JPH0440839A - Culture of large amount of adventitious embryo - Google Patents
Culture of large amount of adventitious embryoInfo
- Publication number
- JPH0440839A JPH0440839A JP2147293A JP14729390A JPH0440839A JP H0440839 A JPH0440839 A JP H0440839A JP 2147293 A JP2147293 A JP 2147293A JP 14729390 A JP14729390 A JP 14729390A JP H0440839 A JPH0440839 A JP H0440839A
- Authority
- JP
- Japan
- Prior art keywords
- embryos
- culture
- embryo
- shaped
- differentiation
- 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.)
- Pending
Links
- 210000001161 mammalian embryo Anatomy 0.000 title abstract description 32
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 85
- 238000012258 culturing Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 230000000392 somatic effect Effects 0.000 claims description 42
- 210000004748 cultured cell Anatomy 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 12
- 230000006698 induction Effects 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 abstract description 42
- 241000196324 Embryophyta Species 0.000 abstract description 13
- 241001233957 eudicotyledons Species 0.000 abstract description 3
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 20
- 244000000626 Daucus carota Species 0.000 description 6
- 235000002767 Daucus carota Nutrition 0.000 description 6
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 2
- 239000003375 plant hormone Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Pretreatment Of Seeds And Plants (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は植物組織培養による双子葉植物の大量増殖方法
に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for mass propagation of dicotyledonous plants by plant tissue culture.
双子葉植物の不定胚に関して最も知見が多く、研究の進
んでいるニンジンを例にとると、培養細胞（カルス）誘
導用の培地、すなわち植物ホルモンとしてオーキシンの
１つである２、４Ｄ（２，４−ジクロロフェノキシ酢酸
）を含む培地で誘導、増殖した培養細胞を不定胚分化用
の培地、すなわち植物ホルモンを含まない培地で培養す
ることにより誘導できることはよく知られていることで
ある。Taking carrot as an example, which has the most knowledge about somatic embryos of dicotyledonous plants and which is the most advanced in research, we use 2, 4D (2, 4D, It is well known that cultured cells induced and proliferated in a medium containing (4-dichlorophenoxyacetic acid) can be induced by culturing in a medium for somatic embryo differentiation, that is, a medium that does not contain plant hormones.
このとき不定胚分化の最終段階である魚雷型胚への分化
率、すなわち用いた培養細胞に対して得られる魚雷型胚
数は他の双子葉植物に比べ分化し易いとされているニン
ジンでも約半数で残りの不定胚は魚雷型胚への発達途中
である球状型胚や心臓型胚の状態にある。また、魚雷型
胚への分化にはニンジンでおよそ２週間を要し、ニンジ
ン以外の双子葉植物では分化に要する期間も最短でも２
週間もしくはこれ以上である。At this time, the differentiation rate into torpedo-shaped embryos, which is the final stage of somatic embryo differentiation, that is, the number of torpedo-shaped embryos obtained for the cultured cells used, is about 100% even in carrots, which are said to be easier to differentiate than other dicotyledonous plants. Half of the embryos and the remaining somatic embryos are in the state of globular or heart-shaped embryos, which are in the process of developing into torpedo-shaped embryos. In addition, differentiation into a torpedo-shaped embryo takes about two weeks for carrots, and for dicotyledonous plants other than carrots, the minimum time required for differentiation is about two weeks.
Weeks or more.
双子葉植物から不定胚を誘導する方法は先に示したよう
に公知であるが、この方法では（１）魚雷型胚にまで分
化する割合は分化開始時に用いた培養細胞のうちの約半
数以下であるため、魚雷型胚への分化効率が低かった。As mentioned above, the method of inducing somatic embryos from dicotyledonous plants is well known, but in this method (1) the proportion of cells that differentiate into torpedo-shaped embryos is approximately less than half of the cultured cells used at the start of differentiation; Therefore, the efficiency of differentiation into torpedo-shaped embryos was low.
