Betaine Aldehyde Dehydrogenase Gene Research Articles

Genome-wide identification, characterization and evolutionary analysis of betaine aldehyde dehydrogenase (BADH), mitogen-activated protein kinase (MAPK) and sodium/hydrogen exchanger (NHX) genes in maize (Zea mays) under salt stress

AbstractBetaine aldehyde dehydrogenase (BADH), mitogen-activated protein kinase (MAPK) and sodium/hydrogen exchanger (NHX) play important roles in the response to salt stress. This is the first study to identify the BADH and NHX genes in maize (Zea mays) via genome-wide analysis. The qRT‒PCR results indicated that ZmNHX was upregulated by 4.38-fold, while a significant difference was not observed in ZmBADH or ZmMAPK, with fold changes of 0.96 and 1.06, respectively, under salinity stress. Genome-wide analysis revealed 8 ZmBADH, 19 ZmMAPK and 11 ZmNHX proteins in Z. mays. Domain analysis confirmed the presence of the aldehyde dehydrogenase superfamily (ALDH-SF), protein kinase and Na_H_Exchanger domains in the ZmBADH, ZmMAPK and ZmNHX proteins, respectively. Motif analysis indicated that the phylogenetic relationships were similar to the conserved motif distributions within the clade. The Ka/Ks ratio indicated that the ZmBADH, ZmMAPK and ZmNHX genes were influenced primarily by purifying selection. This study provides comprehensive identification, characterization, and evolutionary analysis for a better understanding of the ZmBADH, ZmMAPK and ZmNHX genes in maize.

Breeding exceptionally fragrant soybeans for soy milk with strong aroma.

Knockout of the soybean (Glycine max)betaine aldehyde dehydrogenase genes GmBADH1 and GmBADH2 using CRISPR/Cas12i3 enhances the aroma of soybeans. Soy milk made from the gmbadh1/2 double mutant seeds exhibits a much stronger aroma, which consumers prefer; this mutant has potential for enhancing quality in soy-based products.

CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline

2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the betaine aldehyde dehydrogenase 2 (BADH2) gene results in the accumulation of 2AP. However, the function of BADH genes in tobacco (Nicotiana tabacum L.) remains poorly understood. In this study, we successfully obtained four betaine aldehyde dehydrogenase (BADH) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (Lycium barbarum) BADH gene (LbBADH1), so we named them NtBADH1a and NtBADH1b, respectively. The other two BADH genes were orthologues of the tomato (Solanum lycopersicum) aminoaldehyde dehydrogenase 2 (SlAMADH2) gene, and were named NtBADH2a and NtBADH2b, respectively. Expression analysis revealed that the biological functions of NtBADH1a and NtBADH1b were different from those of genes NtBADH2a and NtBADH2b. We introduced mutations into NtBADH1a, NtBADH1b, NtBADH2a and NtBADH2b in tobacco using the CRISPR/Cas9 system and identified transgenic Ntbadh mutant tobacco lines. Single mutants (Ntbadh1a, Ntbadh1b, Ntbadh2a and Ntbadh2b) and double mutants (Ntbadh1a-Ntbadh1b and Ntbadh2a-Ntbadh2b) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the Ntbadh2a-Ntbadh2b double mutant, but not from any single mutant or the Ntbadh1a-Ntbadh1b double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the Ntbadh2a-Ntbadh2b double mutant. These findings indicate that only the combined inactivation of NtBADH2a and NtBADH2b results in 2AP accumulation in tobacco, which was not related to NtBADH1.

Variability in 2-acetyl-1-pyrroline production and associated mutations in BADH2 gene in aromatic rice cultivars of Bangladesh

Aroma is a special trait and aspect of the end-use quality of rice and its principal component is 2-acetyl-1-pyrroline (2AP). Sequence variants of betaine aldehyde dehydrogenase (BADH2) gene on rice chromosome 8 are associated with fragrance, and the most prominent mutation is an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7. In this study, we analyzed sequence variations in exon 7 of BADH2 in four locally popular aromatic rice cultivars and six non-aromatic high-yielding rice varieties in Bangladesh. Sequencing and alignment analysis revealed that two aromatic rice cultivars, Kataribhog and Sakkorkhana, had an 8-bp deletion (discontinuous) and one SNP (A/T) in exon 7 of the BADH2 gene. However, deletions were absent in the other two aromatic rice cultivars (Chinigura and Ranisalut) and all high-yielding varieties (BRRI dhan28, BRRI dhan29, BRRI dhan86, BRRI dhan87, BRRI dhan92 and BINA dhan10). Gene expression analysis using specific markers also supports these findings. Our results suggest that aromaticity in local rice cultivars in Bangladesh may be regulated by differential genetic mechanisms. Further studies are needed to elucidate the genetic mechanisms and fragrant compound(s) involved in the fragrance traits of Chinigura, Ranisalut, and other locally cultivated aromatic rice varieties that are critical for the development of high-yield aromatic rice by knocking out the targeted gene(s) using CRISPR-Cas genome editing.

