Mohammad Hossein Alemkhoumaram; Amir Hossein Keshtkar; Asghar Mirzaie Asl
Abstract
Introduction: Garlic is native to central Asia, with a three thousand years history of human consumption and use. Global consumption has increased fourfold during the 24-years period from 1989 to 2013. Area under garlic cultivation, average yield/ha and total production of the world were 1,468,811 ha, ...
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Introduction: Garlic is native to central Asia, with a three thousand years history of human consumption and use. Global consumption has increased fourfold during the 24-years period from 1989 to 2013. Area under garlic cultivation, average yield/ha and total production of the world were 1,468,811 ha, 18,092 kg/ha and 26,573,001 tons, respectively in 2016; and China produces about 80% of world garlic production. The maximum global record has been registered at 2012 with an average bulb yield of 45,270 tons per hectare belongs to Uzbekistan. The area under cultivation of this plant in Iran is about 9000 hectares with an average yield of 10 tons per hectare. Garlic has been used both as a food flavoring and as a traditional medicine, and these characteristics are affected by sulfur compounds. Alliin (C6H11NO3S) with 177.22 molecule weight is a sulfoxide that is a natural constituent of fresh garlic. It is a derivative of the amino acid cysteine. When fresh garlic is chopped or crushed, the alliinase enzyme, which contains pyridoxal phosphate (PLP), cleaves alliin, generating allysulfenic acid, pyruvate (C3H4O3), and ammonium (NH3). At room temperature two moles of allysulfenic acid as an unstable and highly reactive compound that through a dehydration reaction form one mole of allicin (C6H10OS2) with 162.26 molecule weight, which is responsible for the aroma of fresh garlic. In general, alliinase needs few minutes time to effect on every two molecules of alliin to form one molecule of allicin, two molecules of pyruvate and two molecules of ammonium. The generated allicin is unstable and quickly breaks down, for example during sixteen hours at 23°C it converts to a series of other sulfur-containing compounds such as diallyl disulfide. Allicin is part of a common defense mechanism in garlic plants against pest attacks. It is produced and activated after causing physical injuries. Iranian garlic has a good flavor, while with the exception of Mazand cultivar so far there is no other introduced bred cultivar. In this effort, diversity of agromorphological traits, bulb yield and allicin content of some Iranian landraces were studied to breed promising cultivar/s by single-bulb selection method for Hamedan climatic condition.
Materials and Methods: The experiment was carried out on the basis of a randomized complete block design (RCBD) with four replications in 2015-16 agronomy season at the Agricultural Research, Education and Natural Resources Center of Hamedan Province Located about 6 km north of Hamedan at the altitude of 1740 meters above sea level, longitude 48°, 52̍ E, latitude 34°, 52̍ N. The soil texture of the test site was loam with 24.4, 40.6 and 35 percent of clay, silt and sand, respectively. The soil tillage operation included plowing with the moldboard plow, disc and leveler. The furrow and stack were created with a width of 50 cm. Nine landraces from Hamedan province (Maryanaj, Bahar, Sheverin, Soolan, Toyserkan, Heidareh, Toeen, Hossein Abad and Ali Abad, and other nine ones from Gorgan (Golestan), Oromyeh (West Azarbaijan), Arak (Markazi), Bardsir (Kerman), Amol (Mazandaran), Ramhormoz (Khuzestan), Isfahan (Isfahan), Tarom (Zanjan) and Zabol (Sistan) cultivated on plots having 2 ridges with 4 meters long. Two rows were planted on the ridge with 25 and 10 cm spacing between rows and plants on the row, respectively (40 plant m-2). Bulb planting was carried out on November 17th, and first irrigation was rain-fed on 18th and 19th of November, with 19 and 9.9 mm rain, respectively, and during the fall and winter seasons, all water requirements were met through precipitation. Different landraces germinated from March 12th to March 16th. Pressure irrigation system was installed by re-growing in spring and plants were irrigated every seven to 10 days, depend on air temperature severity, until June 5th. Harvesting of different landraces was carried out by hands from June 26th to July 6th. Evaluated traits for each plot included weight of 30 seed clove, date of germination and maturity, number of plant per plot, chlorophyll a and b and carotenoids content of leaf, chlorophyll index by SPAD, relative water content of leaf, bulb yield, and weight of 30 produced clove, total pyruvic acid, and non-enzymatic pyruvic acid. The evaluated traits for five normal random plants (healthy plants on both sides, and on the opposite side of cultivation lines adjacent to the healthy plant) of each plot were including height, crown diameter, number