Genetic transformation of Indian bread (T. aestivum) and pasta (T. durum) wheat by particle bombardment of mature embryo-derived calli

BACKGROUND: Particle bombardment has been successfully employed for obtaining transgenics in cereals in general and wheat in particular. Most of these procedures employ immature embryos which are not available throughout the year. The present investigation utilizes mature seeds as the starting material and the calli raised from the hexaploid Triticum aestivum and tetraploid Triticum durum display a high regeneration response and were therefore used as the target tissue for genetic transformation by the biolistic approach. RESULTS: Mature embryo-derived calli of bread wheat (Triticum aestivum, cv. CPAN1676) and durum wheat (T. durum, cv. PDW215) were double bombarded with 1.1 gold microprojectiles coated with pDM302 and pAct1-F at a target distance of 6 cm. Southern analysis using the bar gene as a probe revealed the integration of transgenes in the T0 transformants. The bar gene was active in both T0 and T1 generations as evidenced by phosphinothricin leaf paint assay. Approximately 30% and 33% primary transformants of T. aestivum and T. durum, respectively, were fertile. The transmission of bar gene to T1 progeny was demonstrated by PCR analysis of germinated seedlings with primers specific to the bar gene. CONCLUSIONS: The transformation frequency obtained was 8.56% with T. aestivum and 10% with T. durum. The optimized protocol was subsequently used for the introduction of the barley gene encoding a late embryogenesis abundant protein (HVA1) in T. aestivum and T. durum. The presence of the HVA1 transgene was confirmed by Southern analysis in the T0 generation in case of Triticum aestivum, and T0 and T1 generation in Triticum durum

Structural Characterization and Expression Analysis of the SERK/SERL Gene Family in Rice (Oryza sativa)

Somatic embryogenesis (SE) is the developmental restructuring of somatic cells towards the embryogenic pathway and forms the basis of cellular totipotency in angiosperms. With the availability of full-length cDNA sequences from Knowledge-based Oryza Molecular Biological Encylopedia (KOME), we identified the leucine-rich repeat receptor-like kinase (LRR-RLK) genes from rice (Oryza sativa), which also encompasses genes involved in regulating somatic embryogenesis. Eight out of eleven of the rice SERK and SERL (SERK-like) genes have the TIGR annotation as (putative) brassinosteroid insensitive 1-associated receptor kinase (precursor). Real-time polymerase chain reaction analysis was undertaken to quantify transcript levels of these 11 genes. Most of these genes were upregulated by brassinosteroids although only a few of these displayed auxin induction. The expression profile of these genes is nearly uniform in the zygotic embryogenic tissue, but the expression pattern is more complex in the somatic embryogenic tissue. It is likely that OsSERKs and OsSERLs may be involved in somatic embryogenesis and also perform a role in morphogenesis and various other plant developmental processes. Functional validation of these somatic embryogenesis receptor-like kinase genes may help in elucidating their precise functions in regulating various facets of plant development

Characterization of three somatic embryogenesis receptor kinase genes from wheat, Triticum aestivum

We report here the isolation and characterization of three SOMATIC EMBRYOGENESIS RECEPTOR KINASE (TaSERK) genes from wheat. TaSERKs belong to a small family of receptor-like kinase genes, share a conserved structure and extensive sequence homology with previously reported plant SERK genes. TaSERK genes are in general auxin inducible and expressed during embryogenesis in cell cultures. We show here that somatic embryogenesis in Triticum aestivum is associated with high SERK expression which could be enhanced with auxin application and is calcium dependent. TaSERK transcripts could also be enhanced by epibrassinolide and abscisic acid. TaSERK1 and TaSERK2 may have a role in somatic embryogenesis, whereas TaSERK3 appears to be a brassinosteroid-associated kinase (BAK) lacking an SPP motif but shares a characteristic C-terminal domain with other SERK proteins. Also, the transcripts of all the three TaSERK genes could be induced in zygotic and somatic tissues. Although our analysis suggests them to be involved in somatic embryogenesis, they may have a broader role in acquiring embryogenic competence in wheat

