Glad to see I'm not the only one using tobacco plants for their landscapingAnd if we make a "bigger" tobacco, then I may need a taller step ladder (with hand rails).
Garden variety allo-tetraploid tobacco (2015).
Bob
Oldfella
Well-Known Member
Why not just lean out of the window? It's safer than the ladder.And if we make a "bigger" tobacco, then I may need a taller step ladder (with hand rails).
Garden variety allo-tetraploid tobacco (2015).
Bob
Oldfella
Tutu
Well-Known Member
It’s been a long time since I started this thread and it kept me busy for a good while. Bob, thanks for opening the thread once again. The idea of making a cross had me very excited for a while but i was too optimistic. At the time i thought that by making a large number of attempts it would sooner or later work out. I guess that i turned from optimist to realist and will now go down a different path.
I have ordered half a laboratory setup. Microscope, still air box, Petri dishes, scalpels, tweezers and a whole bunch of other items.
The plan is as follows. I will make a sexual cross of N. rustica x N. tabacum. Rather than waiting for sheer luck (all previous attempts) i will harvest immature seeds early. I will then grow them in vitro in an MS medium with sugar and agar.
I am sure i will still need to make many attempts before I’ll succeed, but in theory this has a much higher success rate compared to what I’ve been doing before.
After growing various samples in vitro, hopefully i will be able to bring some specimen to adulthood. The next step would be to then get offspring from the plant which will involve some other steps. Earlier in the thread you guys mentioned using cholchicine for doubling chromosomes.
My plan is to get my hands on another herbicide for doing that, Oryzalin.
We’re still far away from those later stages. For now I’m excited for planning the setup of my provisional lab and which crosses i will be attempting. My main goal would be to cross CT Broadleaf as the paternal line and probably go with Aztec, Istela Puebla and Limonka as the maternal lines.
Stay tuned!
I have ordered half a laboratory setup. Microscope, still air box, Petri dishes, scalpels, tweezers and a whole bunch of other items.
The plan is as follows. I will make a sexual cross of N. rustica x N. tabacum. Rather than waiting for sheer luck (all previous attempts) i will harvest immature seeds early. I will then grow them in vitro in an MS medium with sugar and agar.
I am sure i will still need to make many attempts before I’ll succeed, but in theory this has a much higher success rate compared to what I’ve been doing before.
After growing various samples in vitro, hopefully i will be able to bring some specimen to adulthood. The next step would be to then get offspring from the plant which will involve some other steps. Earlier in the thread you guys mentioned using cholchicine for doubling chromosomes.
My plan is to get my hands on another herbicide for doing that, Oryzalin.
We’re still far away from those later stages. For now I’m excited for planning the setup of my provisional lab and which crosses i will be attempting. My main goal would be to cross CT Broadleaf as the paternal line and probably go with Aztec, Istela Puebla and Limonka as the maternal lines.
Stay tuned!
Tutu
Well-Known Member
With some delay on the original schedule, I was finally able to get to it.
Getting the N. rusticas I was growing to flower at the same time as N. tabacum was hard.
Then once they were overlapping, I was away and couldn't get it done.
But I finally had Istela Puebla and Corojo x Sancti Spíritus flowering.
Five days ago after getting back from a trip to Connecticut I managed to open up some young unripe rustica flowers.
I've taken polen from the tabacum and gotten it on there fresh.
This first batch is a bit of a trial. I did cut off all other ripe rustica flowers to avoid cross polination.
With hindsight I should continue to clip flowers maturing days after the fact as well, to avoid chances of cross polination.
So this first batch isn't risk free, but this first batch is mostly set to try out the set-up I have made for early ovule rescuing.
We will get to that in the next post!
* Istela Puebla, it's a stalk grown from a sucker, the original plant I suckered and it had decent size leafs
* Corojo x Sancti Spíritus is also a plant grown from a sucker, the original harvest is still hanging under the stairs and is mostly cured
* Clipped open rustica flowers with stigmas ready to receive some tabacum pollen
* I realize this is tough to make photos off and get into the right perspective
Getting the N. rusticas I was growing to flower at the same time as N. tabacum was hard.
Then once they were overlapping, I was away and couldn't get it done.
But I finally had Istela Puebla and Corojo x Sancti Spíritus flowering.
Five days ago after getting back from a trip to Connecticut I managed to open up some young unripe rustica flowers.
I've taken polen from the tabacum and gotten it on there fresh.
This first batch is a bit of a trial. I did cut off all other ripe rustica flowers to avoid cross polination.
With hindsight I should continue to clip flowers maturing days after the fact as well, to avoid chances of cross polination.
So this first batch isn't risk free, but this first batch is mostly set to try out the set-up I have made for early ovule rescuing.
We will get to that in the next post!
* Istela Puebla, it's a stalk grown from a sucker, the original plant I suckered and it had decent size leafs
* Corojo x Sancti Spíritus is also a plant grown from a sucker, the original harvest is still hanging under the stairs and is mostly cured
* Clipped open rustica flowers with stigmas ready to receive some tabacum pollen
* I realize this is tough to make photos off and get into the right perspective
Tutu
Well-Known Member
So we are five days ahead of time, the ideal day count to rescue unmature ovules from rustica x tabacum cross.
