NEWS NO.3

　　In Aug., 2002, I found the very intersting article as follows in magazine New Orchids NO. 121 published by Shinkikaku publishing com. The title of article is ' The specilal report, we have succeeded paph. armeniacum and micrnthum seedlings inflasks artificially ' by Murayama Agricultural high school in Yamagata Ken. In Dec. 2001, they reported ' Kakiran seedling has arrived at PLM ' also in orchid magazine. I'll introduce the article now.

We have succeeded the sterile seedlings of some Paphs which is said to be very difficult it artificially.

Murayama Agricultural High School N. Saitho & M. Misawa (Teachers)
11 students of the 3rd year of Bio-technology Class including Mr. M. Takahashi.

NEW Orchids, NO.121 p48. Shinkikaku Publishing Com.

＜Sunnary＞

We have succeeded to propergated seedlings of some Paphs, Paph. armeniacum and micranthum which is said to be very difficult to make sterile seedlings in flask artificially. The culture medium including hormones must be effective for growing them.

＜Materials＞

Unmatured seeds of Paph. armeniacum and micranthum : The healthy plants were selected to be made cross. After about 6 months after crossing, the seeds were used for the following experiments.

The seed pod harvested about 6 or 8 months after crossing.

The seeds harvested in a glass dish.

Culuture Medium：
LS Medium　(1/3 or 1/2 dilution)、White Vitamin solution 、BA(Wako Chemical Com.)、NAA(Wako Chemical Com.)、Sucrose、Glafite(Wako Chemical Com.)、Gelangum(Wako Chemical Com.)、Hyponex　(6.5:6:19)、Peptone(BACTO)、myo-inositol(Wako Chemical Com.)、Thiamine-HCl(Wako)、Coconut water (Biochemical reserch Lab, Japan )

Medium for seedlings
・A：LS Medium
LS Medium　(1/3dilution)、White Vitamin solution　0.5mg/L、BA　2.0mg/L、NAA　0.1mg/L、sucrose 15g/L、Glafite　1g/L、gelangum　2.0g/L、ｐH　6.1
・B：hyponex Medium
ハイポネックス　(6.5:6:19)　1g/L、peptone　1g/L、BA　2.0mg/L、NAA　0.1mg/L、myo-inositol　0.033g/L、Thiamine-HCl (100ppm)　1.3ml/L、Sucrose　15g/L、Glafite　1g/L、gelangum 　2.0g/L、ｐH　6.0
・Liquid medium for seedling　
LS Medium　(1/3 dilution)、White Vitamin solution　0.5mg/L、BA　2.0mg/L、NAA　0.1mg/L、Sucrose　15g/L、ｐH　6.0

Medium for passage
・A：LS Medium
LS Medium　(1/3〜1/2 dilution)、BA　2.0mg/L、NAA　0.1mg/L、Thiamine - HCl　0.2mg/L、myo-inositol　0.05g/L、Sucrose　12g/L、Coconut water　150ml/L、Glafite　1g/L、Gelangum　1.8g/L、ｐH　6.0
・B：Hyponex Medium
Hyponex　(6.5:6:19)　2g/L、peptone　1g/L、Sucrose　20g/L、Menedale　1ml/L、BA　2.0mg/L、NAA　0.1mg/L、myo-inositol　0.05g/L、Thiamine = HCl(100ppm)　1.3ml/L、Glafite 　1g/L、Gelangum　2.0g/L、ｐH　6.0

Seedling

＜Methids＞

(1) sterilization
・ Unmatured seeds 6 months after crossing ： The seed pod was not broken. The seed pod was sreilized in NaHCl2 ( 1% ) and a drop of Tween 20 for 10 min. The seed pod was transfered in a sterile dish and was cut by a sterile knife. Seeds were sowing on a medium.
・ Unmatured seeds 8 months after crossing： The seed pod was broken already or near braking. The seeds were harvested in a few sterile tubes from pod and sterilized in 10 ml of NaHCl2 ( 1% ) and a drop of Tween 20 for 5 min. After throwing out the solution, the seeds wrrte washed with 3 ml of the medium solution for seedling. The seeds were sonicated for 5 min.

(2) Protocome's growing on medium agar
　Seeds innflask was covered by tin foil and was kept in dark at 18 ~ 16 C. The seeds from 8 month seee pod sawed in medium solution was shaked at 55 rpm. The medium was exchanged every 15 days.

The protocomes were recognized after about 40 ~ 50 days.

(3)Passage of protocomes
　The protocomes were passaged in good timing. The medium for passage was the same as one for seedling basically. The seedlings in medium solution were transfered on agar medium. In this time, the excessive medium on agar was thrown by pipetting. In order to check effetive medium , two or three times dilution of LS Medium was used for experiments. The seedlings were growings at 3000〜4500 lux.


　After 5 months starting from culture in dark, protocomes whose diameter was about 5mm were observed. The protocomes were transferred to a new culture agar medium and moved to the new condition at 22℃ and at 3000〜4500 lux. By light, they will developed their leaves and then extend roots.

