A highly efficient method for Agrobacterium mediated transformation in elite rice varieties (Oryza sativa L. spp. indica)

An Agrobacterium mediated transformation method was developed for the Thai rice variety,  Pathumthani 1 (PT1), and the Indian rice variety, Pokkali (PKL). Various aspects of the transformation method, including callus induction, callus age, Agrobacterium concentration and co-cultivation period were examined, in order to improve transformation efficiency. Optimized transformation conditions were established using Agrobacterium strain EHA105, which carries a virulent plasmid, pCAMBIA1301.  A modified Murashige and Skoog (MS) medium supplemented with 1 mg/l 2, 4-D and 0.5 mg/l picloram was optimized for callus induction. Three week old calli were used to co-cultivate with 0.8 -1 OD600  Agrobacterium for 30 min and the culture was continued on agar medium without antibiotics for 2 days. This method can be used to induce high quality calli within three weeks. Based on GUS determination, it was demonstrated that the transformation method was improved significantly, with a high level of transformation efficiency.Keywords: Agrobacterium tumefaciens, indica rice, mature seed-derived callus, rice transformation, transgenic riceAfrican Journal of Biotechnology Vol. 9(34), pp. 5488-5495, 23 August, 201

Production of Salt Tolerance Dwarf Napier Grass (Pennisetum purpureum cv. Mott) Using Tissue Culture and Gamma Irradiation

ABSTRACT As high as 95% of callus was induced when young leaves of dwarf Napier grass were cultured in MS medium supplemented with 5% coconut water and 2 mg/l 2,4-D. These calli were of highly compact type. Upon transferring these calli to grow on the plantlet induction medium, it was found that the MS medium containing 5% coconut water, 1 mg/l NAA, 0.5 mg/l BAP and 0.5 mg/l 2,4-D gave the best average plantlet production of 58.8%. Irradiated calli at the levels of 0, 10, 20, 30, 40 and 50 Gy were subject to culturing growth on the selected plantlet induction medium containing different NaCl concentrations of 0, 0.5, 1.0, 1.5 and 2.0%. The total of 108 clones could survive in the salt supplemented medium. These surviving clones were then transferred to grow in the field at National Corn and Sorghum Research Center, Nakorn Rachasima province. Twenty-one of good characteristic clones were selected from the field to be further grown at high salinity soil in Borabue district, Maha Sarakarm province and 8 clones were finally chosen as salt tolerance. DNA fingerprinting patterns of the 21 selected clones were analyzed using AFLP technique with 11 pairs of primers. The patterns were significantly different at high percentage of polymorphism (94.75%). The similarity index among these samples ranged from 0.569 to 0.784 indicating that the morphological difference among them was the result of true mutation induced by irradiation and tissue culturing

Transfer of Drought Resistant Character from Wild Rice (Oryza meridionalis and Oryza nivara) to Cultivated Rice (Oryza sativa L.) by Backcrossing and Immature Embryo Culture

ABSTRACT Drought is an abiotic stress to be continuing threat to rice production. The characteristics supporting to drought resistance are restricted in cultivars while wild species of Oryza are an important reservoir of useful genes for rice improvement. The objective of this study was to transfer drought resistant character from wild species (O. meridionalis and O. nivara) to cultivated rice varieties RD23 and CN1 by backcrossing in combination with immature embryo culture. Hybridization between cultivated rice and wild species was made to produce six interspecific hybrid crosses. Crossability between cultivated rice and wild species of all crosses varied from 21.2 to 50% with an average of 35.3%. The F 1 hybrid embryos obtained were rescued by culturing on the half-strength MS medium. Germination ability of the hybrid embryos of all crosses ranged from 36.2 to 62.1% with an average of 52.0%. The F 1 hybrid plants obtained showed low pollen fertility and relatively poor agronomic characters. They were then backcrossed to their cultivated recurrent parents to restore fertility and good agronomic performance from cultivated rice. The 299 BC 1 F 1 plants were produced from six backcrosses of which 235 plants could set BC 1 F 2 seeds. The 452 BC 1 F 2 progenies from six backcrosses were screened for drought resistance at vegetative growth stage by visual scoring of leaf rolling, leaf drying and plant recovery. Selection was made for 39 BC 1 F 2 plants resistant to drought and having high seed yield per plant and good agronomic performance. They will be grown to be BC 1 F 3 lines for further evaluation on drought resistance, yield and agronomic performance

