Mannose-Based Selection with Phosphomannose-Isomerase (PMI) Gene as a Positive Selectable Marker for Rice Genetic Transformation

Penna, Suprasanna,Ramaswamy, Manjunatha Benakanare,Anant., Bapat Vishvas. The Korean Society of Crop Science 2008 Journal of crop science and biotechnology Vol.11 No.4

A positive selectable marker system was adapted for transformation of mature embryo-derived calli of Indica rice (Oryza sativa L.) utilizing the PMI gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. The transformed cells grew on medium supplemented with 3% mannose as carbon source and calli were selected on media containing various concentrations of mannose. Molecular analyses showed that the transformed plants contained the PMI gene. The results indicate that the mannose selection system can be used for Agrobacterium-mediated transformation of mature embryo in rice to substitute the use of conventional selectable markers in genetic transformation.

Mannose-Based Selection with Phosphomannose- Isomerase (PMI) Gene as a Positive Selectable Marker for Rice Genetic Transformation

Suprasanna Penna,Manjunatha Benakanare Ramaswamy,Bapat Vishvas. Anant 한국작물학회 2008 Journal of crop science and biotechnology Vol.11 No.4

A positive selectable marker system was adapted for transformation of mature embryo-derived calli of Indica rice (Oryza sativa L.) utilizing the PMI gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. The transformed cells grew on medium supplemented with 3% mannose as carbon source and calli were selected on media containing various concentrations of mannose. Molecular analyses showed that the transformed plants contained the PMI gene. The results indicate that the mannose selection system can be used for Agrobacterium-mediated transformation of mature embryo in rice to substitute the use of conventional selectable markers in genetic transformation. A positive selectable marker system was adapted for transformation of mature embryo-derived calli of Indica rice (Oryza sativa L.) utilizing the PMI gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. The transformed cells grew on medium supplemented with 3% mannose as carbon source and calli were selected on media containing various concentrations of mannose. Molecular analyses showed that the transformed plants contained the PMI gene. The results indicate that the mannose selection system can be used for Agrobacterium-mediated transformation of mature embryo in rice to substitute the use of conventional selectable markers in genetic transformation.

Partial Desiccation of Embryogenic Calli Improves Plant Regeneration in Sugarcane (Saccharum Spp.)

Desai Neetin Shivajirao,Suprasanna Penna,Bapat Viswas Ananat The Korean Society of Plant Biotechnology 2004 Plant molecular biology and biotechnology research Vol.6 No.4

Partial desiccation of embryogenic calli cultures or somatic embryos leads to different physiological changes and maturation of somatic embryos, leading to improved plant regeneration. Embryogenic calli was induced from immature inflorescence segments and young leaf rolls of sugarcane (Saccharum officinarum hybrids CoC-671) on Murashige and Skoog's basal medium enriched with different concentrations of 2,4-D ($1-4\;\cal{mg/l}$), L-glutamine ($100\cal{mg/l}$), malt extract ($100\cal{mg/l}$), casein hydrolysate ($1000\;\cal{mg/l}$) and coconut milk ($5\%$) and solidified with $0.2\%$ gel rite. The embryogenic calli were subjected to desiccation for 1-8 h. Desiccation of the calli for 6-7 h resulted in enhancement of plant regeneration frequency ($83-96\%$) as compared to control ($12\%$). Plantlets exhibited vigorous growth to maturity in the greenhouse. Partial desiccation of embryogenic calli offers as a simple method for improving plant regeneration frequency in sugarcane.

Short-term Salt and PEG Stresses Regulate Expression of MicroRNA, miR159 in Sugarcane Leaves

Vikas Yadav Patade,Penna Suprasanna 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.3

MicroRNAs (miRNAs) are involved in the regulation of stress-responsive gene expression and stress adaptation. Transcript expression of mature miR159 was studied in sugarcane leaves stressed for long-term (15 days) NaCl (150 mM) or iso-osmotic PEG 8000(20% w/v) and for short period (up to 24 h) with NaCl (200 mM) or PEG (20% w/v). The results revealed no significant changes in transcript levels of the miRNA in response to the long-term stress however, short-term salt or PEG stress led to significant up-regulation over the control. Progressive increase in transcripts of the miRNA was observed under short-term PEG stress with exposure period. Using computational tools, various members of the MYB transcription factor family were predicted as the potential targets of miR159. The transcript expression of the MYB-related gene indicated up-regulation at 1 h of salt stress with concomitant slight downregulation of the miRNA. In addition, under short-term PEG stress, the transcript levels of MYB and miR159 were the opposite of each other, suggesting MYB as a potential target of miR159. To our knowledge, this is the first preliminary evidence on the involvement of miR159 in abiotic stress responses in sugarcane.

Short-term Salt and PEG Stresses Regulate Expression of MicroRNA, miR159 in Sugarcane Leaves

Patade, Vikas Yadav,Suprasanna, Penna 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.3

MicroRNAs (miRNAs) are involved in the regulation of stress-responsive gene expression and stress adaptation. Transcript expression of mature miR159 was studied in sugarcane leaves stressed for long-term (15 days) NaCl (150 mM) or iso-osmotic PEG 8000 (20% w/v) and for short period (up to 24 h) with NaCl (200 mM) or PEG (20% w/v). The results revealed no significant changes in transcript levels of the miRNA in response to the long-term stress however, short-term salt or PEG stress led to significant up-regulation over the control. Progressive increase in transcripts of the miRNA was observed under short-term PEG stress with exposure period. Using computational tools, various members of the MYB transcription factor family were predicted as the potential targets of miR159. The transcript expression of the MYB-related gene indicated up-regulation at 1 h of salt stress with concomitant slight down-regulation of the miRNA. In addition, under short-term PEG stress, the transcript levels of MYB and miR159 were the opposite of each other, suggesting MYB as a potential target of miR159. To our knowledge, this is the first preliminary evidence on the involvement of miR159 in abiotic stress responses in sugarcane.

