Setting Up Crosses

Choosing Parent Plants

To avoid confusion, it is preferable to design crosses such that the Fx progeny are easily distinguishable from self-progeny of the female parent. An easy way to do this is to use a female parent that carries a recessive mutation and a male parent that lacks the mutation (i.e., is wild type for this trait). For example, a female parent that is homozygous for gll (i.e., lacking trichomes) can be crossed to a wild-type male parent (i.e., with trichomes), resulting in F progeny with trichomes. Any plants that arise from self-fertilization of the female parent will exhibit the mutant phenotype (i.e., lacking trichomes) and can be easily eliminated.

Choosing Flowers

Once suitable parent plants have been chosen, it is important to select the best flowers to use in the cross. To reduce the possibility of self-fertilization, flowers from the female parents must be used before the anthers begin to shed pollen onto the stigma. For the male parent, choose an open flower that is visibly shedding pollen. The appearance of flowers at the appropriate developmental stage varies among eco-types and developmental mutants; for wild-type Columbia, flowers in which the tips of the petals are just visible are the best choice for the female parent. The first two or three flowers on the first inflorescence shoot are often infertile, and these should be avoided. The next few flowers are the best. As the plants get older, the flowers tend to become smaller, and the failure rate for crosses increases.

Materials

Suitable parent plants

Jeweler's forceps (such as Dumont model #5) A magnifying device, such as a dissecting microscope or jeweler's glasses (optional) Sewing thread or labeling tape in various colors

Procedure

1. Identify the most suitable flowers on the female parent. Healthy plants usually have three flowers on the main shoot suitable for crossing, although expert crossers can sometimes find five. Use a scissors or a jeweler's forceps to remove the flowers just above and below those selected for the cross. Remove any siliques that might be present on the stem. Do not damage the stem when removing the flowers and siliques.

2. For each flower to be crossed on the female parent, remove the sepals, petals, and each anther, but leave the carpels intact. It may be helpful to use a magnifying device. Inspect the flower to ensure that all the anthers have been removed and that they have not shed pollen onto the pistil. The end result should be a stem with three to five exposed carpels.

Expert crossers remove only anthers and leave the perianth (sepals and petals) intact. This increases seed set.

3. Remove an open flower from the male parent and squeeze it near the base with the forceps. This will spread out the flower parts, separating some of the anthers from the other organs. Brush the convex surface of the anthers against the stigmatic surface of the exposed carpels on the female parent. Use a magnifying device to see the pollen on the stigma.

4. Label the crosses by tying a piece of sewing thread around the stem of the female parent. This will allow the seed to be identified when it has matured. Keep track of the parent genotypes using a color code, or by writing on a piece of tape and wrapping it around the ends of the thread. Be sure to record the date that the cross was made.

5. Check the plants regularly during the next few days. If the cross was successful, the siliques will have elongated after 3 days.

6. After 2-3 weeks, seed should be ready for collection, depending on the growing conditions. Maturation time is generally quite consistent within one set of growing conditions. Collect the siliques when they begin to turn yellow. If the silique dries on the plant, it may split open, and the seed will be lost.

Store the siliques in paper envelopes or plastic tubes (such as microfuge tubes). In the latter case, perforate the top of the tube with a syringe needle, allowing moisture to evaporate and thus preventing fungal growth,

7. Dry the siliques at room temperature for 2 weeks before planting. Damp seeds have poor germination rates.

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