Major Areas

Division > Plant Biotechnology > Major Areas

Tissue Culture

Laboratory focuses on micropropagation of economically important forestry species like Eucalyptus,Melia dubia and bamboos with the major objective of cloning for large-scale multiplication. Superior performing clonal individuals of E. camaldulensis and E.tereticornis with different rhizogenic capacities are maintained for in vitro germplasm storage. Clones with low frequency rooting in conventional cutting methodology were subjected to tissue culture conditions for rejuvenation to enhance the rooting frequency under in vitro, wherein various approaches are adopted to overcome the recalcitrance. The micropropagated plants are planted clonal multiplication garden to form the source for cuttings for large scale production. Work is in progress for understanding the process of differentiation and expression of roots from the mature tissues of Dendrocalamus giganteus to control shoot necrosis associated with root formation which will subsequently lead to successful multiplication of plantlets. Cultures of Nicotiana tobaccum are maintained for transgene expression studies. Callus cultures in Aegle marmeloswas initiated with different explants from seedlings. CCA of Aegle marmelos was established and cell suspension cultures are currently being maintained for the analysis of secondary metabolites.

Activities carried out in the laboratory

Micropropagation of important tree species and genetic fidelity testing: Micropropagation methods were developed for the multiplication of important species like Euclyptus tereticornis, E.camaldulensis, E.torrelliana X E. citriodoraAzadirachta indica, E.pellita, E.urophylls X grandis, Acacia hybrids and Tectona grandis. In the case eucalyptsaxillary buds from the coppice shoots of the selected trees were used as explants and the microshoots were rooted under in vitro conditions. High frequency survival during hardening of in vitro rooted plants was achieved. In the case of teak the seedlings raised from CSO seeds were the source of explants. Ex vitro rooting was standardized for rooting of teak microshoots. Commercial viability of the protocols developed was tested by transferring the starter cultures of teak to the SPICAgrobiotech Ltd, Coimbatore and the plants produced were supplied to Andhra Pradesh Forest Development Corporation.

The eucalypts plantlets generated through micropropagation were tested for their genetic fidelity and genetic uniformity to maintain the quality planting stock production using RAPD and AFLP techniques.

Micropropagation of bamboos:

Various species of bamboos like Bambusa bambos, B.bambos vargiganteusD. strictusD.membranaceous were micropropagated through axillary bud proliferation technique from the seedling explants. In the species like D. giganteus, B.nutans and Pseudoxytenanthera stocksii mature clump derived shoots were used for shoot multiplication. In the DBT funded R & D program of bamboos, a high-frequency in vitro plant production of method was developed for mature clumps of Bambusa nutans. Effect of carbohydrate source (glucose and sucrose) and auxins (IAAIBA and NAA) on the in vitro rooting response of B.nutans was evaluated and found that 88mMglucose along with 49.0 mM IBA during the root induction phase gave 85% rooting success. In vitro rooting of the microshoots of D.giganteus originated through callus morphogenesis resulted in shoot necrosis immediately after root induction. However, addition of low levels of auxin in the root formation stage was found to be effective in controlling shoot necrosis. Biochemical studies during root formation in D.giganteus showed high peroxidase and IAA oxidase activity and low levels of internal IAA, favorable for rooting promoted shoot necrosis during root elongation stage.

Patent: A patent was obtained for the process developed for in vitro multiplication of Pseudoxytenanthera stocksiiwith the financial support from National Research Development Council (NRDC), New Delhi.

Field demonstration trials: Under the Department of Biotechnology funded program on field growth performance of bamboo produced through tissue culture methods, demonstration trials were established for B.bambosD. strictusand P.stocksiiMicropropagated, seed raised and cuttings propagated plants show similar growth in the field conditions. Initially, cuttings raised plants showed lesser mean number of shoots, however, no significant variation was noticed after three years of planting. Twenty two species of bamboos were collected throughout the country and assembled as a germplasm bank.

Somatic embryogenesis in D.strictus: Ontogeny of somatic embryos in bamboos was elucidated and revealed that the embryogenic callus originated from scutellar region, where the mode the development of embryos was graminaceous type including globular, scutellar andcoleoptular stages.

Regeneration of protoplasts in E.camaldulensis: A complete protocol for isolation, purification, culture and regeneration of protoplasts from E.camaldulensis was developed. Suspension cultures developed from embryogenic callus originated from cotyledons (in vitro germinated) was found to be most suitable for the production of uniform and totipotent protoplasts. The protoplasts were cultured in KM8p medium and micro callus was regenerated.

Facilities/ Equipment available

Tissue culture laboratory consists of separate rooms for

  1. Washing
  2. Media preparation
  3. Inoculation
  4. Culture incubation
  5. Facilities for hardening of plants include mist chamber, polytunnels and shade house 

Tissue culture laboratory is equipped with

Nanopure water system 
Microwave oven
Refrigerator
Autoclaves

DNA markers

  • The application of DNA markers in tree species include DNA fingerprinting for clonal identification, phylogenetic and diversity analysis, hybrid confirmation, genome mapping, gene tagging and marker assisted selection.

