Scottish Rock Garden Club Forum
Specific Families and Genera => Pleione and Orchidaceae => Topic started by: Guus on April 27, 2009, 09:20:26 PM
-
Hi all, last year I made a simple glovebox to be able to sow orchids on the campsite from green pods. From the pictures you can get the idea of how to copy this divice. Don't hesitate to ask further details!
Goodluck!
Greetings, Guus
-
Ooh you are a tease Guus. ;D
-
Hi Guus
I think the reason the pictures are not showing is that your profile is set to private on the 'photo' hosting website.
Dave
-
Thanks David, I have set the pics to 'public' ...pasted the links again.... but still teasing.
I will keep on trying and ask my daughter for help.
Greetings, Guus
-
Thanks David, I have set the pics to 'public' ...pasted the links again.... but still teasing.
I will keep on trying and ask my daughter for help. Greetings, Guus
Please Guus, don't post the link to a photo hosting site, just post the picture here directly.
-
Yrs, Guus, I would advise using the direct method of uploading photos to the Forum by uploading the pix from your own pc files to the page.... this is not at all difficult and has the benefit that if anything goes wrong with your photo hosting site, as has happened with other forumists using, for instance,"Photobucket" in the past, then your photos disappear, never to be seen again.
-
Thanks Maggi, I even did not need the help of my daughter! It won't happen again...
Greetings, Guus
-
Excellent, Guus, well done for solving your difficulties in this way.......and an intriguing invention from you..... I had wondered quite what the "glove box" was meaninhg! Now I see very well, Thanks!
I am sure we would all like to hear more about how exactly to use this device.... ???
-
Hi all, I took the glovebox on holiday as I said. At home I prepared petridishes with medium in the laminar flowcabinet. Packed those tight in the plastic they are in when sold. Put some ethanol in to keep clean. You need scalpel and forceps and a plastic container (icecream-box). A small hand-operated spray. A small plastic container to put chlorine in to sterilise the seedpods. An old toothbrush. A jar with sterilised water. (sterilised in the pressurecoocker)
Cover the bottom of the box with an old towel.
Fill the spray with 1% chlorine in water with a drop dishwash-stuff. Fill the ice-box with 4% chlorine and a few drops dishwash... put all the 'tools' in the box. Don't forget the seedpods! Put those in the 4% chlorine together with the forceps, toothbrush and scalpel.
Close the box and put your hands into the gloves. Take the spray and go around with it in the box to make sure inside everything is in contact with the chlorine (1%). Leave the box for at least 15 min.. Than you can start brushing, rubbing, brushing, ..... until you think the pods are sterile. Then open the jar with sterile water and rinse the pod for a few minutes. Open a petridish and cut the pod open, spread the seed in the dish (if necessary, put a few drops of water in). Use several dishes for one pod to not overpopulate and to spread the risk of infection. If you are done and when you re-packed the dishes, you can open the box again and write details on the pack.
I wish you lots of success!
Greetings, Guus
-
Gus,
Great invention but what is clorine? You cannot mean Cl2 gas??
Göte
-
Gus,
Great invention but what is chlorine? You cannot mean Cl2 gas??
Göte
It will be a sodium hypochlorite (NaClO) solution. Household bleach is 3-6% strength.
-
Very right! That is it. Just buy in the supermarket. GG
-
Thank you both.
The stuff seem sto be by far more common in your places than in mine but I understand it can be bought.
Göte
-
Thank you both.
The stuff seem sto be by far more common in your places than in mine but I understand it can be bought.
Göte
Domestos (http://www.unilever.co.uk/ourbrands/homecare/domestos.asp) is the obvious chlorine bleach in the UK, but I find the cheapies better for this kind of purpose as they wash off more easily.
-
Guus thanks a lot for this explanation. This box can even be used at home in the garage if we do not have a "proper one". This seem to be very good and thanks for explaining all the steps.
Göte I think it is called "klorin" in Sweden, that is the stuff but with maybe a drop of added dishwashing detergent. The pure stuff is used to add wells to kill of the bacteria in all the countries that have "clorinated water". In swedish "natrium hypoklorit" is the name and it might be bought at paint shops or maybe also at stores aimed at farmers. I have not looked after it in Sweden.
Oops Anthony told You similar when I type my answer.
Hope this inspires people to try this!