Improved salt-tolerance of transgenic soybean by stable over-expression of AhBADH gene from Atriplex hortensis.

The salt-tolerance of transgenic soybean cleared for environmental release was improved by stable over-expression of AhBADH gene from Atriplex hortensis, which was demonstrated through molecular analysis and field experiments. An effective strategy for increasing the productivity of major crops under salt stress conditions is the development of transgenics that harbor genes responsible for salinity tolerance. Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the biosynthesis of the osmoprotectant, glycine betaine (GB), and osmotic balance in plants, and several plants transformed with BADH gene have shown significant improvements in salt tolerance. However, very few field-tested transgenic cultivars have been reported, as most of the transgenic studies are limited to laboratory or green house experiments. In this study, we demonstrated through field experiments that AhBADH from Atriplex hortensis confers salt tolerance when transformed into soybean (Glycine max L.). AhBADH was successfully introduced into soybean by Agrobacterium mediated transformation. A total of 256 transgenic plants were obtained, out of which 47 lines showed significant enhancement of salt tolerance compared to non-transgenic control plants. Molecular analyses of the transgenic line TL2 and TL7 with the highest salt tolerance exhibited stable inheritance and expression of AhBADH in progenies with a single copy insertion. TL1, TL2 and TL7 exhibited stable enhanced salt tolerance and improved agronomic traits when subjected to 300mM NaCl treatment. Currently, the transgenic line TL2 and TL7 with stable enhanced salt tolerance, which have been cleared for environmental release, are under biosafety assessment. TL 2 and TL7 stably expressing AhBADH could then be applied in commercial breeding experiments to genetically improve salt tolerance in soybean.

A non-synonymous SNP in homolog of BADH2 gene is associated with fresh pod fragrance in dolichos bean (Lablab purpureus var. lignosus (Prain) Kumari).

Fresh pods are harvestable and marketable economic product in dolichos bean. Fresh pod fragrance is one of the 'farmers' and 'consumers' preferred traits in dolichos bean varieties. The pods with high fragrance fetch a premium price in the market. In breeding programmes, pod fragrance is routinely assessed by organoleptic (sensory) means, which is highly relative and subjective. Identification of linked DNA markers not only offer an objective means but also enable selection of fragment genotypes at seedling stage itself. Betaine aldehyde dehydrogenase (BADH) is known to be the key gene responsible for fragrance in other legumes such as vegetable soybean and mung bean. Taking cues from highly conserved domains in proteins coded by BADH genes, we isolated dolichos bean homolog (LpBADH2) of soybean GmBADH2 gene using reported degenerate primers designed to conserved domains. Analysis of the translated amino acid sequence of LpBADH2 showed high degree of similarity (97.30%) with those of soybean homolog (GmBADH2). Conserved amino acid sequence of aldehyde dehydrogenase-super family were also identified in LpBADH2. Multiple sequence alignment of nucleotide sequences of LpBADH2 with those of related legumes using "ClustalW" revealed the presence of a single non-synonymous single nucleotide polymorphic (SNPs) and three synonymous SNP sites in LpBADH2. The substitution of the amino acid tyrosine in (fragrant genotypes) with phenyl alanine (non-fragrant genotypes) in protein coded by LpBADH2 appeared to be the cause for switch over from fragrance to non-fragrance in dolichos bean. These results are discussed in relation to strategies to breed dolichos bean cultivars with desired level of pod fragrance.

