Plant Crosse Protocol

Monday, November 10, 2003

Description
Plant Crosse Protocol

Procedure
weed out the plant pot, leave only about 3 to 8 tall and vigorous plants (in a regular 5 cm pot)
on an individual inflorescence, cut off all the opened flowers (older than stage 13), the youngest developing siliques and any side branches that may come into contact with the flowers to be used as pollen acceptors (usually located on the main inflorescence)
emasculate flower: using forceps slighly squeeze open the bud at a level about a third from the bottom, then carefully remove all floral organs except the gynoecium. Use only floral buds that did not yet open (stage 12 and younger), check rigorously that absolutely no pollen is present. In case of any doubt discard the floral bud. Three to five floral buds can be regularly used per inflorescence at a given time point
remove the center part of the inflorescence leaving only the floral buds to be pollinated on the inflorescence
allow the plants to grow for another 1 to 1 1/2 days at the appropriate temperature (e.g., 23癈), keep them separate, take care that no cross pollination is possible; the cross works best when the papillar cells are nicely erect
with forceps remove a flower from the pollen donor plant and squeeze open the flower, the anthers with the pollen should be clearly visible
gently streak the anthers over the stigma of the pollen acceptor, make sure that a lot of pollen is covering the stigma, if not enough pollen is present use a second flower of the pollen donor plant
label the inflorescence just beneath the flowers used for the cross. A single cross refers to the 3 to 5 flowers of the inflorescence pollinated with pollen from the same pollen donor. Never mix genotypes of pollen donors when crossing flowers of the same inflorescence
label the bottom of the main inflorescence if necessary
seeds from selfed flowers on the main branch or side branches can be used to test the genotype of the pollen acceptors/donors if necessary
seed collection typically takes place about 2 to 2 1/2 weeks (@ 23 癈) after the cross: wait until siliques are dry

Recipes

Supplies

Tips
Use a dissecting scope (10x magnification) with a ring light source. When emasculating the flowers or doing the crosses take care not to keep the flowers too long under the disscecting scope. They tend to dry out relatively quickly when kept under the strong light. In addition, don't squeeze the plants between your fingers during handling! The same holds true when labelling the plants. We use a ca. 3 mm wide piece of colored sticky tape as label. Always make sure that the tape is not directly attached to the stem. Prepare a label with a "stem-loop" structure and control that the "loop" part of the label very losely encircles the stem. Use sharpened forceps

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Plant Crosse Protocol