JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2020 | Volume:12 | Issue:2|Papers Count:10

Objective Roses are one of the most important cut flowers in the world and therefore many studies have focused on keeping post-harvest quality and longevity. Ethylene is one of the major factors that limits post-harvest longevity. Therefore, genetic manipulation has been considered to reduce the adverse effects of this hormone and the present research investigates transgenic roses containing the mutant etr1-1 gene. In addition, according to antagonist effects, the change of gibberellin concentration to ethylene in favor of higher gibberellin concentration in the present research has been studied. Materials and methods The content of phytohormones in long-lasting line (transgenic) and control (nontransgenic) after application of ethylene (0 and 1 μ L L-1) and gibberellin (80 mg L-1) treatments were investigated in bud and half-open stages. Application of ethylene treatment was performed by injecting pure gas into polyethylene bags by syringe. For the application of gibberellin treatment, cut flowers were placed in gibberellin solution at the desired concentration. The treatments were applied for 24 hours and samples were taken from the outermost ring of petals. Experiments were conducted as factorial in a completely randomized design with four replications in order to measure phytohormones. Results The results showed a decrease in the amount of gibberellin, benzyl adenine and indole acetic acid hormones and an increase in the amount of abscisic acid and ethylene hormones in bud and half-open stages in long-lasting line were significant compared to the control. In addition, the highest content of gibberellin, indole acetic acid and benzyl adenine hormones and the lowest content of ethylene and abscisic acid belongs to the GA3-treated long-lasting line. Conclusion Therefore, etr1-1 gene appears to be the right candidate for delaying ethylene-dependent aging in sensitive flowers and with GA3 treatment could be considerably improved longevity by reduction of oxidative damage.

Objective Plant diseases can be a limiting factor in planting in an area. One of the new methods of producing disease resistant plants is the use of molecular markers. Molecular markers are able to detect and analyze important genes of resistance. Therefore, due to the severity of fire blight, the apple tree germplasm evaluation is necessary, so this research was carried out with the aim of determining resistance genes in some apple genotypes in Isfahan province using SCAR and SSR markers. Materials and methods Fresh and young leaves of 70 samples of apple trees in Isfahan were collected in early April and DNA was extracted by CTAB method. Then polymerase chain reaction was performed and DNA fragment was detected and DNAs lengths were measured. Results Results indicated that in all populations, none of the primers have a rare band or a typical band replication in 25 or less than 25% population, and 50 or less than 50% population. Semirom-Hana population had the greatest genetic diversity (Nei diversity index, Shannon, effective and different number of alleles) which shows greater genetic diversity in this population rather than other populations. Molecular analysis indicated that diversity among populations is not statistically significant and 92% of the diversity is related to diversity within the populations. The longest distance was between Semirom-Hana and Semirom-Padena populations, and in this disease, the distance between the farthest and closest populations is too low and this result was confirmed by analysis of molecular variance. Conclusion The six primers, which were used in this study, have been well produced and display the distinctions in population. Moreover, results indicate that inter-population diversity was very low but by the cross of the farthest and closest populations, then the cross of progeny can pyramidize the target genes and produce a stronger and more diverse recombination.

Objective This experiment was conducted in order to assess genetic variation, determine of the best genetic structure and association analysis of safflower to identify markers associated with different agronomic traits. Materials and methods In this study, 17 genotypes of safflower were planted as the randomised complete block design at research farm of Shahid Bahonar University of Kerman in 2016. Traits including seed yield, plant height, number of capitulum per plant, number of seed per capitulum, 1000-seed weight, capitulum diameter, day to 50% flowering and day to maturity were measured. The AFLP Technique was performed by the eight EcoR1/Mse1 primer combinations. Results In total, 147 polymorphic bands were generated with average 81. 58 polymorphic percentage. Cluster analysis using UPGMA and Jacard as similarity index discriminated safflower genotypes into two groups. Based on GLM and MLM model, 45 and 39 markers had significant association with the studied traits, respectively. M14/E6-10, M14/E11-16, M14/E11-13, M3/E10-14 and M4/E36-12 markers with seed yield, M3/E10-12, M3/E36-29, M59/E36-21 and M14/E11-10 with capitulum number per plant, M4/E36-8, M59/E36-21, M3/E10-9, M14/E11-14 and M14/E11-13 with number of seed per capitulum, M4/E36-19, M4/E36-12, M14/E11-1 and M4/E10-1 with 1000-seed weight, M4/E10-2, M59/E36-21, M3/E36-30, M4/E10-11 and M4/E36-24 with plant height, M3/E36-24, M3/E36-6, M3/E10-20 and M4/E36-18 with capitulum diameter, M14/E11-10, M3/E36-30 and M59/E36-21 with day to 50% flowering, M14/E11-10 and M59/E36-22 markers with day to maturity in both models. Conclusions Detected AFLP markers with strong effects in this study could be desirable candidates for the marker-assisted selection in breeding programs and conversion into other specific markers.