An early auxin-responsive Aux/IAA gene from wheat (Triticum aestivum) is induced by epibrassinolide and differentially regulated by light and calcium

The plant hormone auxin plays a central role in regulating many aspects of plant growth and development. This largely occurs as a consequence of changes in gene expression. The Aux/IAA genes are best characterized among the early auxin-responsive genes, which encode short-lived transcriptional repressors. In most plants examined, including Arabidopsis, soybean, and rice, the Aux/IAA genes constitute a large gene family. By screening the available databases, at least 15 expressed sequence tags (ESTs) have been identified from wheat (Triticum aestivum), which exhibit high sequence identity with Aux/IAA homologues in other species. One of these Aux/IAA genes, TaIAA1, harbouring all the four conserved domains characteristic of the Aux/IAA proteins, has been characterized in detail. The expression of TaIAA1 is light-sensitive, tissue-specific, and is induced within 15-30 min of exogenous auxin application. Also, the TaIAA1 transcript levels increase in the presence of a divalent cation, Ca2+, and this effect is reversed by the calcium-chelating agent, EGTA. The TaIAA1 gene qualifies as the primary response gene because an increase in its transcript levels by auxin is unaffected by cycloheximide. In addition to auxin, the TaIAA1 gene is also induced by brassinosteroid, providing evidence that interplay between hormones is crucial for the regulation of plant growth and development

Carotenoid biosynthesis genes in rice: structural analysis, genome-wide expression profiling and phylogenetic analysis

Carotenoids, important lipid-soluble antioxidants in photosynthetic tissues, are known to be completely absent in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthesis genes, have been performed to get carotenoid-enriched rice grain. Study of genes involved in their biosynthesis can provide further information regarding the abundance/absence of carotenoids in different tissues. We have identified 16 and 34 carotenoid biosynthesis genes in rice and Populus genomes, respectively. A detailed analysis of the domain structure of carotenoid biosynthesis enzymes in rice, Populus and Arabidopsis has shown that highly conserved catalytic domains, along with other domains, are present in these proteins. Phylogenetic analysis of rice genes with Arabidopsis and other characterized carotenoid biosynthesis genes has revealed that homologous genes exist in these plants, and the duplicated gene copies probably adopt new functions. Expression of rice and Populus genes has been analyzed by full-length cDNA- and EST-based expression profiling. In rice, this analysis was complemented by real-time PCR, microarray and signature-based expression profiling, which reveal that carotenoid biosynthesis genes are highly expressed in light-grown tissues, have differential expression pattern during vegetative/reproductive development and are responsive to stress

Somatic embryogenesis from mature caryopsis culture under abiotic stress and optimization of Agrobacterium-mediated transient GUS gene expression in embryogenic callus of rice (Oryza sativa L.)

