Conservation of Citrus Germplasm 1 страница

Although conservation of citrus genetic resources in situ is valuable and extremely important, conservation ex situ is vital for the utilization of these resources by breed- ers, horticulturists, phytopathologists, etc. The establishment of ex situ collections of citrus germplasm permits the efficient maintenance of appropriate amounts of specific true-to-type, (potentially) pathogen-tested varieties.

The composition of ex situ collections of citrus genetic resources varies with the mission of the collection. Some collections consist solely of commercial varieties, which provide propagative material to growers. Varieties maintained in these col- lections may be pathogen tested and uti- lized in a certifi cation programme, or they may simply be trees of local varieties. These collections cannot be strictly considered as germplasm banks since they have very little variability and their objective is not the conservation of genetic resources. Other collections have the specifi c mission of conserving genetic resources. These collec- tions also include non-commercial types of Citrus for utilization in agricultural research, as well as other genera or species within the Rutaceae (generally within the Aurantioideae).

This section reviews some concepts in the management of citrus ex situ collec- tions. Some general information regarding conservation of the so-called ‘clonal’ crops (at least as practised in the USA) was reviewed some time ago by Westwood

(1989). However, as with many other aspects of citriculture, management of citrus genetic resources differs from that of most other crops, including other clonals. The intent of this section is to provide an overview with general information on man- agement of citrus germplasm resources. An in-depth discussion of the many subjects involved in citrus germplasm resource management is beyond the scope of this chapter. Due to the focus of this book, more information will be provided in the areas utilizing biotechnological techniques. Other areas will be discussed and some general references provided.

Much of this section is based upon the collections with which the authors are most familiar, the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates (NCGRCD)/UCR Citrus Variety Collection at the University of California, Riverside, and the Citrus Germplasm Bank of the Instituto Valenciano de Investigaciones Agrarias (IVIA) in Moncada, Valencia, Spain. Some aspects of the Repository’s functioning have been described by Krueger (1997a, b, 1999) and Williams (1991, 1992a, b); more information about the IVIA germplasm bank can be found in Navarro (1976), Navarro et al. (1980, 1981, 1988, 2002), and at the webpage < https://ivia.es/deps/ biot/germop.htm >.

Overview of citrus genetic resource conservation

Conservation of citrus genetic resources may be thought of as a series of six main steps, not all of which are mutually exclu- sive or in strict sequence: acquisition; intro- duction; maintenance; characterization and evaluation; documentation and databases and utilization. The basic relationships between these various steps are shown in Fig. 4.1.

In order to establish a collection of ex situ citrus genetic resources, acquisition of germplasm resources is necessary. This can occur by plant exploration in an area of genetic diversity; by selection of cultivated

Fig. 4.1. Overview of the management of citrus genetic resources.

or new genotypes originating in an area; or by exchange of germplasm between cooper- ating persons, programmes or agencies. Introduction of germplasm into a germplasm bank has the attendant risk of also introducing exotic pests or pathogens for the area where the germplasm bank is located. If germplasm is introduced from another country, the importation may be regulated by a phytosanitary legislation that could include a mandated quarantine system to protect the phytosanitary health of a country, crop or industry. Even when the germplasm is acquired from within the country where the bank is located, it is advisable to apply therapy methods in order to introduce only healthy plant mate- rial, thus avoiding the presence of

pathogens that may cause the loss of geno- types and/or interfere with their evaluation and utilization.

After introduction, citrus germplasm is maintained in various types of collections in order to conserve it and to make avail- able propagative or other type of material for distribution and utilization. Effi cient utilization is based upon thorough charac- terization and evaluation of pertinent traits. Adequate documentation (nowadays usu- ally in a database) of the germplasm, its provenance and its characteristics is neces- sary for these activities. Because citrus germplasm is generally maintained as living trees, it is appropriate to have back- up collections or plantings. Ideally, long- term preservation of a base collection of

citrus germplasm as a back-up to the active

or working collection is desirable.

Acquisition of citrus germplasm

The fi rst step in the acquisition of citrus germplasm is the identifi cation of the source of the desired genotypes, either domesti- cally or in a foreign country. In this last case, it will be necessary to satisfy legal and political requirements for its introduction, including possible property rights according to international agreements on germplasm exchange. In addition, the phytosanitary and/or quarantine regulations must be met during the following introduction phase.

