Conservation of Citrus Germplasm 4 страница

these broader issues. It is these broader issues that may make a formal effort at con- serving genetic resources necessary or desirable. If a species is well represented in a stable environment, it is less vulnerable to extinction or genetic erosion than if it is represented by only a few specimens in an area undergoing rapid degradation.

The methods as well as the style of documentation are undergoing rapid change due to the infl uence of technology. Early documentation of citrus germplasm was based on personal observation and handwritten notes, with accessions listed in an accession book. Today, observations may be supplemented by information gath- ered from a global positioning device, with visual information provided by a digital camera and entered into a portable com- puter. Evaluation of accessions has also been enhanced by advances in instrumenta- tion and electronic data analysis and main- tenance. This statement is made with the realization that, while the cost of computer technology is decreasing and use of com- puters is increasing, in any individual instance, a ‘database’ may indeed still con- sist of handwritten records. Furthermore, just as a handwritten observation may be lost or deteriorate, electronic data may become deleted or their format or medium become obsolete. Thus, both hard copy and electronic records should be backed up and, in addition, care must be taken that the format of electronic data is updated period- ically so that it may be accessed by cur- rently available machines. Even if data are backed up in several locations electroni- cally, original handwritten records should be retained. They are often invaluable in correcting mistakes or solving various mys- teries associated with the collection. Even multiple copies of the same printed form, with notes taken by different workers, should be retained.

Databases are useful for maintaining, manipulating and reporting data. However, today, most database records are still based upon handwritten observations or notes. There are a few scientists who may take a portable computer into the fi eld or green-

house and make observations directly into it there, but most still make their observa- tions on a datasheet or in a notebook. So, the transcription of original data to the maintained form still offers a potential step for miscopying, whether to a written or an electronic record. Thus, each step in the record-keeping process must be done care- fully and with an eye towards quality con- trol.

instances is the justifi cation for investing in genetic resource conservation. Citrus germplasm banks are or can be used for breeding and research, and directly for commercial propagation through certifi ca- tion programmes.

Utilization of citrus genetic resources for breeding

Often, data are maintained in both local and public databases. The public data- base generally contains non-confidential data that are of interest to the general public and potential users. These often consist of accession information, passport data and a limited amount of descriptor data. Many important types of data, particularly man- agement data, are critical to local users but may not be important to potential collabo- rators. Therefore, complete and well-organ- ized local databases are critical to the functioning of a germplasm collection. However, some types of local data may be of interest to colleagues. For instance, a sci- entist requesting germplasm will often request data from pathogen testing that may have taken place. Accessions or potential accessions should be assigned accession (or pre-accession) numbers that are maintained in the local database. In addition, individ- ual trees within each accession number should have inventory or tracking numbers assigned so that unique properties, prob- lems, etc. associated with individual trees will not become associated with the acces- sion in general or with other trees of the same accession. These numbers should be assigned and recorded as early in the life of the plant as possible. In addition to general information on accessions, local databases should precisely and thoroughly document such attributes of individual trees as root- stock, propagation source, date of propaga- tion, pathogen status, location and notes.

Utilization

Utilization of genetic resources is a primary goal of germplasm banks and in many

Citrus is affected by important abiotic stresses, such as acidic, alkaline and saline soils, fl ooding and drought, freezes and high temperatures; by arthropod, vertebrate and nematode pests; and diseases caused by fungi, bacteria, spiroplasmas, phytoplas- mas, virus, viroids and virus-like pathogens. In addition, markets demand fruits of high quality that are diffi cult to produce because of a lack of adequate vari- eties. Existing high-quality varieties may be impossible to grow in many areas due to susceptibility to the above-mentioned biotic and abiotic stresses.

The consequence of this situation is that the number of varieties and rootstocks that can be used in different citrus-growing areas is very limited and the production is not well adapted to market demands. Genetic improvement has a very high prior- ity to solve these problems, and several countries have been carrying out breeding programmes since the end of the 19th cen- tury (Soost and Cameron, 1975; Soost and Roose, 1996). However, the results so far have been very limited as a consequence of the complex reproductive biology of citrus, including several factors such as apomixis, total or partial pollen or ovule sterility of many genotypes of interest, autoincompati- bility and incompatibility among geno- types, high heterozygosity and long juvenile periods (Cameron and Frost, 1968; Frost and Soost, 1968; Soost and Cameron, 1975; Soost and Roose, 1996). For these rea- sons, the number of active breeding pro- grammes for citrus has been much more limited than for other species, and conse- quently the citrus genetic resources have not had an intensive utilization for breed- ing.