Best of Luck
Joakim
-
Hi Guus and others who do sterile in-vitro work. I know that this thread has been inactive for years but it has been viewed many times so I thought that it may still be OK to post a reply. I used to use hypochlorite as it is such an efficient desinfectant, but it is also quite a nasty compound from a toxicological point of view. I have therefore made a cheap and simple sterile cabinet for handling my orchid seedlings, so now I only use very small amounts of hypochlorite for sterilising the seeds before plating them. My sterile cabinet was very easy to build, I have given the details here: http://invitroorchids.dk/en_index.html (http://invitroorchids.dk/en_index.html)
Anders
-
Hi Guus and others who do sterile in-vitro work. I know that this thread has been inactive for years but it has been viewed many times so I thought that it may still be OK to post a reply. I used to use hypochlorite as it is such an efficient desinfectant, but it is also quite a nasty compound from a toxicological point of view. I have therefore made a cheap and simple sterile cabinet for handling my orchid seedlings, so now I only use very small amounts of hypochlorite for sterilising the seeds before plating them. My sterile cabinet was very easy to build, I have given the details here: http://invitroorchids.dk/en_index.html (http://invitroorchids.dk/en_index.html)
Anders
Hi Anders, this is very useful. I have many questions: you do not seem to have a door in front of the cabinet, is that right?
What type of container do you use? Small jars? Glass or plastic? With or without holes and ptfe filter?
Also, you do not seem to sterilise the petri dishes after you have put the medium in them .... is that correct?
The other question is about the growing medium: what type of medium do you use for European epipactis and for red flowered Asian cypripedium? Or shall we start a separate thread for this?
Best,
Corrado & Rina
Regards
-
Hi Corrado and Rina
I have tried to give as many details as possible on my homepage so I will only give some short answers here. I am an environmental microbiologist, so the way I grow orchids from seeds is basically the same as growing bacteria in the lab. There is no door in front of the cabinet, only a polycarbonate glass plate (plexiglass) and an 15 cm open slit at the bottom, 15 cm is enough to handle things inside the cabinet and to get things in and out. The flow of sterile air from the air purifier at the top is enough to keep the kabinet sterile. As containers, I use glass jam jars from the kitchen. No holes and no ptfe filters. The jars are sealed with two layers of clingfilm which is permeable to oxygen and ethylene, but not to water molecules. I do sterilize the jars with the medium in them (each covered with tin foil) by cooking them 30 min in a pressure cooker. But that is only when I make few jars, most of the time I sterilize the tinfoil covered jars for 60 min in a 200 degree C oven, sterilize the medium in 0.5-L bottles covered with tin foil - no closed bottles in the pressure cooker!!! - and pour the medium into the jars in the sterile cabinet by gently lifting the foil a few centimeters. I use the same medium for all orchids, my recipe is simple (see my homepage) and based on liquid fertilizer for indoor plants. Some orchids probably grow better on Malmgrens medium with inorganic nitrogen, I guess you already know Malmgrens homepage where you can find his recipe. I haven't had any success with European Epipactis except palustris from very fresh seeds. For the Cyps, I always cross-pollinate two genetically different clones, selfing gives way bad germination and growth for most cyps.
Anders
PS MAlmgren homepage http://www.lidaforsgarden.com/Orchids/engelsk.htm (http://www.lidaforsgarden.com/Orchids/engelsk.htm)
-
Hi Guus and others who do sterile in-vitro work. I know that this thread has been inactive for years but it has been viewed many times so I thought that it may still be OK to post a reply. I used to use hypochlorite as it is such an efficient desinfectant, but it is also quite a nasty compound from a toxicological point of view. I have therefore made a cheap and simple sterile cabinet for handling my orchid seedlings, so now I only use very small amounts of hypochlorite for sterilising the seeds before plating them. My sterile cabinet was very easy to build, I have given the details here: http://invitroorchids.dk/en_index.html (http://invitroorchids.dk/en_index.html)
Anders
Thanks very much for that. Very detailed and I have spent the entire evening reading and attempting to digest it.
You now have me thinking about making and sowing some seed. Something I had never really considered before.
