Citrus Micrografting for Regrowth After Shoot Tip Cryopreservation
Citrus Micrografting for Regrowth After Shoot Tip Cryopreservation
Gayle M. Volk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521<EMAIL_ADDRESS>Remi Bonnart, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Ashley Shepherd, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Outline
- Introduction
- Prepare the rootstock seedlings
- Warm the cryopreserved shoot tips
- Micrograft the shoot tips onto the rootstocks
- Shoot tip regrowth and trimming
- References
- Associated information
- Acknowledgments
The citrus micrografting procedure is available to download here.
1. Introduction
This chapter demonstrates the process of micrografting as a method to recover citrus shoot tips after cryopreservation. Micrografting is a process whereby shoot tips are attached to seedling rootstocks and then grow into healthy plants. Micrografting is used to recover citrus shoot tips because they failed to grow when they were placed directly on medium, likely because the medium was not optimized for their growth requirements.
The micrografting method is demonstrated as a series of steps including:
- Preparing the seeds to produce rootstock seedlings
- Warming the cryopreserved citrus shoot tips
- Trimming the cryopreserved citrus shoot tips and micrografting them onto the seedling rootstocks
- Regrowth of the micrografted plants
2. Prepare the rootstock seedlings
‘Carrizo’ citrange rootstock seeds (PI 150916 x Citroncirus sp.) were selected as the rootstock cultivar because they are available from nursery sources, they produce relatively thick hypocotyls for grafting, and they are compatible with nearly every species of citrus.
Video 1 demonstrates the process of peeling the seed coats off the ‘Carrizo’ seeds and placing the resulting embryos into water containing Tween 20 (1 drop per 100 mL). When all the seed coats are removed, the seeds are rinsed three times with tap water and then surface sterilized by first treating the embryos with 70% isopropanol for 2 minutes and then rinsed three times with tap water. 20% bleach (1.65% sodium hypochlorite final concentration) + Tween 20 (1 drop per 100 mL) is then added to the flask and the embryos are transferred to a laminar flow hood. The seeds are shaken intermittently for 20 minutes while they are in the bleach solution. They are then rinsed three times with sterile water and embryos are transferred to test tubes containing the growth medium Citrus Seed Germination Medium. The embryos in the test tubes are then placed in a dark cabinet in a growth room and grown for up to six weeks before use.
Video 1. USDA-ARS Technician Remi Bonnart demonstrates the seed coat removal, surface sterilization, and introduction onto medium processes.
3. Warm the cryopreserved shoot tips
When the rootstocks are at least 3 cm tall, they are ready to