JP2562909B2 - A method for multiplying a shoot primordium agglomerate derived from a woody plant from a shoot primordium agglomerate derived from a woody plant, and a method for regenerating a plant from the seed primordium agglomerate - Google Patents
A method for multiplying a shoot primordium agglomerate derived from a woody plant from a shoot primordium agglomerate derived from a woody plant, and a method for regenerating a plant from the seed primordium agglomerateInfo
- Publication number
- JP2562909B2 JP2562909B2 JP62204269A JP20426987A JP2562909B2 JP 2562909 B2 JP2562909 B2 JP 2562909B2 JP 62204269 A JP62204269 A JP 62204269A JP 20426987 A JP20426987 A JP 20426987A JP 2562909 B2 JP2562909 B2 JP 2562909B2
- Authority
- JP
- Japan
- Prior art keywords
- plant
- shoot
- primordium
- agglomerate
- derived
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims description 33
- 230000001172 regenerating effect Effects 0.000 title claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 47
- 239000002609 medium Substances 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 15
- 239000003375 plant hormone Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 5
- 230000002062 proliferating effect Effects 0.000 claims description 4
- 239000012882 rooting medium Substances 0.000 claims description 4
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 claims description 3
- 239000004062 cytokinin Substances 0.000 claims description 3
- 230000008635 plant growth Effects 0.000 claims description 3
- 229930192334 Auxin Natural products 0.000 claims description 2
- 239000002363 auxin Substances 0.000 claims description 2
- 230000012010 growth Effects 0.000 claims description 2
- 241001453233 Doodia media Species 0.000 claims 1
- 102000018997 Growth Hormone Human genes 0.000 claims 1
- 108010051696 Growth Hormone Proteins 0.000 claims 1
- 239000000122 growth hormone Substances 0.000 claims 1
- 229910017053 inorganic salt Inorganic materials 0.000 claims 1
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 9
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 8
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 241000219000 Populus Species 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 239000003617 indole-3-acetic acid Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000006870 ms-medium Substances 0.000 description 3
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 2
- 241000167854 Bourreria succulenta Species 0.000 description 2
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 241000218657 Picea Species 0.000 description 2
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 241000208422 Rhododendron Species 0.000 description 2
- 235000019693 cherries Nutrition 0.000 description 2
- GPXLRLUVLMHHIK-UHFFFAOYSA-N forchlorfenuron Chemical compound C1=NC(Cl)=CC(NC(=O)NC=2C=CC=CC=2)=C1 GPXLRLUVLMHHIK-UHFFFAOYSA-N 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 2
- 229960001669 kinetin Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000003104 tissue culture media Substances 0.000 description 2
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 2
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 2
- 229940023877 zeatin Drugs 0.000 description 2
- 235000009434 Actinidia chinensis Nutrition 0.000 description 1
- 244000298697 Actinidia deliciosa Species 0.000 description 1
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- 244000144730 Amygdalus persica Species 0.000 description 1
- 241000249058 Anthracothorax Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 244000020190 Azalea indica Species 0.000 description 1
- 235000002498 Azalea indica Nutrition 0.000 description 1
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 240000006248 Broussonetia kazinoki Species 0.000 description 1
- 235000006716 Broussonetia kazinoki Nutrition 0.000 description 1
- 240000001548 Camellia japonica Species 0.000 description 1
- 241000258151 Caudina Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 241001672694 Citrus reticulata Species 0.000 description 1
- 240000007154 Coffea arabica Species 0.000 description 1
- 240000006766 Cornus mas Species 0.000 description 1
- 240000005109 Cryptomeria japonica Species 0.000 description 1
- 244000301850 Cupressus sempervirens Species 0.000 description 1
- 241001265525 Edgeworthia chrysantha Species 0.000 description 1
- 244000004281 Eucalyptus maculata Species 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- -1 KT-30 or zeatin Chemical compound 0.000 description 1
- 241000218652 Larix Species 0.000 description 1
- 235000005590 Larix decidua Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 235000014826 Mangifera indica Nutrition 0.000 description 1
- 235000009134 Myrica cerifera Nutrition 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 240000002834 Paulownia tomentosa Species 0.000 description 1
- 235000010678 Paulownia tomentosa Nutrition 0.000 description 1
- 244000025272 Persea americana Species 0.000 description 1
- 235000008673 Persea americana Nutrition 0.000 description 1
- 240000008670 Pinus densiflora Species 0.000 description 1
- 235000000405 Pinus densiflora Nutrition 0.000 description 1
- 241000218981 Populus x canadensis Species 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 241000220324 Pyrus Species 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 240000002044 Rhizophora apiculata Species 0.000 description 1
- 240000003152 Rhus chinensis Species 0.000 description 1
- 235000014220 Rhus chinensis Nutrition 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000109329 Rosa xanthina Species 0.000 description 1
- 244000061457 Solanum nigrum Species 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 241000219995 Wisteria Species 0.000 description 1
- 241001222097 Xenocypris argentea Species 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 235000021018 plums Nutrition 0.000 description 1
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、木本性植物由来の苗条原基集塊を大量に増
殖する方法および木本性植物由来の苗条原基集塊から植
物体を再生する方法に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for multiplying a shoot primordium agglomerate derived from a woody plant and reproducing a plant from the shoot primordium agglomerate derived from a woody plant. On how to do.
