Bio Technology
Biotechnology, which has made significant contributions to the improvement programs of agronomic crops, offers the opportunities to enhance forestry research and accelerate tree improvement. Forest biologists and tree breeders are turning their attentions to these biotechnologies, which enable them to overcome barriers and can be integrated into conventional breeding methods leading to more rapid progress in tree breeding. Plant biotechnology currently comprises a range of activities, such as vegetative propagation and tissue culture, genome analysis and gene cloning, Deoxyribonucleic acid (DNA) recombination and gene transfer, and DNA-based selection. Although application of biotechnology in forest trees and ornamental woody plants is just in its infancy, micro-propagation is rapidly becoming a standard tool for tree improvement (Huang et al).
Photo Courtesy: Dr. Geeta Joshi.
Tissue Culture
Tissue culture is a technique in which small tissue pieces or organs are removed from a donor plant and cultured aseptically on a nutrient medium. By manipulating the chemical composition of the nutrient medium and other environmental parameters, the growth and development of the tissues in culture can be directed into different channels (Bonga, 1982).
The potential of plant tissue culture includes:
1. Enhanced production of natural products,
2. Rapid clonal multiplication of select genotypes,
3. Production of disease-free plants,
4. Germplasm preservation,
5. Genetic manipulation.
In the recent decades, much more progress has been made in sandalwood tissue culture plants.
Macropropagation
Macropropagation from mature trees is generally difficult and usually employs grafting older scions onto younger rootstocks for rejuvenation of the scion and subsequent propagation. The rejuvenation from mature trees may require serial grafting before vegetative propagation, and this can become labour intensive and time consuming. Further, by conventional methods of vegetative propagation, the numbers of plants that can be multiplied from selected trees in a growing season are often relatively small. The limiting factors for large scale macropropagation of selected material may be restricted availability of improved genotypes as planting material, seasonal restrictions, and available space. To overcome these and related problems, biotechnological approaches offer opportunities for not only mass cloning of elite genotypes throughout the year, but also for in vitro rejuvenation, and genetic manipulation of trees (Mátyás, 1997).
Vegetative propagation is done through air layering or through root suckers. Techniques of tissue culture of sandal using different types of tissues like nodal, internodal segments from young shoots, and suspension culture, using different organs have been standardized.
Micropropagation:
Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.
Sandalwood (Santalum album) was the first angiospermic tree species to show somatic embryogenesis (Minocha and Jain).
Tree sandalwood has proved to be a very versatile material for tissue culture research. Success has been achieved in regenerating plantlets from somatic embryos differentiated from callus tissues originating from hypocotyl, stem and protoplasts cultures. Plantlets could be regenerating from synthetic seeds prepared by encapsulating somatic embryos in a proper matrix. Viable plantlets were recovered from somatic embryos grown in a bioreactor (Bapat, & Rao, 1992).
Hypocotyl segments excised from seedlings of Santalum album (sandalwood tree) grown in-vitro were cultured aseptically on various nutrient media. Regeneration of shoot buds on explants was observed on basal media supplemented with auxins like IAA, IBA, NAA, or NOA. Shoot bud formation was completely suppressed on 2,4-D or pCPA medium. Cytokinins stimulated and enhanced shoot bud formation better than auxins. Excised stem segments from adult plants did not respond to any hormonal treatment. The importance of this method in the propagation of the sandalwood tree is emphasized (Rao & Bapat, 1978).
The combination of high induction frequencies, many embryogenically competent cells and relative uniformity of regeneration responses across genotypes and explants of S. album make them attractive starting materials in initiating efforts to develop a reliable Agrobacterium- based transformation (Acharee & Jones, 1998).
Leaf disc explants of sandalwood (S. album) carry a high potential for rapid multiple shoot regeneration and subsequent micro-propagation. This protocol provides a successful and rapid technique that can be used for mass in-vitro propagation of elite species (Bele, et al., 2012).
Efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg l−1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg l−1TDZ. The WPM medium supplemented with 2.5 mg l−1 BA + 0.4 mg l−1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg l−1 BA and 3.0 mg 1−1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg l−1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants (Singh, et al., 2013).
