I am working on tobacco and tomato plant tissue culture. one variety of tobacco giving very good result in regeneration.
other variety causing some problem when it start shoot regeneration. its leaves were very thick and when transfer into 1/2MS it dos not produce roots.
I am uploading a picture of my plant it will help you to understand the problem.
From the look of the leaf appearance, it looks like that your shoots/leaves have experienced 'hyperhydricity' (another word for it is 'vitrification'). Hyperhydricity is the excessive hydration of tissue cultured plant tissues. The leaves of such plant usually have translucent appearance with thick, brittle leaves. Probably this tobacco variety is sensitive to high humidity in the tissue-culturing boxes.
Please see the paragraph (5.10.2) from this book at link below:
https://books.google.com/books?id=AmSpCwAAQBAJ&pg=PA124&lpg=PA124&dq=transparent+leaves+in+high+humidity+plant+tissue+culture+vessels&source=bl&ots=433hk5fS7A&sig=8uHWe1cIAOeD58H1GlWUYHk-UVE&hl=en&sa=X&ved=0ahUKEwim54qWmdTSAhVCwiYKHcsQAkAQ6AEIOzAJ#v=onepage&q=transparent%20leaves%20in%20high%20humidity%20plant%20tissue%20culture%20vessels&f=false
This is a paper for you:
"Flooding of the apoplast is a key factor in the development of hyperhydricity"
1. Use the liquid medium.
2. Increase the sugar percentage in media are vital for root initiation and growth.
3- Induction of roots from shoots in ½ MS medium containing 0.5 mg/L IBA OR Induction of roots on hormones free media
For more details , you can see the following links:
file:///C:/Users/pc/Downloads/5873-21125-1-PB.pdf
http://scialert.net/fulltext/?doi=biotech.2007.561.566
There are more info and references from Wiki. The sites include some good publications. https://en.wikipedia.org/wiki/Hyperhydricity
Hyperhydricity (previously known as vitrification) is a physiological malformation that results in excessive hydration, low lignification, impaired stomatal function and reduced mechanical strength of tissue culture-generated plants. The consequence is poor regeneration of such plants without intensive greenhouse acclimation for outdoor growth [1]. Additionally, it may also lead to leaf-tip and bud necrosis in some cases, which often leads to loss of apical dominance in the shoots [2]. In general, the main symptom of hyperhydricity is translucent characteristics signified by a shortage of chlorophyll and high water content. Specifically, the presence of a thin or absent cuticular layer, reduced number of palisade cells, irregular stomata, less developed cell wall and large intracellular spaces in the mesophyll cell layer have been described as some of the anatomic changes associated with hyperhydricity [3].
I agree its due to hyperhydricity. I think growing these in little more salt concentration may help.
more salt concentration means only micro and macro nutrients or all components of MS
Did you use 1x or 1/2x for 'shoot' formation for this tobacco variety (I know you use 1/2x for root induction)?
We also use 3M MicroporeTMsurgical tape (not parafilm) to wrap our plant tissue-culturing plate and boxes. Those tapes improve air exchange, which reduce the humidity inside the vessels. However, the tapes are more costly than parafilm. See attachments (1. 3M Micropore tape, 2. parafilm).
If you google it with keywords "3M Micropore tape for plant tissue culture", you will find quite a few papers describing using it.
There are also so-called 'venting' vessels for plant tissue culture. See attached picture. On the vessels, there is a microfilter for air exchange in and out of the vessel.
http://www.tissuequickplantlabs.com/amdpg5c.htm
http://www.tissuequickplantlabs.com/andpccpgc.htm
This is another type of 'venting' vessel. I grabbed the picture from this researcher's post (see below link). You should read the story about his experience on venting issue of plant tissue culture boxes (link below).
http://www.phalaenopsis.net/phpBB/viewtopic.php?f=13&t=2574&start=15
Thank you for pictures and I used 1x for shoot formation and 1/2x for root formation..
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