نشریه بیوتکنولوژی و بیوشیمی غلات
سال دوم شماره 1 (پیاپی 5، بهار 1402)

Heat and its effects are considered one of the serious threats and damages to the production of agricultural and horticultural crops in many regions of the world. Therefore, it has led to pervasive changes in terms of morphological, physiological, anatomical, and molecular, which causes a sharp decrease in the economic performance of agricultural products. Nowadays, in order to reduce these inhibitory effects, in addition to classical and conventional breeding methods, modern and molecular methods can be used to identify heat-tolerant genes and use them in breeding programs to improve plant tolerance to environmental stresses.

The selection for heat tolerance among four local cultivars Hovyzeh, Hamar, Red-Anbori, Champa and three promising heat tolerant lines V1, V2 and V3 obtained from the international breeding programs of Khuzestan Agricultural Research and Training Center and Natural Resources were evaluated by osHATS and HSP70 genes qPCR on two experimental cultivation dates: May 20 (unfavorable) and June 20 (favorable) 2022 in Ahvaz.

The results obtained from the analysis of gene expression by qPCR on the date of the first cultivation confirmed the presence of the osHTAS gene in rice varieties and lines so that the local variety Champa, Hovyzeh, and line V2 has a high relative gene expression compared to other varieties and lines studied and they were significantly different from each other. The analysis of the osHTAS gene expression on the second planting date (June) in Ahvaz also shows the presence of the high-temperature tolerance gene in Hovyzeh, Hamar and V2 lines; therefore, the mentioned varieties could be used as parental lines for transfer heat tolerance gene to be used in multi-breeding programs and terms of the relative expression level of the gene, they showed a statistically significant difference with each other. The results of the relative gene expression of the HSP70 gene in the cultivation date of May showed that the local cultivars Champa, Red-Anbori and Hovyzeh had a remarkable superiority over the others. Examining the HSP70 gene expression pattern of rice cultivars and lines on the second planting date also revealed that the local cultivars Red-Anbori, Champa, Hovyzeh, and Hamar showed superiority. The tolerance of local cultivars to heat stress is due to their rapid growth and the creation of a large green cover in the early stages of growth, and their great adaptation to the region's environmental conditions.

Local cultivars can withstand heat stress well with rapid growth and creating much vegetation due to their high adaptability to environmental conditions and the presence of heat stress tolerance genes. They could be used as parental lines in breeding programs.

The use of seeds to create genetic mutation brings problems because the use of seeds increases the duration of the research, and the study of traits requires plant cultivation, making it impossible to test more than once a year. Another point is the presence of shimmer in mutant plants. Biotechnology techniques, especially tissue culture, solve these problems today. In this method, a repeatable and efficient regeneration system is needed to create mutations in rice plants to obtain fertile plants from the changed tissues.

To determine the appropriate dose of mutation induction to create genetic changes in the callus tissue obtained from the mature and immature embryos of Nemat and Taram rice varieties, a factorial experiment was carried out within a completely randomized design using doses of 20, 30, 40, 50 and 60 Gy (GY) of Gamma radiation in four replications. For callus generation, MS medium with 0.5 mg/liter 2, 4-D and 1 mg/liter Kasein was suitable for both cultivars. Also, for the regeneration of calluses obtained from both types of embryos of the Tarom cultivar, MS base medium containing 4 mg/liter of BAP, 0.5 mg/liter of IAA, 0.5 mg/liter of NAA, and 5 mg/liter of Kasien were used. In the final stage, 30-day-old calluses were subjected to gamma radiation treatment with five doses of 20, 30, 40, 50 and 60 Gy. They were compared with the control treatment in the regeneration stage regarding moisture percentage, growth rate and regeneration percentage.

After irradiation, the effect of variety on regeneration percentage, growth rate based on weight and callus diameter, the effect of embryo type on regeneration percentage and moisture percentage, the effect of radiation on all studied traits, the effect of variety × embryo only for regeneration percentage and moisture percentage, the effect of variety × radiation for all traits and the effect of embryo × radiation and cultivar × embryo × radiation were significant only for the percentage of reproduction. The LD50 obtained in Nemat and Tarom cultivars with mature embryos was related to doses of 26.78 and 41.83 Gray, respectively; with immature embryos, it was 14.55 and 35.30 Gray, respectively.