of leaf, number of fallen leaf, length and width and leaf area, dry weight of plant leaf, dry weight of plant root (to a radius of five centimeters around the bulb) height and width and length of bulb, dry matter of bulb, number of skin layers, number and weight of clove, dry matter percentage. Assessment were also considered based on traits such as, moisture percentage of bulb, leaf area index, sum of chlorophyll a and b, loss weight of cuddle, harvest index, sphericity coefficient of bulb, total number of plant leaf, days number of germination to maturity, enzymatic pyruvic acid and allicin. Traits measurements were done based on "Descriptors for Allium". To measure pyruvic acid, 25 g of cloves with 25 ml of water were completely homogeneous in a mixer and passed through a filter paper after ten minutes. Ten ml of filtrated extract was increased into 100 ml volume with water and a half-milliliter of it was taken and added to 1.5 ml of 5% trichloroacetic acid solution, and after one hour, 18 ml of water was added to sample. Then one ml of resulting solution and each standard solution were transferred to separate test tubes. One ml of water and dinitrophenyl hydrazine 0.0125%, were added to each sample, and were heated in bain-marie bath at 37°C for ten minutes. At the end, by adding five ml of 0.6% normal caustic soda to the test tubes, the concentration of samples were determined in the presence of standard solutions (0.01, 0.225, 0.05, 0.1 and 0.2 μmol / ml sodium pyruvate) at 420 nm wavelength using spectrophotometer Pharmacia-Biotech model Novaspec II. To assess non-enzymatic pyruvate activity, alinase enzyme must become inactivated, so 40 to 60 grams of garlic clove is initially heated in microwave at 900 watts for three seconds for every gram of sample (120 to 180 seconds).
To find relationship between variables, the correlation coefficients between them were calculated. The study of the relationship between traits with bulb yield and alicin yield was done by stepwise regression method. Causal relationships and direct and indirect effects of traits on bulb and allicin were determined by path analysis method. Analysis of variance, mean comparison of traits by LSD method at 1% probability level, correlation determination, stepwise regression analysis and cluster analysis were done by Minitab software version 17.3.1, while Microsoft Word and Excel 2003 softwares were used to draw the graphs and path analysis, respectively.
Results and Discussion: The results shown that Iranian garlic landraces have noticeable diversity of agro-morphological traits, such as weight of 30 seed clove from 21 (Ramhormoz landrace) to 177 g (Hossein Abad landrace), bulb yield from 2059 (Ramhormaoz landrace) to 12090 kg ha-1 (Soolan landrace), Allicin yield from 4.9 (Ramhormoz landrace) to 53 kg ha-1 (Ali Abad landrace), bulb weight from 12.8 (Ramhormoz landrace) to 48 g (Bardsir landrace) and bulb diameter from 28 (Isfahan landrace) to 35 mm (Hossein Abad landrace), which are necessary for breeding programs. It is also confirmed that the adapted landraces from one region may have acceptable production in other regions. According to the present results, landraces from Oromyeh (10866 kg/ha), Arak (9941 kg/ha) and Bardsir (11444kg/ha) produced high yield in Hamedan climatic condition, so that the maximum allicin content were produced by landraces from Ali Abad (53 kg/ha), Maryanaj (51 kg/ha) and Oromyeh (51 kg/ha). Allicin content is calculated based on the bulb yield, and enzymatic pyruvic acid content and its molar relationship with allicin. In the recommended equilibrium of chemical reaction, the alliin coefficient on the one side is two and the coefficients of allicin and pyruvic acid on the other side are two and one, respectively. Bulb weight directly and crown diameter indirectly had highly correlation with bulb yield; and leaf area index, harvest index, bulb diameter and dry weight of plant leaf showed a high correlation with allicin content. The cluster analysis used standardized traits to divide landraces into two main groups as suitable and unsuitable sets to plant under Hamadan climatic conditions.
Conclusion: According to the results of the present study, it is recommended that Oromyeh, Soolan and Heidareh landraces can be used as valuable populations for breeding and introducing the suitable cultivars for Hamedan climatic condition through single-bulb selection method. Characteristics with easy and low cost assessment, such as crown diameter, leaf area index, bulb diameter under field condition; and after harvest measurements, such as bulb weight, harvest index, dry weight of plant leaf traits can also be considered as suitable selection indicators in breeding programs. Thirdly, the current study indicated that the cost of preserving garlic germplasm resources can be decreased by reducing the amount of stored materials, without the significant decline in genetic diversity.
