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jinianI made quite a few crosses last week, which was both great (undergrads don't usually get to make crosses, I am all special) and stressful (PI said my last crosses looked good, so she didn't do any duplicates to back me up this time, eep).
As I mentioned in my seed-collecting post, making crosses is somewhat difficult because Arabidopsis thaliana flowers are tiny and you have to catch them when they're immature, before they self-fertilize. This means I get to wear the sexy magnifying visor and use the very pointy tweezers marked "cross only." We have any number of former cross forceps in the lab; one drop to the floor and the $40 tweezers are bent all to hell. They really have to be perfect, which means they're so pointy they catch on my first layer of skin if I'm not careful.
The overall idea is to get pollen from one plant's flower onto the stigma (sticky receptive part of the pistil) of another plant's flower. So, the first step is to get the seed parent ready to accept pollen. Any flower that's completely open has already self-fertilized, though. I noticed during my last crosses that the stigma actually pokes out of the petals a tiny bit before the petals open, so the self-fertilization is not quite as anti-sex as you might think. The flower's own stamens don't grow long enough to touch the stigma until a little later, which means there's a window in which theoretically some outcrossing could happen. It's probably plenty to keep the gene pool a little agitated. So, I need to expose the stigma completely, which means the petals have to come off. The sepals protect the petals, so I slice/squish/pull them off with the forceps first, then petals, and then the stamens, since they'd be happy to grow longer, as planned, and drop their pollen on the stigma too. That's bad for getting seeds from the actual cross I'm going to all this trouble to make, so off they come. (Icon check: We really do call this "emasculating" the flower.)
While preparing three or four flowers, which is usually all that are at a usable stage on a given stem, I also remove the older flowers and siliques from the stem I'm making the crosses on. There are exceptions to that, if we really want the self-fertilized seed too, but it's useful to force the plant's energy into developing the crossed seeds. Plus, it's a lot easier to find the seeds from my cross if there aren't any others on that stem. I also remove any smaller flower buds and the floral meristem (differentiating cells at the stem tip that produce more flower buds) for the same reasons.
So I have my little cluster of sad, naked pistils, which I always feel a little bad for. Now I am responsible for their reproductive fulfillment! So I take a flower from the pollen parent -- usually I literally take the flower off -- and strip its sepals and petals. A good pollen-donor flower is fully open and under the magnifying visor I can see the powdery texture of pollen on its stamens, so I know they've dehisced (opened up) and I'm not just going to be bumping nonfunctional plant tissues together. I also snip off the top half of the pistil on the pollen donor, which I don't think is necessary (any problems would be due to self-incompatibility chemicals, and A. thaliana selfs all the time) but does clear up my view of the relevant bits. Then I take the pollen parent's flower and brush the stamens against the bare pistils of the seed parent's flowers. Ideally I see a little puff of pollen on first contact. I have to brush maybe a dozen times, going for the good hit when a stamen is actually slightly stuck to a stigma for a moment, and often I repeat with another flower's pollen on the same seed-parent cluster. I have to be fairly quick with all this; even using the room-temperature fiber-optic spotlight, the procedure can be stressful for the plant, and if I do too much violence to the stem the plant will abort the seeds. It's awfully easy to tug the stem, since it sticks to bare fingers, but that doesn't spell automatic doom unless I do it a lot.
The pollinated flowers need to be tagged with the identity of the pollen parent and then tied securely in such a way that they won't be touched by other stems or people watering the plants. Ripe siliques burst right open if touched, and a plant with ripe siliques is too old to make more crosses. (PI got a little worked up about some of her cross-bearing plants that we needed to touch last week, and she was totally justified. It can be a real time problem to raise more plants from seed just to repeat a cross.) If I've done the cross correctly, the pistils that were ready to accept pollen change to a slightly brownish-purple color the next day. Some of the flowers were younger, so their stigmata weren't receptive yet; they'll change color when the pollen tube reaches the ovule, it just takes longer for that to happen.
The good news is that at least one developing silique turned color for each type of cross I made last week! Success! Things could still go horribly wrong if we mess up watering, since plants under water stress often abort their seeds, but generally we're very much on top of that. Wish me and my plants luck!