苗条原基は優れた、かつ有用な遺伝子を持った植物
を、その性質を変異させることなく大量に増殖させる事
が可能な細胞集塊であるが、このような苗条原基の集合
体である苗条原基集塊を短期間で多数増殖し、さらにこ
れから完全な植物体を再生する方法に関するものであ
る。The shoot primordium is a cell mass that allows a plant having an excellent and useful gene to be proliferated in a large amount without mutating its properties. It is an aggregate of such shoot primordia. The present invention relates to a method for growing a large number of shoot primordium agglomerates in a short period of time and then regenerating a complete plant body.
（従来の技術） 苗条原基は田中隆荘等（Jap.J.Genet.Vol.,58:65〜7
0,1983）がキク科の一年生植物であるハプロパップスに
ついて、その生長点を含む茎頂部を摘出して一定の組成
を持つ人工培地に植え付け、一定の温度と照度そして回
転数の下で回転培養して得た細胞集塊である。(Prior art) Takanori Tanaka et al. (Jap.J.Genet. Vol., 58: 65〜7)
(0,1983) was an annual plant of the Asteraceae family, Hapropapus, and the shoot apex containing the growth point was extracted and planted in an artificial medium with a constant composition, and then spin-cultured at a constant temperature, illuminance, and rotation speed. It is a cell mass obtained by
この苗条原基は単独で得られるものではなく、金平糖
状をした苗条原基集塊表面にある突起物であり、苗条原
基から植物体を再生させる場合には、苗条原基を切り出
して苗化培地に移して培養されている。This shoot primordia is not obtained alone, but is a protrusion on the surface of the agglomerate of seed primordia that has a sugar-like shape.When regenerating a plant from the shoot primordia, the shoot primordia are cut out to obtain seedlings. It is transferred to a conditioned medium and cultured.
特開昭59−132822号公報には一年生植物ハプロパップ
ス、特開昭59−132823号公報には有用一年生植物、特開
昭61−96994年公報にはステビアへの応用例が開示され
ている。Japanese Patent Application Laid-Open No. 59-132822 discloses an annual plant Hapropapus, Japanese Patent Application Laid-Open No. 59-132823 discloses a useful annual plant, and Japanese Patent Application Laid-Open No. 61-96994 discloses an application example to stevia.
また、田中隆荘等は植物の生長点を含む茎頂部を摘出
し、これを一定の温度、照度および回転数にて回転培養
して得られる苗条原基法以外にも、カルス（未分化の細
胞集塊）から懸濁細胞を遊離し、それを回転培養系を用
いて苗条原基化する方法を提案している（昭和61年11月
20日，第58回日本遺伝子学会編集発行、第９回日本分子
生物学会プログラム・講演要旨集第189頁参照）。In addition to the shoot primordia method obtained by extracting the shoot apex containing the plant growth point and culturing it at a constant temperature, illuminance and rotation speed, Tanaka Ryoso and others callus (undifferentiated cell We have proposed a method of releasing suspended cells from agglomerates and using them as a shoot primordium using a rotary culture system (November 1986).
20th, edited and published by the 58th Annual Meeting of the Genetic Society of Japan, 9th Annual Meeting of the Molecular Biology Program of Japan, 189).
また本発明者らは特開昭62−55020号公報で苗条原基
法が木本性植物の大量増殖法に有効であることを提案し
ている。Further, the present inventors propose in Japanese Patent Laid-Open No. 6255020/1987 that the shoot primordium method is effective for a method of mass-growing woody plants.
この特開昭62−55020号公報において提案した方法は
木本性植物であるポプラの茎頂部を液体培地で30〜50日
間、一定の条件下で回転培養して苗条原基を作出し、さ
らにこの苗条原基を苗化培地に移植して静置培養し、植
物体を再生させる方法である。The method proposed in this Japanese Laid-Open Patent Publication No. 62-55020 is to cultivate the shoot apex of a poplar, which is a woody plant, in a liquid medium for 30 to 50 days under a constant condition to produce shoot shoot primordia. This is a method of regenerating a plant by transplanting shoot shoot primordium to a seedling medium and culturing statically.