Identification of native Dendrobium species in Thailand by PCR-RFLP of rDNA-ITS and chloroplast DNA

ABSTRACT: The PCR-restriction fragment length polymorphism (PCR-RFLP) approach was successfully developed to identify 25 native Dendrobium species in Thailand. PCR-RFLP of the rDNA-ITS with six restriction enzymes and three chloroplast (cp) DNA regions with five primer-enzyme combinations produced 24 types of DNA patterns altogether. Twenty-three out of the 25 species determined in this study were found to belong to unique classes and were successfully differentiated. Two species, D. crumenatum and D. formosum, possessing the same DNA pattern, however, were identified after cutting the chloroplast DNA fragment amplified by psbC-trnS primer with MboI enzyme. An effective procedure for identifying each Dendrobium species was developed. PCR-RFLP of the rDNA-ITS with TaqI, which is the most informative enzyme, was used for the early detection of 16 Dendrobium species. To identify the remaining Dendrobium species, PCR-RFLP analysis was performed using one more primer-enzyme combination. Our study provides a rapid, simple, and reliable identification method for these Dendrobium species

Production of Salt Tolerant Ruzi Grass (Brachiaria ruziziensis) by Tissue Culture

ABSTRACT The best medium formula for multiple shoot induction was determined for seed culture of Ruzi grass (Brachiaria ruziziensis). Murashige and Skoog (MS) medium containing 10 mg/l BAP gave the highest average of 7.49 shoots. These shoots were irradiated at different doses of 0, 10, 30 , 50, and 70 Gy and transferred to grow on the selected multiple shoot induction medium having different concentrations of NaCl at 0, 0.5,1.0, 1.5 and 2.0%. It was found that the percentages of shoot survival decreased as the radiated doses increased. However, at the high salt concentration of 2.0% the growth was completely inhibited. All 173 clones of surviving plant were transferred to grow in the natural field at the National Corn and Sorghum Research Center, Nakhon Ratchasima province. A set of replicated plants from this field was also grown under high saline soil at Borabue district, Maha Sarakham province for comparison. Fifteen healthy clones were chosen from these two sites and grown in the high saline soil for one year. Only ten surviving clones were collected from this second growth and propagated as salt tolerant Ruzi grass. Upon testing the DNA fingerprints of the original 15 healthy clones using AFLP technique with 10 pairs of primers, there was no differences in the banding patterns found among them

Characterization of five complete Cyrtodactylus mitogenome structures reveals low structural diversity and conservation of repeated sequences in the lineage

Mitochondrial genomes (mitogenomes) of five Cyrtodactylus were determined. Their compositions and structures were similar to most of the available gecko lizard mitogenomes as 13 protein-coding, two rRNA and 22 tRNA genes. The non-coding control region (CR) of almost all Cyrtodactylus mitogenome structures contained a repeated sequence named the 75-bp box family, except for C. auribalteatus which contained the 225-bp box. Sequence similarities indicated that the 225-bp box resulted from the duplication event of 75-bp boxes, followed by homogenization and fixation in C. auribalteatus. The 75-bp box family was found in most gecko lizards with high conservation (55–75% similarities) and could form secondary structures, suggesting that this repeated sequence family played an important role under selective pressure and might involve mitogenome replication and the likelihood of rearrangements in CR. The 75-bp box family was acquired in the common ancestral genome of the gecko lizard, evolving gradually through each lineage by independent nucleotide mutation. Comparison of gecko lizard mitogenomes revealed low structural diversity with at least six types of mitochondrial gene rearrangements. Cyrtodactylus mitogenome structure showed the same gene rearrangement as found in most gecko lizards. Advanced mitogenome information will enable a better understanding of structure evolution mechanisms

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent “a hypothetical ancestral super-sex chromosome” or random distribution?

Background Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. Results We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. Conclusions The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes

Table_S6

Nucleotide sequence divergence (Kz) in intron10 and intron 11 of male-derived CTNNB1Z sequences among eight snake species (below diagonal), and female-derived CTNNB1Z sequences (above diagonal). Standard errors are indicated for all values. Table is also available in PDF format in the "Supplementary file" PD