  • We have estimated the genetic diversity existing in different levels of populations including species, provenances and clones of Casuarina and Eucalyptus using RAPD, ISSR, FISSR and AFLP.

  • Simple Sequence Repeats (SSRs) were identified in Casuarina equisetifolia and used in clonal and species identification. The sequence data was deposited in National Centre for Biotechnology Information (NCBI) with Genbank Accession number AY839233.

  • Species – specific ISSR markers were developed in CasuarinaAllocasuarina and Eucalyptus species. The genetic fidelity of micropropagated plants was assessed using RAPD and AFLP markers in Eucalyptus species.

  • Presently we are working towards developing trait specific SSR markers for adventitious rooting in Eucalyptus species and allele – specific gene markers targeting pulping trait in eucalypt species.
  • Genetic Transformation and Functional Genetics

    Mission

    To evolve transgenic approaches in forestry species for genetic modification of desired traits and functional analysis of genes.

     Worldwide, transgenic options are increasingly contributing to development of improved plant varieties. At IFGTB, a breeding-integrated transgenic programme is ongoing for development of varieties and clones with increased salt and pest tolerance. Development of methods for regeneration and transformation of tree species are challenging and significant advances have been made in Eucalyptus camaldulensis. Salt tolerant tree species like Casuarinas are being prospected for important genes conferring salt tolerance for use in marker assisted selection and transgenic programmes. Project on using gene silencing techniques for functional validation of these genes is ongoing. Initiatives are underway in using RNAi for incorporation of resistance against the Eucalyptus pest, Leptocybe invasa. As these take shape, initiatives for engineering other traits like improved productivity under drought conditions, and improved pulping traits will also be taken up through national and international collaboration, in the process, making IFGTB a world-class centre for transgenic studies in trees.

    Achievements

    • In vitroprocess for generation of transgenic composite plants in Eucalyptus for rapid functional analysis of genes and promoters was developed.
    • Standardised regeneration and transformation methods for Eucalyptus.
    • Partially sequenced sodium transporter gene homologues, viz., NHX1 and HKT gene from salt tolerant tree species.
    • Partial sequenced homologues of chitin synthase gene from Leptocybe invasa and Hyblaea pura

    Projects  

    International projects

    One of the four international partners for "Transcriptome analysis of salt tolerance in Casuarina trees" by the  Joint Genome Institute, US along with  the principal collaborator from Research Institute for Development, France, and other partners including Université Chiekh Anta Diop, Dakar (UCAD), Senegaland University of New Hampshire, US.

    Intramural Projects

    • Development of methods for functional analysis of genes involved in salt tolerance in Eucalyptus tereticornis. ICFRE: 30.93 lakhs  (2009-14)
    • Determination of target genes in Leptocybe invasa for engineering resistance in Eucalyptus through gene silencing approaches. ICFRE: 23.50 lakhs (2010-2014)

    Extramural Projects

    • Web enabled database and analysis of gene sequences implicated in abiotic stress tolerance for screening gene homologues in salt tolerant tree species. DBT: 20.096 lakhs (2009-2012)
    • Identification and functional analysis of genes of the teak insect pest Hyblaea puera Cramer (Hyblaedae: Lepidoptera) for the development of gene silencing based pest control. CSIR student project ( 2009-2014)
    • Genetic transformation of Casuarina for functional analysis of genes conferring salt tolerance. CSIR student project (2010-2015)

    Gene mining

    A program on identification and functional characterization of novel genes from tropical tree species is underway.

    The first project initiated in this direction targeted the identification and isolation of pathogen defense related genes from Casuarina equisetifolia. Transcript profiling of the pathogen elicitor treated and untreated cDNA pools using the procedure of differential display of mRNA was conducted and several transcripts were cloned and sequenced. The transcripts over expressed during elicitation included Resistance or R genes, cytochrome oxidase involved in apoptosis and Hypersensitive reaction (HR); cell wall proteins like arabinogalactans; genes involved in systemic acquired resistance (SAR) like chitinase and glucanase; genes induced during symbiosis like nodulin; other pathogen defense –related genes like 26S proteasome, signal recognition particle, cyclin dependent kinase and genes involved during drought stress were also identified. 52 EST sequences have been submitted to NCBI, USA (Gen Bank Acc. No. GR228669 - GR228718 & GR312925 - GR312926). This is the first report on molecular defense in C. equisetifolia and has provided a pool of candidate genes for detailed molecular dissection of the defense mechanisms which may further broaden the knowledge on tree - pathogen interactions.

    Another initiative on identification of differentially expressed cellulose synthase (CesA) genes from the developing secondary xylem tissues of Eucalyptus tereticornis is under progress. Transcripts showing similarity to all the six groups of cellulose synthase super family have been identified. One of the transcript amplified only in the secondary xylem tissues implicating its specific expression during wood formation. This study will open a new dimension of research using CesA as a candidate gene in association analysis for pulping trait.