ここで得られた植物は、その表現型だけでなく遺伝子
型および染色体型も親植物と全く同一であった。しかし
ながら、この方法で植物体を再生するためには長い期間
培養を続けなければならないだけでなく、培地に添加す
る植物ホルモン濃度の許容範囲が狭く極めて厳密に調製
しなければならない。さらに、再生された植物体の数も
少数である等の問題点があった。The plant obtained here was completely identical to the parent plant not only in its phenotype but also in genotype and chromosome type. However, in order to regenerate the plant body by this method, not only must the culture be continued for a long period of time, but also the concentration range of the plant hormone to be added to the medium must be narrow and must be adjusted very strictly. Further, there are problems that the number of regenerated plants is small.
このために、本発明者等はさらに短期間に多数の苗条
原基を得る方法について鋭意検討したところ、木本性植
物の茎頂部を液体培地で回転培養して得た苗条原基集塊
を一度細片化して、次いで液体培地で振盪培養すること
によって苗条原基集塊を大量に得ることができることを
見いだし本発明をなすに至った（第１番目の発明）。For this reason, the present inventors have made extensive studies on a method for obtaining a large number of shoot primordia in a shorter period of time, and once obtained a shoot primordium agglomerate obtained by spin-culturing the shoot apex of a woody plant in a liquid medium. The inventors have found that a large amount of shoot primordium agglomerates can be obtained by slicing and then shaking culture in a liquid medium, and completed the present invention (first invention).
さらに引き続いて、このようにして得られた苗条原基
集塊から植物体を再生する方法について実験を重ねた結
果、苗条原基集塊を苗化培地に移して培養を続けること
によって、従来行われたように最初に得られた苗条原基
から直接苗化させる方法に比べて苗条の出現までの期間
が短く、また多数の苗条と植物体を得ることができるこ
とを見いだし本発明を完成させるに至った（第２番目の
発明）。Subsequently, as a result of repeated experiments on a method of regenerating a plant from the thus obtained shoot primordium agglomerates, by transferring the shoot primordia agglomerates to a seedling medium and continuing the culture, As described above, it was found that the period until the appearance of shoots is shorter than the method of directly seedling from the shoot primordium obtained first, and that a large number of shoots and plants can be obtained, and the present invention is completed. It came (the second invention).
（本発明が解決しようとする問題点） 本発明は、従来行われてきた摘出した植物の茎頂部を
回転培養して得られた苗条原基集塊、あるいはカルス
（未分化の細胞集塊）由来の苗条原基集塊から直接植物
体を再生する方法においては、長い期間培養を続けなけ
ればならないだけでなく、培地に添加する植物ホルモン
濃度の許容範囲が狭く極めて厳密に調整しなければなら
ず、さらには、再生された植物体の数も少数である等の
問題点を解決すべくさらに有効な方法について検討を行
った。(Problems to be Solved by the Present Invention) The present invention provides a shoot primordium agglomerate or callus (undifferentiated cell agglomerate) obtained by spin-culturing the shoot apex of a conventionally-extracted plant. In the method of directly regenerating a plant body from the shoot shoot primordia agglomerates derived from it, not only must the culture be continued for a long period of time, but also the permissible range of the plant hormone concentration to be added to the medium must be narrow and adjusted very strictly. Moreover, a more effective method was studied to solve the problems such as the small number of regenerated plants.
この結果、木本性植物の茎頂部を液体培地で回転培養
して得た苗条原基集塊を一度細片化し、この細片化され
た苗条原基集塊をさらに液体培地で振盪培養することに
よって再び苗条原基集塊を作出でき、これから植物体を
再生することによって従来の方法に比べて苗条の出現ま
での期間が短く、また多数の苗条と植物体を得ることが
できることを見いだし、本発明を完成するに至った。As a result, the shoot primordium agglomerates obtained by spin-culturing the shoot apices of woody plants in a liquid medium are once fragmented, and the fragmented shoot primordia agglomerates are further shake-cultured in a liquid medium. It was found that the shoot primordium agglomerates can be produced again by this method, and by regenerating the plant from now on, the time until the appearance of shoots can be shortened compared to the conventional method, and a large number of shoots and plants can be obtained. The invention was completed.