As I mentioned in my seed-collecting post, making crosses is somewhat difficult because Arabidopsis thaliana flowers are tiny and you have to catch them when they're immature, before they self-fertilize. This means I get to wear the sexy magnifying visor and use the very pointy tweezers marked "cross only." We have any number of former cross forceps in the lab; one drop to the floor and the $40 tweezers are bent all to hell. They really have to be perfect, which means they're so pointy they catch on my first layer of skin if I'm not careful.
The overall idea is to get pollen from one plant's flower onto the stigma (sticky receptive part of the pistil) of another plant's flower. So, the first step is to get the seed parent ready to accept pollen. Any flower that's completely open has already self-fertilized, though. I noticed during my last crosses that the stigma actually pokes out of the petals a tiny bit before the petals open, so the self-fertilization is not quite as anti-sex as you might think. The flower's own stamens don't grow long enough to touch the stigma until a little later, which means there's a window in which theoretically some outcrossing could happen. It's probably plenty to keep the gene pool a little agitated. So, I need to expose the stigma completely, which means the petals have to come off. The sepals protect the petals, so I slice/squish/pull them off with the forceps first, then petals, and then the stamens, since they'd be happy to grow longer, as planned, and drop their pollen on the stigma too. That's bad for getting seeds from the actual cross I'm going to all this trouble to make, so off they come. (Icon check: We really do call this "emasculating" the flower.)
While preparing three or four flowers, which is usually all that are at a usable stage on a given stem, I also remove the older flowers and siliques from the stem I'm making the crosses on. There are exceptions to that, if we really want the self-fertilized seed too, but it's useful to force the plant's energy into developing the crossed seeds. Plus, it's a lot easier to find the seeds from my cross if there aren't any others on that stem. I also remove any smaller flower buds and the floral meristem (differentiating cells at the stem tip that produce more flower buds) for the same reasons.
So I have my little cluster of sad, naked pistils, which I always feel a little bad for. Now I am responsible for their reproductive fulfillment! So I take a flower from the pollen parent -- usually I literally take the flower off -- and strip its sepals and petals. A good pollen-donor flower is fully open and under the magnifying visor I can see the powdery texture of pollen on its stamens, so I know they've dehisced (opened up) and I'm not just going to be bumping nonfunctional plant tissues together. I also snip off the top half of the pistil on the pollen donor, which I don't think is necessary (any problems would be due to self-incompatibility chemicals, and A. thaliana selfs all the time) but does clear up my view of the relevant bits. Then I take the pollen parent's flower and brush the stamens against the bare pistils of the seed parent's flowers. Ideally I see a little puff of pollen on first contact. I have to brush maybe a dozen times, going for the good hit when a stamen is actually slightly stuck to a stigma for a moment, and often I repeat with another flower's pollen on the same seed-parent cluster. I have to be fairly quick with all this; even using the room-temperature fiber-optic spotlight, the procedure can be stressful for the plant, and if I do too much violence to the stem the plant will abort the seeds. It's awfully easy to tug the stem, since it sticks to bare fingers, but that doesn't spell automatic doom unless I do it a lot.
The pollinated flowers need to be tagged with the identity of the pollen parent and then tied securely in such a way that they won't be touched by other stems or people watering the plants. Ripe siliques burst right open if touched, and a plant with ripe siliques is too old to make more crosses. (PI got a little worked up about some of her cross-bearing plants that we needed to touch last week, and she was totally justified. It can be a real time problem to raise more plants from seed just to repeat a cross.) If I've done the cross correctly, the pistils that were ready to accept pollen change to a slightly brownish-purple color the next day. Some of the flowers were younger, so their stigmata weren't receptive yet; they'll change color when the pollen tube reaches the ovule, it just takes longer for that to happen.
The good news is that at least one developing silique turned color for each type of cross I made last week! Success! Things could still go horribly wrong if we mess up watering, since plants under water stress often abort their seeds, but generally we're very much on top of that. Wish me and my plants luck!
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no subject
Date: 2006-04-10 05:24 am (UTC)no subject
Date: 2006-04-10 05:46 am (UTC)yay happy plant biology girl!
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Date: 2006-04-10 05:56 am (UTC)no subject
Date: 2006-04-11 05:45 am (UTC)no subject
Date: 2006-04-11 05:55 am (UTC)Also, whenever you post, I get new band names in my head. I think Naked Pistils would be a great name for one.