Induction and development of embryogenic callus from mature caryopsis culture of rice (Oryza sativa cv. ADT41) was performed by placing sterilized seeds on MS medium supplemented with 2,4-dichlorophenoxyacetic acid or 2,4-D (2.5, 5.0 and 10 mg/l). Morphogenesis in terms of somatic embryogenesis was recorded and 53% embryogenic callus formed by caryopsis culture indicates high chance of regenerating plants eventually. Furthermore, for water stress treatments, sterilized caryopses were cultured on semisolid MS medium supplemented with (5.0mg/l) of 2, 4-D and various concentrations of mannitol (2.5M, 5M and 10M) were added. Present study indicates that potentials of tissues for callus induction and embryo differentiation gradually decline if the level of mannitol is being increased in the nutrient media. Significantly, at high level of mannitol (5M), direct somatic embryo differentiation from the epicotyl tissues was also evident along with embryogenic callus formation at the basal region of the seedling. Similarly for salt stress treatments, MS medium was supplemented with various concentrations of NaCl (50 mM, 100 mM and 250 mM) along with 2, 4-D (5.0 mg/l). Results obtained on salt–stress treatments indicate that cultured rice cells may respond variously depending on the concentration of salt-stress present in the culture medium. Also, transformation experiments were conducted to optimize the transient GUS gene expression in mature caryopsis derived embryogenic callus by employing the various strains of Agrobacterium tumefaciens carrying the same plasmids or others. Calli were co-cultivated with Agrobacterium strains GV2260 (p35SGUSINT), LBA4404 (p35SGUSINT) and LBA4404 (pCAMBIA 3301) in the presence of acetosyringone (200 μM). Transformation events were best recorded in calli treated with Agrobacterium strain LBA4404 harbouring the plasmid p35SGUSINT, as evidenced by maximum frequency (29%) of transient GUS gene expression on histochemical assay and it was followed by strain GV2260 (p35SGUSINT), however, Agrobacterium strain LBA4404 (pCAMBIA3301) could be proved the least effective in terms of frequency of transient transformation and expression of GUS reporter gene in target tissues

Reconstructing an ancestral genotype of two hexachlorocyclohexane-degrading Sphingobium species using metagenomic sequence data.

Over the last 60 years, the use of hexachlorocyclohexane (HCH) as a pesticide has resulted in the production of >4 million tons of HCH waste, which has been dumped in open sinks across the globe. Here, the combination of the genomes of two genetic subspecies (Sphingobium japonicum UT26 and Sphingobium indicum B90A; isolated from two discrete geographical locations, Japan and India, respectively) capable of degrading HCH, with metagenomic data from an HCH dumpsite (∼450 mg HCH per g soil), enabled the reconstruction and validation of the last-common ancestor (LCA) genotype. Mapping the LCA genotype (3128 genes) to the subspecies genomes demonstrated that >20% of the genes in each subspecies were absent in the LCA. This includes two enzymes from the 'upper' HCH degradation pathway, suggesting that the ancestor was unable to degrade HCH isomers, but descendants acquired lin genes by transposon-mediated lateral gene transfer. In addition, anthranilate and homogentisate degradation traits were found to be strain (selectively retained only by UT26) and environment (absent in the LCA and subspecies, but prevalent in the metagenome) specific, respectively. One draft secondary chromosome, two near complete plasmids and eight complete lin transposons were assembled from the metagenomic DNA. Collectively, these results reinforce the elastic nature of the genus Sphingobium, and describe the evolutionary acquisition mechanism of a xenobiotic degradation phenotype in response to environmental pollution. This also demonstrates for the first time the use of metagenomic data in ancestral genotype reconstruction, highlighting its potential to provide significant insight into the development of such phenotypes

A Novel approach for Agrobacterium-mediated germ line transformation of Indian Bread wheat (Triticum aestivum) and Pasta wheat (Triticum durum)

Recalcitrance of wheat towards tissue culture procedures has hampered the wide use of conventional transformation techniques for its improvement. In the present study, a novel, non-tissue culture, cost effective approach has been established for the introduction of transgenes in wheat. Dry, mature seeds of two Indian varieties of wheat, Triticum aestivum cv. HD2329 (bread wheat), and Triticum durum cv. PDW215 (pasta wheat), were co-cultivated with Agrobacterium strain GV2260 (p35SGUSINT) and LBA4404 (pCAMBIA 3301), respectively, in the presence of 200 μM acetosyringone. The plantlets testing gus positive were raised till maturity in garden pots. T0 lines were screened by PCR for presence of selectable markers in the transformed plants followed by confirmation with Southern hybridization. In bread wheat, nptII was detected in five primary transformed lines (T0) (ws1, ws2, ws3, ws4, ws5) and the bar gene in three putatively transformed durum wheat lines (wsb1, wsb2, wsb3). The transformation efficiency was calculated as 1.16%, and 0.84% for T. aestivum and T. durum, respectively.Â