（問題点を解決するための手段） 本発明は、木本性植物由来の苗条原基集塊を細片化処
理し、次いで細片化された苗条原基集塊を液体振盪培養
して苗条原基集塊を大量に増殖することを特徴とする木
本性植物由来の苗条原基集塊を大量に増殖する方法（第
１番目の発明）、 ならびに 木本性植物由来の苗条原基集塊をホモジナイザーによ
り回転数10,000〜30,000r.p.m.で５〜20秒の条件下で細
片化処理し、次いで細片化された苗条原基集塊を液体振
盪培養して苗条原基集塊を大量に増殖し、得られた苗条
原基集塊を苗化培地で培養して苗化させ、得られた苗条
を発根培地に移植して完全な植物体を再生することを特
徴とする木本性植物由来の苗条原基集塊から植物体を再
生する方法（第２番目の発明） である。(Means for Solving the Problems) The present invention is to treat a shoot primordium agglomerate derived from a woody plant into pieces, and then culture the crushed shoot primordium agglomerates with liquid shaking to produce a shoot origin. A method for proliferating a large amount of shoot original substrate agglomerates derived from woody plants, which comprises proliferating a large amount of basic agglomerates, and a homogenizer for the shoot original substrate agglomerates derived from woody plants. With a rotational speed of 10,000 to 30,000 rpm for 5 to 20 seconds, the shredded shoot primordium agglomerates are cultured by liquid shaking to multiply the shoot primordium agglomerates in large quantities, A shoot derived from a woody plant characterized by culturing the obtained shoot primordium agglomerate in a seedling medium to form a seedling, and transplanting the obtained shoot into a rooting medium to regenerate a complete plant body. A method of regenerating a plant from a primordium agglomerate (second invention).
本発明で使用する植物は特に限定されるものではない
が、ユーカリ、マングロープ、アカシア、パラゴムノ
キ、コーヒー等の常緑広葉樹類、ポプラ、キリ、コナ
ラ、キハダ、コウゾ、ミツマタ、クヌギ、ウルシ等の落
葉広葉樹類、マツ、エゾマツ、トドマツ、スギ、ヒノ
キ、モミ、トウヒ、カラマツ等の有用針葉樹類、さらに
ミカン、レモン、リンゴ、モモ、アンズ、カリン、アボ
カド、キウイ、カキ、クルミ、ブドウ、イチヂク、ヤマ
モモ、サクランボ、ナシ、アーモンド、マンゴウ等の果
樹類やバラ、ツバキ、ウメ、サクラ、ハナミズキ、フ
ジ、サツキ、ツツジ、シヤクナゲ、等の花木類である。The plant used in the present invention is not particularly limited, but eucalyptus, mangrove, acacia, Hevea brasiliensis, evergreen broad-leaved trees such as coffee, poplar, kiri, oak, yellowfin, kozo, mitsumata, kunugi, deciduous broad-leaved trees such as sumac Useful coniferous trees such as pine, pine, spruce, Japanese pine, Japanese cedar, cypress, fir, spruce, larch, etc., and also mandarin orange, lemon, apple, peach, apricot, karin, avocado, kiwi, oyster, walnut, grape, fig, bayberry, Fruit trees such as cherries, pears, almonds, and mangos, and flowering trees such as roses, camellias, plums, cherry trees, dogwood, wisteria, satsuki, azaleas, rhododendrons, and the like.
以下本発明の苗条原基集塊の作出法ならびに植物体の
再生法等について詳しく説明する。The method for producing shoot shoot agglomerates and the method for regenerating plant bodies according to the present invention will be described in detail below.
苗条原基集塊の作出法 すでに確率されている方法（特開昭62−55020号公
報）によって苗条原基集塊を作出する。Method of producing shoot primordium agglomerate A shoot primordium agglomerate is produced by a method already established (Japanese Patent Laid-Open No. 6255020/1987).
すなわち、植物の茎を70％のエタノールならびに７倍
に希釈したアンチホルミン液で殺菌した後に、生長点を
含む茎頂部分約0.5mm程度を無菌的に切り出し、これを
植物の組織培養培地、例えばガンボーグのB5倍地あるい
はムラシゲ、スクーグのMS培地等に、植物ホルモンとし
て例えば、ナフタレン酢酸（NAA）、2,4−ジクロロフエ
ノキシ酢酸（2,4−Ｄ）あるいはインドール酢酸（IAA）
等のオーキシン類、そしてベンジルアデニン（BA）、カ
イネチン、KT−30あるいはゼアチン等のサイトカイニン
類を添加した液体培地に植え付ける。これを20〜30℃の
温度、2,000〜20,000ルクスの照度、そして１〜10r.p.
m.の回転数で30〜50日間回転培養して５〜10mmの大きさ
の苗条原基集塊を得る。That is, after sterilizing a plant stem with 70% ethanol and a 7-fold diluted antiformin solution, about 0.5 mm of the shoot apex portion including the growing point is aseptically cut out, and this is cut into a plant tissue culture medium, for example, As a plant hormone, for example, naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) or indole acetic acid (IAA) in B5 medium of Gumbog or Murashige, MS media of Skoog, etc.
Etc., and cytokinins such as benzyladenine (BA), kinetin, KT-30, or zeatin are added to the liquid medium. Apply this to a temperature of 20-30 ° C, illuminance of 2,000-20,000 lux, and 1-10 r.p.
The culture is cultivated at a rotation speed of m. for 30 to 50 days to obtain shoot primordium agglomerates having a size of 5 to 10 mm.
