Introduction. Citrus graft-transmissible diseases pro- duced by viruses, viroids, some bacteria, spiroplasmas and phytoplasmas produce very important economic losses in

Citrus graft-transmissible diseases pro- duced by viruses, viroids, some bacteria, spiroplasmas and phytoplasmas produce very important economic losses in most citrus-growing areas. In general, they cause decline, loss of vigour and short commer- cial life of trees, low yields and poor fruit quality, and they restrict the use of some rootstocks. Thus, they have the potential to become primary limiting factors of produc- tion. Only preventive measures are useful for control of graft-transmissible pathogens, such as use of tolerant or resistant germplasm, exclusion of potential diseases from the citrus area, and establishing orchards using pathogen-free, high quality nursery trees.

Frequently pathogen-free plants of many cultivars were not available and it was necessary to recover healthy plants from infected ones. The procedure used in the past to recover pathogen-free citrus plants was the selection of nucellar seedlings of polyembryonic or apomictic cultivars (Weathers and Calavan, 1959; Roistacher, 1977). Nucellar embryony is effective because most citrus pathogens are not trans-

mitted through the process of embryogene- sis, and because nucellar plants are pro- duced by asexual embryogenesis in vivo, and thus have the same genotype as the mother plants that produced the seeds. The limitation of the procedure is that nucellar plants are juvenile, and consequently they are excessively vigorous, thorny and late in bearing, and they have to be grown for many years until these characters disappear and are acceptable for commercial propagation (Roistacher, 1977). In addition, seed trans- mission of psorosis-like pathogens and citrus leaf blotch virus has been reported (Guerri et al., 2004). Recovery of pathogen- free nucellar plants of monoembryonic cul- tivars by nucellus culture in vitro has been reported (Rangan et al., 1968; Juá rez et al., 1976), but most of the plants are not true to type (Navarro et al., 1985).

Thermotherapy has been used to recover pathogen-free citrus plants without juvenile characters. However, this tech- nique is not effective for the elimination of pathogens which replicate well under warm conditions, such as citrus exocortis, cachexia and other citrus viroids, citrus stubborn or citrus leaf blotch virus (dweet mottle) (Roistacher 1977).

© CAB International 2007. Citrus Genetics, Breeding and Biotechnology (ed. I.A. Khan) 353

In this situation, a method to recover citrus plants free of all graft-transmissible pathogens and without juvenile characters was required to produce healthy trees for commercial plantings. The initial attempts to accomplish this objective were made by shoot-tip culture in vitro, a technique widely used to recover healthy plants of many plant species, particularly herba- ceous ones. However, all attempts failed (Murashige et al., 1972), and still today it has not been possible to regenerate citrus plants from small shoot-tips cultured in vitro (L. Navarro and J. Juá rez, unpublished results). Murashige et al. (1972) were able to recover a few citrus plants by grafting shoot-tips from diseased plants on young rootstock seedlings growing in vitro. Some of these plants were free of the exocortis viroid and did not have juvenile characters. This technique, named shoot-tip grafting in vitro (STG), was studied in detail by Navarro et al. (1975), who developed a rou- tine procedure that allowed a 30–50% inci- dence of successful grafts that were transplanted to soil with over 95% survival. The resulting plants did not have juvenile characters, and most of them were free of graft-transmissible pathogens.

Several reviews of the STG technique have been published previously (Navarro 1981, 1988, 1992; Jonard, 1986). In this

chapter, the different steps of this tech- nique are described in detail, and the elim- ination of pathogens and its research and industry applications are reviewed.