Screening for aroma in rice genotypes with African and Asian backgrounds

AbstractAromatic rice is highly preferred by consumers and has an important market worldwide. The demand for aromatic rice is high in East African countries, mainly in urban areas. Among the rice varieties that are grown in Burundi, few of them have a slight aroma which is not enough to satisfy the consumers’ demand and preference. There is no published report on the status of aroma in locally grown varieties and breeding lines in Burundi yet. This study aimed to identify aromatic genotypes that can be used in the breeding program to meet preferences of consumers. Two hundred fifteen genotypes were subjected to kompetitive allele‐specific PCR genotyping and sensory evaluation methods. Fifty‐three genotypes had the betaine aldehyde dehydrogenase (BADH2) gene for fragrance and displayed different levels of aroma expression. The sensory evaluation revealed two strongly aromatic genotypes, 43 moderately aromatic genotypes, 131 slightly aromatic genotypes, and 32 nonaromatic genotypes.The results of this study showed that BADH2 gene is significantly associated with rice aroma in accordance with previous reports. Furthermore, aroma and taste were positively and significantly correlated. Strongly aromatic genotypes (SUPA KATRIN and SUPA DE NYANZA –LAC) having the BADH2 gene for fragrance are of great interest for use in the breeding program to improve rice varieties to meet consumers' preferences in Burundi.

Development of Two Functional Markers of <i>Badh2</i> Gene in Guangxi Fragrant Rice

Fragrance in rice is one of the most important quality traits, which resulted from the loss of function of betaine aldehyde dehydrogenase ( Badh2 ) gene on chromosome 8. The mutation of  Badh2  leads to accumulation of 2-acetyl-1-pyrroline (2-AP), which is known as the main volatile in fragrant rice. At least 18 allelic variations have been identified in  Badh2  genes in rice. Marker assisted selection has proved to be an effective way of fragrant rice breeding. Traditional marker detection methods, such as Sanger sequencing or SSR molecular marker, are found to be low efficient. To develop a more dynamic method, we adopt Real-time PCR method to detect the common mutation site in Guangxi. In this study,  Badh2  gene of 40 fragrant rice accessions collected from Guangxi province was sequenced. Most of the fragrant rice accessions belonged to 806 bp deletion between exon 4-5 ( badh2-E4-5.1 ) and 8 bp deletion in exon 7 ( badh2-E7 ). Two Real-time PCR SNP molecular markers were developed, and were used to verify the 40 sequenced fragrant rice accessions. 93.75% of the detection results using Real-time PCR were consistent with the results of Sanger sequencing. Further, 50 local varieties were examined by Real-time PCR. A total of 24 accessions carry  badh-E4-5  allele and 22 accessions detected with  badh-E7 . The two functional SNP molecular markers common in Guangxi fragrant rice were developed and proved to be useful in rice breeding. These functional markers will improve the efficiency of fragrant rice breeding.

Betaine Aldehyde Dehydrogenase (BADH) vs. Flavodoxin (Fld): Two Important Genes for Enhancing Plants Stress Tolerance and Productivity.

Abiotic stresses, mainly salinity and drought, are the most important environmental threats that constrain worldwide food security by hampering plant growth and productivity. Plants cope with the adverse effects of these stresses by implementing a series of morpho-physio-biochemical adaptation mechanisms. Accumulating effective osmo-protectants, such as proline and glycine betaine (GB), is one of the important plant stress tolerance strategies. These osmolytes can trigger plant stress tolerance mechanisms, which include stress signal transduction, activating resistance genes, increasing levels of enzymatic and non-enzymatic antioxidants, protecting cell osmotic pressure, enhancing cell membrane integrity, as well as protecting their photosynthetic apparatus, especially the photosystem II (PSII) complex. Genetic engineering, as one of the most important plant biotechnology methods, helps to expedite the development of stress-tolerant plants by introducing the key tolerance genes involved in the biosynthetic pathways of osmolytes into plants. Betaine aldehyde dehydrogenase (BADH) is one of the important genes involved in the biosynthetic pathway of GB, and its introduction has led to an increased tolerance to a variety of abiotic stresses in different plant species. Replacing down-regulated ferredoxin at the acceptor side of photosystem I (PSI) with its isofunctional counterpart electron carrier (flavodoxin) is another applicable strategy to strengthen the photosynthetic apparatus of plants under stressful conditions. Heterologous expression of microbially-sourced flavodoxin (Fld) in higher plants compensates for the deficiency of ferredoxin expression and enhances their stress tolerance. BADH and Fld are multifunctional transgenes that increase the stress tolerance of different plant species and maintain their production under stressful situations by protecting and enhancing their photosynthetic apparatus. In addition to increasing stress tolerance, both BADH and Fld genes can improve the productivity, symbiotic performance, and longevity of plants. Because of the multigenic and complex nature of abiotic stresses, the concomitant delivery of BADH and Fld transgenes can lead to more satisfying results in desired plants, as these two genes enhance plant stress tolerance through different mechanisms, and their cumulative effect can be much more beneficial than their individual ones. The importance of BADH and Fld genes in enhancing plant productivity under stress conditions has been discussed in detail in the present review.

