♦ Fluorescent in situ hybridization revealed that Erianthus arundinaceus x S. spontaneum hybrid CYM 04-420 (2n = 62) had 30 chromosomes from E. arundinaceus and 32 chromosomes from S. spontaneum. From putative inter-generic hybrids involving Erianthus sp. and S. robustum, genuine hybrids were identified using microsatellite, ITS 1, ITS 2 and 5s rDNA primers. The microsatellite markers could be used for identification of hybrids of the inter-generic hybrids S. officinarum x Erianthus, S. robustum x Erianthus and Erianthus x S. officinarum with sugarcane. Sugarcane microsatellite primers were used for association mapping and few markers specific to red rot resistant and susceptible varieties were identified. Many inter-specific and inter-generic hybrid clones were evaluated at Karnal for top borer and stalk borer resistance and less susceptible clones were identified.
♦ 27 progenies obtained from sugarcane X Sorghum hybrids were characterized morphologically and using Sorghum specific markers, confirming their hybridity.
♦ Three new sugarcane specific drought responsive candidate genes viz. DREB360 LEA385 CALMOD400 were identified based on their presence or absence in drought tolerant parent and progeny through RT-PCR techniques.
♦ Species and genus-specific micro satellite markers were developed and used in the identification of inter-specific and inter-generic hybrids.
♦ Isolation and characterization of Resistance Gene Analogue sequences from sugarcane was done by designing primers on conserved domains of resistance gene sequences. Twenty nine primers were designed and were used to amplify genomic sequences of two varieties Bo-91 and CoC 671, that are resistant and susceptible to red rot respectively. About 54 RGA sequences have been submitted at GSS database in NCBI genbank.
♦ Identification of candidate genes for drought resistance is under progress.
♦Identification of Erianthus specific markers has been carried out that will be useful in identifying true hybrids among the progenies of crosses involving Erianthus spp.
♦ A multiplex reverse transcription polymerase chain reaction (M-RT-PCR) was developed for the detection of sugarcane mosaic virus (SCMV), sugarcane streak mosaic virus (SCSMV) and sugarcane yellow leaf virus (SCYLV), three of the major RNA viruses widely prevailing in the sugarcane growing regions around the world.
♦ Molecular variability based on 5.8s-ITS in C.falcatum distinguished 80 isolates into three different groups.
♦An exhaustive study on molecular basis of red rot resistance in sugarcane was carried out.
♦ Molecular variation in sugarcane viruses – Detailed characterization of sugarcane mosaic virus (SCMV) from India revealed that our isoloates do not share close homology with established type strains SCMV-A, B, D,E and SC reported from other countries. We have grouped Indian SCMV isoloates into nine new SCMV strains and occurrence of these strains inother parts of the world is not known. Characterization of 22 new sugarcane yellow leaf virus (SCYLV) from India based on coat protein sequencing established the occurrence of three SCYLV genotypes viz., CUB, IND and BRA-PER in India. Among them, SCYLV-IND is found only in India and the other two genotypes are reported from other countries.
♦ The first confined field trial to evaluate sugarcane transgenics expressing insecticidal proteins (cry1Ab and aprotinin) was conducted in which the yield and quality parameters at harvest did not show variation among transgenics and control. Due to the general low level of borer incidence in the habitat, the differential response of transgenics could not be established. The dead-heart levels showed significant negative relationship with the leaf Cry content indicating the ability of the toxin to inhibit borer activity even at the moderate range of pest activity. The study on inter-node borer incidence in the trial indicated the possibility that Cry1ab was not antagonistic to inter-node borer.
♦ A new constitutive promoter isolated and characterized from a wild species is being studied for its expression pattern. Besides, two other stem and root specific promoters each were designed and the constructs made were transformed in sugarcane, rice and tobacco for expression studies. High expression of wound inducible gene PR10 was observed and cloning and sequencing of the upstream of this gene showed the presence of different putative promoter elements.
♦ Expression profiling of genes involved in sucrose metabolism indicated differentially regulated transcript levels of SPS, SPP and invertases (SAI and CWI) between high and low sugar commercial sugarcane hybrids.
♦ Out of three 5’ upstream sequences of the three different ubiquitin genes isolated, two were cloned in pCAMBIA vector in place of the CaMV35s promoter so as to get a gene regulatory region – gus fusion. Constructs with regulatory region-I were used for transforming sugarcane, rice, tobacco and Arabidopsis through Agrobacterium / biolistics with appropriate controls for validation studies. This regulatory region could drive the gus gene expression in rice and sugarcane tissues but not in tobacco and Arabidopsis, indicating monocot specific nature of regulatory sequence. With a view to understand the role of different domains of 5’ upstream region in gene expression, six deletions of this region were cloned in pCAMBIA vector for transformation studies.
♦ Thirty-nine transgenics of two varieties i.e. Co 86032 and CoJ 64 with cry1Ab alone or aprotinin genes, whose cry1Ab expression was demonstrated through Western analysis of which the toxin was quantified using ELISA earlier, were bioassayed against shoot borer under green house conditions. In tests with neonate larvae of shoot borer, compared with the untransformed control plants transgenics produced considerably lower percentage of deadhearts, despite suffering feeding damage by the borer. Transgenics produced through particle bombardment or Agrobacterium mediated transformation did not differ in the extent of shoot borer damage, despite higher expression ofcry1Ab in the latter method. Reduction in borer damage in aprotinin-expressing sugarcane pyramided with cry1Ab indicated compatibility of the two genes. Expressed cry1Ab content was negatively related to dead-heart damage caused by the borer in some groups of transgenics.
♦ Cloning and tissue specific expression studies of a new promoter – Upstream of one of the ubi genes with 1929 bp was cloned and sequenced. Analysis of the sequence data has shown that the immediate upstream of the start codon of the gene has introns and exons consisting of 1653 bp and 37 bp of leader sequence. Above it is the promoter sequence of 239 bp. All together formed the regulatory system and the promoter sequence has the promoter elements – TATA box and CAT box- and also cis activating sites for root, guard cell and xylem specific expression. For validation studies tobacco were transformed with the pCAMBIA 1305, where gus gene is driven by the new promoter. In tobacco, some tissue specific expression (guard cells, xylem and root) of the gus gene was observed.
An improved method of plant tissue culture with beneficial bacteria for faster multiplication has been identified. These beneficial bacteria are capable of producing wide range of amino acids, phyto-hormones and vitamins supporting and enhancing growth of plants under tissue culture with no harmful effect on tissue cultured plants. This procedure resulted in 20% reduction in production cost with faster rate of in vitro multiplication, and more shoots produced with substantial reduction in the media ingredients such as mineral salts and phytohormones.