In recent years, several biotechnology techniques have been established for citrus breeding that solve some of the problems of traditional breeding (Navarro et al., 2004). This includes symmetric and asymmetric protoplast fusion procedures that overcome the heterozygosity problem and allow the recovery of somatic hybrids among sexually incompatible parents, including citrus rela- tives (Grosser et al., 2000). The optimiza- tion of technologies for embryo rescue and ploidy analysis by fl ow cytometry is allow- ing establishment of large triploid breeding programmes to recover seedless cultivars (Navarro et al., 2003; Chapter 8). Citrus transformation technologies are opening up completely new and expanding possibili- ties for citrus improvement (Peñ a and Navarro, 1999; Chapter 15). The develop- ment of molecular markers for qualitative and quantitative traits is also advancing very quickly (Chapter 12) and this will accelerate breeding programmes through marker-assisted selection. In addition, the development of large citrus genomic proj- ects that is already underway in some coun- tries such as Japan, Brazil, Spain and the USA will undoubtedly produce a lot of information that will contribute to citrus improvement. Utilization of genetic resources maintained in germplasm banks for citrus breeding is increasing signifi - cantly with the initial application of these new technologies. In the near future, it is expected that utilization for this purpose will be expanded along with further devel- opments in biotechnology.

Utilization of citrus genetic resources for research

Probably the major use of collections of citrus genetic resources is for breeding. However, there are other research uses for materials from these collections. Germplasm collections are continuously being used by physiologists, biologists, plant pathologists and investigators in other disciplines. For instance, the identifi - cation of ‘Dweet’ tangor as an indicator for psorosis (Wallace, 1945) and ‘Parson’s

Special’ mandarin for cachexia (Calavan and Christiansen, 1965) was possible because of citrus genetic resources main- tained in Riverside.

However, the distinction between research to identify materials for utilization in breeding programmes and other types of research is not always clear. For instance, some of the most common investigations carried out are screening of accessions for useful traits, such as disease resistance/tol- erance or adaptation to soil conditions. This information may be utilized in an improvement programme as well as more directly. An example is the trifoliate orange. Early on, its reaction to CTV was known (Costa et al., 1949; Grant and Costa, 1948; Grant et al., 1949, 1951). This led to its direct use both as a rootstock and as a source of CTV resistance in breeding pro- grammes. More recently, the genetic basis of this reaction has been characterized (Gmitter et al., 1996; Mestre et al., 1997a, b, c; Fang et al., 1998a; Fang and Roose, 1999; Deng et al., 2001; Yang et al., 2001). This information may be useful in improvement as well as being interesting as purely basic information.

Many traits documented in Table 4.3 have overlapping bases and results. Not all of these documentations are the result of investigations utilizing formal collections of citrus genetic resources, but some are. Conversely, investigation into and docu- mentation of these types of traits from sources outside a collection can lead to inclusion of certain accessions in collec- tions of genetic resources.

Utilization of citrus genetic resources for certifi cation programmes

The subject of certifi cation programmes for citrus is linked to the area of germplasm resources and will be briefl y addressed. Certification programmes are primarily designed to control graft-transmissible and other important diseases. They only use advanced commercial varieties and in most cases they operate independently of germplasm banks. In citrus, there are only a

few cases where there is a direct connection between certification programmes and germplasm banks, although some of the cer- tifi ed varieties may come from germplasm banks. For more information on certifi ca- tion programmes in general, a recent review has been produced by Waterworth (1998). For citrus in general, Lee et al. (1999) and Navarro (1993) offer good overviews.

The devastating nature of citrus diseases has been noted above. Roistacher (1993), in arguing for mandatory certifi cation pro- grammes for citrus, quite rightly notes that ‘Citrus stands alone among the tree and fruit crops of the world for having the largest number of virus and virus-like diseases which have the capacity to debilitate or destroy its industry. There are very few or no comparable diseases of stone, pome, or vine fruits which can match the rapacity of... citrus diseases …’. Therefore, certifi cation programmes are highly recommended to sup- port citrus production and help maintain the phytosanitary status of the citrus industry.

Citrus was one of the fi rst crops for which various types of clean stock and cer- tification programmes were devised. Probably the fi rst proto-certifi cation pro- gramme for citrus was the ‘Psorosis Free’ programme started in California in 1937 (Calavan et al., 1978). As additional dis- eases were shown to be caused by viruses or other graft-transmissible pathogens, the programme evolved to the form in which it is found today (Gumpf et al., 1997; Bash, 1999; Krueger, 2001). In its current form, clean source trees are established by the Citrus Clonal Protection Program (CCPP), which is run by the University of California. After release to the industry, propagation and distribution of trees are regulated by the California Department of Food and Agriculture (CDFA).

The California programme is the oldest and longest-running certification pro- gramme for citrus. Other well-established programmes include those of Spain (Pina and Navarro, 2001), Australia (Forsyth, 1990), South Africa (Lee and von Broembsen, 1990) and Florida (Castle et al., 2001). The protected collection of the

Spanish germplasm bank is totally inte- grated in the certifi cation programme, since it is the Protected Foundation Block of Initial Material for certifi cation (Navarro et al., 2002; see also Chapter 17). Many other countries are establishing certifi cation pro- grammes or taking the fi rst step in starting phytosanitation programmes. Many of these programmes are supported technically by the FAO and often start foundation plant- ings with materials obtained from estab- lished programmes in California or Spain. Frequently, expert consultations from the FAO or other entities provide the impetus for start up of local indexing and sanitation programmes to complement the introduced germplasm. In addition to the FAO, various other NGOs have taken an active role in promoting or establishing regional certifi ca- tion programmes. These notably include IPGRI, which through its relationship with the International Agricultural Research Centers and the Consultative Group of International Agricultural Research, seeks to promote phytosanitary standards in the exchange of germplasm for research pur- poses (Frison and Diekmann, 1998), and the European/Mediterranean Plant Protection Organization. More recently, the North American Plant Protection Organization has become involved in this area.

