4(7) December 2011 issue
December 2011 issue
Southern Cross Publishing Group©2011

December 2011 issue
Improved germination conditions in Cycas revoluta L. by using sulfuric acid and hot water

Mohammad Zarchini, Davood Hashemabadi, Behzad Kaviani, Parvaneh Rafiei Fallahabadi and Naser Negahdar


Germination percentage (GP) is normally very low in Cycas revoluta seeds. In an effort to improve germination, omission of seed dormancy and acceleration of sexual propagation, sulfuric acid (H2SO4) (0, 10, 25 and 50%) and hot water (60, 80 and 100°C) pretreatments for different lengths of time were tested. An experiment was carried out using a factorial randomized complete block design (RCBD). Based on statistical analysis, significant differences were observed between various levels of sulfuric acid and hot water on germination at 5% level of probability. The highest acceleration of seed germination (171.3 days) was recorded in seeds pretreated with hot water at 80°C for 12 h.  The most germination rates (GR) (95%) and germination value (GV) (44.47) were obtained in seeds pretreated with hot water at 100°C for 1 h along with 25% sulfuric acid for 2 h.

Pages 350-353
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In-silico approaches in comparative genomics, structure prediction and functional characterization of secondary
metabolite proteins of Mentha sp.

Sudeep Roy, Rashi Chauhan, Nidhi Maheshwari, Sanchita Gupta, Dwijendra K Gupta and Ashok Sharma


Mentha species are the main source of a number of natural aroma chemicals. In this paper, an attempt has been made to study some important Mentha species, viz., M. piperita, M. canadensis and M. arvensis. In-silico approaches were used for EST assembly, comparative genomics, homology modeling, molecular threading, domain and fold recognition, secondary structure analysis, physicochemical and functional characterization. Totally, 164 different proteins were reported from contigs which were subjected to modeling. Detailed functional and structural characterizations were carried out for 53 different secondary metabolite proteins of Mentha. Physicochemical characterization of the proteins revealed their aliphatic index instability index and extinction coefficient values. These findings will help in further annotation of Mentha from EST level to protein level.

Pages 354-363
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DNA methylation alterations of rice in response to cold stress

Yajiao Pan, Wensheng Wang, Xiuqin Zhao, Linghua Zhu, Binying Fu and Zhikang Li


Rice is sensitive to cold stress, and cold tolerance in rice is a complex trait. In the present study, DNA methylation alterations induced by cold stress were examined in two contrasting rice genotypes, Li-Jiang-Xin-Tuan-Hei-Gu (LTH) and IR64, by methylation-sensitive amplified polymorphism analysis. At the seedling stage, a remarkable increase in DNA methylation under cold stress was detected only in the roots of the cold-tolerant genotype of LTH. Demethylation in the panicles of LTH under cold stress and subsequent recovery were determined. An increase in DNA methylation in the leaves and demethylation in the panicles were observed in IR64 at the booting stage under cold stress. These results indicate that cold-induced DNA methylation alterations are specific to genotypes and are dependent on the growth stage and tissue/organ types. Further analysis suggests that a number of cold-induced DNA methylation changes in both genotypes cannot be reverted after recovery, implying that these loci with alterations of DNA methylation may be mitotically heritable and involved in cold stress responsiveness.

Pages 364-369
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Morphological and genetic diversity in olive (Olea europaea subsp. europaea L.) clones and varieties

Hayat Zaher, Belkassem Boulouha, Mohamed Baaziz, Lhassane Sikaoui, Fatima Gaboun and Sripada M. Udupa


