Applications of STG

Control of graft-transmissible pathogens

To control diseases caused by graft-trans- missible pathogens, it is necessary to use healthy and high quality trees in the new plantings. The production of these trees requires the establishment of three different but related programmes: sanitation, quaran- tine and certification (Navarro, 1993). Sanitation programmes have the objective to recover healthy plants from local vari- eties, and quarantine programmes have the objective of importing foreign varieties avoiding introduction of new pests and dis- eases. STG plays a key role in these two programmes that produce healthy plants for the certifi cation programmes, which have the objective to guarantee that the sanitary status of the initial material is maintained during the process of commercial propaga- tion at the nurseries. Figure 17.2 shows a diagrammatic representation of these pro- grammes.

Sanitation programmes

The most important application of STG is the recovery of healthy plants. It has been effective to recover plants free from all pathogens assayed, including the causal agents of the following diseases: cachexia, canker, concave gum, cristacortis, dweet mottle, exocortis, huanglonbing (ex green- ing), impietratura, infectious variegation- crinkly leaf, leaf blotch, psorosis A and B,

Fig. 17.2. Diagrammatic representation of the citrus sanitation, quarantine and certifi cation programmes.

ringspot, rough lemon incompatibility, seedling yellows-tristeza, stubborn, tatter leaf, tristeza and yellow vein (Navarro et al., 1975, 1976, 1980a, b, 1981, 1988, 1991,

2002; Roistacher et al., 1976; Roistacher

and Kitto, 1977; Arroyo, 1984; Koizumi 1984; Su and Chu, 1984). These include diseases produced by viruses, viroids, mycoplasma, phloem-restricted bacteria and graft-transmissible agents of unknown nature.

The most important factors that infl u- ence elimination of citrus pathogens by STG are the pathogen itself, the size of the shoot-tip and the growing temperature of the shoot-tip source plants. Some pathogens are very easy to eliminate, such as citrus viroids (cachexia, exocortis), infec- tious variegation-crinkly leaf, stubborn, huanglonbing (ex greening), tristeza and vein enation, and almost 100% of the plants recovered by STG are free of these diseases. Seedling yellows-tristeza and yellow vein are easy to eliminate by STG, and around 80% of micrografted plants are free of these

diseases. Finally, concave gum, cristacortis, dweet mottle, leaf blotch, impietratura, psorosis, ringspot and tatter leaf are diffi - cult to eliminate, and usually fewer than 25% of the micrografted plants are free of these diseases. These data were obtained when shoot-tips were collected from fi eld trees or plants growing in a greenhouse at 18–25°C.

Shoot-tip size has an important infl u- ence on the incidence of healthy plants recovered by STG. It was shown that increasing shoot-tip size resulted in higher incidence of successful grafts, but with an important decrease of the incidence of healthy plants (Navarro et al., 1976). The relatively low frequency of healthy plants obtained in some laboratories is probably due to the use of larger shoot-tips. As men- tioned above, a shoot-tip composed of the apical meristem and three leaf primordia measuring 0.1–0.2 mm gives a realistic fre- quency of successful grafts and healthy plants, and is recommended for routine work.

The incidence of recovery of plants free from diseases difficult to eliminate increased by growing the shoot-tip source plants under warm conditions (Navarro et al., 1980b; Koizumi, 1984). As mentioned above, in the routine procedure in the CVIPS, shoot-tips are isolated from plants growing in a growth chamber at constant 32˚ C or from budwood cultured in vitro at 32˚ C. With this procedure, more than 90% of micrografted plants are usually free of pathogens, including those diffi cult to elim- inate (Navarro et al., 1988, 2002). The only exception is citrus leaf blotch virus, that is eliminated in fewer than 50% of micro- grafted plants.

STG is the best available technique to recover pathogen-free citrus plants for sani- tation programmes, since it is possible to recover plants free of pathogens that cannot be eliminated by thermotherapy, the result- ing plants do not have juvenile characters, as occurs with plants recovered by nucellar embryony, and all recovered plants are true to type. STG is being used in citrus sanita- tion programmes in all the major citrus- growing countries.

In Spain, there is a very extensive citrus sanitation programme based on STG (Navarro, 1976; Navarro et al., 1980a, 1981, 1988, 2002). About 120 million healthy cer- tifi ed nursery trees, originally recovered by this technique, have already been planted in the field. These healthy trees have 15–20% more production of fruit of higher quality that the original infected trees. Diseases produced by graft-transmissible diseases, that were the main limitation of the Spanish citrus industry in the past, do not now produce damage in the new plant- ings.

Quarantine procedures

Movement of citrus species and varieties between different citrus areas for commer- cial and scientifi c purposes is often desir- able. However, uncontrolled importation of budwood carries the risk of introducing new pests and pathogens that in some instances may be devastating or may cause

very important economic damage. This risk may be minimized by controlled introduc- tion through quarantine stations, that have the objective of importing foreign varieties while avoiding the introduction of new pests and diseases that may be carried in the original material. In citrus there are two different quarantine procedures that can be safely used for importation of plant mate- rial (Frison and Taher, 1991)

The classic method consists of propa- gating the imported budwood in quarantine greenhouses located far away from citrus- growing areas. Then the newly propagated plants can be indexed or submitted directly to STG followed by indexing. This proce- dure requires the availability of facilities and trained personnel on citrus pests, dis- eases and cultural practices in areas not directly involved with citrus research. It is used in some countries with a long tradi- tion in quarantine that have central facili- ties and personnel for importation of plant material of several crops, but it is very expensive and diffi cult to establish only for citrus in most countries.

