Biotechnology in forestry
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Unlike crops, only a small percentage of the current world forest plantings comprise material resulting from genetic improvement of any type. Genetic improvement of forest species has essentially been limited to selection and multiplication of well-adapted genotypes, although for industrial forestry species, especially Eucalyptus and Pinus, recurrent selection for population improvement has been widely adopted. Reproductive biology and patterns of variation of these species is reasonably well documented. Vigour, form and wood quality are the priority selection criteria. As regards non-industrial tree species, the knowledge of biology and patterns of variations are poorly understood and selection criteria are also diffused. The breeding work is essentially confined to species testing, the assessment of the biological features and genetic conservation activities. The use of biotechnologies to the various tree species would thus vary according to the current and potential use of and level of previous genetic improvement work done in the species.
In-vitro culture techniques
Successful "slow growth" storage of cultured tissues and organs has been reported for several forest species. The major bottleneck in several cases, however, is the regeneration of plants from the cryopreserved materials. ln vitro conservation can be effective for threatened gene pools. The main advantage of cryopreservation would be the maintenance of the juvenile state to facilitate capture of genetic gains through clonal forestry with industrial species and a few other species for which good breeding programmes are already in progress. In vitro cultured materials would also find favour for germplasm transfer of certain species.
In vitro rejuvenation or accelerated maturation has been obtained in a few forest species and could be exploited for clonal forestry and accelerated breeding cycles, respectively. Micropropagation protocols are known for more than 100 forest species, but commercial exploitation of the technique lags behind because of paucity of data economics of the vitro plantations. Nonetheless, the technique is being used for micropropagation of outstanding clones in breeding programmes, i.e. of tropical eucalypts and for early multiplication of selected varieties prior to release. The efficacy and efficiency of somatic embryogenesis and "artificial seed" is being analysed and improved both in selected public and private institutions and the approach holds promise under certain circumstances. Cryopreservation of somatic embryos with field evaluations of emblings and scaled-up bioreactor-based multiplication of selected cryopreserved embryos is expected to be commercialized for several tree species within the next five to ten years (Gupta et al., 1993).
In agroforestry or community forestry programmes where intensive cropping of forest species is undertaken, micropropagation of desired clones assumes still greater economic importance. Eucalyptus plantations are one of the leading forestry systems. About ten years ago, Gupta and colleagues at the National Chemical Laboratory (NCL), Pune, India, spotted extremely promising trees of Eucalyptus camaldulensis and E. torelliana in the forests of South India and developed micropropagation techniques for their multiplication. Some 50 000 plantlets of E. torelliana and 20 000 plantlets of E. camaldulensis could be produced within a year from just one nodal segment taken from an adult tree of each species. More work is needed on the standardization of micropropagation techniques to render them more cost effective.
Some of the developing Asian countries have made considerable progress in exploiting biotechnology for forestry. For instance, Indian scientists at the NCL were the first to standardize in vitro propagation of 100-year old teak (Tectona grandis), and 30-year old Eucalyptus citriodora and other Eucalyptus species. Pilot projects on teak, eucalypts, bamboo, sandal and salvadora plantations based on in vitro cultured materials are in progress and it is hoped that micropropagation of other useful species such as Tamarindus indica, Azadirachta indica, Santalum album, and Morus alba will also be standardized soon (Bapat and Rao, 1990). An interesting success of the Indian scientists was in vitro flowering of bamboo - a species that flowers once in a decade or more, which is a significant development for bamboo breeding. Indian scientists have succeeded also in standardizing the technique for several nitrogen-fixing tree species such as Acacia nilotica, Albizia lebbek, Dalbergia latifolia, D. sissoo, Leucaena leucocephala, Prosopis cineraria, Sesbania grandiflora, and S. sesban. The work on nitrogen-fixing species is particularly significant from the point of view of nitrogen economy of the soil. Selection of efficient strains of Rhizobium, Azotobacter and Mycorrhizae should also be emphasized concomitantly.
In China, during the past 15 years or so, useful work on the culture of anther, shoot, embryo, endosperm, ovary, suspension cell and protoplast of woody species has been done. In vitro culture plantlets were reported in about 100 woody species, including poplar, eucalypts, paulownia, and Chinese fir. ln vitro culture techniques are being used for developing fast-growing, disease-resistant, and proficient nitrogen-fixing Casuarinas for establishing new shelterbelts and rehabilitating old ones.
Somatic embryogenesis, which allows automation of micropropagation and renders it highly cost effective, can play an important role in reforestation. So far, somatic embryogenesis has been reported in at least 60 species of woody trees belonging to 44 genera and 25 families. In some cases the process has been patented by private companies for commercial exploitation. Greater research and development efforts are needed to standardize the technique in developing countries for large-scale reforestation.
Franclet (1989) observed rather striking homogenety in progenies of certain distant hybrids of Eucalyptus. The uniformity was postulated to be due to apomictic seed setting in the F, plants. This needs to be confirmed and if found true, the finding will revolutionize clonal propagation of hybrid and other populations of Eucalyptus.
Pollen plantlets from about 30 woody species have been obtained in China. Indian scientists also produced haploids of a few tree species, including Albizia lebbeck. The haploids were used by Chinese scientists to produce homozygotes and further breeding work. Given the long life cycle of trees, haploid technology is particularly suitable for breeding improved varieties of forest species.