In flask, the leaves developed of 1 cm N.S. and extended roots strongly. The young seedlings were transferred to a new agar culture medium.
　
　　When N.S. of young seedlings became about 2〜4cm, they were transferred to 6 cm pots. In good situation, it will take about 1 and 8 months or 2 years until de flasking. Corrugated cardboard cut in small pieces by a shredder. Cut corrugated cardboard was laid about 5 ~ 10 mm in depth and over laid Akatama-do, Kanuma-tuchi, sphagnum moss and Keisan=hakudo. Be careful whem they will be transfered because their roots were not so amny and were vaey easy to be taken off.

(4) Results and Conclusion
　The doiluted 2 or 3 times LS medium was better for their growing compared with theusual medium used to use when they were germinated before. And LS Medium was better than Hyponex Medium. Protocomes were observed about 60days after seedling. On the other hand, it was about 45〜50 days after seedling when the seedlings in olution mediumi ? Be care shaken. The protocomes continued to grow in dark further a few months and transfered to a new agar madium. It will be about 6 months afater sawing. When the protocomes continued to keep in darekfurther, their color will be turned to brown suddenly and be die. when the protocomes were trwnsferd to higher density of medium like from 1/3 diluted to 1/2 diluted medium. But sometimes, someprotocomes were developed so larger than we expected. 1/3 diluted LS Medium was desided as our standar ones. In this medium, the seedlings were grown better in higer efficience. When the higher density of medium was used, the seedlings became unusuall.The young seedlings in pots were developed in good and be expected to flower in near future.

Comment form Dr. Tanaka　In Paphiopedilum germination artifically, any species will not grow in the same situation. especially in paph. species, we can not expect the same results from the same species and the same methods even if weパtried to the same species orthe same clones. Sometimes we will get the larger amounts of seedlings orsometime not. On the other hand, some species germinated in good effiency or others not so. Especially, Paph . armeniacum and micranthum is ones of the most difficult to germoinate in flask, we say around the world. ESometimes. we could get a few plantlets but not so many.

界各国のラン仲間と議論されたことが幾度もあったが、低温処理や暗黒環境での培養などのアイデアは出されたものの効果的ではなかった。しかし、頻繁に行われた議論のなかで培地についてほとんど話題にならなかった。なぜなら、ランの播種培地は糖と基本的な塩が含まれた培地で十分であるという共通の認識があったからだ。この報告で、まず驚いたことは、あまり議論されなかった培地の組成が大きく異なっていたことである。ここでは、植物組織の培養培地を基本としてBA(ベンジルアデニン)やNAA(ナフタリン酢酸)等のホルモンを含んでいる。未成熟の胚を育てるのだから、組織培養が基本となるのは当たり前のような話であるが、目から鱗ともいえるこの内容は、我々が固定された概念に捕らわれていたことを深く反省させるものである。そのような意味では、画期的な方法と行っても過言ではないだろう。この内容は、以前、パフィオペディルムのメリクロンが成功したという情報を得たとき、培地には通常の何倍ものホルモンが含まれていたことに驚いたことを思い出させる。パフィオペディルムの胚の生育条件は、他のランとは大きく異なるようだ。この情報をもとに、多くの方々がミクランサムやアルメニアカムの播種に成功できることが期待され、これらの原種が自由に増殖できるばかりでなく、優良な個体が作出されるようになるだろう。さらにこの成功により、自然からの希少なランの違法な採集を止めることに大きく貢献することを信じてやまない。iパフィオペディルムの播種で、特に原種においては、全てが同じ方法で、同じように発芽することはない。まず、国内で種子の形成においても、種子が採れやすいものと採れにくいものがある。また、播種しても、発芽しやすいものと発芽しにくいものもある。そのなかでも、ミクランサムとアルメニアカムについては、良好にフラスコ苗が採れたという話を聞いたことがない。ヨーロッパ、ＵＳＡ，アジア（日本と台湾）などで、同様の結果が聞かれる。全くゼロというわけではないのだが、十分な種子の形成が確認されていても、種子から苗が順調に生育することが極めて少ない。世界各国のラン仲間と議論されたことが幾度もあったが、低温処理や暗黒環境での培養などのアイデアは出されたものの効果的ではなかった。しかし、頻繁に行われた議論のなかで培地についてほとんど話題にならなかった。なぜなら、ランの播種培地は糖と基本的な塩が含まれた培地で十分であるという共通の認識があったからだ。この報告で、まず驚いたことは、あまり議論されなかった培地の組成が大きく異なっていたことである。ここでは、植物組織の培養培地を基本としてBA(ベンジルアデニン)やNAA(ナフタリン酢酸)等のホルモンを含んでいる。未成熟の胚を育てるのだから、組織培養が基本となるのは当たり前のような話であるが、目から鱗ともいえるこの内容は、我々が固定された概念に捕らわれていたことを深く反省させるものである。そのような意味では、画期的な方法と行っても過言ではないだろう。この内容は、以前、パフィオペディルムのメリクロンが成功したという情報を得たとき、培地には通常の何倍ものホルモンが含まれていたことに驚いたことを思い出させる。パフィオペディルムの胚の生育条件は、他のランとは大きく異なるようだ。この情報をもとに、多くの方々がミクランサムやアルメニアカムの播種に成功できることが期待され、これらの原種が自由に増殖できるばかりでなく、優良な個体が作出されるようになるだろう。さらにこの成功により、自然からの希少なランの違法な採集を止めることに大きく貢献することを信じてやまない。

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