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Taxonomic significance of transverse sections of roots of six Citrus species

Taxonomic significance of transverse sections of roots of six Citrus species BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw004 Research article Taxonomic significance of transverse sections of roots of six Citrus species Chinelo A. Ezeabara* and Clement U. Okeke Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria *Corresponding author: Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. Email: ca.ezeabara@unizik.edu.ng; e.chinelo5@yahoo.com Supervisor: Clement U. Okeke, Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. Transverse sections (T/S) of the roots of Citrus aurantifolia (Christm.) Swingle, C. grandis Osbeck, C. limon (L.) Burm.f., C. paradisii Macf., C. reticulata Blanco and C. sinensis (L.) Osbeck were investigated using standard plant anatomical procedures. The pores of roots of all the species were oval in shape. The sizes of the pores found in C. sinensis were the lowest range (117–27 µ m in length; 108–27 µ m in width). The distributions of axial parenchyma were confluent parenchyma in Citrus aurantifolia, C.  grandis, C. paradisii, C. reticulata and C. sinensis, whereas it was vasicentric aliform parenchyma in C. limon. Citrus limon, therefore, possessed an advanced feature which indicated that it evolved at a different rate compared with others. The study revealed ana - tomical characters of these Citrus species and hence shed light on their affinity. The characters of secondary wood, therefore, could be applied as additional characters by plant taxonomists in resolving the ongoing controversy in the taxonomy of Citrus. Key words: Citrus, root anatomy, secondary wood, transverse section, intercellular canals, axial parenchyma Submitted on 14 July 2015; accepted on 16 May 2016 Introduction characters that could be useful in solving taxonomic problems. Sharma (1993) stated that the characters of secondary wood Citrus aurantifolia (Christm.) Swingle (Lime), C. grandis were the most important anatomical features that have been used Osbeck (Shadock/Pummelo), C. limon (L.) Burm.f. (Lemon), in resolving problems of taxonomy and phylogeny. Evidence C. paradisii Macf. (Grapefruit), C. reticulata Blanco from wood anatomy could therefore be employed as additional (Mandarin/Tangerine) and C. sinensis (L.) Osbeck (Sweet characters in solving the problem of Citrus taxonomy. orange) belong to the genus Citrus, of the family Rutaceae. The objective of this research, therefore, was to investigate Citrus originated from South-east Asia, including South the transverse section of the roots of these six Citrus species, China, northeastern India and Burma (Mabberley, 1997). the literature on which is very scanty, to compare them and Citrus has a branched tap root system. Over 70% of Citrus reveal their affinities, with a view to providing additional tree roots are in the top 1.1 m of soil (Manner et al., 2006). characters that might be useful in resolving the problems of Taxonomists are perplexed as to how to classify the various taxonomy of this genus. kinds of Citrus that have existed since antiquity (Moore, 2001). This is probably due to availability of many new varieties of Materials and methods Citrus, which whether all of them deserve the distinction of true species or not is still under discussion. It is also desirable to Sources of materials understand the affinity existing among the species of Citrus. Plant anatomy is simply the study of internal features of plant The root samples used for this work were taken from the organs with the aid of a microscope. This can provide additional major root systems. They were collected from Agricultural © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons • Volume 9 2016 and Natural Resources Department Market Garden, (Erma-Tokyo, Japan) at ×10 magnification for measuring the Amawbia, Nigeria, in the month of June. The plants were length and breadth of the pores. The photomicrographs of the field -grown trees with matured fruits at the time of collection informative slides were then