Citrus Germplasm Indexing Protocol
Bureau Chief: Dr. Greg Hodges, Greg.Hodges@freshfromflorida.com
Biological Administrator III: Dr. Tim Schubert, Timothy.Schubert@freshfromflorida.com
Biological Scientist IV: Lisa Williams, Lisa.Williams@freshfromflorida.com
PO Box 147100 Gainesville, FL 32614-7100
352.372.3505
ENPP Home | Plant Pathology Home
Citrus Germplasm Program Links
There are two indexing protocols available when germplasm is introduced into the CGIP: 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 & 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 & 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 immediately grafted onto a propagating rootstock and to several citrus indicators. The indicators include C. excelsa, citron, Duncan grapefruit, dweet tangor, Eureka lemon, key lime, Parson’s special, Rusk citrange, sour orange, and sweet orange. These are grown out for 6-24 months. 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. For pathogens where a more rapid testing procedure is available (ELISA, PCR etc.), these tests are performed using material from the shoots of the introduced plant. Plant sap is also rubbed out on various herbaceous indicator plants such as red kidney bean and Chenopodium quinoa.
Once scions on propagating rootstocks reach a size that can tolerate being stripped of most of their leaves, preparations for shoot-tip micrografting (STG)ing begin. STGing is a method of therapy capable of eliminating all graft-transmissible plant pathogens, while maintaining the integrity of the original plant material. Shoot-tip meristems with 2-3 leaf primordia, theoretically too young to be infiltrated by vascular-limited pathogens, are harvested and grafted onto in-vitro grown Troyer citrange seedling rootstocks. Surviving micrografts are then regrafted to an established rootstock seedling and grown-out in the greenhouse in preparation for a second round of bio-indexing & laboratory testing to confirm elimination of any pathogens that may have been detected in the preliminary indexing. Due to the uneven distribution of many citrus viruses or virus-like pathogens in their hosts and to the variation in the success among individual shoot tip grafts, several shoot tip grafts are chosen for post-therapy indexing.
Heat or “thermaltherapy” is the second method used to inactivate plant pathogens. Although this method is not effective on heat-tolerant pathogens such as citrus viroids and some bacteria, it is effective on citrus viruses which may be otherwise difficult to eliminate by shoot-tip micrografting. Dormant buds are harvested from previously shoot-tip micrografted plants, grafted onto heat-tolerant seedling rootstocks and subjected to five months of thermaltherapy. One month preconditioning in a greenhouse where temperatures range from 26-36°C, followed by four months in a growth chamber at constant 30/40°C, night/day, 8/16hours. Once treatment is complete, buds are unwrapped and exposed, callus formation is cut away and the buds are forced by lopping over the rootstock.
Once final bio-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 where horticultural evaluations and budwood increase are conducted.
Expedited Protocol
Citrus germplasm received as shoot tip grafts or originating from an institution that maintains healthy pathogen-free citrus material in an insect-free environment may enter Florida under an expedited testing program. The use of this protocol requires that the practices and procedures of the donor institution must be approved by the Florida Citrus Budwood Technical Advisory Committee and it must be certified free of virus and virus-like pathogens by the donor institution. A record of testing methods employed must accompany the germplasm shipment.
Budwood received under the expedited indexing protocol essentially undergoes only level 3 of the full protocol. Since most graft-transmissible pathogens of citrus are not seed-transmitted, germplasm intended for use only as seed should be imported as seed.