（２）培養を開始してから一定の時期に魚雷型胚のみを
集めようとすると不定胚への分化が非同調的に起こるた
め、種々の発達段階の不定胚が混在した中から（＝球状
型胚や心臓型胚が混在した中から）魚雷型胚をメツシュ
（網）などでふるい分ける等の手段により選別する必要
があった。(2) If you try to collect only torpedo-shaped embryos at a certain time after starting the culture, differentiation into somatic embryos will occur asynchronously, so from a mixture of somatic embryos at various developmental stages (= spherical It was necessary to select torpedo-shaped embryos (from a mixture of heart-shaped and heart-shaped embryos) by sifting through a mesh.
不定胚は産業上、種苗生産などとくに人工種子の作製に
利用されるが、このとき上記のような不定胚発達の制御
、すなわち均一な不定胚を一定の時期にし功も大量に得
ることや不定胚の成長度を揃えるなどの技術は極めて重
要であるにもかかわらず、殆ど知られていないのが現状
である。Somatic embryos are used industrially for the production of seedlings, especially for the production of artificial seeds. Although techniques such as adjusting the growth rate of embryos are extremely important, little is currently known about them.
上述した人工種子とは茎頂や胚のよう植物体へ再生し得
る組織をアルギン酸等のゲル化剤でカプセル状に埋包し
たもので、用いる組織としては再生能力の点から不定胚
が最も有力とされている。The artificial seeds mentioned above are tissues that can be regenerated into plants, such as shoot tips and embryos, which are embedded in a capsule shape with a gelling agent such as alginic acid, and somatic embryos are the most likely tissue to be used in terms of regeneration ability. It is said that
本発明者は双子葉植物の不定胚分化にはその発達に培地
量当りの培養細胞数又は不定胚数、すなわち培養密度が
極めて大きく影響することを以下に示すことから確認し
た。The present inventor has confirmed from the following that the number of cultured cells or the number of somatic embryos per medium volume, that is, the culture density, has a very large influence on the development of somatic embryo differentiation in dicotyledonous plants.
〔１〕不定胚分化の同調化を図るに当っては、まず成長
度に差が生ずる原因を考察した。培養細胞から魚雷型胚
へ至る分化の培養においてその変化を経時的に観察、把
握し、下記のことが明らかになっかだ。[1] In attempting to synchronize somatic embryo differentiation, we first considered the causes of differences in growth rate. By observing and understanding changes over time in the culture of differentiation from cultured cells to torpedo-shaped embryos, we have clarified the following.
（１）培養細胞から球状型胚への発達までは用いた培養
細胞の約９０％が分化し、しかも成長度が揃っているこ
と。(1) Approximately 90% of the cultured cells used should be differentiated until they develop into spherical embryos, and the growth rate should be uniform.
（２）不定胚分化の後期すなわち球状型胚から魚雷型胚
への発達の段階ではｐＨが培養開始時の５．７０から６
．７１にまで上昇していること。(2) In the later stages of somatic embryo differentiation, that is, the stage of development from globular to torpedo-shaped embryos, the pH changes from 5.70 at the start of culture to 6.
．． It has risen to 71.
これらの結果から成長度に差が生ずる原因として次のこ
とを予想した。Based on these results, we predicted the following as the cause of the difference in growth rate.
■ 培養終了後に球状型胚或いは心臓型胚の状態にある
不定胚は分化能が低下もしくは死滅している。(2) Somatic embryos that are in the state of globular or heart-shaped embryos after completion of culture have decreased differentiation ability or are dead.
■ 用いる培地中の栄饗が不足し、全ての不定胚が魚雷
型胚にまで発達できない。■ Not all somatic embryos can develop into torpedo-shaped embryos due to lack of fertilization in the medium used.
■ 培養中ｐＨの上昇が不定胚の分化を阻害する。■ Increase in pH during culture inhibits differentiation of somatic embryos.