Identifi cation of citrus germplasm for introduction as new accessions may occur by several means. When a particular type of citrus germplasm is maintained by govern- mental or academic researchers or institu- tions, its existence is often public knowledge. This does not necessarily imply that its existence is widely known, but rather that it is knowable. That statement is meant to contrast the existence of citrus genetic resources in collections (both ex situ and in situ) with those that might be found in the wild or under local cultiva- tion. In the latter circumstances, the exact nature of citrus genetic resources may be known only in very general terms or poten- tially not at all. This is particularly true for areas in which there may be civil unrest or political instability. New and interesting citrus germplasm may also originate by spontaneous budsport mutations in the fi eld.

Citrus germplasm in governmentally or academically maintained collections should be well documented as to its back- ground, availability, disease status and other pertinent facts. Unfortunately, this is not the case in many instances, particularly when a collection has its beginnings in the remote past. The germplasm and its charac- teristics, use, etc. may be discussed at sci- entific meetings, during exchange programmes, in correspondence and other similar professional activities. The same is

true of commercial plantings. The public nature of information concerning these types of citrus germplasm makes identifi ca- tion of useful new accessions a matter of the citriculturist keeping up with events in their fi eld. Obviously, a single individual cannot know of the existence of all types of germplasm. However, an informed researcher will make an attempt to know in general terms of the existence of these resources and will utilize their network of professional contacts to broaden the range of materials with which they are acquainted.

Acquisition of genetic resources from established persons, organizations or enti- ties is termed exchange. There is an impli- cation here of free exchange; however, this is becoming less clear-cut in recent years due to the political factors discussed previ- ously. There have always been legal requirements in germplasm exchange as far as obtaining the necessary permissions, appropriate permits or licences, payment to donors or governmental entities, etc. However, the political factors noted have complicated matters further, and exchange of genetic materials is becoming less free in general than it has been in the past. In many instances, special permission, a Material Transfer Agreement spelling out conditions of use, or other documents must be signed and adhered to where in the past materials were sent freely and could be used without restriction. These requirements are some- times complicated and may interfere with the acquisition of genetic resources. However, the laws of the countries ‘owning’ the genetic resources must be adhered to. An in-depth discussion of these ever- changing issues is beyond the scope of this review, but up-to-date information is avail- able at the IPGRI website (< https://www.ipgri.cgiar.org/>).

Acquisition of citrus genetic resources in a foreign country from wild populations or local plantings owned by indigenous groups is more complicated. As stated, it is often diffi cult or impossible to determine the nature of the genetic resources present in an area. Sometimes information is found in gov-

ernmental surveys, fl ora and other writings. However, these are often not available to researchers from outside the country or, in some cases, outside the immediate area. In this case, it is valuable to have cooperators in the ‘exporting’ country or region who can help identify the potential resources. In some cases, the existence of wild popula- tions is not known to the government or to scientists but only to local inhabitants, and identifi cation of genetic resources in an area becomes almost a matter of serendipity. This is the case in countries such as China and India, where ex situ collections are main- tained but remote sources of uncollected germplasm may still exist.

When genetic resources are to be acquired by plant exploration in a foreign country, it is perhaps even more important to adhere rigidly to whatever guidelines are applicable. It is advisable to secure permis- sion to collect plants from the local author- ities as well as from established governments. Guidance on some of these issues, along with information on the exten- sive planning necessary for a plant collect- ing expedition, are available in Guarino et al. (1995). The remoteness of some desir- able types of germplasm also complicates the physical act of acquiring new materials. This is especially true of germplasm that is often best obtained as perishable vegetative material (i.e. budwood), as is often desir- able with Citrus and related taxa.

Form of propagative material

Citrus germplasm has usually been acquired as either budwood or seed. There are advantages and disadvantages to both forms, and there are often different phy- tosanitary legal requirements for the intro- duction from other countries of seed as compared with budwood. Recently it has been possible in special cases to exchange citrus germplasm as nucellar embryogenic callus cultured in vitro.

Budwood is in many cases the pre- ferred form for exchange. Budwood will be true to type and will not have juvenile char- acteristics associated with it. However,

citrus budwood potentially harbours pests and graft-transmissible pathogens. Therefore, the risk of introducing exotic pests and diseases is much greater with budwood than with seeds. Consequently, introduction of budwood is highly regu- lated, and often an extensive, expensive and time consuming post-entry quarantine process is legally required (Frison and Taher, 1991). This may limit the introduc- tion of new germplasm. This is particularly true in the case of monoembryonic types, which will not come true to type from seed, and for elite varieties or types with unique characteristics, which are best acquired as budwood.