The difference in this experiment can not only be caused by genetic differences, but it can also be due to the difference in callus humidity and the effect of gamma rays.

Among cereals, durum wheat (Triticum turgidum L. var. durum) is one of the most important protein and energy sources after bread wheat. It has a significant role in human nutrition in most countries.

In this study, in order to identify and localization of QTLs controlling traits related to flag leaf and peduncle, 118 durum wheat recombinant inbred lines obtained from the cross between the local genotype of Iran-249 originated from the west of Iran and the local cultivar of Zardak from Kermanshah were evaluated in three environments under rainfall conditions using randomized complete block design along with parents in three replications. QTL analysis for each trait was performed using the inclusive composite interval mapping method to identify genomic regions that significantly affected the studied traits.

Based on the analysis of variance, the effect of genotype (recombinant inbred line) was significant in terms of traits related to flag leaf and peduncle in three environments, which indicates the existence of appropriate levels of genetic diversity in the studied population for measured traits. The results of QTL analysis using the inclusive composite interval mapping method led to the identification of 33 QTL for the measured traits. The common and stable QTLs were located in at least two environments on chromosomes 1A, 1B.1, 2B.1 and 5A for peduncle length and diameter traits and on chromosomes 2A, 5A and 7A for flag leaf length, flag leaf width and flag leaf area. Identification of repeatable QTLs in the different environments can indicate the existence of stable genomic regions related to flag leaf and peduncle. On the other hand, a positive and significant correlation was observed between the traits, probably due to the linkage or pleiotropic effect of their controlling QTLs. Also, based on QTL analysis using the two-locus method, the additive main effect, as well as the interaction effect of the QTL× environment, was significant for all traits except for the middle and bottom peduncle diameter. In total, six gene loci with significant additive× additive epistasis effects were identified for the flag leaf area-related traits and top peduncle diameter.

Identification and localization of stable QTLs in different environments for some traits related to flag leaf and peduncle can assist in understanding better the genetic basis of these traits to be used in breeding programs, especially in MAS projects.

Wheat, like other plants, uses antioxidant enzymes to deal with the high production of free oxygen radicals. Antioxidant enzymes are proteins or polypeptides that are activated in cooperation with cellular substances or mineral elements and are responsible for detoxifying reactive oxygen species in organisms. Superoxide dismutase (SOD) is an important antioxidant enzyme responding to plants' abiotic stresses. This study was performed to identify significant regulatory elements within the promoter regions of superoxide dismutase genes in the wheat genome.

To investigate the regulation of gene expression and the effective factors in regulating superoxide dismutase enzyme using bioinformatics methods, the promoter region of Mn-SOD, Cu/Zn-SOD and Fe-SOD genes was investigated. The reason for selecting these genes is their ability to scavenge the free radicals of oxygen in plants. Also, their gene expression pattern are crucial under stressful conditions. In order to perform the promotor analysis of these genes, first, the sequence of the genes encoding this enzyme from wheat was downloaded using the NCBI site. After receiving the desired gene sequence, all three sequences of this gene were searched to find different versions of the SOD gene in the wheat genome against the genomic sequence of this plant that has been identified and reported on this site so far. Each received promoter sequence was analyzed and their regulatory elements were identified. Fifteen genes encoding superoxide dismutase were identified and annotated in different chromosomes of A and D genomes of wheat. Then, the promoter regions of these genes, including 1500 bp upstream sequences, were separated to identify the corresponding regulatory elements. The promoter sequences were analyzed to detect the statistically significant over-represented regulatory elements.

The results showed that various regulatory elements involved in different biochemical pathways might influence wheat's transcription of superoxide dismutase genes. G-Box, I-box, MBS, TGA-element, A-box, ABRE, and ARE, are regulatory elements annotated by individual and grouping promoter analysis. In this study, genes encoding superoxide dismutase were mainly identified on different chromosomes of wheat's A and D genomes. This indicates that these genes were probably transferred from the parents of wheat that had these two main genomes to wheat.