taken with a photomicroscope of the root samples. They were identified; and the voucher (2.0 model, China). specimens were authenticated by Prof. C.U. Okeke and depos- ited in the herbarium, Department of Botany, Nnamdi Azikiwe Results University, Awka, Nigeria. The transverse sections are shown in Fig. 1A–F. The vessels of Anatomical analysis all the species were diffuse-porous, not occluded by tyloses and oval in shape (Fig. 1A–F; Table 1). The sizes of the vessels var- The method described by Ezeabara et al. (2013a and b) was ied; the length ranged from 162 to 45 µ m in Citrus aurantifolia, adopted with some modic fi ations. Ten root samples of each of C. paradisii and C. reticulata (Table 1). In addition, the sizes of these six species of Citrus were collected in vials containing the pores found in C. sinensis were the lowest range (117– formaldehyde, glacial acetic acid and alcohol in the ratio of 27 µ m in length; 108–27 µ m in width) in comparison with oth- 1:1:18, respectively. The specimens were dehydrated in etha- ers. The arrangements of the pores found in all the species were nol series (30%, 50%, 70% and 95%) each for 2 hs. Complete radial multiples (Fig. 1A–F). Distribution of axial parenchyma dehydration of specimens was effected by storing the speci- was confluent parenchyma in Citrus aurantifolia, C. grandis, C. mens in absolute (99.6%) ethanol, overnight. The specimens paradisii, C. reticulata and C. sinensis (Fig. 1A, B, D, E and F), were then cleared in 3:1, 1:1 and 1:3 ethanol/chloroform each while it was vasicentric aliform parenchyma in C. limon for 3 h. Wax was melted at 70°C in an oven. The cleared spec- (Fig. 1C). Structures of rays were both uniseriate and multiseri- imens were then put in molten anatomical wax and alcohol, ate in all the species (Fig. 1A–F). In addition, the rays were more and allowed to stay at 70°C for 24 h for effective infiltration than half width of pores in all the species (Fig. 1A–F). The inter- of wax into the specimen to replace the chloroform which was cellular canals of C. limon and C. paradisii were traumatic gradually lost by evaporation. (Fig. 1C and D), whereas they were normal in C. aurantifolia, C. Embedding was carried out after infiltration. This was grandis, C. reticulata and C. sinensis (Fig. 1A, B, E and F). done by smearing the inside of clean molds with glycerin, pouring the molten wax into the molds with appropriate ori- entation in a position suitable for the type of section to be cut. Discussion The wax in the mould was allowed to cool into a block. The The results obtained from this investigation revealed some wax blocks were freed from the mould, stuck on a wooden similarities in the anatomical characters of these Citrus species holder and labelled, pending sectioning. which suggested a very close affinity among them and could Wax blocks already freed from the holder and trimmed probably be regarded as the anatomical generic identities. were affixed on the sledge microtome and sectioned at 20 However, the length of pores of Citrus aurantifolia, C. paradisii microns thickness. Ten thin sections of each root sample of the and C. reticulata suggested a very close affinity among them specimens were selected and fixed on clean slides already while the lowest range of sizes of pores of C. sinensis implied smeared with a thin film of egg albumen. They were stretched a moderate affinity with other five species. The most striking by passing them over hot plate. This also made them to difference was the vasicentric aliform parenchyma detected in become attached to the slides. Slides bearing sections arranged the root of C. limon, which probably indicated that the root in a slide rack and placed in an oven at 70°C to melt off wax of C. limon evolved at different rate compared with others. from the sections. This lasted for 12 h. The vasicentric aliform parenchyma has been reported to be a very advanced condition (Roy, 2006). This, therefore, implied For staining, sections were dehydrated by passing slides that C. limon was advanced. In previous studies, radial longi- across xylene and xylene/absolute ethanol series (3:1, 1:1 and tudinal sections (RLS) of stems of these six species of Citrus 1:3 v/v), absolute ethanol, 95%, 70%, 50% and 30% ethanol. revealed that they possessed procumbent ray cell which is a Slides were briefly immersed in water, then stained with 0.1% specialized and advanced character (Ezeabara et al., 2013a). alcian blue and counter stained with 1% safranin. Stained In addition, transverse section of the stem showed the pres- slides were rinsed briefly in tap water, dehydrated through the ence of confluent paratracheal parenchyma in Citrus grandis, ethanol series and cleared across the xylene/absolute ethanol C. limon, C. paradisii, C. reticulata and C. sinensis with the series. exception of C. aurantifolia which possessed vasicentric ali- Mounting was carried out by placing one drop of Canada form parenchyma (Ezeabara et al., 2013b). An advanced char- balsam on a clean slide and carefully covering the sections acter that was found in the tranverse section of the root of with the coverslip in such a way that the Canada balsam C. limon, while a primitive feature was reported present in the spread and covered the specimen sections overlaid by the cov- tranverse section of the stem (Ezeabara et  al., 2013b), sug- erslip. The slides were observed under an OLYMPUS (XSZ- gested that the root evolved, whereas the stem was static. 107BN, China) light microscope as well as a Motic F series It has been stated that among the general principles adopted (China) simple light microscope fitted with eyepiece graticule for the classification of flowering plants, evolution does not 2 Bioscience Horizons • Volume 9 2016 Research article Figure 1. Photomicrographs of T/S of roots of Citrus species ×100. (A) Citrus aurantifolia; (B) Citrus grandis; (C) Citrus limon; (D) Citrus paradisii; (E) Citrus reticulata; (F) Citrus sinensis. Scale bar = 18 µ m. ve, pore/vessel; Ra, ray; Pc, parenchyma cells. necessarily involve all organs of the plant at the same time, Table 1. Size (µ m) of vessels of root (T/S) of Citrus aurantifolia, C. grandis, and one organ or set of organs may be advanced while another C. limon, C. paradisii, C. reticulata and C. sinensis set is stationary or retrograding (Esau, 1977). Moreover, the intercellular canals of all the species studied were normal with S/N Citrus species Length Width the exception of C. limon and C. paradisii. This probably indi- 1 Citrus aurantifolia 162–45 126–54 cated a close affinity among these species, whereas C. limon and C. paradisii were more closely related. 2 C. grandis 162–27 144–36 Various workers have reported that Citrus medica, C. gran- 3 C. limon 162–63 126–63 dis and C. reticulata are the only true species of Citrus and that C. aurantifolia, C. limon, C. paradisii and C. sinensis are 4 C. paradisii 162–45 162–63 hybrids (Katz and Weaver, 2003; Liang et al., 2006). The sim- 5 C. reticulata 162–45 126–36 ilarities in the shapes and arrangements of the pores; struc- tures and arrangements of rays of all the species implied that 6 C. sinensis 117–27 108–27 they have close affinity; while the exhibition of an advanced 3 Research article Bioscience Horizons • Volume 9 2016 character by C. limon probably indicated that it had a moder- Department, and Mr Emmanuel Ikechukwu, Assistance Chief ate affinity with others. Laboratory Technologist, Department of Crop Science, all in University of Nigeria Nsukka, for their technical assistance. Conclusion References Interestingly, the evidence obtained from the transverse sec- Esau, K. (1977) Anatomy of Seed Plants, 2nd edn, Wiley Incorporated, tion of the roots of these Citrus species generally proposed a Canada, pp. 550. close affinity among them . However, C. limon possessed an advanced character that indicated a moderate affinity with Ezeabara, C. A., Okeke, C. U., Aziagba, B. O. et al. (2013a) Taxonomic C.  aurantifolia, C. grandis, C. paradisii, C. reticulata and importance of radial longitudinal section in stem characters of six C. sinensis. One would, therefore, suggest that the characters Citrus species of Southeastern Nigeria, International Journal of of the T/S of the roots of these Citrus