tobacco tissue culture - (Jul/29/2008 )
Hi everyone
I want to start a new project in my lab and unfortunately no one in the lab has any experience with this-----so maybe someone here could help me. I am interested in isolating tobacco prtoplasts so that I can transform them (electroporate) to study a specific protein. I want to greminate tobacco seedlings aseptically for friable callus production so that I then can start a suspension culture and ultimately isolate protoplasts from it.
1. Is this recipe OK? The gernimation media recipe: MS with 2% sucrose, thiamine, pyridoxine, nicotinic acid, 2-(N-morpholino) ethanesulphonic acid MES ph 5.7 (w 1MKOH), 0.8% agar----autoclave
2. I have a protocol to aseptically germinate seedling but once they germinate and grow I am confused as to what explant to use for a friable callus----some protocols say that specific plant parts will produce frianble callus ( ie leaf petiole or the leaf itself) others say to generate friable callus you should alter what is in the growth medium (ie friable: kinetin and 2,4-D compact: 2-IP and IAA). So I'm not sure what I have to do.
3. If I need leaf petiole should I germinate seeds in a petri dish then transfer the seedlings to an aseptic growth container with different recipe for growth and the transfer the explant back to a petri dish for callus production
thanks for any info
Lisa
I want to start a new project in my lab and unfortunately no one in the lab has any experience with this-----so maybe someone here could help me. I am interested in isolating tobacco prtoplasts so that I can transform them (electroporate) to study a specific protein. I want to greminate tobacco seedlings aseptically for friable callus production so that I then can start a suspension culture and ultimately isolate protoplasts from it.
1. Is this recipe OK? The gernimation media recipe: MS with 2% sucrose, thiamine, pyridoxine, nicotinic acid, 2-(N-morpholino) ethanesulphonic acid MES ph 5.7 (w 1MKOH), 0.8% agar----autoclave
2. I have a protocol to aseptically germinate seedling but once they germinate and grow I am confused as to what explant to use for a friable callus----some protocols say that specific plant parts will produce frianble callus ( ie leaf petiole or the leaf itself) others say to generate friable callus you should alter what is in the growth medium (ie friable: kinetin and 2,4-D compact: 2-IP and IAA). So I'm not sure what I have to do.
3. If I need leaf petiole should I germinate seeds in a petri dish then transfer the seedlings to an aseptic growth container with different recipe for growth and the transfer the explant back to a petri dish for callus production
thanks for any info
Lisa
1. Your recipe is similar to what we use but doesn't have inositol which is usually included
2. We get friable callus tissue by transferring explants (petioles, etc) onto MS media containing 2,4-D and kinetin.
3. Germinate the seedlings in a growth container on MS media and transfer explants to petri dish for callus induction using modified MS media.
Thank you
I want to start a new project in my lab and unfortunately no one in the lab has any experience with this-----so maybe someone here could help me. I am interested in isolating tobacco prtoplasts so that I can transform them (electroporate) to study a specific protein. I want to greminate tobacco seedlings aseptically for friable callus production so that I then can start a suspension culture and ultimately isolate protoplasts from it.
1. Is this recipe OK? The gernimation media recipe: MS with 2% sucrose, thiamine, pyridoxine, nicotinic acid, 2-(N-morpholino) ethanesulphonic acid MES ph 5.7 (w 1MKOH), 0.8% agar----autoclave
2. I have a protocol to aseptically germinate seedling but once they germinate and grow I am confused as to what explant to use for a friable callus----some protocols say that specific plant parts will produce frianble callus ( ie leaf petiole or the leaf itself) others say to generate friable callus you should alter what is in the growth medium (ie friable: kinetin and 2,4-D compact: 2-IP and IAA). So I'm not sure what I have to do.
3. If I need leaf petiole should I germinate seeds in a petri dish then transfer the seedlings to an aseptic growth container with different recipe for growth and the transfer the explant back to a petri dish for callus production
thanks for any info
Lisa
1. Your recipe is similar to what we use but doesn't have inositol which is usually included
2. We get friable callus tissue by transferring explants (petioles, etc) onto MS media containing 2,4-D and kinetin.
3. Germinate the seedlings in a growth container on MS media and transfer explants to petri dish for callus induction using modified MS media.