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.


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.


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



(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.