High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

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.

Molecular and Morphophysiological Characterization of Superior Cluster Bean (Cymopsis tetragonoloba) Varieties

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.