-
Hi Corrado and Rina
I have tried to give as many details as possible on my homepage so I will only give some short answers here. I am an environmental microbiologist, so the way I grow orchids from seeds is basically the same as growing bacteria in the lab. There is no door in front of the cabinet, only a polycarbonate glass plate (plexiglass) and an 15 cm open slit at the bottom, 15 cm is enough to handle things inside the cabinet and to get things in and out. The flow of sterile air from the air purifier at the top is enough to keep the kabinet sterile. As containers, I use glass jam jars from the kitchen. No holes and no ptfe filters. The jars are sealed with two layers of clingfilm which is permeable to oxygen and ethylene, but not to water molecules. I do sterilize the jars with the medium in them (each covered with tin foil) by cooking them 30 min in a pressure cooker. But that is only when I make few jars, most of the time I sterilize the tinfoil covered jars for 60 min in a 200 degree C oven, sterilize the medium in 0.5-L bottles covered with tin foil - no closed bottles in the pressure cooker!!! - and pour the medium into the jars in the sterile cabinet by gently lifting the foil a few centimeters. I use the same medium for all orchids, my recipe is simple (see my homepage) and based on liquid fertilizer for indoor plants. Some orchids probably grow better on Malmgrens medium with inorganic nitrogen, I guess you already know Malmgrens homepage where you can find his recipe. I haven't had any success with European Epipactis except palustris from very fresh seeds. For the Cyps, I always cross-pollinate two genetically different clones, selfing gives way bad germination and growth for most cyps.
Anders
PS MAlmgren homepage http://www.lidaforsgarden.com/Orchids/engelsk.htm (http://www.lidaforsgarden.com/Orchids/engelsk.htm)
Hi Anders,
First of all thanks for the very detailed answer!
I had read you very informative homepage, but this summarizes it even better.
I had downloaded and read your recipe for growing medium and I know Svante's home page and his growing media, and had some email exchanges with Svante. I am interested in reproduction of botanic orchids, in particular red Eurasian Cypripedium and Epipactis, and Svante suggests in his talks that those do not like the pineapple juice in the medium and need specific forms of nitrogen. I was wondering whether you had been able to adapt your recipe or Svante's recipe for those species. From your answer I understand you have had no luck with red Eurasian epipactis. Have you had any luck with red Eurasian Cypripedium?
Best
Corrado
Concerning the medium,
-
Hi Corrado
I haven't tried any of the red Eurasian Cyps (macranthos, smithii, tibeticum, corrugatum, etc.), but if pineapple juice is a problem, I would substitute it with orange juice or simply add pure citric acid (200 mg/L) as a pH buffer. When I add fresh orange juice, I always use organic oranges (and organic potatoes) to avoid any pesticides that may interfere with germination and development of the seedlings. Send me a PM if I should try to germinate some of your seeds next summer :-)
Anders
-
Hi Anders,
We use organic fruit as well, more to avoid pesticides entering us than the growing media ;D ;D ;D ;D.
How do you use citric acid? Is it to lower your solutions' PH because it is too high? And why would you add orange juice, does it contain enzymes? Apologies, questions from a novice!
Regards
-
The addition of fruit juice (mostly orange or pineapple) serves at least four purposes. The most important is to add buffer capacity so that the pH remains constant during seedling growth. Plant cells excrete H+ ions (acid) when they take up nutrients, this means that the medium will soon be very acidic and toxic if there is no pH buffer. Fosfate has some buffer capacity but is relatively small at pH 5.8. Fruit juices contain citric acid and other organic acids that are very efficient buffers at pH 5.8. It is of course very important to adjust the pH of the medium to around 5.8 AFTER adding the fruit juice. The second purpose is to keep micronutrients, especially iron, in solution so that they are available to the plant cells. Citric acid and other organic acids form soluble complexes with iron and other micronutrients. Without the citric acid, iron will precipitate as insoluble iron hydroxides (rust) that the plants cannot take up. The third purpose is that fruit juices contain a complex mixture of growth factors (hormones, vitamins, trace elements etc), but the extra plant hormones are not always beneficial, some species can be very sensitive to the wrong combination of hormones. And least important, both orange juice and pineapple juice contain enzymes (proteases) that hydrolyzes proteins into free amino acids and peptides that can be taken up by the plant as an organic nitrogen source. This effect counts only when there is a protein source the medium, for instance in the commercial modified Malmgrens medium where free amino acids are substituted with peptone (partly hydrolyzed protein) or if blended vegetables are added. The enzymes are only active before the medium is heated in the pressure cooker. Canned orange juice and canned pineapple juice havealso been heated which inactivates the enzymes.
Pheew, this became a little too technical.
