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post #1 of 12 (permalink) Old 04-16-2013, 06:20 PM Thread Starter
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Boredom turned productive

I want to start this out by saying something: if you do not enjoy science, and by science I mean detailed research, experimentation, and speculation upon current theory, stop reading now.

I went ahead and filed a grant proposal for a research project I have been considering for months now. The title is "Stomatic Hybridization of Anubais Nana sp. 'White'". The goal of this project is to create a new hybrid species of anubais, one that displays both the color of A. Nana "white" and the size of normal Barteri. As a side project I will also attempt to create a hybrid anubias coffeefolia that maintains its dark brown coloration (not sure how I plan on doing this, so it's on the back burner for now).

Within 2 weeks I should hear back on my proposal and go from there; in the mean time I plan on acquiring my specimens (hopefully without spending a small fortune) and finishing my final draft of the procedure and controls.

Following this I will attempt to do some work with buce and possibly erio provided I cannot find a way to permanently stabilize the dark coloration of anubais coffeefolia. Should I find a way to stabilize the coloration of coffeefolia I will first attempt to darken the color to as close as is possible to black, then attempt to cross it to anubais nana sp. "white".

Hopefully this will be a productive and worthwhile research project, and hopefully I will soon have more anubais sp. white than I know what to do with.

Basic overview:
Stage 1: attempt to successfully propagate anubais nana sp. white and anubais Barteri via tissue culturing. Upon successful tissue culture procedure being established (as proven by the existance of successful cultures) proceed to stage 2.
Stage 2: Attempt Stomatic hybridization via the introduction of cell wall disolving enzyme(s) to samples of both Anubais Barteri and anubais nana sp. "white" and the combination of samples through a number of different methods (not sure which method(s) I will use yet).
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post #2 of 12 (permalink) Old 04-16-2013, 06:36 PM
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This sounds like a great project. Definitely interested to see how it turns out.

So you're planning on incorperating genes from the one species into the genome of the other? Are you targeting DNA or RNA?

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post #3 of 12 (permalink) Old 04-16-2013, 06:46 PM Thread Starter
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Originally Posted by Clemsons2k View Post
This sounds like a great project. Definitely interested to see how it turns out.

So you're planning on incorperating genes from the one species into the genome of the other? Are you targeting DNA or RNA?
The process of Stomatic hybridization saves me much of the trouble that comes with what most people consider "gene splicing". Stomatic hybridization at its most basic level is using cellulase to disolve the cell walls of plants, then forcing these two cells to fuze their membranes via electric shock or chemical manipulation. Stomatic hybridization does not focus upon forcing individual strands of DNA or RNA but instead focusing on combining whole cells, which then are bound within a single cell wall and reproduce asexually.

The benefit of Stomatic hybridization is that there is no need to consider sexual compatibility or even sterility a roadblock.
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post #4 of 12 (permalink) Old 04-16-2013, 07:10 PM
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Originally Posted by pmcarbrey View Post
The process of Stomatic hybridization saves me much of the trouble that comes with what most people consider "gene splicing". Stomatic hybridization at its most basic level is using cellulase to disolve the cell walls of plants, then forcing these two cells to fuze their membranes via electric shock or chemical manipulation. Stomatic hybridization does not focus upon forcing individual strands of DNA or RNA but instead focusing on combining whole cells, which then are bound within a single cell wall and reproduce asexually.

The benefit of Stomatic hybridization is that there is no need to consider sexual compatibility or even sterility a roadblock.
Oh ok I gotcha. That makes it heck of a lot easier. Let the cells do the work of combining their genes. I've seen similar techniques done in bacterial cells during school.

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post #5 of 12 (permalink) Old 04-16-2013, 07:16 PM Thread Starter
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The techniques are actually remarkably similar. Plant cells devoid of a cell wall (protoplasts) have a proclivity for combining with other protoplasts. The cell wall can then be regenerated using hormone treatments and the cell will reproduce asexually from that point given it has favorable conditions.
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post #6 of 12 (permalink) Old 04-16-2013, 07:19 PM
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Umm... intrigued. The Wanna be Scientist in me will watch eagerly.
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post #7 of 12 (permalink) Old 04-16-2013, 07:20 PM
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Interesting project. Subscribed.


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post #8 of 12 (permalink) Old 04-16-2013, 07:24 PM Thread Starter
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Assuming I do manage to successfully created a hybrid sp. I have the sneaking suspicion that all I get at first will be a stolon with 1-2 leaves. The medium in which I plan on growing along with the excess of light (16h cycles) may encourage growth patterns resulting in few leaves but large central growth. Not that it matters.
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post #9 of 12 (permalink) Old 04-16-2013, 07:27 PM
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Originally Posted by pmcarbrey View Post
The techniques are actually remarkably similar. Plant cells devoid of a cell wall (protoplasts) have a proclivity for combining with other protoplasts. The cell wall can then be regenerated using hormone treatments and the cell will reproduce asexually from that point given it has favorable conditions.
Yea their plasma membranes are pretty much made of the same thing. Its amazing how the extreme hydrophobic nature of phospholipids will make them self assemble without any help in solution.

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post #10 of 12 (permalink) Old 04-16-2013, 07:31 PM
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where did you submit the grant proposal?
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post #11 of 12 (permalink) Old 04-16-2013, 10:35 PM
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Somatic Hybridization will be a fascinating project. Which enzyme do you plan to use to hydrolyze the cell membrane isolating the protoplast? When isolated why not use a Polyethlylene glycol rather than electro fusion? I think anubias is a good choice for macrophytes since it's easily grown emersed. This sounds very interesting. Sorry for all the questions. I've only read about this. Never had the opportunity to ask someone that is doing it.
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post #12 of 12 (permalink) Old 04-17-2013, 01:39 AM Thread Starter
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Originally Posted by Zorfox View Post
Somatic Hybridization will be a fascinating project. Which enzyme do you plan to use to hydrolyze the cell membrane isolating the protoplast? When isolated why not use a Polyethlylene glycol rather than electro fusion? I think anubias is a good choice for macrophytes since it's easily grown emersed. This sounds very interesting. Sorry for all the questions. I've only read about this. Never had the opportunity to ask someone that is doing it.

My first attempt will be done using just cellulase, theoretically I see no reason why cellulase would not work, but theory and practice don't always match up perfectly, hence the (s) after enzyme. if need be i will experiment with other enzyme treatments, but I hope that will be unnecessary.

PEG is one method I am considering, this is another theory meets practice moment; Theoretically I should not even need to use electrofusion techniques, the protoplasts should combine on their own, the electrofusion is just intended to expedite the process. I plan on keeping procedure as simple and "fool proof" as possible; I am a firm believer of the best procedure being the simplest and most concise possible!
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