Wolf also studied characteristic differences observable in cross-sections of one year-old roots of the same three species . The trifoliate orange is easily distinguished from the sour orange and the Yuzu by the large vessels in the wood, these being much larger and more numerous than similar vessels in the other two species, and by the more numerous groups of bast fibers in the bark, which form three or four broken concentric rings. In the Yuzu, only a few scattered groups of bast fibers are present in the bark, whereas in the sour orange the groups of bast fibers are numerous and close together in the inner row, with only a few scattered groups farther out, a condition intermediate between that of Yuzu and the trifoliate orange, but clearly differentiating the sour. In the differentiation of trifoliate stock from the Yuzu, Wolf also found that the Yuzu roots, when bruised, emitted a strong penetrating odor, disagreeable to many, and that the odor of the trifoliate is fainter and milder. The author also noticed some differences in the color of roots and their morphological appearance. In Israel, Cossman also studied the anatomy of citrus roots, including the root structure of sweet lime, Rough lemon, sour lemon, citron, ‘Baladi’ sweet orange, ‘Shamouti’ sweet orange, sour orange, grapefruit and shaddock. Cossman felt that characters which might be of taxonomic importance were the mode of lignification of the pith, the configuration of the protoxylem strands, suberization in the endodermis, and the thickening of the walls of the epiblema . Later, Hayward and Long described in detail the anatomy of the seedling and roots of the Valencia orange. In Israel, Green, Vardi, and Galun studied the plastomes of various citrus species and several citrus relatives. They found a resemblance between the plastomes of cultivars of lemon, orange, sour orange, grapefruit and pummelo. The plastomes of other citrus species, such as mandarin and citron,drainage gutter differed from each other as well as the plastomes of the above citrus species.
Furthermore, within the citrus relatives examined, the plastomes of the trifoliate orange and Microcitrus spp. were distinct from each other as well as from the citrus cultivars tested. They felt the result of their study constituted a useful tool for the identification of plastomes in hybrid plants of Citrus developed from protoplast fusion, i.e., somatic hybridization. Some excellent work on the anatomy of Citrus has been published by Schneider . However, most of this is developmental anatomy. He has also published crucially important papers on the seasonal production of xylem and phloem in the sweet orange tree trunk and the ontogeny of lemon tree bark , the relationship of the phloem to certain destructive diseases such as the Buckskin disease of peach and cherry to tristeza , and the incompatibilities and decline of lemons . Schneider et al. were extremely helpful in the early detection and diagnosis of these pathological and physiological problems. However, for some reason, Schneider ignored the structure of the xylem as it might differ between citrus species and how it might aid in identification. Some excellent work has been done on the structure of wood as an aid to identification with forest trees, both conifers and deciduous. One might consult Jane and [text incomplete] . The importance of knowledge of wood structure cannot be emphasized more than the convincing and convicting evidence provided by a wood expert in the trial of the kidnapper of the Charles Lindbergh baby in the early 1930’s. The expert from the regional U.S. Forest Products Laboratory at Madison, Wisconsin, successfully established that the wood in the ladder used by the kidnapper came from the attic of the kidnapper’s home, from which several pieces of wood were missing.Wagnon, Dobbins, and Breece used foliar gland characters in the identification of peach and nectarine varieties. It is possible that the nature, size, number, and arrangement of oil glands in Citrus leaves may also be useful in identification. Hirano , and Gianotti reported on the numbers and variation in stomata in Citrus and some related genera. This technique also might be of some benefit.
Nothing has been done recently with anatomical structure as an aid to root stock identification or taxonomic relationships. Clearly this method will become more complicated as more hybrids involving bigeneric and even trigeneric crosses are made. Furthermore, a microscope, good laboratory, technique, and a thorough knowledge of plant anatomy are required. The first attempt to identify root stocks by colorimetric chemical reactions was apparently that made by Henricksen , who based his method on the presence of varying quantities and kinds of glucosides containing phenol in all citrus roots. He used extracts from root pieces and, with ferric chloride as an indicator, found that the different color reactions or precipitations obtained were more or less characteristic for the four species he worked with, namely sour orange, grapefruit, sweet orange, and Rough lemon. Color density was greatest on sour orange and lightest with Rough lemon. Some confusion existed between sweet orange and Rough lemon. One, of course, needs known standard samples for comparisons. Halma and Haas developed a similar but more extensive method of identifying citrus species by employing colorimetric chemical tests with samples of dried bark since most of the reactive agent seemed to be concentrated there. A number of tests were used in these experiments, but the one that gave most consistent results was the Almen test developed by Cohn for carbolic or salicylic acid, which is practically the same as Millon’s reagent for albumens and phenols. Their experiments also indicated that three other reagents in various forms, molybdic acid, titanium chloride, and ferric chloride, were of value when identification was doubtful. The results obtained by these investigators was sufficiently uniform within commonly accepted limits of the species to lead Halma and Haas to “suggest the possibility that these colorimetric differences may be useful in citrus classification.” In a later paper, Halma described the preparation and use of the Almen test as it had been modified since its first use by Halma and Haas . The tests were only carried out with lemon, Rough lemon, grapefruit, sweet orange, and sour orange. Marloth , in South Africa, made extensive studies and experiments on the use of the four colorimetric reagents in identifying Citrus species, working mainly by the methods suggested by Halma and Haas . Both groups were able to distinguish between the commercial lemon and Rough lemon, and Marloth was able to separate grapefruit and pummelo. As the inroads of tristeza in Brazil became more prevalent and the relationship of root stock-scion combinations became more evident, Bacchi also used these colorimetric tests to distinguish root stocks.
