TERIZATION of HOLOPARASITIC PLANT, Cistanche Phelypea L. in SIWA OASIS, EGYPT
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MORPHOLOGICAL AND MOLECULAR GENETICS CHARAC- TERIZATION OF HOLOPARASITIC PLANT, Cistanche phelypea L. IN SIWA OASIS, EGYPT H. E. ELWAKIL1, N. R. ABDELSALAM1, REHAM M. ABD EL-AZEEM2, A. A. HEMEIDA3, NERMEEN Y. ABASS1 AND A. NASSAR 1. Department of Agricultural Botany, Faculty of agriculture-Saba Basha, Alexandria Univ., Egypt 2. Dept. of Environ. Biotechnology, Institute of Genet. Eng. and Biotechnology, Univ. of Minufiya 3. Dept. of Bioinformatics, Institute of Genetic Engineering and Biotechnology, Univ. of Minufiya 4. Plant Protection Dept., Faculty of Agricultural, Damnhour Univ. gypt is famous for a vast desert is represented in Egypt by four species E spaces. About 4/5 the total area is namely C. phelypaea C. tubulosa, C. desert. Only about one-fifth of the total violacea and C. salsa (Vivi Tackholm, area is cultivated land. Most of the soils 1974; Boulos 2002). In the observed and of this desert are sand dunes, sand sheets, collected areas,Cistanche phelypaea re- sand bed rock, sandstone, limestone and stricted its host range in two families only, salt marshes. In the western desert of Chenopodiaceae and Zygophyllaceae. Egypt there are a series of oases which This holoparasitic species which parasitic stretching longitudinally from the north to on the roots of other plants lacks chloro- the far south in a line almost parallel to the phyll and therefore cannot performs pho- Nile valley. This represents natural de- tosynthesis. So it is an obligate parasite pressions rich in natural springs and wells which totally depends on its host for wa- which are used in agriculture. One of the ter, minerals and organic nutrients advantages of this desert is the presence of (Boulos, 2002). Cistanch philypea L. wild flora species adapted to a biotic grows in Egypt in low density but scat- stresses such as drought, high temperature tered on a large area of the country. It can and salinity. Despite the rarity of wild be recognized in the Nile region including plant cover in such region, this existing Faiyum, the Oases of the western desert flora is a treasure of genetic resources. including Siwa, the Red Sea coastal strip, Most of these flora species are considered and the entire Sinai Peninsula including as the ancestors of domesticated plants, the coastal of the Mediterranean. It grows others have medicinal benefits either on in sandy and alluvial soil, edges of culti- the popular level or on the documented vated fields as a parasite on plant roots medicinal treatments. (Vivi Tackholm, 1974; Boulos, 2002). One of these species is the Angio- Cistanche species have many med- sperm Dicotyledonous holoparasitic plant; ical benefits. Medical tests proved their Cistnche phelypeae. The genus Cistanche beneficial effects as follows: exhibited _______________________________________ Egypt. J. Genet. Cytol., 41: 181-194, July, 2012 182 H. E. ELWAKIL et al. hypocholesterolemic activity in diet- describe the nature of the ecological nich- induced hypercholesterolemia mice es where it lives. (Shimoda et al., 2009), enhanced antibody production in human lymph node lympho- B. Chemical analysis cytes (Maruyama, 2008). Cistanche may delay aging, which may be related to an- The concentration of macro and tagonizing free radical injury and enhanc- micro elements in the parasite and its host ing immunity of aging mice (Zhang et al., species were estimated by soil, water and 2008). Anti-fatigue properties have been plant analysis Unit, Department of Soil shown in mice. Cistanche appeared to and Agricultural Chemistry, Faulty of enhance the swimming capacity of mice Agriculture-Saba Basha, Alexandria Uni- by decreasing muscle damage, delaying versity. Atomic absorption was used for the accumulation of lactic acid and by estimating micro elements; zinc, iron, improving the energy storage and a recent manganese and cupper. Macro elements; clinical study, a memory was found to calcium, magnesium were estimated by enhance properties in Cistanche (Choi et titration method, while sodium and potas- al., 2011). The objective of this research is sium were estimated by flame photometry. to describe the environmental condition, C. Biochemical genetic analysis host range, morphological feature and (isozymes analysis) determine the extent of genetic variation among individuals of this species in two Twenty different plant stems of different populations from two different Cistanche phelypaea from each locality localities by applying biochemical and were examined individually for their molecular genetic analyses. isozyme patterns. A combination of agar- starch gel electrophoresis and enzyme MATERIAL AND METHODS activity attaining was used to screen for polymorphisms of peroxidase. The labora- A. Surveying localities tory methods were performing according Cistanche phelypaea plants were to Jonathan and Norman (1989). Accord- surveyed and photographed combined