species might be consid- Agriculture and Biosciences, 2 (5), 188–191. ered as additional characters in delineating the species of Ezeabara, C. A., Okeke, C. U., Aziagba, B. O. et al. (2013b) Transverse sec- Citrus. A taxonomic conclusion, however, cannot be drawn tion of stem of Citrus and their taxonomic significance, International from these characters alone; hence, a combination with DNA- Journal of Plant Research, 3 (3), 23–26. based genetic characteristics is required, to lead to more sound conclusions. Katz, S. H. and Weaver, W. W. (2003) Encyclopedia of Food and Culture, 1st edn, Schribner, New York, pp. 861. Author’s biography Liang, G., Xiong, G., Guo, Q. et al. (2006) AFLP analysis and the taxonomy of Citrus, Acta Horticulturae, 760, 27–32. C.A.E. got her MSc degree (Plant Taxonomy) from Nnamdi Azikiwe University, Awka, Nigeria in 2012. Prior to this, she Mabberley, D. J. (1997) A classification for edible Citrus (Rutaceae), graduated with a Second Class Honours Upper Division in Telopea, 7 (2), 167–172. BSc Botany from the same university. Her interest lies in Manner, H. I., Buker, R. S., Smith, V. E. et al. (2006) Citrus species (citrus), in Phytomedicine, Plant Anatomy and Morphology. C.A.E. con- Elevitch, C. R., ed, Species Profiles for Pacific Island Agroforestry , ducted the experiments on plant anatomy, wrote up the paper Permanent Agriculture Resources (PAR), Hawaii, pp. 1–35. and have primary responsibility for final content. Prof. C.U.O. designed the study and assisted in the research. Moore, G. A. (2001) Oranges and lemons: clues to the taxonomic of Citrus from molecular marker, Trends in Genetics, 7 (9), 536–540. Acknowledgements Roy, P. (2006) Plant Anatomy, 1st edn, New Central Book Agency (P) Ltd, New Delhi, pp. 389. We appreciate Prof. M.O. Nwosu, Plant Anatomist, and Mr Ezeaku, Laboratory Technologist, Department of Plant Sharma, O. P. (1993) Plant Taxonomy, 1st edn, Tata McGraw—Hill Science and Biotechnology; Prof M.I. Uguru, Head of Publishing Company Limited, New Delhi, pp. 482. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Taxonomic significance of transverse sections of roots of six Citrus species

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BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw004 Research article Taxonomic significance of transverse sections of roots of six Citrus species Chinelo A. Ezeabara* and Clement U. Okeke Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria *Corresponding author: Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. Email: ca.ezeabara@unizik.edu.ng; e.chinelo5@yahoo.com Supervisor: Clement U. Okeke, Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. Transverse sections (T/S) of the roots of Citrus aurantifolia (Christm.) Swingle, C. grandis Osbeck, C. limon (L.) Burm.f., C. paradisii Macf., C. reticulata Blanco and C. sinensis (L.) Osbeck were investigated using standard plant anatomical procedures. The pores of roots of all the species were oval in shape. The sizes of the pores found in C. sinensis were the lowest range (117–27 µ m in length; 108–27 µ m in width). The distributions of axial parenchyma were confluent parenchyma in Citrus aurantifolia, C.  grandis, C. paradisii, C. reticulata and C. sinensis, whereas it was vasicentric aliform parenchyma in C. limon. Citrus limon, therefore, possessed an advanced feature which indicated that it evolved at a different rate compared with others. The study revealed ana - tomical characters of these Citrus species and hence shed light on their affinity. The characters of secondary wood, therefore, could be applied as additional characters by plant taxonomists in resolving the ongoing controversy in the taxonomy of Citrus. Key words: Citrus, root anatomy, secondary wood, transverse section, intercellular canals, axial parenchyma Submitted on 14 July 2015; accepted on 16 May 2016 Introduction characters that could be useful in solving taxonomic problems. Sharma (1993) stated that the characters of secondary wood Citrus aurantifolia (Christm.) Swingle (Lime), C. grandis were the most important anatomical features that have been used Osbeck (Shadock/Pummelo), C. limon (L.) Burm.f. (Lemon), in resolving problems of taxonomy and phylogeny. Evidence C. paradisii Macf. (Grapefruit), C. reticulata Blanco from wood anatomy could therefore be employed as additional (Mandarin/Tangerine) and C. sinensis (L.) Osbeck (Sweet characters in solving the problem of Citrus taxonomy. orange) belong to the genus Citrus, of the family Rutaceae. The objective of this research, therefore, was to investigate Citrus originated from South-east Asia, including South the transverse section of the roots of these six Citrus species, China, northeastern India and Burma (Mabberley, 1997). the literature on which is very scanty, to compare them and Citrus has a branched tap root system. Over 70% of Citrus reveal their affinities, with a view to providing additional tree roots are in the top 1.1 m of soil (Manner et al., 2006). characters that might be useful in resolving the problems of Taxonomists are perplexed as to how to classify the various taxonomy of this genus. kinds of Citrus that have existed since antiquity (Moore, 2001). This is probably due to availability of many new varieties of Materials and methods Citrus, which whether all of them deserve the distinction of true species or not is still under discussion. It is also desirable to Sources of materials understand the affinity existing among the species of Citrus. Plant anatomy is simply the study of internal features of plant The root samples used for this work were taken from the organs with the aid of a microscope. This can provide additional major root systems. They were collected from Agricultural © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Research article Bioscience Horizons • Volume 9 2016 and Natural Resources Department Market Garden, (Erma-Tokyo, Japan) at ×10 magnification for measuring the Amawbia, Nigeria, in the month of June. The plants were length and breadth of the pores. The photomicrographs of the field -grown trees with matured fruits at the time of collection informative slides were then taken with a photomicroscope of the root samples. They were identified; and the voucher (2.0 model, China). specimens were authenticated by Prof. C.U. Okeke and depos- ited in the herbarium, Department of Botany, Nnamdi Azikiwe Results University, Awka, Nigeria. The transverse sections are shown in Fig. 1A–F. The vessels of Anatomical analysis all the species were diffuse-porous, not occluded by tyloses and oval in shape (Fig. 1A–F; Table 1). The sizes of the vessels var- The method described by Ezeabara et al. (2013a and b) was ied; the length ranged from 162 to 45 µ m in Citrus aurantifolia, adopted with some modic fi ations. Ten root samples of each of C. paradisii and C. reticulata (Table 1). In addition, the sizes of these six species of Citrus were collected in vials containing the pores found in C. sinensis were the lowest range (117– formaldehyde, glacial acetic acid and alcohol in the ratio of 27 µ m in length; 108–27 µ m in width) in comparison with oth- 1:1:18, respectively. The specimens were dehydrated in etha- ers. The arrangements of the pores found in all the species were nol series (30%, 50%, 70% and 95%) each for 2 hs. Complete radial multiples (Fig. 1A–F). Distribution of axial parenchyma dehydration of specimens was effected by storing the speci- was confluent parenchyma in Citrus aurantifolia, C. grandis, C. mens in absolute (99.6%) ethanol, overnight. The specimens paradisii, C. reticulata and C. sinensis (Fig. 1A, B, D, E and F), were then cleared in 3:1, 1:1 and 1:3 ethanol/chloroform each while it was vasicentric aliform parenchyma in C. limon for 3 h. Wax was melted at 70°C in an oven. The cleared spec- (Fig. 1C). Structures of rays were both uniseriate and multiseri- imens were then put in molten anatomical wax and alcohol, ate in all the species (Fig. 1A–F). In addition, the rays were more and allowed to stay at 70°C for 24 h for effective infiltration than half width of pores in all the species (Fig. 1A–F). The inter- of wax into the specimen to replace the chloroform which was cellular canals of C. limon and C. paradisii were traumatic