Citrus Germplasm Indexing Protocol
There are two indexing protocols available when germplasm is introduced into the Citrus Germplasm Introduction Program: full and expedited. The choice of protocol used is determined by risk assessment.
Full Protocol Summary
Level 1: New Germplasm
- Establish new germplasm on several seedling rootstocks.
- Pre-index and test for pathogens: citrus greening, citrus leaf rugose, citrus stubborn, citrus tristeza, citrus variegation, citrus vein enation, citrus viroids including cachexia and exocortis, concave gum, cristacortis, impietratura, Satsuma dwarf, seedling yellows, tatterleaf-citrange stunt, Xylella fastidiosa.
Source of Meristems for Shoot-tip Grafting
- Shoot-tip grafting.
- Confirm pathogen elimination detected in pre-index.
- Return to shoot-tip grafting if pathogen persists.
Level 2: Shoot-Tip Grafted Plant
- Increase size.
- Begin final bio-indexing and testing.
- Source of dormant buds for thermaltherapy.
- Thermaltherapy.
Level 3: “Thermal” Plant
- Increase size.
- Complete bio-indexing and testing for pathogens: citrus greening, citrus leaf rugose, citrus stubborn, citrus tristeza, citrus variegation, citrus vein enation, citrus viroids including cachexia and exocortis, concave gum, cristacortis, impietratura, Satsuma dwarf, seedling yellows, tatterleaf-citrange stunt, Xylella fastidiosa.
Level 4: Transfer to Bureau of Citrus Budwood Registration
- Horticultural evaluation.
- Budwood increase.
Full Protocol
Once the budwood is received in Gainesville, it is grafted into rootstocks to preserve the germplasm which is grown in quarantine. The remaining budsticks are set up in culture and are incubated at a heat therapy temperature and the resulting shoots are shoot-tip grafted (STG) to remove any potential pathogens. Laboratory real time PCR (qPCR) tests are performed using leftover incoming budwood material to determine the pathogen status. The resulting STGs are grafted onto rootstock seedlings and grown out in the greenhouse, then tested by qPCR. The horticulturally superior STG is grown out and budded into citrus indicators including: C. excelsa, citron, Duncan grapefruit, Dweet tangor, Eureka lemon, key lime, Rusk citrange, sour orange, and sweet orange. Grafts on citrus indicator plants are observed for symptoms of known graft-transmissible citrus pathogens. Control plants grafted with known pathogens are grown alongside the citrus indicator plants for comparison. Plants are cut back three times to look for symptoms in new flushes of growth. Laboratory real time PCR tests are performed using material from the shoots of the introduced plant. Once biological indexing and laboratory testing are complete, found negative for any known graft-transmissible plant pathogens, and transfer approved, plants are moved to the Bureau of Citrus Budwood Registration foundation in Chiefland where budwood is distributed.