Molecular characterization of glycine betaine biosynthesis genes from deep sea halophilic bacteria, Bacillus atrophaeus NIOT-DSB21

Glycine betaine (N, N, N-trimethylglycine) is an effective compatible solute, which maintains fluidity of membranes and protects the biological structure of the organisms under stress. In this study, betaine aldehyde dehydrogenase ( GbsA ) and betaine alcohol dehydrogenase ( GbsB ) genes encoding for glycine betaine biosynthesis were PCR amplified from genomic DNA of Bacillus atrophaeus NIOT-DSB21 isolated from deep sea sediment of the active volcanic Barren Island, Andaman and Nicobar Islands. The amplified genes were cloned and nucleotide sequences were determined. The sequencing results showed that GbsA and GbsB genes contain ORF of 1473 bp and 1209 bp long, encoding 490 and 402 amino acids respectively (GenBank accession nos. MW045210 and MW045211). In silico sequence analysis revealed that the GbsA and GbsB sequences of B. atrophaeus were conserved in many eubacteria.

Drought tolerance in high-generation transgenic maize inbred lines overexpressing the betaine aldehyde dehydrogenase gene

Abiotic stress such as drought seriously endangers maize growth and production. Glycine betaine (GB) accumulation in transgenic crops following heterologous overexpression of the BADH gene has been shown to dramatically improve the tolerance to salt, cold, and oxidative stresses, but it is unclear if GB accumulation leads to improved drought tolerance of maize. In this study, we analyzed the drought tolerance of high-generation BADH transgenic maize inbred lines at different growth stages using the hyperosmotic solution and water-withholding methods. Molecular detection revealed that exogenous BADH was successfully introduced into the maize plant genome and overexpressed in three transgenic maize inbred lines. Under osmotic stress, transgenic maize held better germination ability than the unmodified Dan988 (WT) line. In addition, transgenic maize contained higher levels of antioxidant enzymes and osmotic regulatory substances compared with WT, and thus accumulated less harmful substances and this alleviated the negative effects of drought. We used the membership function method to quantify the drought tolerance and found that Dan988-BADH-4 showed the best tolerance, followed by Dan988-BADH-2 and Dan988-BADH-1 while WT ranked lowest. This was consistent with the statistical analysis of the experiments. From the indoor and field survey, we observed that the agronomic traits of transgenic maize were not affected by the overexpression of BADH. In conclusion, BADH overexpression in maize is beneficial for drought tolerance and the three transgenic maize lines can be used for further breeding experiments.

Molecular Characterization of Betaine Aldehyde Dehydrogenase (BADH) Gene and Proline Estimation in Hordeum vulgare L. in Response to Abiotic Stress

Barley possesses the highest level of salt tolerance among cereals, thus it is used as an important species to investigate mechanisms involved in salt tolerance. The present investigation was conducted to study the effect of salt stress by different NaCl concentrations (0, 9000, 12000, and 15000 ppm) on proline production and it was found that there is a positive correlation between proline production and salinity. Also study the effect of salinity on BADH-1 gene expression by Real-Time PCR,where this gene was performed and it was aligned in GenBank. The sequencing was translated into amino acid sequences and these amino acid sequences were aligned in GenBank. Finally, the secondary structure of BADH-1 enzyme was determined from the most tolerant barley (Giza 2000 and Wadi Sedr) and sensitive barley Giza 129 cultivars, then sequencing translated into amino acid sequences and these sequences were aligned in GenBank. Finally, the secondary structure of BADH-1 enzyme was determined. These isolated genes were submitted in GenBank under accession numbers KX433169, KX342849, KX342850, KX342851, KX433170, and KX433171.

