Technique of Shoot-tip Grafting In Vitro

The standard procedure described by Navarro et al. (1975) is routinely used in most laboratories. It includes the following steps: rootstock preparation; scion prepara- tion; grafting; culture in vitro of grafted plants; and transfer to soil.

Rootstock preparation

Seedlings recovered by seed germination in vitro are used as rootstocks. Seeds are peeled, removing both seed coats (Fig. 17.1a), surface sterilized and sown in 25 ´ 150 mm culture tubes containing 25 ml of the plant cell culture salt solution of Murashige and Skoog (1962), solidified with 1% Bacto agar. Cultures are incubated at 27°C in continuous darkness for 2 weeks (Fig. 17.1b).

The age of the rootstock has an impor- tant infl uence on grafting success (Navarro et al., 1975). The highest rate of successful grafts using Troyer citrange (Poncirus trifoli- ata (L.) Raft. ´ Citrus sinensis (L.) Osb) as rootstock was obtained with 2-week-old seedlings. Most shoot-tips grafted on younger seedlings (1 week old) were cov- ered with precocious callus formation by the rootstock, whereas most shoot-tips grafted on older rootstock (3 and 4 weeks old) dried, turned brown and died. These results suggest that grafting success depends on the degree of tissue differentiation of the rootstock, which is affected by age.

Age may be only indicative of seedling rate of development, which is infl uenced by embryo size, germination temperature, seed batch and storage period. Stem height and diameter are parameters more appropriate to determine the optimal stage of seedling for grafting. We routinely use Troyer cit- range seedlings 3–5 cm tall with a diameter of 1.6–1.8 mm at the point of grafting. This size is reached in 12–16 days at 27°C.

Seedlings can be stored at 4°C for at least 2 weeks without any detrimental effect on grafting success. This allows an easy selection of uniform seedlings for experimental purposes.

Troyer citrange is the most commonly used rootstock for STG. It has the advantage that its trifoliate leaves serve as a morpho- logical marker for identifi cation of adventi- tious shoots produced by the rootstock. However, any rootstock graft compatible with the shoot-tip scion variety could be used for STG. At least the following root- stocks have been successfully used for STG:

Troyer and Carrizo citranges, Rough lemon (C. jambhiri Lush.), P. trifoliata, Alemow (C. macrophylla West), Etrog citron (C. medica L.), sour orange (C. aurantium L.), Rangpur lime (C. limonia Osb.), Volkamer lemon (C. volkameriana Pasq.), sweet orange (C. sinensis (L.) Osb.) and Cleopatra mandarin (C. reshni Hort. ex. Tan.). Differences in grafting success have been observed among these root- stocks, but detailed comparative studies have not been done. In our laboratory, we routinely use Troyer citrange as rootstock for STG.

The rootstock seedling is removed from the test tube under aseptic conditions and it is decapitated, leaving about 1.5 cm of the epicotyl. The root is cut to a length of 4–6 cm and the cotyledons and their axillary buds removed (Fig. 17.1c). Grafting can be done by several methods, but it is recom- mended to place the shoot-tip in an inverted-T incision (Navarro et al. 1975) (Fig. 17.1i). This is made by a 1 mm long vertical incision, starting at the point of decapitation, and a 1–2 mm wide horizon- tal cut. The cuts are done through the cortex, and the fl aps of the incision are slightly lifted to expose the cortex.

Scion preparation

Shoot-tips can be excised from growing veg- etative fl ushes of fi eld or greenhouse plants, or from budwood cultured in vitro. Shoot- tips can also be excised from dormant buds or shoots from buds cultured in vitro, but these are not recommended sources for rou- tine work, since the incidence of successful grafts and pathogen elimination was reduced in comparison with other sources (Navarro et al., 1975, 1976).

Growing vegetative fl ushes from the different origins are the recommended source of shoot-tips for STG. Field trees are the most direct source of shoot-tips, but have the disadvantage of the seasonal fl ush- ing and the lower rate of elimination of some pathogens. Greenhouse plants have the advantage that fl ushes can be induced

when necessary, avoiding the seasonal dependency of fi eld trees. In addition, they can be subjected to a warm pre-treatment to improve the effi ciency of elimination of some pathogens (Navarro et al., 1980b). Infected citrus plants growing in pots in the greenhouse could be completely defoliated by hand and placed in a warm green- house, or in a growth chamber. As a routine procedure in our laboratory, the infected cultivars are propagated and grown in containers in a greenhouse at 18–25˚ C. They are defoliated by hand when needed, placed in a growth chamber at constant 32˚ C and exposed to

350 mE m–2 s–1 illumination 16 h daily.

After 8–12 days, new fl ushes are produced (Fig. 17.1d) and used as source of shoot-tips for STG.

Budwood cultured in vitro at constant 32˚ C and exposed 16 h daily to 80 mE m–2 s–1 illumination in a culture medium con- taining the plant cell culture salt solution of Murashige and Skoog (1962) solidifi ed with 1.2% Bacto agar is also an excellent source of shoot-tips for STG. New fl ushes are pro- duced in 8–16 days (Fig. 17.1e) and used for isolation of shoot-tips for STG. This source is routinely used in our laboratory for plant introduction through the Citrus Quarantine Station (see below) (Navarro et al., 1984) and is being increasingly used for local genotypes, since it is faster than propaga- tion of plants in the greenhouse, and it gives a better grafting success (Navarro et al., 2002).

Grafting

Flushes 3 cm long or shorter are collected to avoid the abscission stage. They are stripped of larger leaves, cut to about 1 cm long (Fig. 17.1f) and surface sterilized by immersion in a mild 0.25% sodium hypochlorite solution. Larger leaves of shoots are removed with the aid of dissect- ing instruments under a microscope, and shoot-tips composed of the apical meristem and three leaf primordia, and measuring 0.1–0.2 mm in length, are then excised with

a razor blade sliver attached to a surgical handle (Fig. 17.1g and h). This shoot-tip does not have a vascular connection with the rest of the plant (Fig. 17.1g), and this explains why most pathogens are elimi- nated by shoot-tip grafting. The shoot-tip is placed inside the incision of the rootstock with its cut surface in contact with the cortex exposed by the horizontal cut of the incision (Fig. 17.1i) made at the top of the decapitated epicotyl (Navarro et al., 1975).

The frequency of successful grafts increases with shoot-tip size, but the inci- dence of recovery of healthy plants decreases (Navarro et al., 1976). Consequently, it is necessary to choose a size that will give a realistic degree of graft- ing success with the highest possible number of pathogen-free plants, since indexing is more expensive and time con- suming than STG. In our laboratory, we routinely use shoot-tips composed of the apical meristem and three leaf primordia. The size, measured from the cut end to the tip of the largest leaf primordium, varies from 0.1 to 0.2 mm, depending on the citrus species.