in vitro first sprout
 

sharing with everyone my low tech set up and my first in vitro neps seeds germination. seeds sterilisation was using clorox and media was home made.

contamination rate was very high for me. There are several reason - from the glass hood ( fish aquarium ) construction method, sterilisation and handlings.

http://i191.photobucket.com/albums/z...u/daf47e92.jpg

jars and containers were placed on window edge where it was bright and warm
http://i191.photobucket.com/albums/z...u/7de3b187.jpg


still no germination
http://i191.photobucket.com/albums/z...u/0997f19c.jpg


the first sprout among all the jar*biggrin2*
http://i191.photobucket.com/albums/z...u/e2f1fc85.jpg

Re: in vitro first sprout
 

can share the advantage using the vitro? i only know that it will grow much faster
BTW, i like this picture, look like a laboratory labhttp://i191.photobucket.com/albums/z...u/daf47e92.jpg

Re: in vitro first sprout
 

wow.. going to next level.. good luck on your new project !

Re: in vitro first sprout
 

where did u find Agar?

Re: in vitro first sprout
 

Wow, nice setup!

Re: in vitro first sprout
 

yeah allen it was a mini lab*biggrin2*. There are many reason if ones searched, as an example :
http://www2.ulg.ac.be/cedevit/english/advantage_e.htm

hee..hee not that fast. I'm still a new hand in this field. Just curious and to try out the media preparation and sterilization method.

Harris the agar² can be bought from any grocery stores or where those specialised in selling ingredients for cakes making.

Thanks rhealk

Re: in vitro first sprout
 

thanks robert.do pm ME hw u sterilize the media and hw u prepare or make the media and hw do u sterilize the seeds.i just won some seeds from the flytrapcare.com.so i wanna try them in vitro.i just need to learn the preparation of media(sterilizng too),and deflasking it

Re: in vitro first sprout
 

Harris, it's fine to answer yor question here since i searched all the info online. There are many, read through, pick up the details and note down.

Media preparation and sterilising procedure were similar to orchid seeds.

http://www.youtube.com/watch?v=TYz77RXs9Ho

http://www.flytrapcare.com/tissue-culture-basics.html

http://www.phytotechlab.com/Technica...e%20Techniques

Instead of using autoclave i use home pressure cooker to cook the media for 20 minutes ( take the reading once the pressure built up ). Dont use/share the pr.cooker for home food preparation. Get a new one. There are many brand to choose. A China made cost approx RM130.00 which, like most for home use, will achieve 15 psi. Any less cooking have to be adjusted.

Cooked media in jar - leave them for 7 days or longer to make sure no mold form.

If still have doubt please post here so every member can discuss.

Re: in vitro first sprout
 

Wow, great effort, Robert. Perhaps, someday later you can market your 'sealed-pre-repared-in-vitro-jar' for commoners like me who want to try in vitro but do not have the expertise and equipments (and patients!).
Thank you for sharing.

Re: in vitro first sprout
 

Hi Robert. Congratulations! Hey, you (and others) might want to check out;
http://www.hometissueculture.org
A friend of mine in the US just took one of their workshops and was impressed. I've seen the website for years but never got into it myself. Carol, who started it is great and Frank (president) has a whole series of Nep TC videos on you-tube as well. The also sell/ship supplies of all sorts.

Best of luck with the rest!
~Shawn

Re: in vitro first sprout
 

Thanks guys!if i go on TC,i will inform.

Re: in vitro first sprout
 

Thanks Shawn. I was in hometissueculture.org and mostly gathering for info*biggrin2*

Re: in vitro first sprout
 

Robert,where did u find the MS vitamins,etc etc?

Re: in vitro first sprout
 

most can be bought from scientific co. There should be plenty around your area.

Re: in vitro first sprout
 

is DIY store a scientific company?can u named me some Scientific co. or stores in Malaysia?

Re: in vitro first sprout
 

i will PM you some addresses

Re: in vitro first sprout
 

pls pm me also

Re: in vitro first sprout
 

khoon pmed you

Re: in vitro first sprout
 

Nice, hopefully you'll gained in experience to archieve much better success rate, I'm sure you will :smile:

Re: in vitro first sprout
 

yeah, i hope and to anyone who is into it.

Re: in vitro first sprout
 

Nice... :) But I don't think its worth doing in-vitro germination of neps, considering that the natural germination rate on moss is quite high anyway...

Re: in vitro first sprout
 

but it get matures fast!

Re: in vitro first sprout
 

and can avoid the seeds from getting mold that kill your seedlings....

Re: in vitro first sprout
 

errr.. if it's not contaminated

Re: in vitro first sprout
 

Actually,TC is usualy used to make hybrids bcoz some plants take a long time to mature by seeds and they're rare.if Lim can Establish on TC,we can get tons of great VFTs!especially those rare cultivar that is hard to find in Malaysia.TC also can be a business starter.

Re: in vitro first sprout
 

Robert congratulations! Did you see the info in kitchenTissueculture or hometissueculture about an additive that is supposed to help fight contaminants? It is called PPM and allows us to do TC at home, I have the kitchen culture kit but still unfortunately no time to dive into trying out TC myself :) So I'm happy that some of our brothers... like you are getting into it.

To others, please be informed that, though one can actually put additives that will allow for faster growth, the main purpose of TC is not really for faster growth but for faster propagation.

If Robert used 10 seeds and 1 germinated, it is not much use to grow this in TC and then plant it outside eventually. As some people mentioned you don't get the advantage, you have the same rate sowing them directly on potting medium.