After reading more and more research I have concluded that it is not possible to make this cross naturally.
The cross can be made, and embryos might develop in the ovules, but the plant will abort them.
Therefore, they need to be taken out and grown in a medium outside of the plant.
Most information I've gotten from this paper by N. Shizukuda and T. Nakajima which is called;
Production of lnterspecific Hybrids between Nicotiana rustica L.and N. tabacum L. through Ovule Culture
So over the past weeks I have bought items to get to a somewhat decent setup to take out the ovules and grow it on a medium.
I've used a Murashige & Skoog (MS) Basal Medium called M519, in combination with lab grade agar powder, distilled water and sugar.
* My still air box setup
* After having sterilized all material in a pressure cooker
* First opened the ovaries with a scalpel and then work out the ovules with a pincer
* They weren't hard to find
* It looks a bit slaugheterd but most of the ovules look healthy on all three flowers
* Now the waiting game begins....
After reading more and more research I have concluded that it is not possible to make this cross naturally.
The cross can be made, and embryos might develop in the ovules, but the plant will abort them.
Therefore, they need to be taken out and grown in a medium outside of the plant.
Most information I've gotten from this paper by N. Shizukuda and T. Nakajima which is called;
Production of lnterspecific Hybrids between Nicotiana rustica L.and N. tabacum L. through Ovule Culture
So over the past weeks I have bought items to get to a somewhat decent setup to take out the ovules and grow it on a medium.
I've used a Murashige & Skoog (MS) Basal Medium called M519, in combination with lab grade agar powder, distilled water and sugar.
* My still air box setup
* After having sterilized all material in a pressure cooker
* First opened the ovaries with a scalpel and then work out the ovules with a pincer
* They weren't hard to find
* It looks a bit slaugheterd but most of the ovules look healthy on all three flowers
* Now the waiting game begins....
Tutu
Well-Known Member
Time for an update on this project as we are a month and a half ahead in time.
I have made a series of attempts at what I described in the above post.
I have varied in several aspects, but never with too many parameters at once.
I rescued ovules at 4 days, 5 days, and 6 days, even though the literature is clearly indicating 5.
The other aspect where I have varied is the amount of MS Medium, the amount of sugar, and agar.
To be completely honest, after trying different things a handful of times without any result, I decided to give in for now.
I am moving to a different apartment in a few weeks/months and wanted to terminate all tobaccos I had growing.
I have a pretty bad flea beetle infestation so I need to rest the soil a bit before moving and setting up again.
That includes discarding the remaining rustica and tabacums for the above experiment.
Even though I did decide to get rid of the plants, I did not yet throw out the petri dishes.
I had not seen any development apart form some mold in a few of them.
But I also couldn't get myself to completely abandon the project just yet.
Today, before going to work, I had a quick look at the petri dishes.
And to my complete surprise, find something "green looking" under the parafilm.
At first I thought it must be some kind of mold developing, but upon closer inspection, we have a single ovule that started developing.
Now of course its very tempting to upon up the dish, but I will refrain from that now.
The medium should have sufficient nutrients to continue to grow for a bit.
I have now placed the dish in a room that has some more indirect sunlight, which should help.
Within the petri dish there is no sign of any contamination, so it should be a safe space for now.
This is very encouraging and unexpected. Will keep you posted!
Below a series of foto's.
* The almost abandoned collection of petri dishes
* The first notion of something green growing inside, hardly visible because of the parafilm
* After pulling away the parafilm, a better visible sprout
* Difficult to see, but it has its first two true leafs and a little root sprouting
I have made a series of attempts at what I described in the above post.
I have varied in several aspects, but never with too many parameters at once.
I rescued ovules at 4 days, 5 days, and 6 days, even though the literature is clearly indicating 5.
The other aspect where I have varied is the amount of MS Medium, the amount of sugar, and agar.
To be completely honest, after trying different things a handful of times without any result, I decided to give in for now.
I am moving to a different apartment in a few weeks/months and wanted to terminate all tobaccos I had growing.
I have a pretty bad flea beetle infestation so I need to rest the soil a bit before moving and setting up again.
That includes discarding the remaining rustica and tabacums for the above experiment.
Even though I did decide to get rid of the plants, I did not yet throw out the petri dishes.
I had not seen any development apart form some mold in a few of them.
But I also couldn't get myself to completely abandon the project just yet.
Today, before going to work, I had a quick look at the petri dishes.
And to my complete surprise, find something "green looking" under the parafilm.
At first I thought it must be some kind of mold developing, but upon closer inspection, we have a single ovule that started developing.
Now of course its very tempting to upon up the dish, but I will refrain from that now.
The medium should have sufficient nutrients to continue to grow for a bit.
I have now placed the dish in a room that has some more indirect sunlight, which should help.
Within the petri dish there is no sign of any contamination, so it should be a safe space for now.