An efficient plant regeneration protocol was developed for Santalum album L. This plant regeneration was achieved using internodal explants on Murashige and Skoog (MS) medium. Effect of Plant Growth Regulators (PGR) like 6-Benzyl Adenine (BA), Kinetin (KN) and 2-Isopentenyl adenine (2-iP) on shoot multiplication; 2-Isopentenyl adenine supplemented with varying concentrations of Coconut Milk (CoM) for shoot elongation and Indole-3-Butyric Acid (IBA) and α-Naphthalene Acetic Acid (NAA) on rooting was observed. Multiple shoots of S. album were significantly induced on internodal explants cultured on MS medium supplemented with 2-isopentenyl adenine (2iP) and Coconut Milk (CoM). The highest shoot multiplication was achieved on MS medium containing 1.0 mg L-1 (2iP) showed better growth response and produced 41.0±2.0 shootlets with an average length of 2.90±0.10 cm after 45 days of culture. The cluster of shoots were cultured on same media with 10% coconut Milk (CoM) showed shoot elongation up to 4.6 cm. Roots were induced after transfer to half strength MS medium supplemented with 0.5 mg L-1 IBA and 0.25 mg L-1 NAA produced 4.2 ± 0.10 roots with an average height of 4.8±0.2 cm after six weeks in culture. The rooted plantlets were transferred for hardening, 70% of plantlets survived and resumed growth in the mixture of soil, vermiculite and farm yard manure (1:1:1) (Janarthanam & Sumathi, 2011).
The explants shoot tip and single node were found to be standard for direct organogenesis and callusing in Santalum album. Treatment with growth regulator BAP with a concentration of 3mg/l was found to be ideal for shoot tip culture. Treatment with growth regulator BAP with a concentration of 5mg/l was found to be ideal for single node culture. The explants suitable for callusing were found to be shoot tip and single node with a growth regulator concentration of 3mg/l (Naga, et al., 2015)
Reference:
Ø Acharee Rugkhla and M.G.K. Jones, Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum, Journal of Experimental Botany, Vol. 49, No. 320, pp. 563–571, March 1998.
Ø Bapat, V. A.; Rao, P. S., 1992: Biotechnological approaches for sandalwood santalum album L. micropropagation. Indian Forester 118(1): 48-54.
Ø Bele, D., Tripathi, M.K., Tiwari, G., Baghel, B.S., and Tiwari, S., (2012) Microcloning of sandalwood (Santalum album Linn.) from cultured leaf discs. Journal of Agricultural Technology 8(2): 571-583.
Ø Bonga, J. M., (1982)Tissue Culture Techniques, Tissue Culture in Forestry, Volume 5 of the series Forestry Sciences pp 4-35.
Ø Janarthanam, B., and Sumathi, E., (2011). High Frequency Shoot Regeneration from Internodal Explants of Santalum album L., International Journal of Botany, 7: 249-254.
Ø Mátyás, C. (ed.) 1997. IUFRO World Series Vol. 6. Perspectives of Forest Genetics and Tree Breeding in a Changing World. University of Sopron, IUFRO Secretariat Vienna.
Ø Minocha, R., and S.M Jain, Tissue culture of woody plants and its relevance to Molecular biology, USDA Forest Service, NERSP.0. Box 640,271 Mast Road, Durham, NH 03824, USA.
Ø Naga Devi, K., Ch. Madhavi, Balasubramanian Sathyamurthy,Studies on the Invitro Regenerative Response of Santalum Album, International Journal Of Innovative Research & Development, March, 2015 Vol 4 Issue 3, Page 58-68.
Ø Rao, P. S., and V. A. Bapat, Vegetative propagation of sandalwood plants through tissue culture, Canadian Journal of Botany, 1978, 56(9): 1153-1156, 10.1139/b78-129.
Ø Singh, C. K., Sandeep R. Raj, V. R. Patil, P. S. Jaiswal, N. Subhash, (2013), Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions, Plant Tissue Culture, In Vitro Cellular & Developmental Biology – Plant April 2013, Volume 49, Issue 2, pp 216-222.
Ø Yinghua Huang, David F. Karnosky and C. G. Tauer, Applications of biotechnology and molecular genetics to tree improvement, Huang et al: Biotechnology & Molecular Genetics, Journal of Arboriculture 19(2): March 1993, pp 84-98.
Ø http://www.frienvis.nic.in/WriteReadData/UserFiles/file/pdfs/Sandal.pdf viewed 12th August 2015.