Objective Glucoraphanine is an aliphatic glucosinolate, which largly found in Lepidium draba from Brassicaceae family. This glucosinolate convert to isothiocyanate, sulforaphane (SFN) in the presence of myrosinase which has different biological activities such as the antioxidant and anti bacterial properties, and also able to prevent proliferating of cancer cells. CYP79F1 is the first enzyme in the biosynthesis pathway of glucoraphanine. Materials and Methods In this study, L. draba seedlings wer grown for 7 days in the presence of different concentration of sucrose and chitosan (0, 25, 50, 100, 200 and 400 mg/L), in fullyrandom designs with three repetitions. After collection of the seedlings, SFN content in the arial parts of them were measured using HPLC apparatus. Furthermore, in the treated seedlings, the gene expression level of CYP79F1 were examined using the Real Time PCR technique. Results The results showed, that SFN content in the sucrose treated seedlings significantly increased compared to the control. While in the chitosan treated seedlings significant increase in SFN content was only seen at the 200 mg/L concentration. The results showed that the expression of CYP79 F1 was significantly increased in the treated seedlings with 50 mg/L sucrose and 50 and 100 mg/L chitosan compared to the control. Conclusions According to the results, it concluded that the mentioned elicitor's concentrations can stimulate aliphatic glucosinolate biosynthesis through expression of CYP79F1 gene. Based on the role of SFN as a valuable medicinal metabolit and also the role of CYP79F1 enzyme in the glucoraphanin biosynthesis, it suggested that the effects of others concentrations and time of treatment of the mentioned elicitors were analyzed on this plant.

Objective Lettuce is considered as a model plant for biotechnology because of its compatibility with stable genetic transformation and tissue culture. GDP-mannose-3’ , 5’-epimerase (GME) is one of the key genes in ascorbic acid biosynthesis pathway in plants. The present study aims to transfer GME gene from Actinidia deliciosa L. into Lactuca sativa L. Materials and Methods To investigate callus induction rate using the effects of explant (cotyledon and true leaves) and 16 plant growth regulator combination including concentrations of 0. 02, 0. 04, 0. 05, and 0. 1 mg/l NAA and 0. 1, 0. 2, 0. 4, and 0. 6 mg/l BAP, and also direct regeneration rate using the effects of explant (cotyledon and true leaves) and 6 plant growth regulator combination including concentrations of 0. 02 and 0. 05 mg/l NAA and 0. 2, 0. 4, and 0. 6 mg/l BAP, a factorial experiment based on completely randomized design with three replications was conducted. In order to transform GME into Lactuca sativa L. using L. sativa L. cv. Setareh and Agrobacterium tumefaciens strain (C58) on two types of explants (cotyledon and true leaves), a factorial experiment with three replications and 2 min and 8 min inoculation was done. Results The results revealed that the highest percentage of callus induction and indirect regeneration (100%) were observed on leaf and cotyledon explants and MS medium containing 0. 1 mg/l BAP and 0. 04 mg/l NAA. The results also confirmed the presence of pBI121+GME in transgenic plants. Conclusions The explant true leaves and 2 min inoculation (with 18 percent transformation ratio) were more suitable for transformation.

Objective Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Posttranscriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana. Material and methods To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests. Results The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 μ g. g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively. Conclusion The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

Objective Rice is a food source for more than half of the world's population and it is important as a model plant for monocots, and also its growth and yield is strongly affected by salinity stress, hence comprehensive research is essential for the development of salt tolerant varieties. In the present study, the expression pattern of several new genes involved in salt tolerance of rice was investigated by Real-Time PCR in sensitive (ARC6578 and Shoemed) and tolerant (Bombilla) rice cultivars. Materials and methods RNA samples were extracted from 20-day-old seedlings treated with NaCl (100 mM) at three times of 24, 48 and 72 h after salt stress. Gene expression analysis was performed on 7 candidate genes (including SOD, CatA, 14-3-3 like protein GF14, Proxidase BP1 precursor, Zinc ion binding protein and Plasma Membrane H+-ATPase, OsSIPK) using real-time PCR. The Actin gene was used as the reference gene. Results The expression of OsSIPK in tolerant cultivar was increased 24 and 48 h after salt stress and was decreased 72 h after stress, while the expression of Zinc ion binding protein was decreased in all cultivars at all times after stress. The expression of PM H+-ATPase at early times after stress in both tolerant and sensitive cultivars was increased but it decreased after 72 h in sensitive cultivar and increased in tolerant cultivar. The expression of 14-3-3 like protein GF14-6 was highly increased at 48 h after stress but it was decreased after 72 h in sensitive cultivars and increased in tolerant cultivar. The expression of Peroxidase BP1 precursor was increased up to 40 times at 72 h after stress in tolerant cultivar. The expression of CatA was significantly increased at 72 h after stress only in tolerant cultivar. The expression of SOD didn't show a special time pattern, although its expression in tolerant cultivar was higher than that in sensitive cultivar at 72 h after stress. Conclusions Some genes in this research did function in general response of the rice plant to salt stress via scavenging the reactive oxygen species (ROS) (such as SOD and CatA). In another hand, a time-cultivar dependent relationship between salt tolerance and expression level of other studied genes (such as OsSIPK, PM H+-ATPase, 14-3-3 like protein GF14-6 and Peroxidase BP1) could be recognized.

