The overall goal of this procedure is to demonstrate how to perform crosses for cter verus a new model system For the grasses. This is accomplished by first selecting, trimming and labeling the pinnacle. The second step is to emasculate the trimmed panicle by dipping it into a 48 degrees Celsius water bath.
Next, the trimmed pinnacle is used as a female for pollination by a pinnacle that is shedding pollen. The final step is harvesting seeds two weeks after the cross pollination. Ultimately, Gus staining of the putative hybrid seeds is used to show the effectiveness of the technique.
The main advantage of this technique over the existing methods like physical emasculation, is that it is less invasive and relatively easy to establish in the lab. This method will help accelerate the adoption of cter verus as a genetic model by making it easier to perform crosses. To begin, remove one cell for watering and then fill a tray with four by nine cells With metro Mix 360.
Next lightly mist the cells and sow seeds on the surface of the soil, one seed per cell. Then cover the seeds with a layer of soil, 0.5 centimeters thick mist the surface of the soil to dampen them, and then water flats from the bottom. Keep watering the cells from the bottom one to two times per day after seed sowing, taking care not to over water.
Set the growth chamber conditions to 31 degrees Celsius during the day and 22 degrees Celsius at night with a preda treatment of 15 degrees Celsius from 8:30 AM to 9:00 AM.Also maintain a 12 hour light dark schedule and set the relative humidity to 50%Under these conditions. Satter verus a 10.1 will flower 21 to 22 days from seed sowing. Ideally, emasculation is performed in the afternoon or evening.
Select the primary pentacle on each plant that has one to five flowers in bloom. Then cut off the tip of the pentacle and remove all the flowers in the lower section of proximal middle and base sections using fine forceps. If a only has one 10th to one fifth of flowers in bloom.
In order to retain the lower section of distal, middle, and upper section of proximal middle for further trimming, cut off the tip and trim the flowers from the base of the panicle. If a panicle has one fourth to one half of flowers in bloom, retain the proximal middle section for further trimming and cut off the distal middle section. Once the pentacle have been cut, use surgical scissors to lightly trim the bristles.
Take care to retain the bristles in the region that will be pollinated in order to protect florets from possible heat damage. For each spikelet, retain the uppermost mature floret. The uppermost floret will have a higher probability of anthesis at the time of pollination.
Then remove all flowers that have bloomed and all immature flowers leaving approximately 20 to 30 flowers per pinnacle that are evenly distributed. Next, mark, one side of each flower with a red permanent marker. And record the number of florets on the pinnacle flag the pinnacle with the following information, the date of emasculation, the duration, and the temperature at which the emasculation is performed.
Once the panicle is trimmed, remove all tillers of each plant to ensure reallocation of more resources to the development of the main pinnacle. One hour before emasculation, set a water bath to 48 degrees Celsius for the heat treatment. Once preheated, gently bend the trimmed pentacles and dipped three to four at a time in warm water for three to six minutes.
Use caution not to break the stem of the pentacle. The flag leaf, which sub tens the pentacle should not come into contact with warm water. This is to prevent heat damage to leaf tissue.
Once emasculation is performed for the desired duration, remove panicles from the water bath and gently shake off the water from the panicle. Then fully cover the emasculated panicle with a customized micro perforated bread bag and secure it with a twist tie. Micro perforated bread bags can be customized according to the size of the panicle.
Finally, place the plants back in the growth chamber until the following day. At 9:00 AM on day two and day three, move emasculated female and non emasculated male parents from the growth chamber to the lab to perform crosses, observe and add another red. to the flowers that have bloomed or are blooming.
Using a red permanent marker, record the total number of bloomed flowers per panicle. Then record the date of pollination and the name of the male parent on the tag to keep track of what crosses have been performed. Observe the peak time of antithesis in male parents under our lab conditions, flowers on the pentacles of the male parent begin synchronous opening around 10:10 AM Once open, the complete exertion of anther and release of pollen will occur after 20 minutes.
Start pollination immediately when yellowish. White anthers are visible on flowers of the male parent. Pollinations are performed on day two and day three by using panicle to panicle pollination or anther to stigma pollination.
Using one detached panicle as the male gently rub the panicle together with the emasculated panicle to facilitate pollination and discard the male panicle to avoid contamination, repeat the pollination process until each emasculated pinnacle has been pollinated by two to three pentacles. The most effective method is anther to stigma pollination using forceps. Pick a flower that is blooming with yellowish white anthers or pick anthers and physically touch the stigma of the flower on the female parent.
Use one flower from the male parent to pollinate one flower on the emasculated panicle between the pollinations of different male parents. Dip the forceps in 95%ethanol, followed by wiping with Kim wipes to avoid contamination due to carryover of pollen between different crosses. Once the pollination has been performed, cover the panicle of the female parent with a customized micro perforated bread bag and secure it at the base using a twist tie.
After pollination until seed harvesting, harvest seeds 14 to 16 days after the day of pollination and place the tag in the same envelope as the seeds. Seeds with red markings on both sides represent the seeds that resulted from the controlled genetic cross. Discard any seeds without red markings as they represent the seeds that result from newly developed flowers.
After emasculation seeds with red markings, only on one side will likely represent the seeds resulting from self pollination. Finally, dry seeds at 30 degrees Celsius for two days. Store dried seeds in the lab, or ideally in a seed chamber.
When ready for further analysis, remove the physes if necessary. The methods described in this video were the results of numerous trials using different temperatures and treatment times to optimize the emasculation process. The success of this process was measured by the number of hybrid seeds that were obtained.
Six seeds from one of the test crossings where the male parent carried a reporter. Gene encoding for beta glucuronidase are shown here. The reporter gene causes the seed to turn blue when stained, making the number of hybrid seeds easy to count Once mastered, as many as 15 pentacles can be crossed per day if the procedure is performed properly.
After watching this video, you should have a good understanding of how to perform crosses in Aria verus.