■ 発達の速い不定胚が成長を阻害する物質を培地中に
放出する。■ Rapidly developing somatic embryos release substances into the medium that inhibit their growth.
〔２〕予想した各項目が原因であるか否かを確認・する
ため各実験を実施し、下記のことが分かった。[2] We conducted experiments to confirm whether each predicted item was the cause or not, and found the following.
■ 不定胚分化のため培養の終了後、球状型胚の状態に
ある不定胚を集め新しい培地で培養したところ、約３割
が心臓型胚に残りの約７割が魚雷型胚へ分化した。従っ
て培養終了後に球状型胚や心臓型胚の状態にある不定胚
は分化能を消失したり、死滅したりしたわけではないこ
と。■ After the completion of culture for somatic embryo differentiation, the somatic embryos in the globular state were collected and cultured in a new medium, and about 30% differentiated into heart-shaped embryos and the remaining 70% into torpedo-shaped embryos. Therefore, the somatic embryos that are in the state of globular or heart-shaped embryos after the completion of culture have not lost their differentiation potential or died.
■ 不定胚分化のための培養の中間、すなわち球状型胚
が得られた時点で、これを新しい培地に移し換えて培養
した。その結果、魚雷型胚への分化率は僅かに７％上が
っただけであり、ｐＨの変化も開始時５．７０に対し５
．８８であった。従って上記〔１〕の■■■の予想が直
接の原因とは考え難いと判断した。■ In the middle of the culture for somatic embryo differentiation, that is, when a globular embryo was obtained, it was transferred to a new medium and cultured. As a result, the differentiation rate into torpedo-shaped embryos increased by only 7%, and the pH change also increased from 5.70 at the start to 5%.
．． It was 88. Therefore, we judged that it is difficult to think that the prediction of ■■■ in [1] above was the direct cause.
［３］ ［２）ｌまでは不定胚分化のための培養のう
ち培地中の栄養源、ＰＨなど化学的な要因について検討
したが、次にこれ以外に考えられる要因として物理的な
要因を検討することにより原因を考察した。[3] [2] Up to I, we considered chemical factors such as nutrients and pH in the culture medium during culture for somatic embryo differentiation, but next we will consider physical factors as other possible factors. The cause was investigated by doing this.
現状の方法では不定胚分化のための培養において培養細
胞の密度は、培養細胞の体積１に対して体積４００〜１
０００倍の体積の培地、もしくは培地１艷に対し培養細
胞５００個である。しかし、これら密度が不定胚分化に
与える影響についての知見はこれまで知られておらず、
そこで密度が与える影響について検討した。In the current method, the density of cultured cells in culture for somatic embryo differentiation is 400 to 1 volume per volume of cultured cells.
000 times the volume of the medium, or 500 cultured cells per medium. However, there is no knowledge of the effects of these densities on somatic embryo differentiation.
Therefore, we investigated the influence of density.
不定胚分化のための培養において高い培養密度、例えば
１０００〜２０００個／ｄで培養すると用いた培養細胞
の約９０％が球状型胚、心臓型胚へ分化した。このとき
球状型胚と心臓型胚が混在した状態でそのまま培養を続
けたが、これ以上分化しなかった。この結果から培養細
胞から魚雷型胚への発達においては培養密度が極めて大
きく影響することを知った。When cultured for somatic embryo differentiation at a high culture density, for example 1,000 to 2,000 cells/day, about 90% of the cultured cells used differentiated into globular or heart-shaped embryos. At this time, culture was continued with a mixture of globular and heart-shaped embryos, but no further differentiation occurred. From this result, we learned that culture density has a very large effect on the development of torpedo-shaped embryos from cultured cells.
