High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)
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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 extra-prolific 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 ± 2°C 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.
KeywordsEastern Cottonwood Internodes Leaf Direct plant regeneration Populus deltoides Root segments
Murashige and Skoog
Scanning electron microscopy
This work was supported by the Department of Biotechnology, Ministry of Science & Technology, Government of India, New Delhi as a research project to Prof. A. Chaudhury. Mr. Rakesh Yadav, Mr. Dharmendar Kumar and Mr. Dinesh Katyal duly acknowledge financial assistance in the form of JRF’s and Technical Assistant in Department of Biotechnology sponsored project. The authors wish to express their gratitude to Dr. H.C. Chaturvedi, Emeritus Scientist, NBRI (CSIR), Lucknow and Dr. Vibha Dhawan, Vice Chancellor, TERI University for providing plant material and rendering help in initial phase of work. The transgenic greenhouse, tissue culture facility were established under the FIST program, and financial assistance in form of color reproduction fee for manuscript figure was provided under BT-19 Project, Department of Science & Technology, Ministry of Science & Technology, Government of India, New Delhi.
- Bolstad PV, Libby WJ (1982) Comparison of radiata pine cutting of hedge and tree form origin after seven growing seasons. Silvae Genet 31:9–13Google Scholar
- Borchert R (1976) The concept of rejuvenility in woody plants. Acta Hortic 56:21–36Google Scholar
- Chaturvedi HC, Sharma AK, Agha BQ, Jain M, Sharma M (2004) Production of clonal trees of Populus deltoides through in vitro regeneration of shoots from leaf, stem and root explants and their field cultivation. Indian J Biotechnol 2:203–208Google Scholar
- Hackett WP (1985) Juvenility, maturation, and rejuvenation in woody plants. Hortic Rev 7:109–155Google Scholar
- Thakur AK, Sharma S, Srivastava DK (2005) Plant regeneration and genetic transformation studies in petiole tissue of Himalayan poplar (Populus ciliata Wall.). Curr Sci 89:664–668Google Scholar
- Tsvetkov I, Hausman JF, Jouve L (2007) Thidiazuron-induced regeneration in root segments of white poplar (P. alba L.). Bulg J Agric Sci 13:623–626Google Scholar
- Vasil V, Vasil IK (1984) Preparation of cultured tissues for scanning electron microscopy. In: Vasil IK (ed) Cell culture and somatic cell genetics of plants. Laboratory procedures and their applications, vol 1. Academic Press, New York, USA, pp 738–743Google Scholar