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Variation of Citrus Cultivars in Egypt Assessed

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Variation of Citrus Cultivars in Egypt Assessed African Journal of Biotechnology Vol. 11(91), pp. 15755-15762, 13 November, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.2259 ISSN 1684–5315 ©2012 Academic Journals Full Length Research Paper Phytohormones content and random amplified polymorphic DNA (RAPD) marker assessment of some Egyptian citrus cultivars Hala F. Ahmed Department of Botany, Faculty of Science, Ain Shams University, Cairo, Egypt. E-mail: [email protected]. Accepted 26 September, 2012 In Egypt, Citrus represents one of the main fruit tree crops for both local and export potentials. In this study, leaf and vegetative bud samples were studied for cultivars of sour orange (seedless, sweet seeded, Brazilian and Spanish), common sweet orange (Florida, Fsido, Shamoty, and Valencia) and navel orange B29 from four different areas in Egypt (El-Qalubaiya governorate, Wadi El-Mollak; Ismailia governorate, El-Salheia; Sharqaia governorate, and El-Minya; South Egypt). Cluster analysis generated by random amplified polymorphic DNA (RAPD) molecular marker showed close similarities of the four sour orange accessions, used as rootstocks, where they were grouped in one cluster. The four cultivars of sweet common orange and sweet navel orange was linked together in a separate cluster. Navel orange cultivated at El-Minya and El-Salheia showed drop in yield due to substantial flower and young fruit abscission, whereas trees of the same cultivar did not suffer from abscission and yielded enhanced crop. Comparison of the four previously mentioned localities speculated that the navel orange accessions at El-Minya and El-Salheia are subjected to drought stress. This could be further verified by the substantially enhanced levels of abscisic acid in the plants showing abscission, as compared to those exhibiting normal flowering and enhanced fruiting at El-Qalubaiya and Wadi El- Mollak. Key words: Abscisic acid, abscission, Citrus, cluster analysis, deoxyribonucleic acid (DNA) random amplified polymorphic DNAs (RAPDs), drought, rootstock, scions. INTRODUCTION Citrus species are among the most widely grown fruit annual production. crops in the world and have a large market worldwide. In In Egypt, Citrus cultivation has doubled in area over the Egypt, some Citrus species have been cultivated tradi- past 25 years. The fruit bearing area reached more than tionally since ancient times. Egypt is considered as the 140, 000 ha in 2004 (Hofer, 2007). During the last 9th largest Citrus producer in the world, with an annual decade, the private sector invested into establishment production approximately estimated as 3, 200, 000 tons, more than 10000 ha new plantations in the desert outside which represents about 2.92% of the total world produc- the Nile valley. More than one fourth of the total Citrus tion (FAO, 2006). production, now, comes from the "new lands" in the The genus Citrus belongs to family Rutaceae, sub- desert. Approximately, half of the total acreage in the family Aurantoideae (Tanaka, 1977). This genus has country is cultivated by large-scale farms, whereas the many species; the main cultivated species are sweet other half is cultivated by small-scale growers. orange (Citrus sinensis (L.) Osbeck), mandarin (C. reticu- The national average Citrus yield in Egypt is very low late Blanco), grape fruits (C. paradisi Macf.), lemons (C. compared to world levels (FAO, 2006). The main reasons limon (L.) Burm., f. (lemon), and sour orange (Citrus for the low productivity are inappropriate cultivation aurantium L.). Ollitrault et al. (2010) stated that Citus is a practices, old trees, lack of regular pruning, off-type diploid genus whose cultivated forms play a major role in cultivars, and poor irrigation practices, as well as graft- the human diet with more than 122 MT of the world transmissible diseases (Hofer, 2007). 15756 Afr. J. Biotechnol. In Egypt, Citrus productivity is determined by diverse (GAs) are known to inhibit flower production in Citrus environmental stimuli. In this context, navel sweet orange species, which is in contrast to its flowering promoting (Citrus sinesis cv. B29) produces a high yield at El- effect in annual plants (Guardiola et al., 1982; Lliso et al., Qalubaiya governorate and Wadi El-Mollak (Ismailia 2004). On the other hand, auxins have been reported to governorate). However, at El-Salheia (Sharqaia governo- act as stimulators of growth and also as abscission rate) and El-Minya (South Egypt), the crop yield is very agents, perhaps through promoting ethylene synthesis low, where the trees are subjected to severe flower and (Iglesias et al., 2007). Enhanced abscisic acid levels young fruit abscission. Main variation among the three coincided the abscission waves in ovaries of Citrus at aforementioned mentioned areas could be attributed to petal fall and during the transition from cell division to cell the moisture content which is markedly lower at the sites enlargement in fruits (Goren, 1993). This hormone is also showing abscission (weather forecast, 2011). high over periods of low