Development and Validation of a Reversed-Phase Liquid Chromatography Method for the Simultaneous Determination of Indole-3-Acetic Acid, Indole-3-Pyruvic Acid, and Abscisic Acid in Barley (Hordeum vulgare L.)

A simple, sensitive, precise, and specific reverse HPLC method was developed and validated for the determination of plant hormones in barley (Hordeum vulgare L.). The method includes extraction in aqueous organic solvent followed by solid-phase extraction, sample evaporation, and reversed-phase HPLC analysis in a general purpose UV-visible (abscisic acid (ABA)) and fluorescence detection (indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPA)), high-performance liquid chromatography system. The separation was carried out on Zorbax Eclipse XDB C8 column (150  ×  4.6  mm I.D) with a mobile phase composed of methanol and 1% acetic acid (60 : 40 v/v) in isocratic mode at a flow rate of 1 ml min−1. The detection was monitored at 270 nm (ABA) and at 282 nm (Ex) and 360 nm (Em) (IAA, IPA). The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification, and robustness. The determined validation parameters are in the commonly acceptable ranges for that kind of analysis

Differential disease resistance response in the barley necrotic mutant nec1

<p>Abstract</p> <p>Background</p> <p>Although ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. <it>Arabidopsis thaliana </it>CNGC4 mutants <it>hlm1 </it>and <it>dnd2 </it>display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant <it>nec1 </it>comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses <it>PR-1</it>, yet it is not known what effect the <it>nec1 </it>mutation has on barley resistance against different types of pathogens.</p> <p>Results</p> <p><it>nec1 </it>mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating <it>nec1 </it>disease resistance demonstrated positive effect of <it>nec1 </it>mutation on non-host resistance against <it>Pseudomonas syringae </it>pv. <it>tomato </it>(<it>Pst</it>) at high inoculum density, whereas at normal <it>Pst </it>inoculum concentration <it>nec1 </it>resistance did not differ from wt. In contrast to augmented <it>P. syringae </it>resistance, penetration resistance against biotrophic fungus <it>Blumeria graminis </it>f. sp. <it>hordei </it>(<it>Bgh</it>), the causal agent of powdery mildew, was not altered in <it>nec1</it>. The <it>nec1 </it>mutant significantly over-expressed race non-specific <it>Bgh </it>resistance-related genes <it>BI-1 </it>and <it>MLO</it>. Induction of <it>BI-1 </it>and <it>MLO </it>suggested putative involvement of <it>nec1 </it>in race non-specific <it>Bgh </it>resistance, therefore the effect of <it>nec1</it>on <it>mlo-5</it>-mediated <it>Bgh </it>resistance was assessed. The <it>nec1</it>/<it>mlo-5 </it>double mutant was as resistant to <it>Bgh </it>as <it>Nec1</it>/<it>mlo-5 </it>plants, suggesting that <it>nec1 </it>did not impair <it>mlo-5 </it>race non-specific <it>Bgh </it>resistance.</p> <p>Conclusions</p> <p>Together, the results suggest that <it>nec1 </it>mutation alters activation of systemic acquired resistance-related physiological markers and non-host resistance in barley, while not changing rapid localized response during compatible interaction with host pathogen. Increased resistance of <it>nec1 </it>against non-host pathogen <it>Pst </it>suggests that <it>nec1 </it>mutation may affect certain aspects of barley disease resistance, while it remains to be determined, if the effect on disease resistance is a direct response to changes in SA signaling.</p

HorTILLUS - a rich and renewable source of induced mutations for forward/reverse genetics and pre-breeding programs in barley (Hordeum vulgare L.)

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platformis the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations.We offer the usage of this valuable resource to the interested barley researchers on cooperative basis

Table2.DOCX

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Table3.DOCX

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Image2.tif

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Image3.tif

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Image1.tif

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Table1.DOCX

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p