ing to the electrophoretic pattern exhibited with their natural hosts in the western de- by each of peroxidase loci under investi- sert of Egypt from two different localities; gation, genotypic frequencies were calcu- south of Siwa Oasis, in the sand sheets lated and used to estimate gene frequen- region and in uninhabited (neglected) Oa- cies of alleles segregating at each locus. A sis (Sheiata) away from Siwa with about simple method of calculating gene fre- 80 kilometers in the north west direction quency of S (allele coding for slow mi- (Fig. 1). Different morphological features grating band) and F (for fast) uses the fol- of different developmental stages of the lowing equations: q (S) = m (S) + 1/2 H species were photographed in its natural and q (F) = m (F) + 1/2 H, where q (S) habitats accompanied with its hosts to and q (F) are the gene frequencies of S CHARACTERIZATION OF HOLOPARASITIC PLANT 183 and F alleles, respectively, m (S) and m gels at 100 volts for 1.30 hrs with 1 x TBE (F) are the frequencies of SS and FF ho- buffer. To detect ethidium bromide/DNA mozygotes, respectively, and H is the fre- complex, agarose gels were examined on quency of heterozygotes (EL-Metainy et ultraviolet transilluminator (302 nm wave- al., 1977). length) and photographed. Using 100 bp DNA ladder (V-gene Biotechnology Lim- D. Molecular genetic analysis ited, shiqao, P. R. China), the lengths of the different DNA fragments were deter- Twelve individual plants were tak- mined. The reproducible DNA fragments en at random from two different localities from two runs were scored for their pres- of Siwa Oasis, six plants were taken from ence (1) or absence (0) for each genome. each locality. One locality is in the west of the Oasis (A), while the other is in the E. Data analysis East (B). The two localities are far away from each other with about 80 Km (Fig. Data matrices were entered into the 1). Genomic-DNA from each population NTSYS program (Numerical Taxonomic was isolated from plants stem using modi- and Multivariate Analysis System) soft- fied CTAB method (3% CTAB, 3% PVP ware package, version 2.1, Applied Bio- and 3% β mercaptoethanol) according to statistics Inc. (Rohlf, 2000). Similarity coefficients were used to construct Doyle and Doyle (1987). dendrograms using the UPGMA (un- The PCR operon primers used for weighted pair group method with arithme- RAPDs are listed in Table (1). These pri- tic average) and the SAHN (Sequential mers were selected from the Operon kits Agglomerative Hierarchical Nested clus- (Operon Technologies Inc., Alabameda tering) routine in the NTSYS. CA). RAPD-PCR analysis was performed RESULT AND DISCUSSION according to the method of Williams et al. (1990). The polymerase chain reaction A. Morphological description mixture (25 µl) consisted of 0.8 U of Taq DNA polymerase; 25 pmol dNTPs; 25 Figure (2) illustrates the morpho- pmol of primer and 50 ng of genomic logical features of different developmental DNA. PCR amplification was performed stages of the inflorescence of the parasitic in a Biometra T1 gradient thermalcycler plant Cistanche phelypaea, it has been for 40 cycles after initial denaturation for surveyed in two different localities in the 3 min at 94C. Each cycle consisted of western desert of Egypt; Siwa and Sheiata denaturation at 94C for 1 min; annealing Oases. The plant is mostly existed in dry at 36C for 1 min; extension at 72C for 2 sandy soil. It is a chlorophyll-free obligate min and final extension at 72C for 10 parasite with stout fleshy flowering stems min (Soliman et al., 2003). Amplification bearing bright yellow flowers and rising to products were separated on 1.5% agarose 30 cm tall from a swollen base. The botan- 184 H. E. ELWAKIL et al. ical description of the species was de- host species. This association was estab- scribed in details by Boulus (2002) as lished with the help of haustoria which follow, it is herbaceous parasitic glabrous then penetrated into the deeper layers of perennial 20-80 cm; stem fleshy, robust the host tissue. However, when the para- scales 1.8-2.5 x 0.3-0.8 cm, ovate- site occurred on Calligonum polygonoides lanceolate, the margin scarious and den- and Calotropis procera, as C. phelypaea, ticulate; inflorescence a dense spike 10-20 it attached itself as on the apex of the root. cm; bract 1.5 x 0.3-0.4 cm, lanceolate; bracteoles 1-1.2 x 0.2 cm, narrowly B. Macro and micro element concentra- lanceolate; flowers are pollinated by in- tions in the parasite and host species sects, sessile; calyx 1.4-1.6 cm, the lobes Table (2) illustrates the concentra- 3-4 mm, broadly ovate; corolla 3-5 cm, tion of the nutrients as macro elements uniform bright yellow; corolla-tube slight- and micro elements in the parasite plant; ly curved, the lobes broadly rounded; fil- C. phelypaea and in the two different aments sparsely hairy at the base; anthers hosts C. monacantha and Z. album. As pubescent; capsule 1.2-0.4 cm narrowly shown in Table (2), it is evident that the ovoid; seeds 0.4 mm.