gradually lost by evaporation. (Fig. 1C and D), whereas they were normal in C. aurantifolia, C. Embedding was carried out after infiltration. This was grandis, C. reticulata and C. sinensis (Fig. 1A, B, E and F). done by smearing the inside of clean molds with glycerin, pouring the molten wax into the molds with appropriate ori- entation in a position suitable for the type of section to be cut. Discussion The wax in the mould was allowed to cool into a block. The The results obtained from this investigation revealed some wax blocks were freed from the mould, stuck on a wooden similarities in the anatomical characters of these Citrus species holder and labelled, pending sectioning. which suggested a very close affinity among them and could Wax blocks already freed from the holder and trimmed probably be regarded as the anatomical generic identities. were affixed on the sledge microtome and sectioned at 20 However, the length of pores of Citrus aurantifolia, C. paradisii microns thickness. Ten thin sections of each root sample of the and C. reticulata suggested a very close affinity among them specimens were selected and fixed on clean slides already while the lowest range of sizes of pores of C. sinensis implied smeared with a thin film of egg albumen. They were stretched a moderate affinity with other five species. The most striking by passing them over hot plate. This also made them to difference was the vasicentric aliform parenchyma detected in become attached to the slides. Slides bearing sections arranged the root of C. limon, which probably indicated that the root in a slide rack and placed in an oven at 70°C to melt off wax of C. limon evolved at different rate compared with others. from the sections. This lasted for 12 h. The vasicentric aliform parenchyma has been reported to be a very advanced condition (Roy, 2006). This, therefore, implied For staining, sections were dehydrated by passing slides that C. limon was advanced. In previous studies, radial longi- across xylene and xylene/absolute ethanol series (3:1, 1:1 and tudinal sections (RLS) of stems of these six species of Citrus 1:3 v/v), absolute ethanol, 95%, 70%, 50% and 30% ethanol. revealed that they possessed procumbent ray cell which is a Slides were briefly immersed in water, then stained with 0.1% specialized and advanced character (Ezeabara et al., 2013a). alcian blue and counter stained with 1% safranin. Stained In addition, transverse section of the stem showed the pres- slides were rinsed briefly in tap water, dehydrated through the ence of confluent paratracheal parenchyma in Citrus grandis, ethanol series and cleared across the xylene/absolute ethanol C. limon, C. paradisii, C. reticulata and C. sinensis with the series. exception of C. aurantifolia which possessed vasicentric ali- Mounting was carried out by placing one drop of Canada form parenchyma (Ezeabara et al., 2013b). An advanced char- balsam on a clean slide and carefully covering the sections acter that was found in the tranverse section of the root of with the coverslip in such a way that the Canada balsam C. limon, while a primitive feature was reported present in the spread and covered the specimen sections overlaid by the cov- tranverse section of the stem (Ezeabara et  al., 2013b), sug- erslip. The slides were observed under an OLYMPUS (XSZ- gested that the root evolved, whereas the stem was static. 107BN, China) light microscope as well as a Motic F series It has been stated that among the general principles adopted (China) simple light microscope fitted with eyepiece graticule for the classification of flowering plants, evolution does not 2 Bioscience Horizons • Volume 9 2016 Research article Figure 1. Photomicrographs of T/S of roots of Citrus species ×100. (A) Citrus aurantifolia; (B) Citrus grandis; (C) Citrus limon; (D) Citrus paradisii; (E) Citrus reticulata; (F) Citrus sinensis. Scale bar = 18 µ m. ve, pore/vessel; Ra, ray; Pc, parenchyma cells. necessarily involve all organs of the plant at the same time, Table 1. Size (µ m) of vessels of root (T/S) of Citrus aurantifolia, C. grandis, and one organ or set of organs may be advanced while another C. limon, C. paradisii, C. reticulata and C. sinensis set is stationary or retrograding (Esau, 1977). Moreover, the intercellular canals of all the species studied were normal with S/N Citrus species Length Width the exception of C. limon and C. paradisii. This probably indi- 1 Citrus aurantifolia 162–45 126–54 cated a close affinity among these species, whereas C. limon and C. paradisii were more closely related. 