Sequential expression of key genes in proline, glycine betaine and artemisinin biosynthesis of Artemisia aucheri Boiss using salicylic acid under in vitro osmotic stress

Artemisia aucheri belongs to the Asteraceae family, which is known for its medicinal and aromatic properties. The present study was conducted to elucidate the influence of salicylic acid (SA) pretreatment on proline and glycine betaine (GB) metabolism as well as artemisinin biosynthesis in A. aucheri under in vitro osmotic stress (˗0.6 MPa) induced by polyethylene glycol (PEG). The expression of ∆1-pyrroline-5-carboxylate synthetase (P5CS) and betaine aldehyde dehydrogenase (BADH) genes and related enzymes were enhanced under osmotic stress. SA pretreatment improved accumulation of proline and GB and increased the expression of BADH and P5CS enzyme activity under osmotic stress. However, proline dehydrogenase (PDH) gene expression and PDH activity were not affected by osmotic stress and SA pretreatments. In addition, SA in combination with or without PEG induced expression of the key genes involved in artemisinin biosynthesis and increased artemisinin content. These findings indicated that, higher accumulation of proline and GB under osmotic stress by SA is possibly due to increasing of osmoprotectants biosynthesis. This study suggested that A. aucheri is one of the potential species for artemisinin biosynthesis under environmental stress and exogenous SA.

Development and application of two novel functional molecular markers of BADH2 in rice

Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase ( BADH2 ) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 ( badh2-E2 ) or an 8-bp deletion and 3-bp variation in exon 7 ( badh2-E7 ). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2-E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F 2 population. Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future. How to cite: Li W, Zeng X, Li S, et al. Development and application of two novel functional molecular markers of BADH2 in rice. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.04.004 .

Salt tolerance of potato genetically engineered with the Atriplex canescens BADH gene

Potato (Solanum tuberosum L.) is among the top staple foods in the world, and salinity adversely affects its yield and quality. To improve salt tolerance in potato, the present study is focused on the Agrobacterium-mediated transformation of potato by the Atriplex canescens betaine aldehyde dehydrogenase (BADH) gene driven by single, double, and triple CaMV 35S promoters. The study led first to the detection of seven lines containing the BADH gene followed by the identification of T-DNA insertions via DNA hybridization and enzyme-linked immunosorbent assays. The salt tolerance was found to be promoter dependent, as the lines with triple promoters showed a higher resistance than those tranformed with single and double promoters. The transgenic lines showed lower content of H2O2 and malondialdehyde and a lower relative electrical conductivity than wild-type plants. Furthermore, these lines also showed higher proline and chlorophyll content. In silico analysis confirmed that the A. canescens BADH protein had a remarkable tendency to interact with sodium ions and water molecules like other BADH proteins. Taken together, the overexpression of BADH under triple CaMV 35S promoters enhanced salt tolerance of potato.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5′-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

Transformation of wheat Triticum aestivum with the HvBADH1 transgene from hulless barley improves salinity-stress tolerance

Studies have shown that the stress tolerance of cereal plants to osmotic or salinity stresses can be improved to varying degrees by the overexpression of an introduced betaine aldehyde dehydrogenase (BADH) gene. In the present study, the HvBADH1 gene from Hordeum vulgare L. var. nudum Hook. f., encoding a cytosolic BADH, was transferred into Triticum aestivum via traditional Agrobacterium tumefaciens-mediated transformation. Molecular methods, such as PCR, Southern blot analysis, and real-time quantitative RT-PCR were used to identify the successful integration and expression of the HvBADH1 transgene in genetically transformed wheat lines. To detect the efficacy of the HvBADH1 transgene in the transformants, some pivotal physiological indicators that reflected abiotic stress tolerance were measured in individual transgenic plant lines. These indicators included intracellular K+ and Na+ contents or K+/Na+ ratio, relative conductivity, and malondialdehyde and glycine betaine (GB) concentrations in cells. The results revealed that all the tested transgenic lines could significantly increase the recruitments of K+ in their cytosol than the wild-type seedlings. Similarly, 11.59- to 21.82-fold greater accumulation of GB, 2.11–2.56 times higher calli relative growth rates, and 26.2–29.1% seedling survival rates were found in transgenic lines under 150 mM NaCl stressed conditions. Our results demonstrated that by overexpressing the HvBADH1 transgene in genetically transformed wheat, the overall salt tolerance of the target plants was significantly increased, and the damaging effects of high salinity were significantly reduced.