Finally, the analysis showed that the important regulatory elements identified in this pathway could include ABRE, ARE, CCAAT-box, G-Box, Skn-1_motif, and circadian, which respectively have a role in the pathway of response to abscisic acid, and response to other reactions such as aerobics reactions, the binding site of elements related to genes encoding MYB transcription factor, response to light conditions, endosperm expression pathway and the pathway of regulation of periodic light responses. Most of the regulatory elements of these genes are involved in light reactions and environmental stresses, which protect the plant against their injury effects.

The rapid increase in the world population and the need to increase agricultural production, and on the other hand, the rapid change of climate conditions, are the significant challenges facing the world in the coming years. Wheat, the most important plant in the cereal family, plays a vital role in the world's food security now and in the future. Therefore, producing new cultivars with high yields and carefully selecting cultivars suitable for cultivation in different climatic zones are important future research priorities.

This study investigated the reaction of different bread wheat cultivars regarding yield and its components. It also aimed to study the growing degree days (GDD) and some physiological traits related to cold tolerance in different bread wheat cultivars. This study was conducted as two independent experiments during the 2013-14 cropping year at the research field, Campus of Agriculture and Natural Resources, Razi University, Kermanshah, Iran. The first experiment was a randomized complete block design with three replications. Treatments included 12 bread wheat cultivars (Norstar, Zarin, Kavir, Baz, Sivand, Alvand, Pishgam, Pishtaz, Orum, Shahryar, Bahar, and Parsi). The second experiment was a factorial experiment in a randomized complete block design with three replications. In this experiment, 12 bread wheat cultivars were evaluated as the first factor and sampling times as the second factor in three levels: early January, early February, early March.

The results of this study showed that Sivand and Parsi spring cultivars with the highest plant growth rate in different stages of development, with 5170 and 5100 kg/ha, respectively, and Norstar winter cultivar with the lowest plant growth rate in various stages of development, with 2230 kg/ha, had the lowest value of grain yield. The correlation between grain yield, growing degree days requirement to flowering, and physiological maturity were negatively significant. Cultivars with lower grain yield had higher growing degree day’s requirements. Examination of the growing degree days requirement of cultivars up to the flowering stage showed that, on average, spring cultivars had lower growing degree day requirements than winter cultivars. The accumulation of soluble sugars in the crown of winter and intermediate cultivars during winter was higher than spring cultivars. The accumulation of soluble sugars in the wheat crown was downward during the winter, so at the beginning of winter, the crown had a higher sugar content than in the middle and late winter. Winter and intermediate cultivars, on average, had higher leaf greenness and maximum photochemical efficiency of photosystem II than spring cultivars.

According to the results obtained in the study area, on average, winter and intermediate cultivars were superior to spring cultivars in terms of physiological traits related to cold tolerance. But, due to the obtained seed yield. However, according to the grain yield obtained in this study, spring cultivars are more recommended among cultivated cultivars than the other two growth types in Kermanshah and similar climate zones.

Today, wheat is grown on more land area than any other commercial crop and continues to be the most important food grain source for humans. Rejaw cultivar is adaptable to the conditions of rainfed cultivation and supplementary irrigation in the cold regions of Iran. Of course, it can play an essential role in increasing and sustaining the yield of rainfed wheat. Regarding the quality of the resulting flour, Rejaw has a high quality, increasing the bread quality and reducing bread waste. Two-dimensional electrophoresis is a combination of isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, which allows the separation of proteins based on electrical charge and molecular weight.

In this study, Rejaw wheat variety was used to investigate the changes of protein spots in two different ranges of linear pH 4 to 7 and nonlinear pH 3 to 10. For this purpose, bread wheat seed sample of Rejaw cultivar obtained from the Dryland Agricultural Research Institute (Sararoud branch), Kermanshah, Iran, was grown in a greenhouse under normal conditions in a pot; during the experiment, the lighting period was 14 hours, and natural lighting and fluorescent lamps were used to adjust this light. The daily temperature was about 20 degrees Celsius and the night temperature was about 10 to 12 degrees Celsius. Finally, leaf samples were used in protein extraction to perform two-dimensional electrophoresis, and their total protein was extracted. The experiment was carried out in the greenhouse of Payam-Noor University and the protein department laboratory of the Medical Biology Research Center of Kermanshah University of Medical Sciences.

