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Chaudhury, Ashok,Pal, Minakshi 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.2
Data presented herein provides a rapid and efficient method for Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima for hairy root cultures as well as for enhancing Shikonin production. Etiolated explants viz. shoot tip, nodal, leaf and internodal segments were co-cultivated with Agrobacterium rhizogenes for induction of hairy root. Among the various explants employed, leaf explant showed maximum 70.7% response followed by shoot tip 48.3%, nodal segment 38.7% and internodal segment 9.3%. Integration of Ri plasmid rolB gene in the transformed hairy root cultures was confirmed by PCR analysis using forward (FrolB) and reverse (RrolB) primers of rolB gene resulting in the amplification of 0 ~ 0.8 kb fragments. Medium compositions have been optimized for in vitro induction of Shikonin in hairy root cultures of Arnebia hispidissima. Hairy roots on hormone-free MS medium showed red spots in the older part of the tissues which turned white after a second subculture. Whereas hairy roots cultured on RC medium showed faster growth and produced large amount of Shikonin. The Shikonin content in transformed hairy root culture was estimated by recording absorbance at 620 nm and quantified against authentic sample of Shikonin. Shikonin content was estimated to be $0.85\;mg\;g^{-1}$ fresh weight of tissue at the end of the 50 days of culture. The results presented herein will help to design strategies for bridging the gap between ever increasing demand and supply of raw products necessary for obtaining Shikonin for cosmetic, dyeing, food, medicinal, and pharmaceutical industries.
Ashok Chaudhury,Minakshi Pal 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.2
Data presented herein provides a rapid and efficient method for Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima for hairy root cultures as well as for enhancing Shikonin production. Etiolated explants viz. shoot tip, nodal,leaf and internodal segments were co-cultivated with Agrobacterium rhizogenes for induction of hairy root. Among the various explants employed, leaf explant showed maximum 70.7% response followed by shoot tip 48.3%, nodal segment 38.7% and internodal segment 9.3%. Integration of Ri plasmid rolB gene in the transformed hairy root cultures was confirmed by PCR analysis using forward (FrolB) and reverse (RrolB) primers of rolB gene resulting in the amplification of 0 ~ 0.8 kb fragments. Medium compositions have been optimized for in vitro induction of Shikonin in hairy root cultures of Arnebia hispidissima. Hairy roots on hormonefree MS medium showed red spots in the older part of the tissues which turned white after a second subculture. Whereas hairy roots cultured on RC medium showed faster growth and produced large amount of Shikonin. The Shikonin content in transformed hairy root culture was estimated by recording absorbance at 620 nm and quantified against authentic sample of Shikonin. Shikonin content was estimated to be 0.85 mg g-1 fresh weight of tissue at the end of the 50 days of culture. The results presented herein will help to design strategies for bridging the gap between ever increasing demand and supply of raw products necessary for obtaining Shikonin for cosmetic, dyeing, food, medicinal, and pharmaceutical industries.
Minakshi Pal,Ashok Chaudhury 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.1
The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg l-¹ KIN, 0.25 mg l-¹ BAP, 0.1 mg l-¹ IAA and 100 mg l-¹ CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg l-¹ IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg g-¹ fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing,food, and pharmaceutical industries.
Pal, Minakshi,Chaudhury, Ashok 한국작물학회 2010 Journal of crop science and biotechnology Vol.13 No.1
The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg $1^{-1}$ KIN, 0.25 mg $1^{-1}$ BAP, 0.1 mg $1^{-1}$ IAA and 100 mg $1^{-1}$ CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg $1^{-1}$ IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg $g^{-1}$ fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing, food, and pharmaceutical industries.
