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jinianThis is not the most exciting thing I do, but it's what I did today, apart from cleaning up. And I find it satisfying -- not quite the moment of zen from packing tips[*], but it's progress in a way tips aren't.
Mature plants of Arabidopsis thaliana, our model organism for plant organ development, are a lot like shotweed, only bigger and with a tendency to sprawl. Our plant trays would look like the last shot here, a wild-grown A. thaliana patch, if we didn't tie the plants' flowering stems up before the seeds were mature. Usually we make a little wreath with the stems from one plant, holding them together with twist-ties or tape, and attach that to a wooden stick so it doesn't flop around too much. Like shotweed, Arabidopsis will shatter its siliques (specific term for its kind of seed pod) all over everything if you touch them once they're ripe, and getting the stems caught up with another plant's is a great way to lose a bunch of seed. Because we're doing genetic crosses and tracking the genotypes of specific plants, mixed-up seeds are usually pretty useless to us and just get thrown out.
So, I've tied my plants neatly (and not too early in their life span or they'll just grow long roaming stems again) and the siliques are looking mature. Normally we take them out of the watering rotation a few days before harvest, which I think is because the siliques will break more easily if they're good and dry.
Once they're ready, I label a non-sterile microtube with the generation of the seed...
[Seed/plant generation digression]
Here's a little background reading on Mendelian genetics, in case you don't remember or never knew about it. I'll also answer questions happily, since I think it's really fun stuff.
The first time you cross two pure lines, the offspring are called the F1 (filial) generation. Generally, F1 Arabidopsis plants give F2 seed, because it self-fertilizes unless a human specifically prevents that. The seed I collected today was B2F2: backcrossed into wild-type parent for two generations, then allowed to self-fertilize. It's not the most useful kind of seed because there's not really any progress being made. We want to backcross to wild-type to "clean up the genome," because the way we got the experimental allele into this line of plants was by exploiting a plant pathogen, Agrobacterium, which inserts chunks of DNA into the plant's genome, and it can often make several insertions. We just want one insertion in the right place, so we cross the transformed plant with the laboratory wild-type strain, which has perfectly normal genes; the result is the B1F1 generation. We look at the genotypes of the B1F1 plants, find the ones that carry the allele we're studying, and cross those with wild-type plants again: B2F1. Normally three or four backcrosses are made before the gene is studied in depth, just in case there's other weirdness brought about the the bacterial transformation. It would be really frustrating to find a great new phenotype and then discover you didn't really know what genes were producing it!
So B2F2 seeds are good to have -- they can grow into plants that will mostly (3:1) carry the allele we're looking for, in case we lose all our other B2 plants to old age -- but don't get us closer to the goal of having a stable line of plants that are only different from the wild-type plants in the one gene we want to study.
Because crosses in Arabidopsis have to be done by hand when the always-tiny flowers are immature, we usually have very few seeds of any F1 cross. Usually a few are enough, but today I discovered a cross that looks like no viable seeds were produced (little flat black specks instead of little round brown shinies, uh-oh) so my B2F2 seeds might be important after all.
[End digression]
... and get two pieces of paper ready. The paper needs to be white on one side, and I usually fold it in half, which helps with pouring the seeds into the storage tube later. I pull the dry plant out of the pot, which may contain up to eight other plants -- this is where having tied up the stems is a huge help! -- and hold it over the paper, then gently rub the stems so that the siliques break open. Arabidopsis seeds are so tiny that we can't wear gloves when we handle them, because the static produced by the plastic tubes they're stored in makes them stick to things and jump around. I have to ground them some with my bare, conductive hands, or there's no working with them at all. They can also be hard to see; hence the clean white paper. Once I've rubbed all the stems, I've got a paper full of silique halves, dead flower buds, dried petals, bits of stem and leaf, immature siliques, and oh yeah some seeds. ("Some" is a thousand or more for a healthy plant, with ten thousand seeds per plant considered a maximum. No plant I've seen has come even close to filling the 1.5-mL tubes we use. I don't know how many seeds that would take.) People's techniques for seed-winnowing differ; I usually dump everything in a heap and pick off as much of the chaff on top as I can, then exploit the fact that the seeds are the roundest thing in the pile to roll them off the paper ahead of the other cruft.
After I have mostly seeds and no giant pieces of other stuff, I pour them into the tube and add "Drierite" (anhydrous calcium salt with cobalt in, sucks water out of air and changes color once it has) to desiccate them. The seeds do best if they dry out for a couple of days before they're planted.
[*] Pour disposable tips for metered pipets from bag into lid of appropriately colored box, then put them into holes in box. Tape both sides with autoclave tape. Calming, with a visible product, which I like.
C'est tout! That was longer than I thought. Was it reasonably clear?
Mature plants of Arabidopsis thaliana, our model organism for plant organ development, are a lot like shotweed, only bigger and with a tendency to sprawl. Our plant trays would look like the last shot here, a wild-grown A. thaliana patch, if we didn't tie the plants' flowering stems up before the seeds were mature. Usually we make a little wreath with the stems from one plant, holding them together with twist-ties or tape, and attach that to a wooden stick so it doesn't flop around too much. Like shotweed, Arabidopsis will shatter its siliques (specific term for its kind of seed pod) all over everything if you touch them once they're ripe, and getting the stems caught up with another plant's is a great way to lose a bunch of seed. Because we're doing genetic crosses and tracking the genotypes of specific plants, mixed-up seeds are usually pretty useless to us and just get thrown out.