さらに培養開始時の培養細胞の大きさ及び魚雷型胚の大
きさから、それぞれ両者の体積を比較したところ、魚雷
型胚のそれは培養細胞のおよそ２０倍であることを知っ
た。従って不定胚が分化するにつれて培地中に占める組
織体の体積が大きくなることからも不定胚分化のための
培養において培養密度が重要であることをさらに認識し
た。Furthermore, by comparing the volumes of the cultured cells and the torpedo-shaped embryos at the start of culture, it was found that the volumes of the torpedo-shaped embryos were approximately 20 times larger than the cultured cells. Therefore, as the somatic embryo differentiates, the volume of the tissue body that occupies the medium increases, and we further recognized that culture density is important in culturing for somatic embryo differentiation.
本発明は以上の知見に基いて完成されたもので、双子葉
植物の一部から培養細胞を誘導し、この誘導細胞を培養
して球状型胚、心臓型胚、魚雷型胚を発達させて不定胚
を分化させる不定胚の大量培養方法において、上記誘導
細胞を高密度状態で培養して球状型胚と心臓型胚を混在
した状態で得た後、同球状胚と心臓型胚の混在物を低密
度状態で培養して魚雷型胚へと発達させることを特徴と
する不定胚の大量培養方法である。The present invention was completed based on the above findings, and involves inducing cultured cells from a part of a dicotyledonous plant, and culturing these induced cells to develop globular-shaped embryos, heart-shaped embryos, and torpedo-shaped embryos. In a method for mass culturing somatic embryos for differentiating somatic embryos, the above-mentioned induced cells are cultured at high density to obtain a mixture of globular and heart-shaped embryos, and then a mixture of globular and heart-shaped embryos is obtained. This is a method for mass culturing somatic embryos, which is characterized by culturing them at low density and developing them into torpedo-shaped embryos.
本発明は双子葉植物の培養細胞を不定胚分化用の培地で
高い培養密度、例えば２０００個／ｉで培養し約９０％
の分化率で球状型胚、心臓型胚を得た後、これらを低い
培養密度、例えば以下の実施例で示すように１５０個／
ａ１！以下で培養すにようにした双子葉植物の不定胚の
大量培養方法である。In the present invention, cultured cells of dicotyledonous plants are cultured in a medium for somatic embryo differentiation at a high culture density, for example, 2000 cells/i.
After obtaining globular and heart-shaped embryos with a differentiation rate of , these were cultured at a low culture density, e.g.
a1! This is a method for mass culturing somatic embryos of dicotyledonous plants as described below.
本発明は不定胚誘導用の培地で培養細胞を誘導・増殖し
、これを例えば２０００個１ｍもの高密度で不定胚分化
用の培地を用いて不定胚を分化させる技術は従来方法に
よるが、不定胚が球状型胚、心臓型胚へ発達した時点で
これらを回収し、低密度例えば１５０個／ｍｌで培養す
ることにより、これ以降魚雷型胚への発達を同調化でき
る。In the present invention, cultured cells are induced and proliferated in a medium for somatic embryo induction, and the somatic embryos are differentiated at a high density of, for example, 2,000 cells per meter, using a medium for somatic embryo differentiation. By collecting the embryos when they have developed into globular-shaped embryos or heart-shaped embryos and culturing them at a low density, for example, 150 embryos/ml, the subsequent development into torpedo-shaped embryos can be synchronized.
分化の初期過程すなわち培養細胞から球状型胚付近まで
は従来の技術によって高頻度に分化させることは可能で
あるが、本発明方法を用いることにより分化の後期すな
わち魚雷型胚まで同調化を図ることができ、しかも用い
た培養細胞のおよそ８０％近い高頻度で分化できる。さ
らに培養期間すなわち分化に要する日数は従来に比べ短
縮できる。なお、得られた魚雷型胚を光照射下で培養す
ると幼植物体を得ることができる。Although it is possible to perform high-frequency differentiation from the early stage of differentiation, that is, from cultured cells to the vicinity of a globular-shaped embryo, by using the method of the present invention, it is possible to achieve synchronization up to the late stage of differentiation, that is, a torpedo-shaped embryo. Moreover, it can differentiate at a high frequency of approximately 80% of the cultured cells used. Furthermore, the culture period, ie, the number of days required for differentiation, can be shortened compared to conventional methods. Note that by culturing the obtained torpedo-shaped embryo under light irradiation, young plants can be obtained.