Anita Punia,Rakesh Yadav,Pooja Arora,Ashok Chaudhury 한국작물학회 2009 Journal of crop science and biotechnology Vol.12 No.3
Cluster bean (Cymopsis tetragonoloba) belongs to tribe Indigoferae of family Leguminosae. India is the world-leader for cluster bean production as it contributes 80% shares of its total production. Cluster bean (guar) is a cash crop for its application in textile, paper, petroleum, mining, pharmaceuticals, explosives, and food industries. Owing to its immense wealth of variable morphophysiological and industrial qualities there is a strong need for appropriate addressing and well documentation of the germplasm. Efforts are to be made to organize research programs on germplasm characterization, utilization, and molecular characterization. Superior cluster bean varieties were selected on the basis of morphophysiological characters and subjected to DNA-based molecular marker analysis. Eighteen of the best genotypes were chosen for DNA extraction, optimization of PCR conditions, and genetic diversity studies using 37 random primers. A total of 381 random amplification fragments were obtained; number of amplifications ranging from 4 to 22 with an average of 10.29 amplified fragments per primer. Evaluation of RAPD data reveals a magnificent range (0.34-0.76) of genotypic similarity coefficients. The UPGMA dendrogram was constructed based on similarity indices which illustrated discrete clustering of different genotypes into groups. Results recorded a positive correlation amongst varieties vis-à-vis cluster analysis generated by NTSYSpc and morphophysiological characteristics. The degree and distribution of genetic diversity in cluster bean would facilitate an evolutionary relationship between numerous accessions that eventually catalogues genetic resources in a more concerted fashion. Cluster bean (Cymopsis tetragonoloba) belongs to tribe Indigoferae of family Leguminosae. India is the world-leader for cluster bean production as it contributes 80% shares of its total production. Cluster bean (guar) is a cash crop for its application in textile, paper, petroleum, mining, pharmaceuticals, explosives, and food industries. Owing to its immense wealth of variable morphophysiological and industrial qualities there is a strong need for appropriate addressing and well documentation of the germplasm. Efforts are to be made to organize research programs on germplasm characterization, utilization, and molecular characterization. Superior cluster bean varieties were selected on the basis of morphophysiological characters and subjected to DNA-based molecular marker analysis. Eighteen of the best genotypes were chosen for DNA extraction, optimization of PCR conditions, and genetic diversity studies using 37 random primers. A total of 381 random amplification fragments were obtained; number of amplifications ranging from 4 to 22 with an average of 10.29 amplified fragments per primer. Evaluation of RAPD data reveals a magnificent range (0.34-0.76) of genotypic similarity coefficients. The UPGMA dendrogram was constructed based on similarity indices which illustrated discrete clustering of different genotypes into groups. Results recorded a positive correlation amongst varieties vis-à-vis cluster analysis generated by NTSYSpc and morphophysiological characteristics. The degree and distribution of genetic diversity in cluster bean would facilitate an evolutionary relationship between numerous accessions that eventually catalogues genetic resources in a more concerted fashion.
Rakesh Yadav,Pooja Arora,Dharmendar Kumar,Neeraj Dilbaghi,Ashok Chaudhury 한국식물생명공학회 2009 Plant biotechnology reports Vol.3 No.3
Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l-1 KIN and 0.25 mg l-1 IAA. Nodal and internodal segments were found to be extraprolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l-1 KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l-1 IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future. Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l-1 KIN and 0.25 mg l-1 IAA. Nodal and internodal segments were found to be extraprolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l-1 KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l-1 IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.