‘Picholine marocaine’ is a predominant olive cultivar in Morocco, widely spread in all the olive growing regions. Clonal selection is one of the breeding methods used for developing varieties and the clones that showed improved traits in field were selected from ‘Picholine marocaine’. In this study, we compare the potential of microsatellite markers with morphological traits to differentiate the varieties and genotypes developed through clonal selections, and to estimate the relationships among the clonal selections, Moroccan local and Mediterranean varieties. For this purpose, we evaluated 7 clonal selections from ‘Picholine marocaine’, five Moroccan local and seven Mediterranean varieties for variation at 15 morphological traits and 20 microsatellite loci. The results clearly showed that microsatellite markers were more efficient compared to morphological traits to differentiate closely related varieties and genotypes developed through clonal selections, in addition to distantly related varieties. Nine out of the 20 microsatellites markers tested were polymorphic, revealing a total of 48 alleles. Average number of alleles per locus was 5.3, ranged from 3 to 9 alleles. Observed heterozygosity ranged from 0.42 to 1 with a mean of 0.79. Upon comparison, the dendrograms, constructed based on morphological traits and microsatellite markers, showed a positive and highly significant relationship. The combination of microsatellite marker profiles and the morphological characters serve as reliable tools for detailed description of olive varieties.
Pages 370-376
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Isolation of two novel isoforms encoding zinc- and copper-transporting P1B-ATPase from Gouan (Aeluropus littoralis)

Leila Rastgoo, Abbas Alemzadeh, and Alireza Afsharifar


Two new isogenes encoding heavy metal ATPase have been partially cloned from Aeluropus littoralis genome. One of them was 849 bp in length and related to an isoform which encodes a copper-transporting P1B-ATPase (AlHMA1), and the other one was 403 bp in length and related to an isoform encoding a zinc-transporting P1B-ATPase (AlHMA2).
When southern blot hybridization was performed under low-stringency, 6 to 7 hybridization bands appeared, indicating that in this plant heavy metal ATPase is encoded by a multigene family. RT-PCR assay displayed that the AlHMA2 highly expresses in leaf and root and its transcription level is the same in both organs. Phylogenetic tree showed that copper transporters from different plants were located in one group and zinc transporters were located in another group, although all heavy metal P1B ATPasees have the same function. It means the different isoforms in single plant that involve in transporting of different heavy metals, may come from different sources. 

Pages 377-383 | PDF Full Text
Does Agro-injection to soybean pods transform embryos?

Muhammad Zia, Waheed Arshad, Yamin Bibi, Sobia Nisa, Muhammad Fayyaz Chaudhary


In planta and agroinfiltration technologies for plant transformation have gained attention in recent years. These technologies pass laborious tissue culture steps but bear low transformation efficiencies. Over-night grown Agrobacterium tumefaciens EHA105 harboring pROKIILFYGUSint plasmid was injected into soybean pods at three developmental stages. The seeds obtained were assessed for transformation through GUS histochemical analysis and PCR. Three plants of NARC-7 and two plants of NARC-4 (transformation efficiencies 14.2 and 6.45%, respectively) showed GUS activity in plant tissue when Agrobacterium was injected after 2-3 days of pod formation; i.e. stage I. Highest GUS expression (39.16 %) was observed on NARC-7 seeds when pods were treated at the late stage of development; i.e. stage III. To our knowledge, this is the first report that demonstrates transformation of developing embryo by pod agroinjection. Using this procedure, transformed seeds can directly be produced and can be further analyzed at progeny level.

Pages 384-390
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Genomic analysis and gene structure of the two invertase families in the domesticated apple (Malus × domestica Borkh.)

Tae Kyung Hyun, Seung Hee Eom, Ju-Sung Kim


Plant invertase (
ß-fructofuranosidase, EC comprises a family of enzymes which plays an important role in the hydrolysis of sucrose to glucose and fructose. Although the specific functions of different invertase families are not clear, they regulate the entry of sucrose into different metabolic pathways in higher plants. Plants contain two unrelated invertase families with different biochemical properties and subcellular localizations. In this study, we identify gene families including vacuolar invertases (designated MdoVIN1-3), cell-wall bound invertases (MdoCIN1-3) and neutral/alkaline invertases (MdoNIN1-12) from the domestic apple (Malus × domestica Borkh.) genome. Based on phylogeny, most neutral/alkaline invertases could be divided into two subgroups, whereas MdoNIN12 was found to be a pseudogene. In addition, specific motifs were discovered in neutral/alkaline invertases, which suggested that different motifs are associated with differences in protein function between subgroups a and ß. Taken together, our comparative genomic analysis of invertase genes and encoded proteins in the domestic apple provides the first step towards the functional dissection of the role of invertase families in heterotrophic metabolism