なお苗条原基集塊は植物の生長点を含む茎頂部細胞由
来の苗条原基集塊以外にもカルス由来の苗条原基集塊も
使用出来ることはいうまでもない。Needless to say, the shoot primordium agglomerates derived from callus can be used in addition to the shoot primordium agglomerates derived from shoot apical cells containing plant growth points.
細片化処理 上記苗条原基集塊の作出法によって得られた５〜10mm
の範囲の大きさの苗条原基集塊を、ガンボーグのB5培地
あるいはムラシゲ、スクーグのMS培地に2,4−ジクロロ
フエノキシ酢酸（2,4−Ｄ）、ナフタレン酢酸（NAA）あ
るいはインドール酢酸（IAA）等のオーキシン類および
ベンジルアデニン（BA）、カイネチン、KT−30あるいは
ゼアチン等のサイトカイニン類及びショ糖を添加した
「洗い液」と共にホモジナイザーで10,000〜30,000r.p.
m.の回転数で５〜20秒処理して0.5〜3mmの範囲の大きさ
に細片化する。これを目の荒いナイロンメッシュで過
して分画し、次に、洗い液で１〜５回十分に洗浄する。Fragmentation treatment 5-10 mm obtained by the above method of producing shoot primordium agglomerates
The shoot primordia agglomerates in the range of the size of B4 medium or Murashige of Gumbog or MS medium of Skoog are 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) or indoleacetic acid. (IAA) and auxins and benzyladenine (BA), kinetin, cytokinins such as KT-30 or zeatin, and a "washing solution" with sucrose added, and a homogenizer 10,000-30,000 rp
It is processed for 5 to 20 seconds at a rotation speed of m. to be fragmented into a size in the range of 0.5 to 3 mm. This is passed through a coarse nylon mesh to be fractionated, and then thoroughly washed with a washing liquid 1 to 5 times.
液体振盪培養 さらにこれを上記洗い液と同様の組成を持つ液体培地
の中で振盪培養する。培養条件は80〜100r.p.m.の振盪
速度、20〜30℃の温度、そして10,000〜20,000ルクスの
照度で12〜16時間の明条件（残りの12〜８時間は暗条
件）が好ましい。Liquid shaking culture Further, this is shake cultured in a liquid medium having the same composition as the above washing solution. The culture conditions are preferably a shaking speed of 80 to 100 rpm, a temperature of 20 to 30 ° C., and an illuminance of 10,000 to 20,000 lux for 12 to 16 hours (dark conditions for the remaining 12 to 8 hours).
このような条件で約１ヵ月培養して２〜5mmの大きさ
の苗条原基集塊を約102〜103個得ることが出来る。By culturing under these conditions for about 1 month, about 10 2 to 10 3 shoot primordium agglomerates having a size of 2 to 5 mm can be obtained.
苗化法 このようにして得られた苗条原基集塊をガンボルグの
B5培地あるいはムラシゲ・スクーグのMS培地に植物ホル
モン類ならびに0.4〜0.6の濃度で寒天を添加してた苗化
培地に植え付けて20〜30℃の温度、12〜16時間の明条件
で15〜25日間培養することによって多数の苗条が得られ
る。Seedling method The shoot primordia agglomerates obtained in this way
B5 medium or Murashige-Skoog MS medium with plant hormones and agar added at a concentration of 0.4 to 0.6 was planted in a seedling medium at a temperature of 20 to 30 ° C and a light condition of 12 to 16 hours for 15 to 25 A large number of shoots can be obtained by culturing for a day.
植物体の再生法 得られた苗条をガンボーグのB5培地あるいはムラシゲ
・スクーグのMS培地に植物ホルモン類および0.4〜0.6％
の濃度で寒天を添加した発根培地に移植して苗条の作出
法と同様の条件で10〜20日間培養することにより完全な
植物体が再生される。Plant Regeneration Method The obtained shoots were added to Gamboorg B5 medium or Murashige-Skoog MS medium with phytohormones and 0.4 to 0.6%.
A complete plant is regenerated by transplanting to a rooting medium supplemented with agar at the above concentration and culturing for 10 to 20 days under the same conditions as in the method for producing shoots.
以下、実施例によって本発明を更に詳しく説明する
が、本発明はこれらの実施例に限定されるものではな
い。Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.
（実施例 １） 供試植物 ポプラ（Populus charkowiensis XP.caudina、OP−2
0）。(Example 1) Test plant Poplar (Populus charkowiensis XP.caudina, OP-2)
0).
