Study of Agromorphological Diversity, and Path Analysis of Bulb Yield and Allicin Content of Iranian Garlic Landraces under Hamedan Region

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.

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.

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.

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.