Pages 391-399
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Expression of OsBADH1 gene in Indica rice (Oryza sativa L.) in correlation with salt, plasmolysis, temperature and light stresses

Supaporn Hasthanasombut, Narongrit Paisarnwipatpong, Kanokporn Triwitayakorn, Chalermpol Kirdmanee and
Kanyaratt Supaibulwatana


The relationship between environmental factors, salt tolerant and the expression of betaine aldehyde dehydrogenase (BADH) gene, salt stress related gene, was investigated in Indica rice. The expression was observed in various rice cultivars as well as under different environmental conditions. Northern blot analysis revealed that salt-tolerant in each rice cultivar is correlated to the expression level of OsBADH1 mRNA. The expression studies showed that OsBADH1 can be induced by a variety of environmental factors such as salinity, drought, cold, heat, light intensity and CO2 concentration. The results demonstrated that the OsBADH1 mRNA expression was up-regulated by salinity, drought, cold and high light intensity but down-regulated by CO2 enrichment and heat stress. The primary response of OsBADH1 gene expression was induced within 24 h after salinity, cold or drought stress treatment. Moreover, these results suggest that the expression of OsBADH1 gene in response to salt stress could be magnified under high light conditions. Interestingly, the effect of salt stress on the expression of OsBADH1 gene was alleviated by CO2 enrichment. This report showed that BADH1 gene not only plays role in the response of indica rice to salt stress but also to plasmolysis, temperature and light stresses. Therefore, BADH1 gene might involve in multifunctional mechanisms in response to environmental stresses of indica rice.  

Pages 400-407
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Evaluation of trigonelline production in Trigonella foenum- graecum hairy root cultures of two Iranian masses

Akbarian Raheleh, Tahereh Hasanloo and Mahmud Khosroshahli


The production of the trigonellin by hairy root cultures of Trigonella foenum was described in two Iranian masses (Zanjan and Borazjan). Different Agrobacterium rhizogenes strains (A4, 9126 and 15834) and infection methods (co-coltivated and injection) were tested to investigate the ability for transformation and production of trigonellin in T. foenum. PCR analysis using rolb gene was used for identification of transformed hairy roots. All the strains of A. rhizogenes used in this study were able to produce hairy roots. The transformation efficiency was %26 in injection method for whole plants. The higher growth index was obtained in hairy roots induced by 2 strains, 15834 and 9126, in Zanjan and Borazjan masses, respectively. Detection and identification of trigonellin for a 28 days period was carried out by high performance liquid chromatography method. The highest amounts of trigonellin in Borazjan and Zanjan hairy roots were 14.89 and 14.03 mM g-1 DW after 28 and 7 days, respectively. The selection of an effective A. rhizogenes strain and T. foenum genotype for the production of trigonellin in transformed root cultures is important. This successful production of trigonellin may be used as a useful system for comparative studies of production capacity of trigonellin in different genetic resources T. foenum.

Pages 408-412
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Alteration of the expression and activation of tomato invertases during Botrytis cinerea infection

Tae Kyung Hyun, Seung Hee Eom, Yeonggil Rim and Ju-Sung Kim


Invertase is a key enzyme that hydrolyzes sucrose into glucose and fructose. In higher plants, invertases occur as a set of isoenzymes with different biochemical properties and sub-cellular locations. Although the expression and activity of invertase are known to be up-regulated when plants are exposed to stress-related stimuli, the interaction between the invertases and the necrotrophic pathogen Botrytis cinerea is not yet fully understood. Therefore, we have analyzed the expression pattern and activation of tomato invertases during an infection of B. cinerea. The expression of Lin5 and Lin6 is up-regulated during a response to B. cinerea, whereas other isoenzymes such as Lin7 and Lin8 are not significantly changed by B. cinerea in mature green fruit. In addition, the infection of B. cinerea results in increasing activity of vacuole and extracellular invertases. When the detached leaves were treated with methyl-jasmonic acid, the increased activities of vacuole and extracellular invertases were observed, and salicylic acid had no influence on the invertases activation. These findings indicate that carbohydrate partitioning via the activation of invertases might be involved in jasmonic acid-dependent defense responses during B. cinerea infections.