He attempted to identify 15 species, hybrids, and cultivars and found that the reactions obtained were somewhat different from those described by Halma and Haas and Marloth . The differences between sweet orange and sour orange were quite apparent, but the situation becomes more complex with other species and varieties. Bacchi therefore proposed the separation of root stock species and varieties into four groups: sour orange, sweet orange, lemon, and “all the others.” When tristeza began to threaten California orchards and a variety of root stocks appeared to be involved, Masters made a review of laboratory tests for the determination of Citrus root stock varieties. He refined the technique somewhat and was more specific in his color chart,macetas para fresas which is perhaps the best available; it is reproduced here for the benefit of those who wish to conduct such colorimetric tests . Masters was the first person to point out that there is a difference in color reaction between above-ground and underground samples, and for these reasons an addendum is attached. Masters also proposed the use of ultraviolet light and fluorescence as an additional aid. Some of the differences between above-ground and below-ground samples may be pointed out, such as: above-ground sour orange extracts are clear with the ferric chloride test, belowground samples may become cloudy; sweet orange extracts show poor fluorescence above-ground and good fluorescence below-ground. Some of the differences in bark sample location may account for the discrepancies between previous investigators. Certainly it makes a difference as to how much the bark sample is scraped or washed to remove soil particles. The presence of contaminants such as fertilizer, pest control residues, fungicides, and other chemical agents may also make a difference in the color reactions. Of course, the importance of having knowns to compare unknowns with is critical to the tests. Furr and Reece also used a modification of the root stock color tests for the identification of hybrid and nucellar citrus seedlings with a reasonable degree of success. Similar tests were also used by Nishiura, Matsushima and Okudai to identify species and also distinguish hybrids from nucellar seedlings. Nakamura and Nakayama and Krishnamurthy, Singh and Deo also used the tests for studying phylogenetic relationships of the citrus species. Although these chemical tests were somewhat primitive by today’s standards, remarkable results were obtained by someone with care and experience. As chemical techniques and procedures improved, so have the diagnostic aids. Selle proposed a method of clearly identifying sour orange root stock from other stocks by paper chromatography. Essentially the method consisted of taking a piece of root stock bark, placing it upon a sheet of filter paper and hitting it with a hammer. Or, he used a bark extract made with a solution of ethyl alcohol-normal butyl alcohol-acetic acid and water, and placed drops of the extract on filter paper. The spots were allowed to dry, sprayed with a dilute solution of ninhydrin and again allowed to dry. The spots were examined with a long wave ultraviolet lamp and a characteristic flame pattern was observed for fluorescence. The hammer technique gave the most striking results. Selle also developed a spot chromatographic method. Root sections were taken and the bark removed, cut into very small pieces, placed in a bottle, and treated with 2,2-dimethoxypropane.
After standing for 30 minutes, single drops were placed on filter paper and the spots observed as they dried. Complete identification of all the root stocks was not obtained by using dimethoxypropane alone, and he got better differentiation by adding anhydrous aluminum chloride to the solution. He was thus able to identify sour orange, sweet orange, grapefruit, tangelo, mandarin, Rough lemon, trifoliate orange and Troyer citrange. The memory of the past is not always too reliable, but the author is quite certain that in conversation with Selle, he indicated he could also tell with the root bark what the scion variety was budded on it. Unfortunately, with Selle’s sudden death, perhaps this information was lost. Pieringer, Edwards, and Wolford and Kesterson et al. studied the application of gas-liquid chromatography to the citrus leaf oils for the identification of kinds of Citrus. Kesterson and his coworkers included eleven kinds of citrus and their data demonstrated that the oil composition for the different species is quite variable. They list the most prevalent and distinguishing features for each type oil in order of importance for sour orange, grapefruit, tangelo, mandarin, sweet orange and Rough lemon. They state that, “The percent of composition within species is shown to be sufficiently different to distinguish one variety from another.” Limits of normal deviation, tree variability, and seasonal variations are all factors which may affect leaf oil properties and they feel additional work will establish these limits. Pieringer, Edwards and Wolford studied the leaf oils of eight different citrus varieties and two sources of sour orange as subjected to four methods of instrumental analyses. These were: infrared and ultraviolet spectrophotometry, gas chromatography, and measurement of refractive indices. Some methods were more effective than others in separating closely related cultivars. They felt gas chromatography more successfully differentiated the varieties, whereas infrared and ultraviolet spectrophotometry appeared to be limited to the identification of Citrus species. The value of the refractive index was not fully determined. Burger , on page 109 of this thesis, obtained chromatograms with high pressure liquid chromatography of the phenolic present in the root stocks he worked with. Using this method, he could distinguish between Troyer and Carrizo citranges, which most other researchers could not do.