Anders
-
Technical ? Maybe. Fascinating? Certainly! :)
-
The addition of fruit juice (mostly orange or pineapple) serves at least four purposes. The most important is to add buffer capacity so that the pH remains constant during seedling growth. Plant cells excrete H+ ions (acid) when they take up nutrients, this means that the medium will soon be very acidic and toxic if there is no pH buffer. Fosfate has some buffer capacity but is relatively small at pH 5.8. Fruit juices contain citric acid and other organic acids that are very efficient buffers at pH 5.8. It is of course very important to adjust the pH of the medium to around 5.8 AFTER adding the fruit juice. The second purpose is to keep micronutrients, especially iron, in solution so that they are available to the plant cells. Citric acid and other organic acids form soluble complexes with iron and other micronutrients. Without the citric acid, iron will precipitate as insoluble iron hydroxides (rust) that the plants cannot take up. The third purpose is that fruit juices contain a complex mixture of growth factors (hormones, vitamins, trace elements etc), but the extra plant hormones are not always beneficial, some species can be very sensitive to the wrong combination of hormones. And least important, both orange juice and pineapple juice contain enzymes (proteases) that hydrolyzes proteins into free amino acids and peptides that can be taken up by the plant as an organic nitrogen source. This effect counts only when there is a protein source the medium, for instance in the commercial modified Malmgrens medium where free amino acids are substituted with peptone (partly hydrolyzed protein) or if blended vegetables are added. The enzymes are only active before the medium is heated in the pressure cooker. Canned orange juice and canned pineapple juice havealso been heated which inactivates the enzymes.
Pheew, this became a little too technical.
Anders
Technical? Just .... perfect!!!! Thanks a lot .... since we are on the subject, I will annoy again .... have you experimented with:
1) different agar content? (as suggested in Ranback 2007)
2) injecting / inoculating the medium with mychorriza (if you can find it)?
3) the effects of organic vs inorganic N?
Best,
-
The agar content should be as low as possible, but the gel should also be strong enough to support the seedlings. The brand I use (from a Chinese food store) is good, 5 g/L is sufficient. For other brands, it may be necessary to add 7 or 8 g/L.
I have never tried any of the fungi that are reported in the scientific literature. I have isolated a few strains from the inside of Dactylorhiza roots, but the result on germination was disappointing. For the symbiotic methods you would need a completely different medium based on oat meal that supports growth of the fungus, and some of the good fungal lab-strains.
I have done some testing with different N-sources. Some species seem to grow better on inorganic N, others grow better on organic N, but in most cases I get an OK result with the combination of dilute inorganic fertilizer and partly hydrolysed protein from potatoes. I have done quite a lot of experimenting with different sources of peptone (partly hydrolysed protein) and the amino acid glutaminate. The problem with peptone is that most sources have a high salt content where especially the chloride is toxic for the seeds. Glutaminate can be bought in any super market as the third spice. Initially, I had good results with glutaminate but it seems that the quality (purity) varies a lot from brand to brand and even between batches of the same brand. In some of my testings, glutamate had a very negative effect, probably because of high salts contents, I have therefore totally given up on glutaminate. Next summer, I will do some testing of laboratory grade glycine which is the most simple of the amino acids and the one with the highest N-content.
Anders
-
The agar content should be as low as possible, but the gel should also be strong enough to support the seedlings. The brand I use (from a Chinese food store) is good, 5 g/L is sufficient. For other brands, it may be necessary to add 7 or 8 g/L.
I am using this in my experiments, [attach=1] again, organic, from seaweed and reasonably cheap, and it is the same one I use for cooking. It helps when you leave one jar in the kitchen cupboard for too long, so it expires and you can then move it to "the other cupboard" without any feeling of guilt or family side effects .... :)
I was wondering whether you had seen this:
Teˇšitelová, T., Teˇšitel, J., Jersáková, J., Rˇíhová, G., Selosse, M.-A., 2012. Symbiotic germination capability of four Epipactis species (Orchidaceae) is broader than expected from adult ecology. Am. J. Bot. 99, 1020–1032. doi:10.3732/ajb.1100503
and this:
Shefferson, R.P., Kull, T., Tali, K., 2008. Mycorrhizal interactions of orchids colonizing Estonian mine tailings hills. Am. J. Bot. 95, 156–164. doi:10.3732/ajb.95.2.156
Best,
Corrado