JP2017525688A - 植物接種法 - Google Patents
植物接種法 Download PDFInfo
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- JP2017525688A JP2017525688A JP2017505560A JP2017505560A JP2017525688A JP 2017525688 A JP2017525688 A JP 2017525688A JP 2017505560 A JP2017505560 A JP 2017505560A JP 2017505560 A JP2017505560 A JP 2017505560A JP 2017525688 A JP2017525688 A JP 2017525688A
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- nitrogen
- composition
- plant
- polysaccharide
- surfactant
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Abstract
Description
刈草への散布
手順
G.ジアゾトロフィクス(G.diazotrophicus)の培養:
pRGS561プラスミド発現GUSがあるG.ジアゾトロフィクス(G.diazotrophicus)IMI501986株(現在のIMI50998)は、必要に応じて、ATGUS培地[0.8%(w/v)寒天、酵母抽出物(2.7gl−1)、グルコース(2.7gl−1)、マンニトール(1.8gl−1)、MES緩衝液(4.4gl−1)、K2HPO4(4.8gl−1)、およびKH2PO4(0.65gl−1)、pH6.5]上で培養された。b−グルクロニダーゼ(gusA)遺伝子の発現は、50mgl−1のX−Gluc(5−ブロモ−4−クロロ−3−インドリル−β−D−グルクロン酸シクロヘキシルアンモニウム塩)を含有するATGUS培地上に播種することで試験され;暗青色コロニーの形成は、gusA遺伝子発現を示唆した。
G.ジアゾトロフィクス(G.diazotrophicus)の水性懸濁液が調製されて、600nmで1.1の光学濃度の1ミリリットルあたり約109個のコロニー形成単位(CFU)が得られた。CFU数は、連続希釈してATGUS培地(必要に応じて抗生物質添加)上に播種し、ペトリ皿内で4日間の培養(28°C、遮光)後に細菌コロニーを数えることで判定された。懸濁液は10−4に希釈されて、1mlあたりおよそ100個の細菌を含有する溶液が生成され、それは下述するように直ぐに噴霧できる。
水+3%スクロースの対照
Gd+水+3%スクロース
Gd+水
Gd+水+3%スクロース
Gd+水+0.1%ツイーン
Gd+水+0.3%アラビアガム
Gd+水+3%スクロース+0.1%ツイーン
Gd+水+3%スクロース+0.3%アラビアガム
Gd+水+3%スクロース+0.1%ツイーン+0.3%アラビアガム
苗はピンセットを用いて寒天から除去され、あらゆる残留寒天が根から洗浄された。各苗は紙バッグに入れて、80°Cのオーブンに48時間入れられ、次に秤量された。
グルコンアセトバクター・ジアゾトロフィクス(Gluconacetobacter diazotrophicus)(Azoticus)によるチャノキ(Camellia sinensis)のコロニー形成
挿し枝からの栄養生殖
チャノキのクローンの栄養繁殖の標準手段は、単葉挿し枝である。およそ4〜6個の節および茎頂を含んでなるより大型の茎から、組織の健康状態(すなわち昆虫および疾患を含まない)に基づいて、茎および葉の切片が選択された。挿し枝のために選択された部分は、Yamasaki et al.Soil and Crop Management,(2008)SCM−23)によって推奨されるように、赤枝と緑枝の間であった。最近、腋芽を積極的に破壊した、成熟葉に隣接するわずかに赤くなった樹皮を含有する成熟苗条が、最良の発根成功をもたらすことが発見されている。
各チャノキ挿し枝の下部は、1%インドール酪酸溶液に浸されて個々のポットに入れられた;挿し枝は、葉が土壌に接触しないように、茎をまっすぐからわずかに傾けて栽植された。各ポットは、砂とJohn Innes 1号挿し木ミックスを比率4:1で含有し、水で飽和された。各挿し枝の切断面には、20μlの水、または20μlの水中の2.5×105cfu/mlのGdのどちらかが塗布されて、各サンプルの湿度は、各ポットをプラスチックシートで覆い、軽く水を噴霧することで保たれた。
草バイオマスに対する処理効果の研究