以下双子葉植物の１つであるニンジンを例にとり実施例
を説明する。用いたニンジンの品種は黒田五寸であり、
この種子を０．８％の寒天を含む培地で発芽させ、約１
週間後に得られた幼植物体の下胚軸を約１ ｃｍの大き
さに切断した。Examples will be described below using carrot, which is one of the dicotyledonous plants, as an example. The variety of carrots used was Kuroda Gosun.
These seeds were germinated in a medium containing 0.8% agar, and approximately 1.
The hypocotyls of the seedlings obtained after a week were cut into pieces of about 1 cm in size.
この下胚軸を第１表に示すような５Ｘ１０（Ｍ）の２．
４− Ｄを含むＬｉｎ ＆ ５ｔａｂａの改変培地（以
下ＬＳ培地）で培養し、培養細胞を誘導した。なお、培
養は暗黒下で温度２５℃とした（以下同じ）。This hypocotyl was divided into 2.5 x 10 (M) cells as shown in Table 1.
Cultured cells were induced by culturing in a modified Lin & 5taba medium (hereinafter referred to as LS medium) containing 4-D. The culture was carried out in the dark at a temperature of 25°C (the same applies below).
得られた培養細胞のうち３０〜５０μｍの大きさの培養
細胞を２０００個／ｒｎｌの培養密度で植物ホルモンを
含まないＬＳ培地で培養した。Among the obtained cultured cells, cultured cells with a size of 30 to 50 μm were cultured at a culture density of 2000 cells/rnl in LS medium containing no plant hormones.
なお、培養は３００ｍ１容フラスコに培養液８〇−であ
った。培養を開始して７日後、培地１ｍｌ当り球状型胚
、心臓型胚がそれぞれ１０７４個、７１８個得られた。The culture was carried out in a 300ml flask with 80ml of culture solution. Seven days after the start of culture, 1074 and 718 globular-shaped embryos and 718 heart-shaped embryos were obtained per ml of culture medium, respectively.
このときの分化率は９０％であった。The differentiation rate at this time was 90%.
得られた球状型胚及び心臓型胚を５０〜５００個／ｒｎ
ｌの培養密度でＬＳ培地で培養した。その結果、第２表
に示すように培養密度１５０個／ｄ以下では用いた球状
型胚、心臓型胚は魚雷型胚へ９０％という高頻度で分化
した（第２表二分化率４）の欄参照）。しかもこの結果
は、例えば５日後、６日後にそれぞれ分化した魚雷型胚
を合わせて９０％というのではなく、培養開始から５日
後という一定時期に得られた分化率であり同調的に分化
させることができた。50 to 500 globular and heart-shaped embryos/rn
The cells were cultured in LS medium at a culture density of 1. As a result, as shown in Table 2, when the culture density was below 150 cells/day, the spherical and heart-shaped embryos used differentiated into torpedo-shaped embryos at a high frequency of 90% (bidifferentiation rate of 4 in Table 2). (see column). Furthermore, this result does not represent a total of 90% of torpedo-shaped embryos differentiated after 5 and 6 days, for example, but represents a differentiation rate obtained at a fixed time, 5 days after the start of culture, and indicates that synchronous differentiation is achieved. was completed.
次に最初に用いた単位培地量＜＝ ｉ ｙｔｅ＞当りの
培養細胞数に対して得られた魚雷型杯数及び分化率も各
培養密度について第２表に併せて示した（それぞれ全魚
雷型杯数５）、培養細胞に対する分化率６）の欄参照）
１５０個／ｍ７！以下では用いた培養細胞をおよそ
８０％近い高頻度で目的とする魚雷型胚へ分化させるこ
とができた。Next, the number of torpedo-shaped cups and differentiation rate obtained for the number of cultured cells per unit volume of culture medium used initially are also shown in Table 2 for each culture density (respectively, all torpedo-shaped cups See columns for number of cups 5) and differentiation rate for cultured cells 6))
150 pieces/m7! In the following, the cultured cells used could be differentiated into the desired torpedo-shaped embryos at a high frequency of approximately 80%.