But because that 1 plant that germinated is sterile, Robert can actually cut it up into several piece, add additive that will make the pieces sprout into new plants themselves.. so if you have 1 plant, you can get say 3 to 5, and the 3 to 5 plants can then be cut up and thus the multiplication factor increases! This is quite nice for rare plants. Which reminds me, Robert what species of seeds did you use? I mean don't get me wrong, once I get to try TC, I will try it out with not so rare seeds first.

Of course VFT is way easier, just get one leaf, disinfect it like crazy, and cut up and then put it in an additive and you should have a lot of VFTs soon. It's Nepenthes that is a big challenge.... good luck Robert!

Hopefully next time you can try out seeds of N. Attenboroughii :)

TTFN
Arvin

Re: in vitro first sprout
 

hey Robert,wanna try VFT seeds on TC?

Re: in vitro first sprout
 

Robert, sorry for bothering, but would you mind PM me the information as well? i am getting interested in TC.....
and may i know so far how much had you spent in this? i mean from TC until the plant can transplant into normal media...thankyou

Re: in vitro first sprout
 

every 4-6weeks,u have to change the media bcoz the plant already absorbed the minerals in it.thats the hardest for me.easy get infected by Germs.a media can only be used around 1month++

Re: in vitro first sprout
 

IMHO, its just not economically worth the investment in creating a setup to germinate nepenthes seeds in vitro because fungal/bacterial contamination is bound to occur while replating (changing media in flasks due to runout of nutrients) and after deflasking anyway. In fact, flask-grown seedlings are more difficult to establish once out of flasks (I'm speaking for orchids, which are similar) because of the drastic environment change (~99% humidity to much lower) and also change of medium. If germinate directly on moss, the seedlings wold have already sent out roots into the sphag and can be transplanted with less shock.

Re: in vitro first sprout
 

u can make them root fast by putting a few of the rooting hormone that u can obtain at local store.not just fast,but the roots will be crowded with each to other strands :)

Re: in vitro first sprout
 

Hi Arvin, i've read in most post ppm was highly recommended. I have problem getting most chemical locally. Suppliers are not keen for small purchase. In most cases it had to be flown from the peninsular, and costly. Thus i use clorox and spirit which turn out as good, and inexpensive. The only problem was the wide margin of error. contamination,i read , can happen due to the age of spirit and clorox. using these to sterilise seeds ones have to adjust the time to sterilise seeds.

thanks Harris, if you have some to spare*biggrin2*

the detail from my pmed, you need to call or email them for the items you required.

Re: in vitro first sprout
 

a great project....your tools are very nice

Re: in vitro first sprout
 

if i really can spare vft seeds, will you try?

Re: in vitro first sprout
 

Hi Robert,

From what I understand, PPM is an additive to the Agar itself, and prevents contamination on the medium... contamination of your Tissue is of course through sterilization such as your mentioned Chlorine, some actually say Hydrogen Peroxide, etc. I also think that once the Tissue is in the TC medium, the PPM will also probably protect it from Contamination as well.

I have a feeling that Kitchen Tissue Culture will be able to ship directly to you, if you order from them. That would already be as simple or as complicated as buying from Peninsular Malaysia.

Just my 2 cents here, I think that because VFT leaves are easy to disinfect... no need to start from seeds, you can just cut a leaf or two and that is the start of your Tissue, just disinfect the leaves or pieces properly. At least this way, no waiting for germination. As mentioned the only CP that is difficult to TC are Neps, because apparently getting Tissues from nepenthes plants and successfully and effectively sterilizing them is almost impossible.

So if you don't mind, and have the time.... May I challenge you or request you to please experiment by cutting up one of your more common nepenthes plant (just a stem cutting don't worry) and try to get a Meristem from it and then try to get that going in TC medium.

TTFN
Arvin

Re: in vitro first sprout
 

Re: in vitro first sprout
 

Thanks allen someone had sent me sarras and vft seeds.

Re: in vitro first sprout
 

if u have success on VFTs and Sarrs,u can sell them!

Re: in vitro first sprout
 

Robert, Aww oh come on! Try Meristem! Cut a piece of nepenthe stem, then sterilize... Then cut off a few layers and then sterilize, until you get to the middle part of the stem (meristem) then sterilize and then TC :) I heard that they said that the hard part is to get a sterile sample... I don't know if that is accurate, I didn't hear anyone say that the tissue does not work, it's that usually you usually find that the Tissue gets contaminated. But as I said that is based on what I heard or read. I might have had some discussion about it here somewhere.

In any case for nepenthes, the norm is to TC the plants that sprout from seeds, at least those are sterile.

TTFN
Arvin

Re: in vitro first sprout
 

maybe one day when i've developed the skilled with all the sophisticated chemicals available i would give a try.

Re: in vitro first sprout
 

orchid in vitro propagation-my successful trial with hand pollination of cross between phalaenopsis bellina x phalaenopsis cornu cervi. It took about 9 months for the seed pods to ripe but uncrack. I only need to sterilise the outer skin by dipping in ethanol, and flame over the burner.

formation of protocorms
http://i191.photobucket.com/albums/z...u/c9c80c75.jpg

2 months later after replating to several jars.
http://i191.photobucket.com/albums/z...u/b6698daf.jpg

Re: in vitro first sprout
 

Congrats on your achievement. I cant even get my phalaenopsis to flower, cnt provide the low temp drop. Any tips?

Re: in vitro first sprout
 

how do you grow your phal ... do you grow them in pot, what media do you use, or they are mounted on wood/fern bark?), they are grown under nursery shade?

Re: in vitro first sprout
 

Grow in pot and under shade. Media i experiment with all charcoal and also all sphag moss, both also the same for me.

Re: in vitro first sprout
 

why not try mounting on wood or fern bark for a change. I have the same problem in the past until i mount them on fern bark. I also fertilise them with mild orchid fertilizer once a week. Light water misting every morning and evening.