7. Terminate the apical meristem and maintain plants until signs of maturation and senescence become evident; diminish and then cut-off watering.

Terminate the apical meristem: This phrase means to remove any incipient flowers beyond those that you reasonably hope will produce useful and non-crappy seeds. We have found that the FPsc line can readily bear 8-12 fully fertilized seed pods (siliques), each holding 15-25 maturing seeds. If you’re selfing to propagate a lineage (e.g., to advance an F1 plant to yield a segregating F2 seed family, or to amplify stocks of true-breeding wild type or homozygous mutant lines) it’s best not to ask or allow your plants to bear more than those numbers of progeny. Not only will your seed quality be diminished, but it will take longer for the plants to reach senescence and for you to be able to harvest seeds and go on to the next generation. Thus, if you’re propagating lineages through selfing, do hand pollinations at 3 and 5 or 6 days after the first flower opens, give the plant another day or two, and then use forceps to cleave the remaining inflorescence buds from the previously fertilized flowers. If you are daring you can do so immediately after the last round of hand-pollination, but we like to allow a bit of stem distance to emerge that will separate the open stem wound resulting from termination to the last-fertilized flower. If you’re doing crosses and are confident that the cross “took”—as evidenced by vigorous elongation of the pistil/silique—let a few more flowers form, hand-pollinate those (always a good practice in genetics experiments), and then terminate the meristem as above.

The rationale behind this recommended strategy is as follows: Plants (as with all organisms) have a drive to survive. Upon transition from vegetative (leaf-forming) development to reproductive (flower-forming) mode, individual plants will continue to flower as long as suitable conditions exist or until the plant reaches a tipping point that balances the resource demands of growing embryos versus new flower/embryo production. It is a well-known phenomenon in the plant research community that male-sterile plants (e.g., those in selfing varieties unable to effect fertilization) flower FOREVER—or at least it seems like forever in researchers’ minds—whereas fertile plants flower for some typical period of time and then cease flower production. While the genetic and molecular details remain almost wholly obscure, the inescapable conclusion is that there exists a feedback mechanism that signals to the plant that some “normal” level of reproductive success has been achieved. Plants thereafter cease reproductive development and, instead, allocate resources derived from photosynthesis to nurturing growth and development of embryos and seeds. As experimental scientists, our estimation of the “normal” level of reproductive success is driven not by innate tendencies of the plant, but by experimental imperatives. By terminating the meristem, we send an unmistakable signal to the plant that it’s time to stop flowering and to devote resources to raising healthy seeds. [If you are as obsessive about hurrying through generations as we are, you might also take care to terminate secondary floral inflorescences that either exist when you terminate the apical meristem, or emerge as a consequence of releasing apical dominance through removal of the primary shoot.] We’re getting pretty deep into the weeds of plant reproductive biology here, so let’s return to first principles: once you are confident that the plant has produced and can support development of a good crop of mature seeds, try to prevent it from devoting any more resources to flowering.

Maintain plants: Be sure to provide adequate water during this critical period but, as noted above, don’t drown the plants. Inadequate watering is one of the surest ways to produce a crappy seed lot. Finding that Goldilocks balance—not too wet and not too dry—isn’t as difficult to do as it may sound as written here. It only takes some time, attention, and common sense. Depending on growing conditions (planting density being most important since widely spaced plants grow taller), it may be necessary to stake the plants to prevent lodging, or, to use the technical term, “falling over”. We use 10”-12” bamboo barbeque skewers that we get from a local hardware, big box store or online sources. Poke the pointed end into the soil near the base of the plant and secure the plant to the stake by using a paper-coated wire twist-tie (the sort you get with loaves of bread). Bend the twist-tie into the shape of a V and position the vertex behind the skewer. Grasping the twist-tie, stake, and stem of the plant just below the inflorescence, use your other hand to put in a couple of twists. It doesn’t take much pressure to keep the tie in place since small slivers of bamboo will give the tie a grip; be sure that you don’t strangle the plant.

Signs of maturation and senescence: As noted above, successful pollination/fertilization is indicated by an elongating pistil. That lengthening should be evident by 3 days post-pollination and will continue until the carpels and enclosed embryos/seeds (collectively, the silique) reach a length of 2”-3” after ~2-3 weeks. During that time there will be a fattening of the silique that results from seed development and enlargement. In time, the contours of seeds within the silique will become apparent. At the end of those 2-3 weeks further elongation and swelling will stop and you might think the process is finished, but it will take ~2 more weeks before seeds are fully mature and ready to harvest. During that time (which can seem painfully long), energy reserves are synthesized and stored in cells of the embryo and the mother plant directs formation of a tough and ultimately hard, red/brown seed coat. Nearing the end of the mother plant life cycle, she mobilizes nutritional resources from leaf tissues, as evidenced by wilting and loss of the formerly deep green pigmentation. It is likely that the only photosynthetically-active tissues late in the plant life cycle are the carpels, so be sure to keep them close to the lights. Ultimately, photosynthesis ceases in the carpels, as well, and they become successively paler and then brown. That browning of siliques typically begins at the tip, or “beak” as it is known in botanical circles and later spreads throughout the silique.

Elongating siliquesSenescent plantsFilling siliques

Figure 12: Maturation and senescence of flowers and siliques (seed pods). Successful pollination/fertilization is indicated by elongation and swelling of the pistil over time (left). Developing seeds become evident 1-2 weeks after pollination (center) and senescence/maturation is complete ~5 weeks after fertilization (right).

During these late stages of the plant life cycle, water needs are substantially reduced so it’s advisable to cut back on watering-- don’t leave them parched, just a little thirsty. When plant tissues, especially the carpels, are ~50% brown, you can stop watering altogether and seeds can be harvested after another week or ten days. It may be helpful to keep in mind the natural life cycle of rapid-cycling B. rapa varieties. Similar to the related and invasive garlic mustard plant that is the scourge of woodlands throughout the upper Midwest, these plants are among the earliest to germinate and begin growing in spring, well before the competition gets started. They thereby face less competition for resources (especially light) and use that window of opportunity to rapidly progress through vegetative and then reproductive development. Lengthening days and warmer temperatures are a signal that the hot summer months are coming, as is a decrease in available moisture. The plants perceive these environmental cues and respond by devoting all resources towards seed production and maturation. We try to mimic those cues, at least with respect to water availability, in order to accelerate the seed harvest.

There is another important reason to avoid excessive watering late in the FPsc life cycle. One of the goals of the breeding program that lead to both the canonical WFP variety and our FPsc plants is “a need for speed”. Time is a precious commodity in both classrooms and laboratories and we hoped that by minimizing the generational (seed-to-seed) time, our teachers and students might be able to conduct experiments in genetics that span multiple generations. One outcome of pursuing that goal is that dormancy in our seed stocks is readily broken. That is, whereas seeds of some plant species can remain dormant in soil through periods of wet and dry, hot and cold, for long periods of time—sometimes decades—our seeds barely see the stop sign at the end of development. Excessive watering towards the end of the FPsc life cycle can lead to the phenomenon of vivipary, wherein seeds actually germinate within the siliques of senescent mother plants (see figure 4). Needless to say, that is a condition to be avoided. 8. Harvest the seeds