The embryo rescue protocol is mandatory for regeneration of immature embryos derived from interspecific crosses between different Cucurbita species. It is most commonly used for crosses between Cucurbita moschata and Cucurbita pepo. The main advantage of this technique is its simplicity.
The protocol uses only MS media with antibiotics. Hence, it can be easily replicated by any lab. It would be possible to modify this technique to investigate different barriers to interspecific or intergeneric hybridization.
Paying attention to the details during every step will be helpful for a successful outcome. Begin with planting the Cucurbita plants by filling 50 cell starting flats of 25 by 50 centimeters using potting medium supplemented with complete nitrogen, phosphorus and potassium fertilizer, containing 1.38 grams per kilogram of nitrogen, phosphorus, and potassium each. Next, sow the seeds to a depth equal to their length and cover them with the potting medium.
Water the flats without creating standing water and keep the media moist by watering them once per day. At the end of the second true leaf stage, transplant the seedlings into 30-centimeter diameter pots supplemented with three tablespoons of complete fertilizer per pot. After every seven days, fertilize each pot with 500 milliliters of liquid fertilizer containing 20, 20, 20 nitrogen, phosphorus and potassium in one gram added to a gallon of water.
Maintain plants in the greenhouse at temperatures between 22 to 28 degrees Celsius under the natural light regime. For vining genotypes, provide a supporting trellis in the greenhouse. Perform the controlled hybridization or pollination as soon as the plants start flowering at six to eight weeks from the seeding.
Check the unopened flowers having a yellow hue on the petals, and identify male flowers and female flowers of Cucurbita pepo and Cucurbita moschata cultivars. To prevent accidental insect pollination, gently tape the closed flower at the top using masking tape. The following morning, carry out the pollination before 10:00 AM by gently removing the taped top portion of the male and female flower.
Separating the petals from the male flower, and transferring the pollens by gently rubbing the anther on the female flower stigma. After pollination, immediately close the pollinated female flower with masking tape, and use a tag to record the date of pollination and to indicate the paternal and maternal parents used in the cross. A successful cross indicated by an expanded ovary will form a small fruit within one week.
Harvest the fruit after 45 to 55 days of pollination. Prepare a cefotaxime stock. Filter it through a sterile 0.22 micron syringe filter.
And make 0.5 milliliter aliquots of 250 milligrams per milliliter cefotaxime before storing them at minus 20 degrees Celsius. Similarly, prepare ampicillin stock. Filter it through a sterile 0.22 micron syringe filter and prepare one milliliter aliquots of 100 milligrams per milliliter ampicillin before storing them at minus 20 degrees Celsius.
Prepare Murashige and Skoog, or MS medium by dissolving 2.45 grams of the medium in 500 milliliters of distilled water in a one liter bottle. Add 1.5 grams of gellan gum to the MS medium and autoclave it at 121 degrees Celsius for 20 minutes. Cool down the medium by placing the bottle in a water bath at 50 degrees Celsius.
Thaw the antibiotic stocks inside the laminar flow cabinet. Transfer the medium bottle to the laminar flow hood and add 0.6 milliliters of cefotaxime stock and 0.25 milliliters of ampicillin stock to the medium with proper mixing. Pour about seven milliliters of the medium into a 60 by 15 millimeters sterile Petri dish.
Allow the medium to solidify in the Petri dishes for approximately 15 to 20 minutes. Close the Petri dishes with sealing wrap and place them into a storage box at room temperature until further use. Harvest the squash fruit by cutting it off the main vine and disinfect the fruit by washing its surface in the lab sink using a liquid detergent like 0.3%chloroxylenol.
Rinse the fruit with ample tap water and dry it with clean paper towels before transferring the fruit to the laminate airflow cabinet. Sterilize the fruit surface inside the cabinet by spraying 70%ethanol. Bisect the fruit with a sterile knife and extract the seeds.
Use sterile forceps or hands with sterile gloves to aseptically open the seed coat and expose the immature embryos before placing them in a Petri dish containing antibiotic-supplemented MS Medium. Seal the Petri plates using wrapping film and place them in a growth chamber having 25 degrees Celsius temperature, 16-hour photo period, and 70%relative humidity. In case of contamination, immediately subculture the uncontaminated embryos into a new plate having the same medium.
Once roots are developed after 10 to 14 days, and the cotyledons are developed after 15 to 21 days, remove the plantlets out of the Petri dishes, and gently wash off the media present around the roots using tap water. Place cleaned plantlets in a 14 by 9 by 4 centimeter plastic container and cover the roots with wet paper towels. Cover the container and re-moisten the paper towels when necessary.
Keep the plastic containers at 25 to 28 degrees Celsius, and in a 16-hour photo period to acclimatize the plantlets while maintaining the moist condition of paper towels. After 7 to 10 days of acclimatization, transfer the three to four centimeter long seedlings into 50 cell flats containing commercial potting mix amended with fertilizer and move them to the greenhouse. Avoid overwatering to prevent rotting and add approximately 10 to 20 milliliters of water per cell as needed.
At the second to the third true leaf stage, transplant the seedlings into 25 centimeter diameter pots filled with potting medium amended with fertilizer and perform controlled hybridization when the plants start flowering. Maintain the plants in the greenhouse at 22 to 28 degrees Celsius under the natural light regime. And evaluate the plants for fruit and seed characteristics.
Four Cucurbita pepo and 11 Cucurbita moschata were chosen for interspecific hybridization. Out of 24 interspecific cross combinations attempted, a fruit set was obtained for 22 crosses representing more than 92%success in the fruit set. The crosses between Cucurbita moschata and Cucurbita pepo involving genotypes O and M, and genotypes E and J, produced no mature fruit.
Whereas the cross between Cucurbita moschata and Cucurbita pepo involving genotypes F and J, produced six fruits. The number of flowers pollinated for different cross combinations ranged from 1 to 11. And the success rate of pollination ranged from 0%to 100%Out of 44 fruits produced from all the cross combinations, only one fruit from a cross between Cucurbita moschata and Cucurbita pepo, genotypes C and J produced immature embryos that could be rescued.
For the development of these poorly developed embryos, they were cultured using the embryo rescue media, which resulted in an 80%success rate for embryo rescue. The two most important procedures are identifying compatible crosses between two species namely, Cucurbita moschata and Cucurbita pepo, and successful embryo regeneration. Since the embryo rescue technique is simple and easily replicable, squash breeders worldwide can use this technique for rescuing immature embryos derived from interspecific crosses between different Cucurbita species.