なお、得られた魚雷型胚を不定胚の分化に用いたものと
同じ培地を用いて光照射下で培養すると幼植物体が得ら
れた。In addition, when the obtained torpedo-shaped embryos were cultured under light irradiation using the same medium used for differentiation of somatic embryos, seedlings were obtained.
表−１
ｉｎ
ｔａｂａ
の改変培地
〔発明の効果〕
双子葉植物の不定胚、ここでは不定胚分化の最終段階で
ある魚雷型胚を大量に得るに当たって
（１）用いる培養細胞を魚雷型胚へ高頻度で分化できる
ため、増殖した培養細胞を無駄なく有効に利用できる。Table 1 Modified culture medium for in taba [Effects of the invention] In order to obtain a large amount of somatic embryos of dicot plants, here, torpedo-shaped embryos, which are the final stage of somatic embryo differentiation, (1) the cultured cells used are raised to torpedo-shaped embryos; Since it can be differentiated at high frequency, the proliferated cultured cells can be used effectively without waste.
（２）成長度の揃った魚雷型胚を得ることができるた杓
、とくにこれのみを回収することなしにそのまま利用で
きる。例えば人工種子の生産においてはとくに魚雷型胚
のみを選別する工程が省け、直接カプセル化の工程へ移
ることができる。(2) A ladle that can obtain torpedo-shaped embryos with uniform growth, and in particular can be used as is without having to be recovered. For example, in the production of artificial seeds, the step of selecting only torpedo-shaped embryos can be omitted, and the process can proceed directly to the encapsulation step.
（３）分化に要する期間が従来よりも短く、培養期間が
短縮できるため不定胚の生産効率を上げられる。(3) The period required for differentiation is shorter than conventional methods, and the culture period can be shortened, so the production efficiency of somatic embryos can be increased.
Claims (1)
を培養して球状型胚、心臓型胚、魚雷型胚を発達させて
不定胚を分化させる不定胚の大量培養方法において、上
記誘導細胞を高密度状態で培養して球状型胚と心臓型胚
を混在した状態で得た後、同球状胚と心臓型胚の混在物
を低密度状態で培養して魚雷型胚へと発達させることを
特徴とする不定胚の大量培養方法。In a method for mass culturing somatic embryos, in which cultured cells are induced from a part of a dicotyledonous plant, and the induced cells are cultivated to develop globular-shaped embryos, heart-shaped embryos, and torpedo-shaped embryos to differentiate into somatic embryos, the above-mentioned induction Cells are cultured at high density to obtain a mixture of globular and heart-shaped embryos, and the mixture of globular and heart-shaped embryos is then cultured at low density to develop torpedo-shaped embryos. A method for mass culturing somatic embryos, characterized by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2147293A JPH0440839A (en) | 1990-06-07 | 1990-06-07 | Culture of large amount of adventitious embryo |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2147293A JPH0440839A (en) | 1990-06-07 | 1990-06-07 | Culture of large amount of adventitious embryo |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0440839A true JPH0440839A (en) | 1992-02-12 |
Family
ID=15426934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2147293A Pending JPH0440839A (en) | 1990-06-07 | 1990-06-07 | Culture of large amount of adventitious embryo |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0440839A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5776999A (en) * | 1994-09-06 | 1998-07-07 | Ciba Vision Corporation | Methods of using and screening extended wear ophthalmic lenses |
-
1990
- 1990-06-07 JP JP2147293A patent/JPH0440839A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| BIOCHEM PHYSIOL PFLANZEN * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5776999A (en) * | 1994-09-06 | 1998-07-07 | Ciba Vision Corporation | Methods of using and screening extended wear ophthalmic lenses |
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