Somaclonal selection has recently been used to produce and select clones of hybrid poplar resistant to Septoria musiva (Ostry and Skilling, 1988). Exposure of cell cultures to disease toxins, saline/alkaline conditions, unusual temperature regimes, etc. facilitate isolation of resistant types. Using this technique, Eucalyptus camaldulensis clones resistant to saline soils were developed. However, no somaclonal tree has yet been put to commercial use.
The value of hybrid populations in terms of growth, vigour and productivity of tree species, including the Eucalyptus hybrid populations described earlier, is well-known. Controlled hybridization of selected parents holds great promise for producing novel and productive populations.
For those species that cannot be hybridized sexually, somatic hybridization - protoplast fusion and regeneration of plantlets from "hybrid" protoplasm is a possible alternative. As regards forest and other woody species, protoplast fusion and regeneration have been achieved only in a few species, such as elms, poplar and rubber. Concerted effort is needed to standardize the protoplast culture, fusion and regeneration technology for important woody species.
RFLPs and RAPDs are being used extensively for quantification of genetic variation, markerassisted selection and genotype verification in several forest species. Transgenic eucalypts are being produced for incorporating cold tolerance to enable expansion of this species into cold-prone zones to which this species has little tolerance. Another objective is to transfer pest resistance in poplars and some tropical hardwoods, while a further attractive proposition is the reduction of lignin biosynthesis in the pulp species.
A recent FAO study (1993) carried out by Haines under the André Mayer Research Fellowship underlines that the use of biotechnologies for the improvement of tree species necessitates intimate knowledge of biology, breeding systems and variations in the pattern of the target species, which is generally poor. While new technologies could be effective and expedient in specific cases, major emphasis on the improvement of most tree species will in the near future remain on taxonomic studies of variation, species and provenance testing, field assessment and genetic conservation. A major constraint in the use of biotechnology in tree improvement programmes is the lack of skilled tree breeders. The study concludes: "In the short term, for industrial species, with known biology and variation patterns, opportunities exist for technology replacement in the incorporation of micropropagation into integrated multiplication systems for intensively bred species, and in the use of molecular markers in genotype verification. For non-industrial species and for presently less well-known hardwoods with potential for industrial use, opportunities exist for the use of markers in studies of genetic variation and breeding systems. In the longer term, significant applications of new technologies are likely. Major research priorities include the acquisition of better understanding of molecular genetic processes in forest trees, particularly those underlying adaptation, and their manipulation. Continuation of research on markers and genetic engineering with model species will lay the foundations for later application of these techniques to both industrial and non-industrial species. For intensively bred industrial species, development of somatic embryogenesis and artificial seed technologies, of cryopreservation as a means of preserving juvenility, and of techniques for the genetic engineering of lignin reduction (in species grown for pulp), will be useful targets for continued research. Genetic engineering of sterility will be an important objective likely to facilitate later deployment of transgenic plants. The development of simple micropropagation protocols for species for which these are not already available will also be of value.
Among woody species, vitroplantation of cardamom is a success story in India not only in terms of increased productivity and income, but also in terms of a link-up between private and public sector. The National Chemical Laboratory, Pune, standardized the tissue culture and micropropagation protocol for cardamom in 1983 and, as a consultant, helped a private company, A.V. Thomas & Co., Cochin, to commercialize this technique. The tissue culture technique led to rapid multiplication: 1 000 plants per year, against five to six plants per year through rhizome propagation. Moreover, vitroplants were precocious by two years and gave much higher yields of uniform and high-quality fruits. In one district of Kerala, using vitroplants, the average yield nearly doubled, from 58 kg/ha in 1982-83 to 114 kg/ha in 1985-86 (Kumar, 1990). Encouraged by this experience, the Department of Biotechnology of the Government of India undertook a project to demonstrate the performance of elite in vitro-derived clones of cardamom in 100 units of one hectare each throughout the main cardamom growing areas of the country (Kerala, Tamil Nadu and Karnataka provinces). Several private companies in the country are now producing vitro-plants of cardamom and the effort has fructified in significantly enhancing national production and export.
Production of somatic embryos using bioreactors was standardized for sandalwood (Santalum album), a commercially important species producing fragrant wood and oil, and its vitroplants are being field evaluated by the Forest Research Laboratory, Bangalore (Bapat and Rao, 1990). The Department of Biotechnology has been supporting a project on biomass, including industrial and non-industrial wood species, using tissue culture techniques. Pilot-plant units at the National Chemical Laboratory, Pune and the Tata Energy Research Institute, New Delhi supported under this project are serving as intermediaries in the transfer of tissue culture technology from the laboratory to the field (Mascarenhas, 1991). About 7 000 vitroplants of bamboo are already being field-tested in the states of Uttar Pradesh, Orissa, Karnataka and Delhi for their growth and biomass production.
Rattan (Calamus sp. especially C. manan), is an important economic and export species of the Philippines, Malaysia and Thailand. The naturally occurring populations are eroding fast due to overexploitation. In order to meet the expanding demand of the raw material, rattan replantation using the selected species and clones is being vigorously pursued in the Southeast Asian countries. For this purpose, micropropagation of rattan has been standardized but is yet to be fully exploited. In addition, the technique is being standardized and even used for micropropagation of Acacia mangium in Malaysia; bamboo, Albizzia falcatoria and Eucalyptus camaldulensis in the Philippines; and teak and Eucalyptus in Thailand.
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