An alternative citrus tissue culture pro- cedure was developed for safe introduction of citrus genotypes (Navarro et al., 1984, 1991) that has been proved to be very effi - cient in excluding citrus pests and diseases (Fig. 17.3). Budsticks that are received from another country are thoroughly cleaned and surface sterilized and then cultured in vitro at 32°C in a growth chamber as described above to induce the sprouting of lateral buds and formation of fl ushes (Fig. 17.1e) from which shoot-tips are isolated and micrografted in vitro. The only material really imported is a small shoot-tip that usually is free of pests and pathogens. This process includes many different controls (Fig. 17.3) and minimizes the possibility of escape of harmful pathogens, and it allows rapid processing of new entries.

This tissue culture method has several advantages over the traditional quarantine method. Pests and diseases that might be in the original material are eliminated at the early stages of introduction, thus shorten- ing the quarantine period. With tissue cul-

Fig. 17.3. Diagrammatic representation of the procedure of in vitro quarantine of citrus.

ture, the test tubes serve as a substitute for the greenhouses located in isolated areas, and thus the quarantine station may be located at citrus research stations. At many of these stations STG is being used for san- itation of local cultivars, and the required facilities and personnel are usually avail- able. Consequently the tissue culture proce- dure can be easily established in many countries for safe import of citrus vegetative material. This method is recommended for exchange of citrus germplasm (Frison and Taher, 1991) and has been legally accepted in several areas, such as the European Union. Due to these advantages, the tissue culture method is recommended for the safe importation of citrus budwood. In Spain, it has been used successfully to import over 200 varieties from different citrus areas.

Research applications

In addition to research with graft-transmis- sible pathogens, STG is becoming a very useful technique for production, propaga-

tion and regeneration of elite genotypes in several areas of research. In vitro grafting for this purpose may be done using larger shoots (at least up to 1 cm) using different types of incisions (Fig. 17.4) with close to 100% grafting success. Some research applications of STG are described below.

Regeneration of somatic hybrids

In protoplast fusion experiments, abnormal embryos are very often produced. These include multiple fasciated cotyledons, embryos that only produce shoots, germi- nating embryos without a good vascular connection between shoot and root, and abnormal shoot proliferation, among others (Fig. 17.5) (Olivares-Fuster, 1988). These embryos do not produce plants that can be established in the greenhouse, thus reduc- ing the effi ciency of recovery of somatic hybrids and losing potentially valuable genotypes. In our laboratory, we routinely graft in vitro shoots produced by these embryos to recover plants that are estab- lished in the greenhouse with high effi - ciency (Fig. 17.5).

Regeneration of plants from irradiated shoots

Irradiation is used in citrus improvement programmes in attempts to reduce the number of seeds produced by high quality genotypes, particularly with mandarins. However, in many cases, unstable chimeras are produced that revert to the original vari- ety after some cycles of propagation. In assays to produce seedless clementines, shoot-tips were isolated, placed facing upwards in a Petri dish with agar media and irradiated. The assumption was that cells of the exposed meristem would be mutated, producing stable plants. After irradiation, whole plants were recovered by STG, transplanted to soil and evaluated (Fig. 17.6). A relatively high number of apparently stable mutants have been pro- duced and a new variety Nulessí n selected and released (Asins et al., 2002).

Regeneration of haploid plants

Haploid plants have interesting applica- tions in citrus genetics and genomics. They can be recovered by in situ parthenogenesis after pollination with irradiated pollen, which induce aborted seeds that in some cases contain haploid embryos, that after in vitro culture produce a few plants very dif- ficult to establish in the greenhouse (Ollitrault et al., 1996). We have used this approach in our laboratory, and haploid embryos produced abnormal clusters of proliferating tissues that did not allow the regeneration of plants. However, grafting in vitro some of these shoot-like tissues allowed us to regenerate several Nules clementine plants that were successfully established in the greenhouse, where they are even fl owering (Fig. 17.7).

Production of stable tetraploid plants of monoembryonic genotypes

Tetraploid plants of monoembryonic geno- types are very important for use as female parents in triploid mandarin breeding pro- grammes. However, this type of genotype is not available. They could be recovered by

colchicine treatment of budwood, but usu- ally only unstable chimeras without any value for breeding are produced. We have attempted the recovery of tetraploid plants by treatment of shoot-tips with colchicine solution and regeneration of plants by STG or by adding a drop of colchicine solution on a shoot-tip two weeks after grafting in vitro (Juá rez et al., 2004). The objective was to induce chromosome duplication in the exposed cells of the meristem. We were able to recover stable tetraploid plants of Nules, Fina and Marisol clementines and Moncada mandarin that are now the most widely used female parents in our triploid breeding programme (Navarro et al., 2003).

Regeneration of plants from somaclonal variation experiments of adult material

Somaclonal variation is an interesting approach for citrus improvement that is being used for this purpose in some labora- tories (Grosser et al., 2003). Clementines are a good candidate for somaclonal variation studies, since they are genetically unstable and frequently produce budsports in the field. We have attempted to recover somaclonal variants through the process of adventitious organogenesis from adult internode tissues, but these only produce very tiny buds that cannot be rooted to pro- duce plants (Fig. 17.8). However, grafting these buds in vitro allowed us to recover more than 450 plants that are under fi eld evaluation (Fig. 17.8).

Regeneration of transgenic plants

One of the major limitations of citrus trans- formation was the diffi culty in recovery of plants from transgenic shoots (Peñ a et al., 2003; see also Chapter 15). With many genotypes, the rooting effi ciency of trans- genic shoots is very low and rooting of shoots from adult material is almost impos- sible. The use of STG to regenerate plants from transformed shoots or buds has become a routine procedure in most labora- tories, allowing an important increase of

the effi ciency of the genetic transformation protocols (Fig. 17.9).