Citrus spp.

Distribution and Importance of Citrus

Citrus is cultivated in many tropical and subtropical regions of the world. Those areas with the highest level of production include parts of the United States (Florida, California, Texas, and Arizona), Asia (Japan, China, India, and other areas), Central America (Mexico), South America (Brazil, Argentina, Chile, Trinidad, and Tobago), the Mediterranean (Spain, Italy, Greece, Cyprus, Israel, and other areas), South Africa, and Australia. These regions help define a “citrus belt” which is contained between 35°N and 35°S. Some of these areas produce citrus on a large scale and export fruit, while others produce fruit mainly for local consumption.

Basically, there are four major commercial species of Citrus, each of which contains different cultivars which are grown in different areas of the world depending upon the climate. Citrus sinensis includes the sweet oranges, and the major cultivars include Hamlin, Navel, Valencia, and Pineapple oranges. The major cultivars of C. paradisi (grapefruit) are Duncan, Marsh, Thompson Pink, and Ruby. The most common lemons (C. limori) include the Bearss, Meyer, and Ponderosa cultivars, while the most common C. aurantifolia variety is the Tahiti or Persian lime.

The nutritive value of citrus has been investigated and well documented. These studies have focused on the availability of vitamins, especially vitamin C, as well as on levels of pectins, fiber, calcium, and other desirable components. Therapeutic effects of citrus biofla-vonoids and of flavonoids in general have also been investigated and the reader is referred to these sources for comprehensive reviews of this topic.

Fruit taste, nutrition, and the development of methods for production of frozen concentrated juice have been responsible for the development of a large citrus industry in many areas of the “citrus belt”. In this type of industry, fruit color and ease in peeling (for the fresh fruit market) or processing (canning and beverage industry) are also important considerations. These factors, in addition to cold hardiness and disease resistance, have dictated much of the selection process in the propagation of citrus.

Propagation of Citrus

Most citrus varieties can be propagated by seeds, and this was a popular method during the 18th and 19th centuries in Europe and the United States. While this method can result in large, productive trees, it has a major drawback in that there is a relatively long juvenility period (up to 10 years) before the trees start producing fruit. In addition, most high quality varieties are not resistant to soil microbes, and therefore they are readily attacked by these pathogens. Neither of these negative features is acceptable or desirable in a commercial operation.

The most actively used method of propagation today is by the process of budding or grafting. These procedures were practiced in Europe during the 16th and 17th centuries, and regained popularity in the late 1800’s in both Europe and the U.S.. Budding involves the insertion of a scion bud under the bark of the stock so that the cambial tissues of the two samples contact each other. This results in a union of the cambia and in the wood itself. The advantages of this method over seedling propagation are tremendous since the resulting plant shows little juvenility, and therefore results in the earlier production of fruit. Secondly, the grower can select a rootstock with specifically desired characteristics (disease-resistant, drought-resistant, etc.) and most plants show uniform fruit quality and seasonal development. Most rootstocks are chosen on the basis of their cold hardiness and disease resistance, while budstocks are chosen on the basis of fruit quality.

Secondary Compounds in Citrus

There are a wide variety of secondary compounds found in Citrus. These include phenolic compounds such as flavonoids, benzoic acids, cinnamic acids, and coumarins as well as terpenoids such as carotenoids and limonoids. Research on the benzoic and cinnamic acids of citrus have focused on physiological as well as antimicrobial studies and will not be discussed here. Coumarins and coumarin glycosides are being utilized in chemotaxonomy and also will not be discussed here. Most recently, research efforts have focused mainly on flavonoids and limonoids in citrus, and the work on these two groups of compounds will be discussed in greater detail.

Conclusions and Directions for Further Research

The inability of callus cultures to accumulate naringin and limonin coupled with the maintenance of very low levels in actively growing cultures would seem to point to the presence of a balanced metabolic system, enzyme levels that are low, or controlled so that their activities are low. The increased levels of the two compounds found in organoid areas (buds) and regenerated shoots suggest that limonin and naringin synthesis and net accumulation is tied to morphological development. Whether it is the production of organized tissues (e.g., leaf) or the other hormonal or metabolic activities which accompany these structures that regulate limonin and naringin synthesis and accumulation is not clear. We are currently investigating the effect of hormonal levels in the medium and of culture conditions on the production of limonin and naringin in grapefruit callus cultures.

Several questions about the biosynthesis of limonin and naringin are unanswered. Do the increased levels of limonin which are observed in young developing tissue and during organogenesis represent de novo synthesis of limonin or conversion of precursors? Do the increased levels of naringin under these same conditions represent glycosylation of a precursor pool (e.g., naringenin, prunin)? Are the increased levels of naringin due to an isomerization of the glycoside moiety from a rutinose, which does not bind to the antibodies used in the RIA, to a neohesperidose (i.e., conversion of narirutin to naringin) which does bind to the antibodies? Is there an increased synthesis of the C-15 ring structure along with the glycosylation, or a mobilization of “reserves” of the C-15 ring nucleus?

Current efforts in our laboratory to clarify these points include the development of immunoassays for both naringenin and prunin. Such assays will enable us to test for the presence of these compounds in tissue cultures and, if found, to follow levels during callus initiation and shoot regeneration. In addition, it will be possible to test for these compounds during the growth and development of Citrus and thus to monitor developmental aspects of the control of naringin biosynthesis.

We are also working on the isolation and characterization of the glycosyl transferase(s) involved in naringin biosynthesis in grapefruit seedlings, since both activities should be present in intact tissues as well as tissue cultures. To date, the flavanone-specific 7-0-glucosyl transferase activity in grapefruit seedlings has been purified and characterized. Once the glycosylating enzymes have been characterized in seedlings, their levels and activity will be studied in cultures from callus initiation through the process of shoot regeneration. Cultures are also being tested for levels of activity of the enzymes involved in the biosynthesis of the C-15 flavonoid ring. Information from this work may provide the basic information necessary for an investigation of the control of the activities of these enzymes at the cellular and at the gene level.

It is apparent that tissue culture has the potential to be a valuable tool in the study of the biosynthesis of citrus bitter compounds. As this process is clarified, it will be possible to consider and evaluate techniques to suppress or enhance this synthesis in plants. Such insight would greatly aid any efforts to use molecular techniques in an attempt to improve this crop.

The screening of plants regenerated from callus culture for individuals with altered limonin- and/or naringin-producing potential could also be conducted. Shoots with desired biosynthetic capacities could be used in a propagation program. It would be important to monitor levels through the propagation phase and in the resultant plant so as to determine if this capacity will hold through these processes. The determination of the relationship between the levels produced in the regenerated shoots and the levels eventually found in the fruits produced by the plants obtained after budding of these shoots would be crucial to an organized breeding effort.

Selections from the book: “Medicinal and Aromatic Plants III”, 1991.