humidity, dehydration and salt or The use of molecular markers has been followed as a drought conditions which are known to induce abscission valuable and precise strategy to physiological and mole- (Mehouachi et al., 2005; Agusti et al., 2007; Tang et al., cular studies on Citrus. Among them, random amplified 2012). Taken together, it is concluded that GA deficiency polymorphic DNA (RAPDs) has been employed most is associated with ABA rise and ethylene release, and widely. Thus, RAPDs have been used for cultivar identify- eventually ovary abscission (Gomez-Cadenas et al., cation, genetic mapping, genetic diversity assessment 1996). and other breeding programs (Dehesdtani et al., 2007). In Thus, the present work intended to investigate: (a) the genus Citrus, information dealing with the generation interrelations of 12 accessions of Citrus that are used in of genomic tools and resources was reviewed by Talon Egypt as root stocks and scions in the breeding pro- and Gmitter (2008) and by Tadeo et al. (2008) on the grams, on the bases of RAPD and (b) measuring phyto- progress in molecular biology and genetics of develop- hormone levels in the plants grown at different localities ment. that underlie variable yield potentials. Navel oranges (characterized by the growth of a second fruit at the apex, which protrudes slightly and This part was moved from MATERIALS AND METHODS resembles a human navel) are economically important the discussion area since they are very popular because of their use as a The current investigation was carried out on 12 accessions of Citrus tasty eating orange, their widespread distribution, and cultivars in Egypt. The plant materials used in the present their long growing season. Navel oranges (resulted from investigation was obtained from the Agriculture Research Centre, a single mutation) are characterized by a rather thicker Giza, Egypt. Fresh young leaves (first and second leaves from skin which is easier to peel than common orange (Kim- branch tops) of Citrus were collected in ice box, transferred to laboratory, and then stored at -20C until use for DNA extraction ball, 1999). Because the mutation left the fruit seedless, and randomly amplified polymorphic DNA polymerase chain reac- and therefore sterile, the only mean of cultivation of new tion (RAPD-PCR). Terminal buds and young leaves at branch tops variety is to graft cuttings onto other varieties of Citrus. were similarly collected in 80% redistilled ethanol [High-perfor- Rootstock influences not only the rate of growth, disease mance liquid chromatography (HPLC) grade] and kept in a deep resistance and productivity of the cultivar but also the freeze (-20C) till use. The 12 accessions under study presented physical and chemical attributes of the crop (Chang-pin et four cultivars of sour orange (seedless, seeded, Brazilian and Spanish), four cultivars of sweet common oranges (Florida, Fsido, al., 2010). Because all navel oranges are clones of one Shamoty, and Valencia). In addition, four accessions of navel another, they are highly susceptible to various environ- orange B29 were similarly collected from farms under supervision of mental factors (Wutscher and Bowman, 1999). Therefore, the Agriculture Research Centre at El-Qalubaiya governorate, Wadi environmental changes in climatic factors, nature of soil, El-Mollak (Ismailia governorate), El-Salheia (Sharqaia governorate) etc. create a demand for new Citrus rootstocks and and El-Minya (South Egypt). All samples were collected in middle assessment of some genetic and physiological traits of June. All chemicals and hormone authentics were perched from Sigma the present Citrus trees, where some of which have Aldrich Company. The DNA primers were obtained from the Operon ambiguous origin and dates of introduction (Dehesdtani Technology Inc., Alameda, CA, USA. et al., 2007; Snoussi et al., 2012). Moreover, adequate genetic diversity is necessary in breeding programs for the development of high yielding varieties (Golein et al., Deoxyribonucleic acid (DNA) extraction 2012). Thus, understanding phylogenetic relationships Isolation of total genomic DNA for random amplified polymorphic and genetic diversity in the Citrus resources in Egypt is DNA (RAPD) was carried out according to Dellaporta et al. (1983), important for optimizing their use; which will help for with slight modification as follows: leaf pieces were washed with tap further physiological and agronomical evaluations and water, rinsed twice in sterile distilled water and ground with liquid breeding programs. nitrogen in a sterile mortar and pestle. The powder obtained (about The elements and mechanisms whereby environmental 200 mg) was transferred to a micro centrifuge 1.5 ml tube and resuspended in 600 ml extraction buffer [120 mM Tris, pH 8.2, 800 and cellular stimuli affect physiological criteria as flower mM NaCl, 50 mM EDTA, 1% sodium dodecyl sulphate (SDS), 0.2% and fruitlet abscission in Citrus are triggered by hormonal β-Mercaptoethanol]. Samples were incubated for 45 min at 65C signals (Iglesias et al., 2007). In this respect, gibberellins water bath and then 300 ml M KCl was added. The tubes were Ahmed 15757 Table 1.
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