Pages 413-417
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Mapping quantitative trait loci for seedling vigour and development in sunflower (Helianthus annuus L.) using recombinant
inbred line population

Robab Davar, Ahmad Majd, Reza Darvishzadeh, Ahmad Sarrafi


The ability of seeds to germinate and establish seedlings in a predictable manner under a range of conditions has a direct contribution to the economic success of commercial crops, and should therefore be considered in plant breeding programs. Quantitative trait loci (QTLs) implicated in seedling vigour and development of sunflower were investigated using a population of recombinant inbred lines (RILs) developed through single-seed descent from the cross ‘PAC2
´ RHA266’. The experiments were conducted in completely randomized design with three replications. Different traits associated with germination, seedling vigour, early growth and development were studied. Analysis of variance for all studied traits revealed significant differences (P<0.01) among RILs and their parents. A large genetic variation and transgressive segregation was observed for all the studied traits. QTL-mapping was performed using a high-density simple sequence repeat/ amplified fragment length polymorphism (SSR/AFLP) linkage map and several QTLs associated with the studied traits were identified. The percentage of phenotypic variation explained by individual QTLs ranged from 5.4% to 35.7%. Based on overlapping support intervals, the co-location of QTLs for all ten traits was determined. QTLs controlling most of the traits were overlapped on different linkage groups, which was in accordance with the phenotypic correlation results among the traits. Although the detected regions need to be fine-mapped to dissect the genes underlying QTLs, the information obtained could help sunflower breeders in marker-assisted breeding programs for developmental traits.

Pages 418-427 | PDF Full Text | Supplementary data
Genomic identification of putative allergen genes in woodland strawberry (Fragaria vesca) and mandarin orange
(Citrus clementina)

Tae Kyung Hyun and Ju-Sung Kim


Although many fruits are plant-derived healthy foods, they may harbor a series of allergenic proteins. Based on biochemical and molecular biological approaches, a number of allergens have been identified and characterized in Rosaceae family fruits, such as apple and peach. However, our understanding of the allergens from the Rosoideae subfamily and the Rutaceae family is very limited. Therefore, in this study, we identified gene families, including pathogenesis-related protein 10 (PR-10), non-specific lipid transfer protein (nsLTP), and profilin as allergens in Fragaria vesca (woodland strawberry) and Citrus clementina (mandarin orange). Using comprehensive analysis, we identified 25 putative allergen genes from woodland strawberry and eight mandarin orange genome sequences. The analysis of functional annotation indicated that these allergens have complex physiological roles that are involved in plant defense and plant development. Although further functional analysis of these allergens though the analysis of cross-reactivity of immunoglobulin E antibody to these allergens will be required, our comparative genomic analysis of the allergen genes and encoded proteins in woodland strawberry and mandarin orange provides a basis for future research on developing low-allergenic cultivars without compromising the natural defense of plants.

Pages 428-434
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In vitro propagation of Matthiola incana (Brassicaceae)-an ornamental plant

Behzad Kaviani, Afshin Ahmadi Hesar, Ardashir Kharabian-Masouleh


Tissue culture techniques are applied for micropropagation and production of pathogen-free plants. Successful in vitro propagation requires an understanding of specific requirements and precise manipulation of various factors. Direct plant production from cultured explants is important to minimize somaclonal variation in regenerated plants. In this study, an efficient protocol for micropropagation of Matthiola incana using shoot tips is presented. Seeds from mother plants were germinated on MS medium without growth regulators. Shoot tips from in vitro germinated seedlings were subcultured on solid MS medium supplemented with kinetin (KIN)(0, 0.5, 1 and 2 mg/L) and naphthalene acetic acid (NAA)(0, 0.5, 1 and 2 mg/L). Four-week-old in vitro plants, obtained from micro-cuttings, showed successful shooting and rooting. MS medium supplemented with 2 mg/L KIN without NAA resulted in the best shoot length (1.166 cm) and largest number of node (4.64). When the shoot tips were inoculated in the medium containing 2 mg/L NAA without KIN and medium containing the combination of 1 mg/L NAA + 2 mg/L KIN, the best result was observed for root number (1.85) and root length (5.2 cm). Moreover, fresh weight, dry weight and chlorophyll content of plants were calculated. 

Pages 435-440
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