Introduction of new citrus germplasm by seeds is simpler from the phytosanitary standpoint, since systemic seed-borne dis- eases of citrus are very rare (Garnsey, 1999; Timmer et al., 2000). (In addition to the old reports on psorosis transmission through seeds (Childs and Johnson, 1966), there have been recent reports of seed transmis- sion of citrus leaf blotch virus (Guerri et al., 2004) and citrus variegated chlorosis (Li et al., 2003).) However, the extended juvenil- ity period of seed-introduced material delays and complicates the use of acces- sions introduced in this form. In addition, monoembryonic types, including the basic species of citron and pummelo, several mandarin types and most species of related genera, will not come true to type. However, if the goal of an introduction is to increase the amount of genetic diversity present in a collection rather than a specifi c type, this may be acceptable. Even polyem- bryonic types may have off-types present that must be carefully rouged. Recently, it has been possible to identify nucellar seedlings using molecular markers (Ruiz et al., 2000; Krueger and Roose, 2003; Krueger et al., 2003). However, this is somewhat complicated and may be beyond the resources available to some programmes.

Citrus germplasm can also be exchanged in the form of embryogenic nucellar callus that can be recovered by ovule culture in vitro (Button and Bornman, 1971; Kobayashi et al., 1983; Grosser and

Gmitter, 1990; Pé rez et al., 1998, 1999). This type of material has the advantage of being free of pests and diseases. Another advantage is that it can be used directly for breeding through protoplast fusion (Grosser et al., 2000; Olivares-Fuster et al., 2000) or genetic transformation (Fleming et al., 2000). The disadvantage is that recovered plants have juvenile characters and that tissue culture laboratory facilities are needed for exchange.

Minimizing phytosanitary risks in the collection and shipping of citrus germplasm

All possible measures should be taken to avoid collecting propagative material that is infested with pests or pathogens, either if selected in the country or imported from other areas. This is not always possible, particularly when collecting material from wild populations or when relying on a col- laborator who may or may not be able to comply with this concept. However, as far as possible, this principle should be adhered to.

If possible, germplasm should be obtained from a ‘healthy collection’. If this is not possible, material should be obtained from the area with the lowest phytosanitary risk. When possible, source trees should be identifi ed to allow future observation. If maintained in a formal collection, any infor- mation on the disease status or pathogen testing of the trees should be documented, as well as the prevalence of pests and dis- eases. Germplasm should not be collected from high risk areas: areas with diseases for which no indexing procedures are available; areas where background information on donor trees is lacking or very limited; areas with diseases of high economic threat; and areas with diseases that are known or sus- pected to be vector transmitted.

When collecting citrus seeds, sound, healthy appearing, true-to-type fruit should be collected from parts of the tree more than 1 m above the ground. The seeds should be extracted from the fruit and treated if nec- essary to remove the pulp from the seed, and then surface sterilized. When collect-

ing citrus budwood, collecting tools should be sterilized by dipping in a dilute solution of sodium hypochlorite between each tree from which budwood is collected. Budwood should be labelled, packed in plastic bags and maintained at 25˚ C or lower until it can be further processed. The budwood should be rinsed, surface steril- ized, re-rinsed, and air- or towel-dried com- pletely. If possible, the budwood should be dipped in a solution containing a fungicide, insecticide and miticide. The ends of the budsticks or the entire stick can be dipped in melted paraffi n wax to help reduce des- iccation. It is important that both budwood and seeds be completely dry before packag- ing or storing.

Movement of citrus germplasm

Acquisition of citrus germplasm involves movement from one location to another. In a few instances, the distances involved may be short, but often the movements are inter- national in nature. Germplasm may be shipped by several different means, or hand carried if possible. It is important to take all possible measures to prevent deterioration or loss of propagative materials during tran- sit. This is particularly true of budwood, but seeds are also vulnerable to deteriora- tion. Some of the phytosanitary considera- tions in this area are discussed in a later section.

Citrus budwood deteriorates rapidly at even moderately warm temperatures. Therefore, after collection (whether from an ex situ collection or from the wild), citrus germplasm should be stored if possible at refrigerator temperature (3–5˚ C) prior to packaging and shipment. If this tempera- ture is for some reason unobtainable, the germplasm should be kept at as low a tem- perature as possible under the circum- stances. Contamination from pathogenic organisms can also result in the deteriora- tion of budwood, and for this reason it is important to maintain as high a state of cleanliness as possible. Generally it is desirable to pack budwood in sealed bags within a sturdier container, such as card-

board boxes. The budwood should be kept as cool as possible by using insulated con- tainers, ‘blue ice’ or other appropriate means.