Shashi Bhushan,Mandakini Gogoi,Abhispa Bora,Sourav Ghosh,Sinchini Barman,Tethi Biswas,Mathummal Sudarshan,Ashoke Ranjan Thakur,Indranil Mukherjee,Subrata Kumar Dey,Shaon Ray Chaudhuri 한국미생물·생명공학회 2019 한국미생물·생명공학회지 Vol.47 No.2
Earlier studies by our group revealed that gallic acid in phytochemicals stimulated biofilm production in epiphytes, while caffeic acid in phytochemicals inhibited biofilm production in non-epiphytes. It is well documented that antimicrobial secretion by some epiphytic bacteria inhibits non-epiphytic bacterial growth on leaf surfaces. These selection criteria help plants choose their microbial inhabitants. Calcium and iron in phytochemicals also stimulate biofilm formation and thus, may be selection criteria adopted by plants with respect to their native epiphytic population. Furthermore, the processing of leaves during phytochemical extraction impacts the composition of the extract, and therefore its ability to affect bacterial biofilm formation. Computation of the Hurst exponent using biofilm thickness data obtained from the Ellipsometry of Brewster Angle Microscopic (BAM) images is an efficient tool for understanding the impact of phytochemicals on epiphytic and non-epiphytic populations when compared to fluorescent microscopy, scanning electron microscopy, and staining techniques. To the best of our knowledge, this is the first report that uses the Hurst exponent to elucidate the mechanism involved in plant microbe interaction.
Pooja Bangar,Neetu Tyagi,Bhavana Tiwari,Sanjay Kumar,Paramananda Barman,Ratna Kumari,Ambika Gaikwad,K. V. Bhat,Ashok Chaudhury 한국작물학회 2021 Journal of crop science and biotechnology Vol.24 No.2
Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] is vital grain legume having nutritional and socio-economic importance, especially in the developing countries. We performed whole-genome re-sequencing of three accessions representing the wild progenitor species, released and landrace of mungbean to identify SNPs with relevance to genetic relationships analysis. Approximately 9.3 million raw reads were obtained using Ion Torrent PGM™ platform and more than 92% of the reads were mapped to the reference mungbean genome. We identified a total of 233,799 single-nucleotide polymorphisms in relation to the reference genome (SNPs: 103,341 in wild, 93,078 in released and 37,380 in landrace accessions) and 9544 insertions and deletions (InDels: 4742 in wild, 3608 in released and 1194 in landrace accessions) in the coding and non-coding regions. In all accessions, genomic variants were unevenly distributed within and across the mungbean chromosomes. Among these 5339, 4739 and 1795 SNPs were non-synonymous in 815, 790 and 317 genes of wild, released and landrace accessions, respectively. These polymorphisms might contribute to the variation in important pathways of genes for abiotic and biotic stress tolerance and important agronomic traits, such as seed dormancy, fl owering time and seed size in mungbean. Among the randomly selected SNPs, a selected subset was validated using Sanger sequencing technique. The genomic variations among mungbean wild, released and landrace accessions constitute a powerful tool to support genetic research and molecular breeding of mungbean.
Bhushan, Shashi,Gogoi, Mandakini,Bora, Abhispa,Ghosh, Sourav,Barman, Sinchini,Biswas, Tethi,Sudarshan, Mathummal,Thakur, Ashoke Ranjan,Mukherjee, Indranil,Dey, Subrata Kumar,Chaudhuri, Shaon Ray The Korean Society for Microbiology and Biotechnol 2019 한국미생물·생명공학회지 Vol.47 No.2
Earlier studies by our group revealed that gallic acid in phytochemicals stimulated biofilm production in epiphytes, while caffeic acid in phytochemicals inhibited biofilm production in non-epiphytes. It is well documented that antimicrobial secretion by some epiphytic bacteria inhibits non-epiphytic bacterial growth on leaf surfaces. These selection criteria help plants choose their microbial inhabitants. Calcium and iron in phytochemicals also stimulate biofilm formation and thus, may be selection criteria adopted by plants with respect to their native epiphytic population. Furthermore, the processing of leaves during phytochemical extraction impacts the composition of the extract, and therefore its ability to affect bacterial biofilm formation. Computation of the Hurst exponent using biofilm thickness data obtained from the Ellipsometry of Brewster Angle Microscopic (BAM) images is an efficient tool for understanding the impact of phytochemicals on epiphytic and non-epiphytic populations when compared to fluorescent microscopy, scanning electron microscopy, and staining techniques. To the best of our knowledge, this is the first report that uses the Hurst exponent to elucidate the mechanism involved in plant microbe interaction.