Amir Hossein Keshtkar; Noshin Fallahi; Mohammad Reza Abdollahi; Hassan Sarikhani; Hooshmand Safari; Zhaleh Mohseni Araghi
Abstract
Introduction: Polyploidy plays an important role in creation of genetic variability. Polyploidy induction by mutagenic chemicals such as colchicine is considered to enhance the potential of secondary metabolites production in herbs breeding. Colchicine is the most effective chemicals used in the polyploidy ...
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Introduction: Polyploidy plays an important role in creation of genetic variability. Polyploidy induction by mutagenic chemicals such as colchicine is considered to enhance the potential of secondary metabolites production in herbs breeding. Colchicine is the most effective chemicals used in the polyploidy induction studies. The effect of colchicine is to form cells with two or multiply number of chromosomes resulting in a lack of germination and death of a large number of plant samples. Flow cytometry analysis and cytogenetic studies were effectively used to assess the ploidy levels for fenugreek (Trigonella foenum-graecum) plants. In the beginning of 90 decade, a new type of adsorption technique called solid-phase micro extraction (SPME) has been developed by Pawliszyn and co-workers. This method compared to the traditional techniques, offers many advantages such as the high sensitivity and reproducibility, does not require solvent, and combines extraction and pre-concentration in a single step without pre-treatment of samples. Moreover, it is a fast and inexpensive method, requires low sample volume and it can be easily automated. Solid-phase micro extraction (SPME) uses a fine rod (fused silica or metal) with a polymeric coating to extract organic compounds from their matrix and it directly transfer them into the gas chromatograph injector for thermal desorption and analysis (Kaykhaii, 2008). The aim of this study was to investigate the effect of colchicine treatment on ploidy levels and compare some of the morphological, physiological, cytogenetical, flow cytometric analysis and biochemical characteristics in diploid and tetraploid fenugreek plants.
Materials and Methods: In order to investigate the effect of polyploidy induction by colchicine on Trigonella foenum-graecum medicinal species, an experiment was planned as a factorial completely randomized design with five concentrations of colchicine (0, 0.05, 0.1, 0.2, and 0.5%) for 12, 24, 48 and 72 hours. In this experiment the effect of colchicine was examined on the percentage of survival and tetraploidy of seed, root and terminal bud samples. Level of ploidy was identified in survival explants through root tip chromosome counting and flow cytometry of leaf samples. In addition to distinguish tetraploid from the diploids plants, morphological, physiological and biochemical characteristics were considered in treated plants. SAS and SPSS software programs were used to analysis of variance and comparison of means by Duncan's multiple test. Graphs were also drawn by EXCEL software.
Results and Discussion: The analysis of variance showed that all characteristic factors for survival percent and mixoploidy percentage were statistically significant. Survival percentage was decreased with increasing of colchicine concentration and increased exposure time of colchicine-treated seed. After the observation of morphological changes, the samples were considered to assess the ploidy levels by flow cytometry system. Results showed that 0.5% colchicine concentration had the highest survival rate after control treatment for the terminal bud. The highest percentage of mixoploidy was also observed in treated terminal buds with 0.1 and 0.2% of colchicine concentrations. Morphological, physiological, cytogenetic, flow cytometric analysis and biochemical studies confirmed that terminal bud treatment with 0.2% colchicine for 72 hours is the most effective treatment to induce tetraploidy in fenugreek plant. The results of GC/MS also indicated an increase in secondary metabolites content, but traits including growth rate and plant height of tetraploid reduced compared to the diploid plants. Result of this study showed a significant increase in chlorophyll a, b and total chlorophyll contents of tetraploid plants, which were higher than the levels of diploid plants.
Conclusion: Polyploidy induction using mutagenic chemicals is one of the methods to enhance the production of plant secondary metabolites. Colchicine is the most effective mutagenic chemical in inducing plant polyploidy. Although, flow cytometry is an expensive method, it is increasingly used for ploidy screening by analyzing of nuclear DNA content. In this study, both flow cytometry and chromosome microscopic examinations were used to test ploidy. The two methods were compared, and it was found that flow cytometry testing was fast and labor saving, especially in case of a large number of samples. Tetraploidy induction significantly affected different morphological, physiological, and biochemical characteristics of Trigonella foenum-graecum. These changes suggested that ploidy manipulation as a rapid and effective method for enhancing genetic diversity and metabolite production for this plant. SMPE method offers a number of practical advantages: smaller sample volume, simplicity of extraction and low cost, when compared to the other methods that are currently being used.