草は、植物グロースチャンバー(Fitotron(登録商標))(23°C/15°Cat65%湿度)内において、John Innes 1号堆肥を使用して、種子トレー内で2週間栽培された。それは、次に8cmの高さで切断され、家庭用噴霧器を使用して、下に記載される10mlの処理剤が即座に噴霧された。
1. 水
2. 3%スクロース+0.1%ツイーン+0.3%アラビアガム
3. 水+Gd(2.5×105cfu/ml)
4. 水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.5×103cfu/ml)
5. 水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.5×104cfu/ml)
6. 水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.5×105cfu/ml)
7. 水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.5×106cfu/ml)
8. 水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.5×107cfu/ml)
草は、同様の成長条件下で、Fitotronにさらに2週間戻された。無作為に選択された5本の植物が、地表面で切断されて1個のサンプルが作成され秤量された。これはさらに5回反復されて、各処理のために全部で6個のサンプルが得られた。
野外実験
水のみで処理された1m2の未接種刈草試験区(対照)との比較で、水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.×105cfu/ml)を含んでなる調合物が、1m2の刈草試験区(確立されたホソムギ(Lolium perenne)芝生)に散布された。
組成物の成分比較
実験で使用された組成物の個々の成分を含む、様々な組成物を使用して、実施例3の方法が繰り返された。具体的には、この実験で使用された組成物は、次のとおりであった。
1.水
2.水+Gd(2.5×105cfu/ml)
3.水+3%スクロース+0.1%ツイーン+0.3%アラビアガム+Gd(2.5×105cfu/ml)
4.0.3%アラビアガム+Gd(2.5×105cfu/ml)
5.3%スクロース+Gd(2.5×105cfu/ml)
6.0.1%ツイーン+Gd(2.5×105cfu/ml)
Claims (33)
- 窒素固定細菌を成長植物の傷に投与するステップを含んでなる、窒素固定細菌を植物に接種する方法。
- 前記窒素固定細菌が、グルコンアセトバクター・ジアゾトロフィクス(Gluconacetobacter diazotrophicus)である、請求項1に記載の方法。
- 前記窒素固定細菌が、テルリバチルス属(Terribacillus)の株と組み合わされる、請求項1または請求項2に記載の方法。
- 前記傷が、芝刈り、草刈り、株出し栽培、剪定、家畜による摂取または収穫の結果である、請求項1〜3のいずれか一項に記載の方法。
- 予備段階において、前記植物が、芝刈り、草刈り、株出し栽培、剪定または収穫の対象である、請求項4に記載の方法。
- 前記窒素固定細菌が、前記予備段階の1〜2時間以内に適切に塗布される、請求項5に記載の方法。
- 前記窒素固定細菌が、組成物の形態で前記植物の傷に塗布される、請求項1〜6のいずれか一項に記載の方法。
- 組成物が、1ミリリットルあたり1〜1×107個の窒素固定細菌を含んでなる、請求項7に記載の方法。
- 前記組成物が、1ミリリットルの組成物あたり50〜200個の窒素固定細菌を含んでなる、請求項8に記載の方法。
- 前記組成物が、農業上許容される界面活性剤をさらに含んでなる、請求項9に記載の方法。
- 前記界面活性剤が、非イオン性合成洗剤である、請求項10に記載の方法。
- 前記界面活性剤の70%が脂肪酸オレイン酸から構成され、残部がリノレン酸、パルミチン酸、およびステアリン酸の組み合わせである、請求項11に記載の方法。
- 前記組成物が、多糖類をさらに含んでなる、請求項7〜12のいずれか一項に記載の方法。
- 前記組成物が、多糖類および農業上許容される界面活性剤の双方を含んでなる、請求項13に記載の方法。
- 前記多糖類が、滲出ガム多糖類である、請求項13または請求項14に記載の方法。
- 前記滲出ガム多糖類が、アラビアガムである、請求項15に記載の方法。
- 前記植物が、多年生、二年生または永続的一年生植物である、請求項1〜16のいずれか一項に記載の方法。
- 前記植物が、果樹または低木、つる植物、飼料作物、観賞用芝、生垣、森林、園芸植物またはハーブである、請求項17に記載の方法。
- 前記植物が、刈草である、請求項18に記載の方法。
- グルコンアセトバクター・ジアゾトロフィクス(Gluconacetobacter diazotrophicus)と、多糖類または界面活性剤またはそれらの組み合わせから選択されるさらなる成分とを含んでなる、農業上許容される組成物。