Differential Osmotic Adjustment to Iso-osmotic NaCl and PEG Stress in the in vitro Cultures of Sesuvium portulacastrum (L.) L.

Lokhande, Vinayak Haribhau,Nikam, Tukaram Dayaram,Penna, Suprasanna 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.4

The influence of iso-osmotic (-0.7 MPa) NaCl and PEG stress on growth, osmotic adjustment and antioxidant defense mechanisms was investigated in the in vitro cultures of Sesuvium portulacastrum (L.) L. The decreased relative growth rate (RGR) and water content of PEG-stressed calli in comparison to NaCl was found to be correlated with differences observed in the energy expenditure for the maintenance of osmotic balance. Osmotic adjustment in the NaCl-stressed calli favored higher accumulation of saline ions and soluble sugars, whereas PEG-stressed calli confirmed increased levels of organic osmolytes (proline, glycine betaine and soluble sugars). Permeability of $Na^+$ ions across the membrane revealed increased relative electrolytic leakage (REL) in NaCl-stressed calli, however non-penetrating and highly viscous solution of PEG amplified the peroxidation of membrane lipids. Increased activities of superoxide dismutase and catalase displayed efficient removal of toxic reactive oxygen species in comparison to ascorbate peroxidase in the calli exposed to iso-osmotic stress. These findings suggest that differential tolerance potential to iso-osmotic NaCl and PEG stress in terms of osmotic adjustment appears to be the prime defense mechanism of Sesuvium for its survival under iso-osmotic stress conditions at the expense of reduced growth and water content.

Differential Osmotic Adjustment to Iso-osmotic NaCl and PEG Stress in the in vitro Cultures of Sesuvium portulacastrum (L.) L.

Vinayak Haribhau Lokhande,Tukaram Dayaram Nikam,Suprasanna Penna 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.4

The influence of iso-osmotic (-0.7 MPa) NaCl and PEG stress on growth, osmotic adjustment and antioxidant defense mechanisms was investigated in the in vitro cultures of Sesuvium portulacastrum (L.) L. The decreased relative growth rate (RGR) and water content of PEG-stressed calli in comparison to NaCl was found to be correlated with differences observed in the energy expenditure for the maintenance of osmotic balance. Osmotic adjustment in the NaCl-stressed calli favored higher accumulation of saline ions and soluble sugars, whereas PEG-stressed calli confirmed increased levels of organic osmolytes (proline, glycine betaine and soluble sugars). Permeability of Na+ ions across the membrane revealed increased relative electrolytic leakage (REL) in NaCl-stressed calli,however non-penetrating and highly viscous solution of PEG amplified the peroxidation of membrane lipids. Increased activities of superoxide dismutase and catalase displayed efficient removal of toxic reactive oxygen species in comparison to ascorbate peroxidase in the calli exposed to iso-osmotic stress. These findings suggest that differential tolerance potential to iso-osmotic NaCl and PEG stress in terms of osmotic adjustment appears to be the prime defense mechanism of Sesuvium for its survival under iso-osmotic stress conditions at the expense of reduced growth and water content.

Optimization of different factors for an Agrobacterium-mediated genetic transformation system using embryo axis explants of chickpea (Cicer arietinum L.)

Suman Kalyan Sadhu,Phanikanth Jogam,Kranthikumar Gande,Raghu Banoth,Suprasanna Penna,Venkataiah Peddaboina 한국식물생명공학회 2022 JOURNAL OF PLANT BIOTECHNOLOGY Vol.49 No.1

In this study, we developed a reliable and efficient Agrobacterium-mediated genetic transformation system by applying sonication and vacuum infiltration to six chickpea cultivars (ICCV2, ICCV10, ICCV92944, ICCV37, JAKI9218, and JG11) using embryo axis explants. Wounded explants were precultured for 3 days in shoot induction medium (SIM) before sonication and vacuum infiltration with an Agrobacterium suspension and co-cultivated for 3 days in co-cultivation medium containing 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Responsive explants with putatively transformed shoots were selected using a gradual increase in kanamycin from 25 mg/l to 100 mg/l in selection medium to eliminate escapes. Results showed optimal transformation efficiency at a bacterial density of 1.0, an optical density at 600 nm wavelength (OD600), and an infection duration of 30 min. The presence and stable integration of the β-glucuronidase (gusA) gene into the chickpea genome were confirmed using GUS histochemical assay and polymerase chain reaction. A high transformation efficiency was achieved among the different factors tested using embryo axis explants of cv. JAKI 9218. Of the six chickpea cultivars tested, JAKI9218 showed the highest transformation efficiency of 8.6%, followed by JG11 (7.2%), ICCV92944 (6.8%), ICCV37 (5.4%), ICCV2 (4.8%), and ICCV10 (4.6%). These findings showed that the Agrobacterium-mediated genetic transformation system will help transfer novel candidate genes into chickpea.