苗条原基集塊の作出法 切り出してきたポプラの茎を70％エタノールおよび７
倍に希釈したアンチホルミン液で殺菌した後に、約0.5m
mの大きさの生長点を含む茎頂部を無菌的に切り出し、
これを植物の組織培養培地であるガンボーグのB5培地
（表−１）に植物ホルモンとしてナフタレン酢酸を0.05
mg/、ベンジルアデニンを0.4mg/の割合で添加し
て、pH5.6に調整した液体培地に植えつけた。Method of producing shoot primordium agglomerate Poplar stems cut out were treated with 70% ethanol and 7
About 0.5m after sterilizing with double diluted antiformin solution
Aseptically cut out the stem apex including the growing point of m size,
This was added to a plant tissue culture medium, Gumbog's B5 medium (Table 1), with 0.05% naphthalene acetic acid as a plant hormone.
mg /, benzyladenine was added at a ratio of 0.4 mg /, and the mixture was planted in a liquid medium adjusted to pH 5.6.
これを28℃の温度、20,000ルクスの照度、そして2r.
p.m.の回転数で回転培養した結果、培養開始後40日で直
径10mmの大きさの緑色をした苗条原基集塊（Ａ）を得
た。This is a temperature of 28 ℃, illuminance of 20,000 lux, and 2r.
As a result of rotation culture at a rotation number of pm, 40 days after the start of culture, a green shoot primordium agglomerate (A) having a diameter of 10 mm was obtained.
細片化処理 得られた苗条原基集塊1gに、ガンボーグのB5培地に0.
04mg/のナフタレン酢酸、0.5mg/のベンジルアデニ
ン、さらに３％のショ糖を加えてpH5.6に調整した洗い
液15mlを加えて、20,000r.p.m.で10秒間ホモジナイザー
で処理して直径約1mmの大きさに細片化した。これを目
の洗いナイロンメッシュで過して分画し、洗い液で２
階洗浄した。 Fragmentation 1 g of the obtained shoot primordium agglomerate was added to B5 medium of Gamboorg.
04 mg / naphthalene acetic acid, 0.5 mg / benzyl adenine, and 3% sucrose were added to add 15 ml of washing solution adjusted to pH 5.6, and treated with a homogenizer at 20,000 rpm for 10 seconds to obtain a size of about 1 mm in diameter. It was shredded. This is filtered with a nylon mesh for eye wash and fractionated.
Washed the floor.
液体振盪培養 洗浄した材料をガンボーグのB5培地に0.04mg/のナ
フタレン酢酸と0.5mg/のベンジルアデニンの両植物ホ
ルモンと３％のショ糖を添加し、pH5.6に調整した人工
培地で振盪培養した。培養条件は10,000ルクスの照度で
12時間の明条件、27℃の温度、そして100r.p.m.の振盪
速度とした。Liquid shaking culture The washed material was shake-cultured in an artificial medium adjusted to pH 5.6 by adding 0.04 mg / naphthalene acetic acid and 0.5 mg / benzyl adenine both plant hormones and 3% sucrose to G5 Borg medium. did. Culture conditions are 10,000 lux illuminance
Light conditions for 12 hours, temperature of 27 ° C., and shaking speed of 100 rpm.
１ヵ月間培養した結果、回転培養して得られた苗条原
基集塊（Ａ）と外見的には類似しているが大きさが２〜
5mmと小さい苗条原基集塊（Ｂ）が苗条原基集塊（Ａ）
１ケ当り約8.5×102個得られた。As a result of culturing for 1 month, it is similar in appearance to the shoot primordium agglomerate (A) obtained by spin culturing, but the size is 2 to
The shoot base lumps (B) as small as 5 mm are the shoot base lumps (A)
About 8.5 × 10 2 pieces were obtained per one.
苗条原基集塊の苗条化 ポプラの茎頂部を回転培養して得た従来法による苗条
原基集塊（Ａ）と、これをさらに細片化処理し、液体振
盪培養して苗条原基集塊を大量に増殖して得た本発明法
による苗条原基集塊（Ｂ）をガンボーグのB5培地にNAA
とBAを種々の濃度で組合せ、濃度３％のショ糖を添加、
さらに寒天濃度が0.6％となる様に寒天を添加し、次い
でpH5.6に調整した苗化培地に置床し、27℃の温度、照
度4000ルクス、16時間の明条件で培養を継続した。Shoot primordium agglomeration of shoots A shoot primordium agglomerate (A) obtained by a conventional method by spin-culturing the shoot apex of poplar, and further shredding treatment and liquid shaking culture to cultivate the shoot primordium. The shoot primordium agglomerates (B) according to the method of the present invention obtained by proliferating a large amount of lumps were NAA-treated with B5 medium of Gamboug.
And BA in various concentrations, and added sucrose at a concentration of 3%,
Further, agar was added so that the concentration of agar was 0.6%, and the mixture was then placed in a seedling medium adjusted to pH 5.6, and the culture was continued under the conditions of 27 ° C, illuminance 4000 lux, and light for 16 hours.