This is very encouraging and unexpected. Will keep you posted!
Below a series of foto's.
* The almost abandoned collection of petri dishes
* The first notion of something green growing inside, hardly visible because of the parafilm
* After pulling away the parafilm, a better visible sprout
* Difficult to see, but it has its first two true leafs and a little root sprouting
Kushtaka
Member
I really like this project. I hope you are able to continue after your move.
Tutu
Well-Known Member
Thanks Kushtaka! I definitely intend on continuing.
I have a feeling that this one hybrid might not make it on its own.
Reading the literature, typically there's a high rate of hybrids that don't develop properly.
I think to have a higher chance of success, I will need far more hybrids.
So once I've moved and have settled down, this should be a next goal.
This first specimen gives me the confidence that I can get it done.
It's probably the difference between continuing and giving up haha.
So for as long as it lasts, let's focus on attempting to get this little seedling to adulthood.
It has been 5 days since I have discovered it and it's been growing slowly.
It's currently located in a room with a lot of light during the day, without receiving direct sunlight.
It's growth is a bit blocked by the top of the pedri dish.
This doesn't help it get shaped but on the other hand, it can grow a bit larger.
Soon it will need to go from a sterile environment to a non-sterile one so the larger it is, the better.
I am thinking about transplanting it soon, one of these days.
Would like to hear what you guys think. Wait a bit longer or transplant now?
I have a feeling that this one hybrid might not make it on its own.
Reading the literature, typically there's a high rate of hybrids that don't develop properly.
I think to have a higher chance of success, I will need far more hybrids.
So once I've moved and have settled down, this should be a next goal.
This first specimen gives me the confidence that I can get it done.
It's probably the difference between continuing and giving up haha.
So for as long as it lasts, let's focus on attempting to get this little seedling to adulthood.
It has been 5 days since I have discovered it and it's been growing slowly.
It's currently located in a room with a lot of light during the day, without receiving direct sunlight.
It's growth is a bit blocked by the top of the pedri dish.
This doesn't help it get shaped but on the other hand, it can grow a bit larger.
Soon it will need to go from a sterile environment to a non-sterile one so the larger it is, the better.
I am thinking about transplanting it soon, one of these days.
Would like to hear what you guys think. Wait a bit longer or transplant now?
StoneCarver
Bureau for Paranormal Research and Defense
I think I'd go ahead and transplant. I'm sure it would be a good idea to at least pasteurize the soil you transplant it into.
Kushtaka
Member
I say transplant. That little thing needs more than agar.
Its always nice when a long shot project gives you a hint that you are on the right path right when you are feeling it is a lost cause. Like a nudge from the universe to keep at it.
Its always nice when a long shot project gives you a hint that you are on the right path right when you are feeling it is a lost cause. Like a nudge from the universe to keep at it.
Tutu
Well-Known Member
Yes, in the end the agar is there mostly for texture, to support the seedling physically.
Its the MS medium that feeds it, this Murashige & Skoog powder contains a lot of mineral nutrients.
It has nitrogen, potassium, calcium, magnesium, phosphorus and sulfur.
It also has micronutrients like iron, manganese, zinc, copper, boron, molybdenum, cobalt and iodide.
Besides that it has quite a lot of vitamins for metabolism.
Then there's some sucrose to give it some additional energy before it starts to have more photosynthesis.
However, I think with its first leafs, it won't really need much of that.
More light is also difficult here. I can keep it in a room that has windows, so it gets indirect sunlight.
Anything other than that I find a bit risky, the sun is very strong here, direct sunlight will most certainly kill it.
What I am considering is to cut with the scalpel around the medium which has the root in it.
Then transfer the cube of the medium around the seedling into a small hole in a soil I've bought that should be pasteurized.
I'm just hoping the excess of agar/ms-medium is not going to promote too much microbial growth.
But by transferring like that I will not damage the root structure and it can slowly grow from the agar into the soil.
I'm again just doubtful on whether to do this now or wait a few more days...
Its the MS medium that feeds it, this Murashige & Skoog powder contains a lot of mineral nutrients.
It has nitrogen, potassium, calcium, magnesium, phosphorus and sulfur.
It also has micronutrients like iron, manganese, zinc, copper, boron, molybdenum, cobalt and iodide.
Besides that it has quite a lot of vitamins for metabolism.
Then there's some sucrose to give it some additional energy before it starts to have more photosynthesis.
However, I think with its first leafs, it won't really need much of that.
More light is also difficult here. I can keep it in a room that has windows, so it gets indirect sunlight.
Anything other than that I find a bit risky, the sun is very strong here, direct sunlight will most certainly kill it.
What I am considering is to cut with the scalpel around the medium which has the root in it.
Then transfer the cube of the medium around the seedling into a small hole in a soil I've bought that should be pasteurized.
I'm just hoping the excess of agar/ms-medium is not going to promote too much microbial growth.
But by transferring like that I will not damage the root structure and it can slowly grow from the agar into the soil.
I'm again just doubtful on whether to do this now or wait a few more days...