- 1ミリリットルあたり1〜100個の細菌を含んでなる、請求項20に記載の組成物。
- 前記多糖類が、滲出ガム多糖類である、請求項20または請求項21に記載の組成物。
- 前記滲出ガム多糖類が、アラビアガムである、請求項22に記載の組成物。
- 前記界面活性剤が、非イオン性合成洗剤である、請求項20〜23のいずれか一項に記載の組成物。
- 前記界面活性剤の70%が脂肪酸オレイン酸から構成され、残部がリノレン酸、パルミチン酸、およびステアリン酸の組み合わせである、請求項24に記載の組成物。
- 0.0005〜10%v/vの界面活性剤を含んでなる、請求項20〜25のいずれか一項に記載の組成物。
- 前記窒素固定細菌のための栄養素をさらに含んでなる、請求項20〜26のいずれか一項に記載の組成物。
- テルリバチルス属(Terribacillus)の株をさらに含んでなる、請求項20〜27のいずれか一項に記載の組成物。
- (i)窒素固定細菌、および(ii)農業上許容される界面活性剤、または多糖類またはそれらの組み合わせを含んでなるさらなる成分を含んでなる、農業上許容される組成物を形成するためのキット。
- 前記窒素固定細菌が、凍結乾燥形態である、請求項29に記載のキット。
- 前記さらなる成分が、希釈されて前記窒素固定細菌と混合されて、農業上許容される組成物を生成してもよい濃縮物である、請求項29または請求項30に記載のキット。
- 植物のクロロフィルレベルを増大させることで、植物の緑色を増大させるために実施される、請求項1〜19のいずれか一項に記載の方法。
- IMI504998(以前のIMI501986)またはIMI504958(以前のIMI504853)から選択される、グルコンアセトバクター・ジアゾトロフィクス(Gluconacetobacter diazotrophicus)の株。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB1413333.4A GB201413333D0 (en) | 2014-07-28 | 2014-07-28 | Plant inoculation |
GB1413333.4 | 2014-07-28 | ||
PCT/GB2015/052170 WO2016016629A1 (en) | 2014-07-28 | 2015-07-28 | Plant inoculation method |
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US10968446B2 (en) | 2012-11-01 | 2021-04-06 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
GB201413333D0 (en) | 2014-07-28 | 2014-09-10 | Azotic Technologies Ltd | Plant inoculation |
CN115418357A (zh) | 2015-07-13 | 2022-12-02 | 皮沃特生物公司 | 改良植物性状的方法及组合物 |
GB201513277D0 (en) * | 2015-07-28 | 2015-09-09 | Azotic Technologies Ltd | Diagnostic kits |
JP2018537119A (ja) | 2015-10-05 | 2018-12-20 | マサチューセッツ インスティテュート オブ テクノロジー | リファクターされたnifクラスターを使用する窒素固定 |
CN110799474B (zh) | 2017-01-12 | 2022-07-26 | 皮沃特生物公司 | 用于改良植物性状的方法及组合物 |
WO2019133923A1 (en) * | 2017-12-28 | 2019-07-04 | Sustainable Community Development, Llc. | Microbial-based composition and method of use |
MX2020013875A (es) | 2018-06-27 | 2021-08-11 | Pivot Bio Inc | Composiciones agricolas que comprenden microbios remodelados de fijacion de nitrogeno. |
TWI692524B (zh) * | 2018-12-28 | 2020-05-01 | 嬌朋生技股份有限公司 | 栽培材料組成物 |
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