両方法によって得た苗条原基集塊（Ａ），（Ｂ）から
の発根の有無、苗条発生の本数、苗化までの日数を調べ
た結果を第２表に示した。Table 2 shows the results of examining the presence or absence of rooting from shoot primordium agglomerates (A) and (B) obtained by both methods, the number of shoot occurrences, and the number of days until seedling formation.
これによれば本発明による方法では従来法に比較して
発根に及ぼすホルモン濃度の許容範囲が広いだけでな
く、苗条の発生本数が多く、さらに苗化日数も少ないこ
とが明らかになった。 According to this, it was revealed that the method according to the present invention not only has a wider allowable range of hormone concentration on rooting as compared with the conventional method, but also has a large number of shoots and a small number of days for seedling formation.
苗条から植物体の再生法 上記の方法で得られた苗条を、ガンボーグのB5培地に
0.01mg/のNAA、３％のショ糖そして0.4％の寒天を加
え、pH5.6に調整した発根培地に移植して、苗条の作出
を行ったのと同様の条件で培養した結果、20日目には完
全な植物体が再生された。しかしながら、従来法では苗
化までの日数が30日であった。Regeneration of plants from shoots Shoots obtained by the above method were added to G5 B5 medium.
After adding 0.01 mg / NAA, 3% sucrose and 0.4% agar and transplanting to a rooting medium adjusted to pH 5.6 and culturing under the same conditions as when producing shoots, 20 Complete plants were regenerated on day one. However, in the conventional method, the number of days until seedling formation was 30 days.
（発明の効果） 本発明によって、有用でかつ優れた遺伝子を持った植
物を、その性質を変異させることなく大量に、また短期
間に、従って安価に作出することが可能になった。すな
わち、苗条原基集塊から苗条の出現までの期間を30％程
度短縮し、また苗条の発生本数も20％程度以上は増大す
る。(Effects of the Invention) According to the present invention, it has become possible to produce a plant having a useful and excellent gene in a large amount, in a short period of time, and at a low cost without mutating its properties. In other words, the time from the shoot root mass to the emergence of shoots is shortened by about 30%, and the number of shoots generated is increased by about 20% or more.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭63−222620（ＪＰ，Ａ) 特開 昭63−301784（ＪＰ，Ａ) 「種苗産業と育種新技術」（昭58−10 −28）シーエムシーＰ．177−183 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-63-222620 (JP, A) JP-A-63-301784 (JP, A) "Seed and Seedling Industry and New Breeding Technology" (SHO-58-10-28) CMC P. 177-183
Claims (4)
イザーにより回転数10,000〜30,000r.p.m.で５〜20秒の
条件下で細片化処理し、次いで細片化された苗条原基集
塊を液体振盪培養することを特徴とする木本性植物由来
の苗条原基集塊を大量に増殖する方法。[Claim 1] A shoot primordium agglomerate derived from a woody plant is fragmented by a homogenizer at a rotation speed of 10,000 to 30,000 rpm for 5 to 20 seconds, and then fragmented A method for proliferating a large amount of shoot primordium agglomerates derived from woody plants, which comprises culturing liquid with shaking.
植物の生長点を含む茎頂部を摘出し、これを無機塩組成
物および植物成長ホルモンを含む液体培地中で光照射下
に回転培養して得られたものである特許請求の範囲第１
項記載の方法。2. A shoot primordium agglomerate derived from a woody plant is extracted from a shoot apex containing a growth point of the woody plant, and is irradiated with light in a liquid medium containing an inorganic salt composition and a plant growth hormone. Claim 1 which has been obtained by spin culturing
The method described in the section.
はムラシゲ・スクーグのMS培地に、植物ホルモンとして
オーキシン類あるいはサイトカイニン類を含む液体培地
で20〜30℃の温度条件、10,000〜20,000ルクスの照度で
12〜16時間の明条件、そして80〜100r.p.m.の振盪速度
で行う特許請求の範囲第１項又は第２項記載の方法。3. Liquid shaking culture in a liquid medium containing G5 or B. medium of Murbige-Skoog and Auxins or cytokinins as plant hormones at a temperature of 20 to 30 ° C. and an illuminance of 10,000 to 20,000 lux.
The method according to claim 1 or 2, which is carried out under light conditions for 12 to 16 hours and at a shaking speed of 80 to 100 rpm.
