上し、また発根した挿し穂1本あたりに生じた根の本数も多かった。 As is clear from Table 1, E.I. In both globulae lines, adventitious root formation was promoted by sonication, and the rooting rate improved compared to the case without sonication, and per rooted shoot. There were also a lot of roots.
[実施例2]
リンゴ(Malus pumila var. domestica)の当年枝2〜5cmを切取って挿し穂として用いた。なお、このときも、上記当年枝についた比較的大きな葉は、半分程度に切断して蒸散作用を抑制すると共に、挿し穂を密植した場合に、隣り合った挿し穂の葉と葉が重なり合わないようにした上で、挿し穂として用いた。
[Example 2]
An apple ( Malus pumila var. Domestica ) 2-5 cm of the current branch was cut out and used as an ear. At this time, the relatively large leaves on the branch of the current year are cut in half to suppress the transpiration, and when the cuttings are densely planted, the leaves and leaves of adjacent cuttings overlap. It was used as a cutting head after there was no such thing.
一方、植物ホルモンとしてIBA2mg/lを添加した4倍希釈MS液体培地にて湿潤させた、スミザーオアシス社製「オアシス(登録商標)」を、ポリカーボネート製の培養容器内に、培養容器1ケースあたり16個用意し、上記のようにして得られた挿し穂を、その1個につき1本づつ挿しつけた。なお、このとき、「オアシス(登録商標)」及びポリカーボネート製培養容器は、実施例1で使用したものと同じ寸法・形状等のものを用いた。 On the other hand, “Oasis (registered trademark)” manufactured by Smither Oasis, which was moistened with a 4-fold diluted MS liquid medium supplemented with 2 mg / l of IBA as a plant hormone, was placed in a polycarbonate culture vessel in 16 cases per culture vessel. One piece was prepared, and the cutting heads obtained as described above were inserted one by one. At this time, “Oasis (registered trademark)” and a polycarbonate culture vessel having the same dimensions and shape as those used in Example 1 were used.
挿しつけ後の挿し穂は、ポリカーボネート製の培養容器ごと、水を張った超音波洗浄機(本多電子製卓上型超音波洗浄機『W−113MKII』)の超音波洗浄槽に、培養容器内に水が入らないように置いて、周波数31khz、出力100wにて、60秒間超音波処理を行ってから、炭酸ガス濃度1000ppm、温度25℃、湿度60%に調節した培養室内で、650〜670nmの波長成分と450〜470nmの波長成分とを、8:2の割合で含む光照射下で培養した。 After insertion, the cutting head is placed in the culture vessel in the ultrasonic washing tank of the ultrasonic washing machine (Honda Desktop Electronics W-113MKII) filled with water. In a culture chamber adjusted to a carbon dioxide gas concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60% after sonication at a frequency of 31 kHz and an output of 100 w for 60 seconds, 650 to 670 nm And a wavelength component of 450 to 470 nm were cultured under light irradiation containing a ratio of 8: 2.
挿しつけから3週間後、目視にて発根状況を観察し、発根した挿し穂の数と、そのときの挿し穂一本当りに生じた根の数を調査した。結果を表2に示す。 Three weeks after the insertion, the state of rooting was visually observed, and the number of rooting cuttings and the number of roots generated per cutting head at that time were investigated. The results are shown in Table 2.
[比較例2]
超音波処理を行わなかった以外は、実施例2と同様にして、リンゴの当年枝より得られた挿し穂を、「オアシス(登録商標)」に挿しつけて培養し、その発根状況を観察し、発根した挿し穂の数と、そのときの挿し穂一本当りに生じた根の数を調査した。結果を表2に示す。
[Comparative Example 2]
Except that no sonication was performed, in the same manner as in Example 2, the cuttings obtained from the current branch of the apple were inserted into “Oasis (registered trademark)” and cultured, and the rooting situation was observed. Then, the number of rooted cuttings and the number of roots generated per cutting head at that time were investigated. The results are shown in Table 2.
表2より明らかなように、E.グロブラスと同様、リンゴの挿し穂も、超音波処理を行うことにより不定根形成が促進され、超音波処理を行わなかった場合と比べ、発根率の向上と、発根した挿し穂1本あたりに生じる根の本数の増加とが認められた。とりわけ、この場合は、発根率の向上に対する効果が顕著であった。 As is clear from Table 2, E.I. Like globulas, apple cuttings promoted adventitious root formation by sonication, and improved rooting rate compared to the case without sonication, and per rooted cuttings. An increase in the number of roots produced was observed. In particular, in this case, the effect on the improvement of the rooting rate was remarkable.
Claims (4)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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JP2005288358A JP4868812B2 (en) | 2005-09-30 | 2005-09-30 | Cutting method of cuttings |
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CN103999706A (en) * | 2014-06-24 | 2014-08-27 | 南京林业大学 | Method for increasing cutting rooting rate of pecans |
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CN105532294A (en) * | 2015-12-08 | 2016-05-04 | 北京农学院 | Cutting breeding method for improving cutting rooting percentage of Liriodendron Chinese |
CN105830716A (en) * | 2016-04-08 | 2016-08-10 | 常州润达科技信息咨询有限公司 | Cutting seedling raising method for Antiaris toxicaria |
WO2020196388A1 (en) * | 2019-03-28 | 2020-10-01 | カゴメ株式会社 | Method for producing nursery plant of inter-genus hybrid within brassica family, and method for suppressing vitrification of adventitious bud of inter-genus hybrid within brassica family |
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