Overexpression of TaBADH increases salt tolerance in Arabidopsis

Soil salinization is an important threat to wheat growth and production. Previous transcriptome analysis showed that the expression of the betaine aldehyde dehydrogenase (BADH) gene differed significantly between cultivars with strong or weak salinity tolerance. Herein, the BADH gene from the wheat cultivar Dongnongdongmai 1 was cloned and transformed into wild-type Arabidopsis to identify its function in salt tolerance. Root length was calculated at 0, 50, 100, 150, and 200 mmol L−1 NaCl for 7 d. The relative electrolytic leakage (REL), GB content, and BADH activity were measured at 150 mmol L−1 NaCl for 1 and 3 d. It was determined that BADH activity and the GB content of TaBADH-overexpressed transgenic (TaBADHOE) lines were significantly higher than in wild-type lines. Salt stress analysis showed that the root length of TaBADHOE lines 4, 18, and 19 were 0.44, 0.54, and 0.35 cm, respectively, which were significantly longer than the 0.24 cm roots of the wild-type line in the media containing 150 mmol L−1 NaCl for 7 d. In addition, the RELs of transgenic lines 4, 18, and 19 were 0.37, 0.33, and 0.42, respectively, which is significantly lower than the 0.63 of the wild-type line in media containing 150 mmol L−1 NaCl for 3 d. These results demonstrate that TaBADH significantly increased plant salt tolerance, indicating that genetic transformation of TaBADH may be an effective and sustainable breeding method for increasing salt tolerance in wheat cultivars.

Agrobacterium-mediated genetic transformation of ajowan (Trachyspermum ammi (L.) Sprague): an important industrial medicinal plant

Biotechnology-based breeding methods can improve useful industrial medicinal plants in a faster way. Tissue culture-based and in planta Agrobacterium-mediated gene transformation methods were applied to enhance drought and salinity tolerance in ajowan medicinal plant using betaine aldehyde dehydrogenase (BADH) gene. The pBI121 binary vector harboring kanamycin resistance gene (nptII) and BADH gene in its T-DNA region was applied to transfer BADH gene. Different concentrations of the selective antibiotic were tested on 15 day-old hypocotyl segments and 75 mg/L kanamycin was identified as the minimum inhibitory concentration to screen for transformed cells of ajowan. The different parameters of gene transformation including Agrobacterium optical density (OD), Agrobacterium strain, Agrobacterium killing antibiotic, acetosyringone concentration, and inoculation duration were tested during tissue culture-based Agrobacterium-mediated transformation. The OD600 = 0.6-0.8 of LB4404 strain of Agrobacterium × 160 mg/L timentin × 250 μmol/L acetosyringone × 30 min inoculation duration were identified as the best gene transformation parameters. Successful integration and expression of transferred gene were confirmed in T0 plants using polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) methods. Five transgenic lines with 2.94% transformation efficiency were achieved by regular tissue culture-based Agrobacterium-mediated gene transformation method, whereas the dip flora in planta transformation method has led to one transgenic line with 1.42% transformation efficiency. Bioassay analysis revealed enhanced drought and salinity tolerance in transformed plants in comparison with non-transformed plants. The morphological characteristics of plant height and seedling fresh weight of BADH-transformed plants were improved under salinity stress. Under drought stress, the proline content and relative water content of transgenic plants were more than non-transformants. The results of gas chromatography mass spectrometry (GC–MS) revealed significant increase in thymol content of BADH-transformed plants (55.07%) compared with non-transformed plants (39.20%) under drought stress condition. The obtained transgenic plants are cultivable in unfavorable environments to give an acceptable level of useful secondary metabolites. The optimized protocol is applicable for gene transformation of other medicinal plants of Apiacea family for different purposes.

Betaine Aldehyde Dehydrogenase (BADH) gene and free amino acid analysis in Rhizophora mucronata Lam. from Thalassery region of Kerala, India.

Mangroves are salt tolerant plants inhabiting saline environment. Multiple factors contribute to their salt tolerance and we need multifaceted approach to reveal the mechanisms of salt tolerance in the plant. In the present study, leaves of the mangrove, Rhizophora mucronata grown in the presence and absence of salt were used, free amino acids and the expression of selected genes were analyzed. Chromatographic technique showed the accumulation of free amino acids like proline, glycine, aspartic acid, valine, leucine and glutamic acid in the presence of salt. RNA was isolated from the leaf sample and cDNA was synthesized. Gene specific primers were designed and standardized. Among the genes studied (P5CS, BADH, NHX1), Betaine Aldehyde dehydrogenase (BADH) gene was found to be expressed.