Objective Rice grain discoloration disease is emerging as a major threat in improved and late ripening varieties in the north of Iran that deteriorates grain quality and physical properties. Breeding for disease resistance is the preferred strategy to manage grain discoloration. An important application of molecular markers in plant systems involves improvement in the efficiency of conventional plant breeding by carrying out indirect selection through molecular markers linked to the traits of interest. In this context, association analysis was used to identify molecular markers associated with grain discoloration resistance in rice genotypes in this study. Materials and Methods In this study, resistance of 94 rice genotypes (Iranian landrace and improved varieties and exotic genotypes) to grain discoloration disease was evaluated during 2017 and 2018. Genotyping was done using 128 pairs of polymorphic microsatellite markers on 94 genotypes of rice. Then association analysis was conducted through general linear model (GLM) and mixed linear model (MLM) by using TASSEL software. Results Phenotypic analysis showed that there is high diversity in the studied genotypes for resistance to grain discoloration disease. The model-based structure analysis classified rice genotypes into two sub-populations. In association analysis based on GLM and MLM models, 12 and 3 QTLs showed significant relations after considering Bonferroni correction with grain discoloration in two years, respectively. Conclusion Both GLM and MLM detected 3 markers (RM242, RM5709 and RM5955) associated with grain discoloration resistance in both 2017 and 2018, suggesting that these markers can be used in rice breeding programs like marker-assisted selection (MAS).

Objective Genetic diversity in field crops and their wild ancestors plays important role in breeding programs. The aim of this study was to investigate the genetic diversity of einkorn (Triticum monococcum L. ssp. boeoticum) wheat genotypes collected from western parts of Iran using SSR markers. Material and methods In this study, genetic variation of 163 genotypes from 34 populations of einkorn wheat collected from western parts of Iran was investigated using 19 SSR loci developed in the A genome of hexaploid wheat. Results In the investigation of 163 einkorn genotypes using 19 microsatellite loci were generated 151 polymorphic alleles with an average of 7. 94 per locus. The content of polymorphic information content (PIC) in the studied einkorn genotypes ranged from 0. 64 for Xgwm480-3A locus to 0. 89 for Xgwm4-4A locus. The mean content of polymorphic information in the studied genotypes was 0. 77. According to the results, Xgwm4-4A, Xgwm610-4A and Xgwm282-7A loci were identified as the most suitable primers for studying genetic diversity and differentiation of einkorn wheat genotypes. The mean of Shannon coefficient of 0. 16 indicated moderate variation in genotypes under study. The average percentage of polymorphic gene loci in the 34 studied populations was 36. 74 and the average heterozygosity was 0. 114. Based on analysis of molecular variance for 34 populations, the variations between and within populations were calculated as 7% and 93%, respectively. Cluster analysis based on Jaccard coefficients and UPGMA algorithm classified the einkorn genotypes into 10 distinct groups. The results of the principal coordinate analysis revealed that the two primary vectors explained 29. 33% and 24. 01% of the total molecular genetic variance, respectively. Conclusion The results indicated the usefulness of microsatellite markers in identifying and grouping einkorn wheat genotypes, so that the obtained information could be used in breeding projects, germplasm conservation planning and collection of einkorn wheat populations.

Objective Around the world, scientists are looking for new animal protein sources and trying to prioritize the animals with the best conversion rates. One of these animals is Camel. Identification of genetic differences in sequences of Mitochondrial Genome by Bioinformatics Tools is a rapid and confident method for identification and categorizing species. This method is one of the most effective ways of evaluating genetic biodiversity and Phylogenetic relationships in different livestock like Camel. This research was done for sequencing of Ribosomal non-coding RNA gene and comparison of its genetic construction between camelus dromedaries and camelus bactrianus of Iran. Materials and Methods We were collecting blood samples from twenty camelus dromedaries and camelus bactrianus. We extracted DNA at the beginning then 12S rRNA with 1326 pair bases was amplified and sequenced by two pair specific primers. Results The Presence of 3 Haplotypes in Bactria camels and one haplotype in Dromedary were proved in the study population. These Haplotypes have two single nucleotide polymorphism (SNP) site in camelus bactrianus and there was not any Nucleotides difference among camelus dromedaries. Phylogeny Tree design with using similar mitochondrial 12S rRNA gene sequencing in other species which are in World Gene Bank. Conclusion Phylogeny Analysis and defining, Genetic diversity was showing camelus dromedaries and camelus bactrianus are placing in two different groups and Iranian camelus bactrianus have more Genetic Diversity which indicates more historical evolution of this specie. More information about Genetic diversity among the different generation of Camels can facilitate developing generations revising Programs and is a necessity for protecting from Genetic Resources.