Navarro (1993) and Lee et al. (1999) differentiate certifi cation programmes from quarantine and clean stock (or sanitation) programmes. In this schema, quarantine programmes (as discussed above) are designed to ensure the safe introduction of new types of germplasm, while clean stock programmes have the aim of producing and maintaining healthy, true-to-type local cul- tivars. In reality, most of the techniques and technology used in a quarantine pro- gramme are also utilized in clean stock pro- grammes, and sub-federal level regulations or quarantines make handling local culti- vars similar from a security standpoint to handling foreign introductions even if the risk of introducing exotic pests or pathogens is lower.

Certifi cation programmes, as the term is used by Navarro (1993) and Lee et al.

(1999), introduce regulatory or legal requirements and restrictions that enforce to some degree the use of clean material. These regulations are based upon local con- ditions and may not regulate all pests and pathogens. For instance, in California the CCPP provides a quarantine programme for introduction of new varieties from outside California, and also functions as a clean stock programme for cultivars already established in California. In providing these functions, CCPP tests for all known graft- transmissible pathogens. However, after release from state (and where applicable, federal) quarantine(s), the propagation of these materials is regulated by the CDFA, and testing is done for only a limited range of endemic diseases.

The most important components of cer- tifi cation programmes are the sources of propagative material. The bases for all fur- ther propagations are Foundation Blocks. These include both Protected Foundation Blocks of Initial Material and fi eld-planted or protected Foundation Blocks usually located at the nurseries. The tendency today is to protect as much as possible all blocks of the programme. In Spain, IVIA holds the Protected Foundation Block of initial material, which is the protected col- lection of the germplasm bank, and the nurseries hold nine foundation blocks that are also protected. The multiplication blocks located in 39 nurseries are also all protected. Protected Foundation Blocks offer greater security as far as avoidance of infection by an insect vector, but infrastruc- ture costs are greater. Foundation plantings should be periodically re-tested for the pathogens included in the regulations, which always include at least the endemic pathogens. Although this is obviously more important for a fi eld-planted Foundation Block than for a Protected Foundation Block, the latter should also be re-tested although regulations usually establish longer testing intervals. The most impor- tant diseases from a re-testing standpoint are endemic diseases that are naturally spread.

Due to the expenses associated with

maintaining and re-testing foundation trees, only a small number of trees from each variety are generally maintained (typically 2–6). This is not enough to supply bud- wood directly to propagate nursery trees. Therefore, material from the Foundation Block is normally used to establish Budwood Increase Blocks, allowing the rapid and effi cient multiplication of buds. Increase Blocks may be protected or estab- lished in the fi eld. The fi nal step in a certi- fi cation programme is the production of certifi ed nursery trees from either founda- tion materials or Increase Blocks. Production may be protected or in the fi eld. Again, the actual requirements for certifi ca- tion are mandated by regulatory agencies and vary from location to location.

Certifi cation programmes may be either mandatory or voluntary. Mandatory pro- grammes are of course more coercive, but in most cases are more effective than volun- tary programmes. In voluntary programmes, the initially higher investment in certifi ed materials may lead some nurserymen to use non-certifi ed materials despite the long- term negative economic impact of using this inferior material. This can lead to greater disease pressure than if a pro- gramme was mandatory and, if endemic vector-transmitted diseases exist, these can be transmitted to trees produced under a voluntary certifi cation programme. Thus, a few non-cooperative nurseries seeking a short-term economic advantage can cause long-term phytosanitary and economic damage to an industry.

Certifi cation programmes are among the best established means of increasing phy- tosanitary health, and some of those for citrus are among the oldest in the world. In con- junction with quarantine and clean stock programmes, they remain important weapons in the ongoing fi ght against citrus diseases.

Germplasm banks may have a very important role in certifi cation programmes by supplying the initial materials for prop- agation, particularly if they maintain healthy plants of commercial varieties. In some cases, such as in Spain, the

germplasm bank is an integrated part of the certification programme and also is in charge of the post-entry quarantine station. Nurseries have already released 100 million plants to growers that originated in the germplasm bank. Healthy material from some germplasm banks, like those located in the USA, Spain and France (Corsica),

have been used by many countries to start certifi cation programmes when resources to establish their own clean stock programmes were not available. This is an example of international collaboration that has pro- duced large benefi ts to the citrus industry worldwide and that shows the importance of maintaining citrus genetic resources.