Citrus seeds are less prone to deteriora- tion than budwood. Their lower sensitivity to temperature makes shipping seeds less complicated than shipping budwood. Seeds should be stored at 3–5˚ C after surface ster- ilization. They can then be taken from stor- age and shipped immediately or after a short period of drying (to remove surface moisture from condensation during stor- age). Citrus seeds can generally be success- fully shipped in an envelope or padded envelope at ambient temperature. When transit times are long, it may be a good idea to use an insulated container. Maintaining a low temperature whenever possible is advisable but is not as critical as it is for budwood.

Citrus germplasm should be packaged with permanent identification labels or tags, the package should be marked to indi- cate that plant material is enclosed, and required documents should be included in the package (see below). Citrus germplasm should be shipped as soon as possible after packaging, using the most rapid and reli- able form of transport available. Clear instructions should be given to the carrier to avoid exposure to extremes of tempera- ture. The receiver (both the consignee and any relevant inspection stations) should be notifi ed in advance of all shipping details.

In some cases, such as collecting germplasm in the wild, it is not possible to adhere to all appropriate packaging and shipping concepts. However, the closer that it is possible to adhere to these concepts, the greater the chance that the citrus prop- agative materials will arrive at their desti- nation in a viable condition.

All required documentation should be included in the shipping container. In some cases, it is required that permits and other documents be affi xed to the outside of the container. Required documentation will vary with the regulations of the importing country or political subdivision thereof. In some cases, no legal documents may be

required. However, movement of citrus between countries (and sometimes between political subdivisions within a country) is, in general, highly regulated. In fact, citrus is one of the most highly regulated crops in regards to restrictions on its free movement. Usually, at a minimum, a phytosanitary cer- tificate is required. In many cases, an import permit issued by the importing country or a political subdivision is required. In some cases, these imports spell out specifi c requirements that must be sat- isfied before the citrus germplasm is allowed in. These requirements may be either pre-entry or post-entry. Pre-entry requirements may include cutting material from pathogen-tested stock, area freedom from a particular pest or disease, specifi c testing results, pesticide treatment, etc. Post-entry requirements may include a spe- cifi c quarantine protocol or test, establish- ment in an isolated area for observation, etc. In addition to the required legal docu- ments, germplasm movements should include identifi cation, passport data and other information required or useful to the consignee or end-user.

Introduction of citrus germplasm

Phytosanitary considerations play a very important role in the conservation of citrus genetic resources since in the majority of cases they determine how new accessions can be introduced in a germplasm bank and also have a strong infl uence in their man- agement and utilization. Of all the tree and fruit crops of the world, citrus is affected by the greatest number of graft-transmissible pathogens in addition to pathogenic fungi and bacteria and pests. These graft-trans- missible pathogens can debilitate trees and devastate industries, due to their virulent nature and transmission by use of infected budwood for propagation, mechanical transmission and insect vectors.

In the 1930s, the viral nature of psoro- sis was demonstrated by Dr Howard S. Fawcett, the father of citrus plant pathol- ogy, and soon thereafter many other impor-

tant diseases of citrus were shown to be caused by viruses. Since those early years, many if not the most important graft-trans- missible diseases of citrus have been estab- lished as being caused by viruses, viroids, mycoplasmas or bacteria. However, the causal agents of some diseases of quaran- tine signifi cance of citrus have not yet been established. New diseases of citrus caused by graft-transmissible agents are occasion- ally reported, as are the causal agents of conditions previously thought to be genetic or physiological.

Citrus is native to the southern/South- east Asia region of India, China and sur- rounding areas. It has spread outward from this centre of origin for many centuries as seed, budwood or small plants. For most of this period, there were no restrictions upon movement of propagative materials, and, along with the citrus material, pathogens and some of their vectors were undoubtedly moved to different parts of the world in which they had not been previously estab- lished. In addition, new severe diseases have appeared in several countries that are not present in the areas of origin.

Graft-transmissible pathogens and cer- tain other plant pathogens of quarantine signifi cance are unable to move on their own, and are not spread by environmental factors. Their dispersal is primarily by humans or insects. Humans may knowingly or unknowingly spread these pathogens by movement of plant parts. The pathogens may be moved inadvertently in the course of normal trade, travel or scientific exchange. This may occur either directly (i.e. in plant parts) or indirectly (i.e. in an insect contaminating a shipment of com- modity). Once moved to a new area, these plant pathogens may infect previously uninfected plants through human activities (e.g. propagation with infected materials) or insects. Under appropriate conditions, these pathogens can become established as endemic diseases and in some cases may cause epidemics of plant diseases.