2 C. grandis 162–27 144–36 Various workers have reported that Citrus medica, C. gran- 3 C. limon 162–63 126–63 dis and C. reticulata are the only true species of Citrus and that C. aurantifolia, C. limon, C. paradisii and C. sinensis are 4 C. paradisii 162–45 162–63 hybrids (Katz and Weaver, 2003; Liang et al., 2006). The sim- 5 C. reticulata 162–45 126–36 ilarities in the shapes and arrangements of the pores; struc- tures and arrangements of rays of all the species implied that 6 C. sinensis 117–27 108–27 they have close affinity; while the exhibition of an advanced 3 Research article Bioscience Horizons • Volume 9 2016 character by C. limon probably indicated that it had a moder- Department, and Mr Emmanuel Ikechukwu, Assistance Chief ate affinity with others. Laboratory Technologist, Department of Crop Science, all in University of Nigeria Nsukka, for their technical assistance. Conclusion References Interestingly, the evidence obtained from the transverse sec- Esau, K. (1977) Anatomy of Seed Plants, 2nd edn, Wiley Incorporated, tion of the roots of these Citrus species generally proposed a Canada, pp. 550. close affinity among them . However, C. limon possessed an advanced character that indicated a moderate affinity with Ezeabara, C. A., Okeke, C. U., Aziagba, B. O. et al. (2013a) Taxonomic C.  aurantifolia, C. grandis, C. paradisii, C. reticulata and importance of radial longitudinal section in stem characters of six C. sinensis. One would, therefore, suggest that the characters Citrus species of Southeastern Nigeria, International Journal of of the T/S of the roots of these Citrus species might be consid- Agriculture and Biosciences, 2 (5), 188–191. ered as additional characters in delineating the species of Ezeabara, C. A., Okeke, C. U., Aziagba, B. O. et al. (2013b) Transverse sec- Citrus. A taxonomic conclusion, however, cannot be drawn tion of stem of Citrus and their taxonomic significance, International from these characters alone; hence, a combination with DNA- Journal of Plant Research, 3 (3), 23–26. based genetic characteristics is required, to lead to more sound conclusions. Katz, S. H. and Weaver, W. W. (2003) Encyclopedia of Food and Culture, 1st edn, Schribner, New York, pp. 861. Author’s biography Liang, G., Xiong, G., Guo, Q. et al. (2006) AFLP analysis and the taxonomy of Citrus, Acta Horticulturae, 760, 27–32. C.A.E. got her MSc degree (Plant Taxonomy) from Nnamdi Azikiwe University, Awka, Nigeria in 2012. Prior to this, she Mabberley, D. J. (1997) A classification for edible Citrus (Rutaceae), graduated with a Second Class Honours Upper Division in Telopea, 7 (2), 167–172. BSc Botany from the same university. Her interest lies in Manner, H. I., Buker, R. S., Smith, V. E. et al. (2006) Citrus species (citrus), in Phytomedicine, Plant Anatomy and Morphology. C.A.E. con- Elevitch, C. R., ed, Species Profiles for Pacific Island Agroforestry , ducted the experiments on plant anatomy, wrote up the paper Permanent Agriculture Resources (PAR), Hawaii, pp. 1–35. and have primary responsibility for final content. Prof. C.U.O. designed the study and assisted in the research. Moore, G. A. (2001) Oranges and lemons: clues to the taxonomic of Citrus from molecular marker, Trends in Genetics, 7 (9), 536–540. Acknowledgements Roy, P. (2006) Plant Anatomy, 1st edn, New Central Book Agency (P) Ltd, New Delhi, pp. 389. We appreciate Prof. M.O. Nwosu, Plant Anatomist, and Mr Ezeaku, Laboratory Technologist, Department of Plant Sharma, O. P. (1993) Plant Taxonomy, 1st edn, Tata McGraw—Hill Science and Biotechnology; Prof M.I. Uguru, Head of Publishing Company Limited, New Delhi, pp. 482.

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