So, I've tied my plants neatly (and not too early in their life span or they'll just grow long roaming stems again) and the siliques are looking mature. Normally we take them out of the watering rotation a few days before harvest, which I think is because the siliques will break more easily if they're good and dry.
Once they're ready, I label a non-sterile microtube with the generation of the seed...
[Seed/plant generation digression]
Here's a little background reading on Mendelian genetics, in case you don't remember or never knew about it. I'll also answer questions happily, since I think it's really fun stuff.
The first time you cross two pure lines, the offspring are called the F1 (filial) generation. Generally, F1 Arabidopsis plants give F2 seed, because it self-fertilizes unless a human specifically prevents that. The seed I collected today was B2F2: backcrossed into wild-type parent for two generations, then allowed to self-fertilize. It's not the most useful kind of seed because there's not really any progress being made. We want to backcross to wild-type to "clean up the genome," because the way we got the experimental allele into this line of plants was by exploiting a plant pathogen, Agrobacterium, which inserts chunks of DNA into the plant's genome, and it can often make several insertions. We just want one insertion in the right place, so we cross the transformed plant with the laboratory wild-type strain, which has perfectly normal genes; the result is the B1F1 generation. We look at the genotypes of the B1F1 plants, find the ones that carry the allele we're studying, and cross those with wild-type plants again: B2F1. Normally three or four backcrosses are made before the gene is studied in depth, just in case there's other weirdness brought about the the bacterial transformation. It would be really frustrating to find a great new phenotype and then discover you didn't really know what genes were producing it!
So B2F2 seeds are good to have -- they can grow into plants that will mostly (3:1) carry the allele we're looking for, in case we lose all our other B2 plants to old age -- but don't get us closer to the goal of having a stable line of plants that are only different from the wild-type plants in the one gene we want to study.
Because crosses in Arabidopsis have to be done by hand when the always-tiny flowers are immature, we usually have very few seeds of any F1 cross. Usually a few are enough, but today I discovered a cross that looks like no viable seeds were produced (little flat black specks instead of little round brown shinies, uh-oh) so my B2F2 seeds might be important after all.
[End digression]
... and get two pieces of paper ready. The paper needs to be white on one side, and I usually fold it in half, which helps with pouring the seeds into the storage tube later. I pull the dry plant out of the pot, which may contain up to eight other plants -- this is where having tied up the stems is a huge help! -- and hold it over the paper, then gently rub the stems so that the siliques break open. Arabidopsis seeds are so tiny that we can't wear gloves when we handle them, because the static produced by the plastic tubes they're stored in makes them stick to things and jump around. I have to ground them some with my bare, conductive hands, or there's no working with them at all. They can also be hard to see; hence the clean white paper. Once I've rubbed all the stems, I've got a paper full of silique halves, dead flower buds, dried petals, bits of stem and leaf, immature siliques, and oh yeah some seeds. ("Some" is a thousand or more for a healthy plant, with ten thousand seeds per plant considered a maximum. No plant I've seen has come even close to filling the 1.5-mL tubes we use. I don't know how many seeds that would take.) People's techniques for seed-winnowing differ; I usually dump everything in a heap and pick off as much of the chaff on top as I can, then exploit the fact that the seeds are the roundest thing in the pile to roll them off the paper ahead of the other cruft.
After I have mostly seeds and no giant pieces of other stuff, I pour them into the tube and add "Drierite" (anhydrous calcium salt with cobalt in, sucks water out of air and changes color once it has) to desiccate them. The seeds do best if they dry out for a couple of days before they're planted.
[*] Pour disposable tips for metered pipets from bag into lid of appropriately colored box, then put them into holes in box. Tape both sides with autoclave tape. Calming, with a visible product, which I like.
C'est tout! That was longer than I thought. Was it reasonably clear?
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no subject
Date: 2006-02-08 03:19 am (UTC)And now I read post. :)
no subject
Date: 2006-02-08 03:25 am (UTC)Yes, very clear, and very nifty. I display my complete ignorance with the question: is the goal here to produce a new strain of or new properties in Arabidopsis thaliana, or to study the genetic sequence itself? Also, is this work, or school? Also also, very very nifty. :)
no subject
Date: 2006-02-08 04:42 am (UTC)What we're studying is what screwing the gene up (with a bad allele, a "knockout mutation") does. Since genes don't do anything unless they're in organisms, we're trying to get a strain that has the mutation so we can examine its phenotype. To complicate things some more, many many genes are redundant, so often you have to get more than one gene knocked out before you observe anything much. Luckily, that's a matter of one cross once the individual strains are established.
It is work and school both, I guess. I started last quarter as a lab assistant, and I'm still doing that this quarter when my 4 credits of Undergraduate Research don't need any work. There's a lot of waiting for plants to set seed, and then I wash dishes and stuff. :) I'm getting to do some stuff undergrads don't usually get to do, like making my own crosses, so that's neat and a little intimidating. It's fun, and I feel really dedicated to it, which is kind of a novel experience!
no subject
Date: 2006-02-08 05:16 am (UTC)you don't need a Terrarium. i keep mine on the windowsill.
http://www.cobraplant.com/drosera-capensis.html
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Date: 2006-02-11 08:04 pm (UTC)