The results showed the changes of the expressed proteins in two different pH 4 to 7 linear and 3 to 10 non-linear. Although the resolution of the image and the separation of the protein spots at pH 3 to 10 were nonlinear due to the wider pH range, the movement of the proteins in two-dimensional electrophoresis showed that these molecules, in addition to the pI difference, have a solvable behavior. Attention in the conditions of SDS-PAGE and two-dimensional electrophoresis, which is not seen in the usual SDS-PAGE method. In other words, in two-dimensional electrophoresis, proteins are separated based on electric charge and molecular weight but in one-dimensional electrophoresis, they are separated based on molecular weight. For this reason, a set of proteins may have the same molecular weight.

However, this degree of protein diversity and relatively heterogeneous electrophoretic behavior of the proteins obtained from the leaves of Rejaw cultivar, which was apparently only shown in this study with two-dimensional electrophoresis, is remarkable and requires more studies in other related species. The results obtained from the two-dimensional electrophoresis study in this study can open the way for proteomic studies in the future.

Global warming has increased the earth's average temperature, and the tensions resulting from extreme heat have increased in different regions. Heat stress occurs in the form of an increase in temperature beyond the critical threshold for a certain period of time in different stages of the growth of crop plants. Therefore, the quantitative and qualitative changes in crop plants, including rice, as a result of heat stress caused by the global increase in temperature is one of the most critical concerns in many rice-growing regions of the world, including our country. Plants were adapted to heat stress by changing physiological, biochemical, and molecular metabolism. A stable photosynthetic system can reduce heat damage and increase grain yield under heat-stress conditions. The damage caused by high temperatures can be different in different rice growth stages, from germination to seedling stage, flowering, and seed ripening. The flowering and seed-filling stages are heat-sensitive stages in rice plants. Thermal damage in these stages can cause significant economic losses to rice production. In this article, an attempt has been made to review the physiological changes of rice under the influence of heat stress. The studied changes include the biochemical reaction of the plant during heat stress (electrolyte leakage, lipid peroxidation level and antioxidant enzyme activity), photosynthesis, membrane changes, plant changes to tolerate or escape from heat stress, changes in metabolism at the physiological, biochemical and molecular levels, adaptation to heat, regulating the activity of transcription factors related to heat stress response and changes in the expression pattern of genes under the influence of heat stress. Also, the relationship between heat stress and the biochemical characteristics that determine the quality of rice and the factors affecting the efficiency of rice grain quality, including the metabolic activities of sugar and starch and the gene expression of quality traits of rice under the influence of heat stress, were studied. Finally, the ability of rice breeding to deal with heat stress was reviewed. In the discussion of breeding for heat tolerance in rice, heritability and genetic diversity of traits related to heat stress tolerance, the results of studies conducted in the field of identifying QTLs for heat tolerance, the use of classical crossing to introduce varieties resistant to heat stress, induced mutation in breeding rice was evaluated for increased heat stress tolerance. The results have shown that rice can tolerate non-lethal heat stress by avoiding, escaping, or physical changes at the cellular level, such as changing the physical state of the membrane, synthesis of specialized heat shock proteins, and enhancing anti-oxidative defense. Changes in the physical state of the membrane and its compounds, the production of heat shock proteins, transcription factors, osmolytes, and increasing the level of antioxidant defense are the key processes to maintain the cellular oxidation-reduction balance in heat stress. Investigating the quality changes of rice under heat stress showed that the decrease in the level of enzymes in the synthesis of sucrose and starch causes significant damage to the yield and quality of the grain in hot weather. Also, the identification of several QTLs for heat tolerance, induction mutation, classical breeding using the crossing of a high-quality variety and a heat stress-resistant variety had favorable results in the identification and introduction of heat stress-resistant cultivars. Based on the reviewed results, it can be concluded that heat stress is an inevitable condition in rice cultivation; therefore, identifying and reviewing its physiological changes can be very important and provide appropriate breeding goals.