Accidental introduction of pathogen- infected citrus budwood can have cata- strophic consequences. For instance, prior

to the 1920s, the citrus industries of Argentina and Brazil fl ourished despite the use of tristeza-susceptible rootstocks. However, 20 years after tristeza-infected nursery stock was imported from South Africa and Australia, 20 million trees had perished. Another example is the case of Spain, where tristeza was probably intro- duced in the 1930s with nursery trees of navel oranges imported from California; up to now, the disease has killed over 40 mil- lion trees grafted on sour orange (Cambra et al., 2000). These and similar catastrophes led to restrictions on the movement of citrus propagative materials and the estab- lishment of quarantines and other regula- tions controlling the movement and propagation of citrus.

The risk of introductions of previously unestablished pests and pathogens is mini- mized by completely prohibiting introduc- tion of the pest or pathogen either directly or indirectly. However, when regulatory agencies have considered the benefi ts of accepting new material into a country, they may allow the entrance of materials under an approved series of safeguards designed to minimize the risk of introducing exotic pests and pathogens. This generally applies to an introduction of limited amounts of material, initially for research purposes and only later released to the general public. The risks associated with large-scale com- mercial importations are unacceptably high in most instances.

In general terms, the steps usually taken to prevent introduction of exotic pests and pathogens include regulation of importations which may result in the intro- duction of an exotic pest or pathogen; for- eign certifi cation of materials before entry; post-entry inspection by domestic plant health authorities; and in some cases post- entry quarantine. Some general information on issues pertinent to quarantine has been reviewed recently by Foster and Hadidi (1998), Frison and Diekmann (1998) and Kahn (1999a, b).

As a consequence of these safeguards, introduction of new accessions from other countries to a germplasm bank often has to

be done through post-entry quarantine sta- tions, which are usually operated by agen- cies different from those operating the gene banks. This may pose important limitations to the germplasm bank’s ability to acquire new germplasm. There are only a few coun- tries in which the quarantine facilities are located in the germplasm banks. In these cases, it is much easier to schedule the importation of new germplasm from other countries. A few countries have post-entry quarantine stations for citrus that do not have any direct relationship with the germplasm banks, e.g. Australia, Brazil, Japan and New Zealand. These programmes are generally concerned more with importa- tion of commercial varieties than with germplasm per se.

Citrus propagative material is highly regulated as compared with propagative material of most other crops. Generally, citrus and other commodities carrying sim- ilar risks require import permits that spell out the requirements for accepting new material and for releasing it. Phytosanitary certifi cations are usually required or pre- ferred. Post-entry inspection is generally required. Some general aspects for the safe introduction of citrus propagative materials from other countries are found in Broadbent (1999), Frison and Diekmann (1999), Frison and Taher (1989), Knorr

(1977), Lee et al. (1999), Roistacher (1977), Roistacher et al. (1977), Lee et al. (1999), Navarro (1992, 1993), and Navarro et al. (1984a, 1991).

In some countries without post-entry quarantine stations, budwood not known to be virus free is completely prohibited from entering a country. Therefore, the mainte- nance of pathogen-free materials is critical not only for phytosanitary health, but also to make possible or facilitate exchange of citrus germplasm. In any case, pathogen- free budwood is highly desirable for exchange as compared with budwood of unknown disease status. This situation complicates the exchange of germ- plasm and has led to the establishment of different collections around the world, since needed germplasm would not be

readily available from a single source. In some instances, importing countries allow the importation of citrus germplasm from programmes which are recognized by the importing country as maintaining high-quality, pathogen-tested, usually protected collections. In these cases, there is usually a periodic review of the programme and re-certification (or de- certifi cation).

A brief comment should be made regarding scientifi c exchange of plant mate- rial or germplasm. Although it may seem that this sort of exchange would be less likely to result in the introduction of exotic pathogens than would commercial importa- tions, this is not necessarily the case. It is unlikely that an exotic virus of citrus would be introduced on a shipment of fruit (although pests and other types of pathogens might be). However, most citrus pathogens of quarantine signifi cance are graft transmissible in budwood, which is the preferred medium of exchange for research purposes in most instances. In the USA, introductions of budwood through approved channels for scientifi c research were routinely contaminated. During the period 1954–1966, 70% of budwood enter- ing California was infected (Roistacher and Nauer, 1968), and from 1968 to 1978, 100% of the budwood entering the USA was infected (Kahn et al., 1979); overall from 1958 through 1993, 65% of the budwood entering the USA was infected (Waterworth, 1993). Although the percent- age of introduced budwood that is infected has decreased somewhat as knowledge of these diseases has increased and more clean stock programmes have been estab- lished, the threat of introduced budwood harbouring pathogens is still very real.