イザーにより回転数10,000〜30,000r.p.m.で５〜20秒の
条件下で細片化処理し、次いで細片化された苗条原基集
塊を液体振盪培養して苗条原基集塊を大量に増殖し、得
られた苗条原基集塊を苗化培地で培養して苗条化させ、
得られた苗条を発根培地に移植して完全な植物体を再生
することを特徴とする木本性植物由来の苗条原基集塊か
ら植物体を再生する方法。4. A shoot primordium agglomerate derived from a woody plant is fragmented by a homogenizer at a rotation speed of 10,000 to 30,000 rpm for 5 to 20 seconds, and then fragmented. Shaking culture liquid to grow a large amount of shoot primordium agglomerates, and the resulting shoot primordium agglomerates are cultured in a seedling medium to form shoots,
A method for regenerating a plant from a shoot primordium agglomerate derived from a woody plant, which comprises transplanting the obtained shoots to a rooting medium to regenerate a complete plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62204269A JP2562909B2 (en) | 1987-08-19 | 1987-08-19 | A method for multiplying a shoot primordium agglomerate derived from a woody plant from a shoot primordium agglomerate derived from a woody plant, and a method for regenerating a plant from the seed primordium agglomerate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62204269A JP2562909B2 (en) | 1987-08-19 | 1987-08-19 | A method for multiplying a shoot primordium agglomerate derived from a woody plant from a shoot primordium agglomerate derived from a woody plant, and a method for regenerating a plant from the seed primordium agglomerate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6447318A JPS6447318A (en) | 1989-02-21 |
| JP2562909B2 true JP2562909B2 (en) | 1996-12-11 |
Family
ID=16487676
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62204269A Expired - Fee Related JP2562909B2 (en) | 1987-08-19 | 1987-08-19 | A method for multiplying a shoot primordium agglomerate derived from a woody plant from a shoot primordium agglomerate derived from a woody plant, and a method for regenerating a plant from the seed primordium agglomerate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2562909B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4099898B2 (en) | 1999-05-07 | 2008-06-11 | 王子製紙株式会社 | Methods for transforming adult Eucalyptus plants |
| CN104620805B (en) * | 2014-12-23 | 2017-10-13 | 杨光云 | A kind of quick tillering propagation method of Paris polyphylla live body stem tuber |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63222620A (en) * | 1987-03-10 | 1988-09-16 | 湧永製薬株式会社 | Preparation of regenerated plant body |
-
1987
- 1987-08-19 JP JP62204269A patent/JP2562909B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 「種苗産業と育種新技術」（昭５８−１０−２８）シーエムシーＰ．１７７−１８３ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6447318A (en) | 1989-02-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shekhawat et al. | Factors affecting in vitro clonal propagation of Prosopis cineraria | |
| David | In vitro propagation of gymnosperms | |
| JP2970277B2 (en) | A method for promoting rooting of cultured shoots in Rhododendron plants | |
| GB2195656A (en) | Mass propagation through shoot primordia and regeneration of plants from protoplasts of shoot primordia | |
| Matani Borkheyli et al. | In vitro multiplication and rooting of GF677 rootstock | |
| JPH06189643A (en) | Large-scale multiplication of rhododendron | |
| JP2562909B2 (en) | A method for multiplying a shoot primordium agglomerate derived from a woody plant from a shoot primordium agglomerate derived from a woody plant, and a method for regenerating a plant from the seed primordium agglomerate | |
| Giovanelli et al. | Micropropagation of Mediterranean cypress (Cupressus sempervirens L.) | |
| US5310673A (en) | Mass propagation through shoot primordia and regeneration of plants from protoplasts of shoot primordia | |
| JPH0346086B2 (en) | ||
| Sharma et al. | Improved protocol for in vitro propagation of gloxinia (Sinningia sp.) | |
| JP2649641B2 (en) | How to regenerate plants from protoplasts | |
| JP3020207B2 (en) | Propagation method of woody plants | |
| Aboel-Nil | Tissue culture of Douglas-fir and Western North American conifers | |
| JPH03277219A (en) | Tissue culture of rose | |
| JP3621611B2 (en) | Regeneration method of coconut plant | |
| EP4477070A1 (en) | Method for producing multiple shoot cluster and method for producing clonal sapling | |
| JPH0642809B2 (en) | How to make shoot base lumps | |
| Bhattacharya et al. | 2, 4, 5-T induced somatic embryogenesis in papaya (Carica papaya L.) | |
| Goldfarb et al. | Douglas-Fir [Pseudotsuga menziesii (Mirb.) Franco] | |
| JPH05236832A (en) | Shoot primordial conglomerate of arboreous plant and method for creating the same and proliferating arboreous plant with shoot primordial conglomerate | |
| JP2967968B2 (en) | Seedling production method of Cineralia plant | |
| JP2623320B2 (en) | Method of regenerating plant from protoplast of woody plant | |
| Deb et al. | Research Article In vitro Immature Embryo Culture and Propagation of Paphiopedilum villosum Var. Boxallii (Rchb. f.) Pfitzer | |
| JP2024180263A (en) | Method for manufacturing multiple shoots, method for manufacturing clone seedlings |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |