While working on genetic diversity studies amongst the mangroves, it was felt that these unique systems offer an amazing potential in harnessing genetic combinations that could help susceptible crop species cope with increasing salinity in the soil due to global warming and sea level rise. Therefore, MSSRFundertook an anticipatory research programme for developing location specific crop varieties offering tolerance/ resistance to coastal salinity. Significant leads have been obtained in this area of research. With limited resources, and sustained funding by the Department of Biotechnology, the group has been able to isolate and characterize a number of promising genes, and promoters with implications on abiotic stress tolerance and has also mobilized some of these genes into transgenic systems. Limited field trials of the transgenic rice varieties containing mangrove genes have been undertaken under contained conditions. The group has already 36 full-length genes isolated from the mangrove species, Avicennia marina; as also a number of partial clones for abiotic stress conditions. Eight patent applications has been filed. This research work has been rated as among the ten most important scientific work carried out during the last decade in India, which can change our lives for better (Outlook -18t11 July 2005).

On a competitive bidding process, MSSRF has been supported under the Centre for Excellence programme of the Department of Biotechnology, Govt. of India.

Genes for Abiotic stress from Avicennia marina, Prosopis juliflora, and Porteresia coarctata : Avicennia is a monotypic pantropical mangrove genus with eight species, of which A. marina is widely distributed both latitudinally and longitudinally. The high salt tolerance of A. marina is a consequence of water use efficiency, which balances the relation between carbon gain, water loss, and ion uptake with the transpiration stream on a low but constant level. A. marina grows in coastal regions where the salt concentration can be as high as 9%. The regulation of inorganic ions occurs partially by exclusion at the roots and also by excretion via salt glands, the excretion rate for sodium and chloride ions being 0.41 mol m-2 S-I and 0.0461 mol m-2 s-1,respectively. It is thus an ideal candidate plant for mining genes for salt tolerance.

As a first step towards the characterization of genes that contribute to combating salinity stress, we have constructed a cDNA library from A. marina. Random expressed sequence tag (EST) sequencing of 1,841 clones produced 1,602 quality reads. These clones were classified into functional categories, and BLAST comparisons revealed that 113clones were homologous to genes earlier implicated in stress responses, of which the dehydrins are the most predominant in this category. Of the ESTs analyzed, 30% showed no homology to previously uncharacterized genes in the public plant databases. Of these 30%,52 clones were selected for reverse Northern analysis: 26 were shown to be up-regulated and five shown to be down-regulated under stress. The results obtained by reverse Northern analysis were confirmed by Northern analysis.

Prosopis Jllliflora, a leguminous a phreatophytic perennial deciduous thorny small tree is now the dominant woody plant found in about 45 million ha of grazing lands worldwide from sea level to 1500 m. It grows in very hot and dry regions with temperatures as high as 48°C and annual precipitation of 150-750 mm. The roots penetrate to great depths in the soil and the tree can grow in a variety of areas including saline, alkaline, sandy and rocky soils. An introduced species in .' India, it has subsequently spread aggressively across the country. P. Jllliflora can withstand high leafto- air vapour pressure deficit (VPD) by reducing metabolic activity and by effective adjustments in the partitioning of electron flow between assimilation and non-assimilation processes, which in turn imposes a strong limitation on the potential carbon gain. P.Jllliflora is also heavy metal tolerant and due to the heavy metal accumulating property of P.Jllliflora, it has been suggested as a green solution for soils contaminated with cadmium, chromium and copper. The plant, therefore, is an ideal candidate for mining genes for abiotic stress tolerance including drought tolerance.

Random expressed sequence tag (EST) sequencing of 1750 clones produced 1467 highquality reads. These clones were classified into functional categories, and BLAST comparisons revealed that 114 clones were homologous to genes implicated in stress response(s) and included heat shock proteins, metallothioneins, lipid transfer proteins, and late embryogenesis abundant proteins. Of the ESTs analyzed, 26% showed no homology to previously pncharacterized genes in the databases. Fifty-two cl,ones from this category were selected for reverse Northern analysis: 21 were shown to be upregulated and 16 downregulated under stress. The results obtained by reverse Northern analysis were confirmed by Northern analysis. Two of the abundant genes coding for a nonspecific lipid transfer protein and late embryogenesis abundant protein were sequenced completely. Northern analysis (after polyethylene glycol stress) of the 2 genes was carried out.

Antiporters

The ion pump, vacuolar H+-ATPase (V-ATPase) plays a major role in the maintenance of cellular pH, and influences the transport of cations into the vacuoles of plant cells. A cDNA clone (PcVHAc1) encoding the c subunit of V-ATPase was isolated from the salt-tolerant wild rice, Porteresia coarctata. The DNA sequence of PcVHA-c1 showed significant homology with V-ATPase subunit c of rice. The deduced amino acid sequence of PcVHAc1 and other reported c subunits were compared, and sequence relationships have been drawn to know their genetic relatedness. Southern analysis suggested the presence of multiple coding regions for subunit c in P. coarctata. Northern and Western analyses of salt-treated P. coarctata plants revealed that subunit c of V-ATPase was upregulated by NaCl treatment at both transcriptional and translational level.

Reactive Oxygen Species genes

Antioxidant enzymes play an important role in conferring abiotic stress tolerance. Superoxide dismutase (SOD) is the first enzyme in the enzymatic antioxidative pathway. Halophytic plants like mangroves have been reported to have a high level of SOD activity, which plays a major role in defending the mangrove species against severe abiotic stresses. We have isolated of Sod 1, a cDNA encoding a cytosolic copper zinc super oxide dismutase from the mangrove plant Avicennia marina and studied its mRNA expression pattern during various oxidative and abiotic stresses. The cDNA was cloned into a binary vector (pCAMBIA 1300) and transformed into indica rice. Southern hybridization analysis of transgenic rice plants revealed stable integration of the Sodl trans gene in the rice genome. The mRNA transcript of Sodl was detected by Northern hybridisation in the transgenic rice plants. SOD isozyme assay of the transgenic rice plants revealed the stable expression of the transgenic Sod1 protein. The transgenic plants withstood salinity stress of 150 mM of NaCI for a period of eight days while the untransformed control plants wilted at the end of the stress treatment in hydroponics. The transgenic plants also revealed better tolerance to drought stress in comparison to untransformed control plants.

To investigate the functions of antioxidative enzymes in a mangrove plant, we isolated three cDNAs encoding cytosolic Cu-Zn SOD (Sod 1), catalase (Cat 1) and ferritin (Fer 1) from Avicennia marina cDNA library. Sod 1, Cat 1and Fer 1 cDNA encoded full-length proteins with 152, 492 and 261 amino acids respectively. We studied the expression of these antioxidant genes in response to salt, iron, hydrogen peroxide, mannitol and light stress by mRNA expression analysis. Cat 1, Fer 1 showed short-term induction while Sodl transcript was found to be unaltered in response to NaCl stress. A decrease in mRNA levels was observed for Sod 1, Cat 1 while Fer 1 mRNA levels remained unaltered with osmotic stress treatment. Sod 1, Cat 1and Fer 1 mRNA levels were induced by iron, light stress and by direct HP2 stress treatment, thus confirming their role in oxidative stress response.

Ascorbate peroxidase has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPXl, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. The peroxisomal localization of Am-pAPXl was confirmed by stable transformation of the GFP- (Ala)lO-Am-pAPXl fusion in tobacco. RNA blot analysis revealed that Am-pAPXl is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron).

Transcription factors

Fifteen putative transcription factor cDNA clones were identified from the sequenced A. marina ESTs. These transcription factors were categorized into five major groups based on the domain it constitute. These transcription factors are MYB, NAC, AP2/EREBP, bZIP and Zinc finger and are known to play a vital role in abiotic and biotic stress tolerance. There is very limited report on role of MYB, bZIP and plant specific NAC transcription factors from mangroves with respect to stress tolerance. Northern analysis was done to further characterize two cDNAs PA272 and PD126 coding for MYB and NAC transcription factors respectively. Genomic clone of Am-AmMYB 1 that is 1500bp in length was isolated using AmMYBlcDNA forward and reverse VTR primers. AmMYBl cDNA coding for MYB transcription factor was cloned in pCAMBIA1301 binary vector under control of 35SCaMV promoter (Constitutive) and mobilized into Agrobacterium tumefaciens strain LBA4404and was used to transform Nicotiana tabacum cv. Petit Havana by the leaf disc method. Preliminary selection of transformed plants were selected using Gus staining and PCR analysis with AmMYBl gene specific primers and further characterizations of this gene in contribution to different abiotic conditions are in progress.

NAC transcription factors are a family of functionally diverse proteins responsive to biotic and abiotic stresses. A full-length cDNA isolated from the salt stressed mangrove plant Avicennia marina showed high sequence identity to NAC proteins induced upon abiotic stress in tomato and potato. The predicted protein sequence had all the highly conserved sub domains characteristic of NAC domain containing proteins. Northern analysis for AmNACl expression under tolerable (250mM) concentration of NaCl revealed up regulation of the transcript after 48 h and higher transcript level after 10 days of treatment. Induction of AmNAC1 after 12 h of ABA treatment was similar to the treatment with stressful (500 mM) concentration of NaCl. The results suggest the involvement of AmNAC1 in early salt stress response and long-term adjustment to salt, besides a role for ABA in its expression under salt stress conditions.

Genes for bioremediation

Prosopis juliflora is a tree species that grows well in heavy metal laden industrial sites and accumulates heavy metals. To understand the possible contribution of metallothioneins (MTs) in heavy metal accumulation in P. juliflora, we isolated and compared the metal binding ability of three different types of MTs (PjMT1-3). PjMT1 and PjMT2 are induced by copper and zinc respectively while copper, zinc and cadmium induce PjMT3. Variation in induction of PjMTs in response to metal exposure and their differential binding to metals suggests that each MT has a specific role in P. juliflora. Of the three MTs analyzed, PjMT1 shows maximum heavy metal sequestration and is thus a potential candidate for use in heavy metal phytoremediation.

Promoters

Promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters are typically located upstream of the genes they regulate. For some traits, the expression pattern desired is constitutive, sometimes constitutive expression may be detrimental and expression limited to specific organs. Wide variety of promoters has been described from crop species, but relatively few have been tested for use in crop plants. Using β-glucuronidase (GUS) reporter gene fusions, we have characterized the expression patterns in transgenic tobacco of several nominally strong constitutive promoters, several number of gene promoters, inducible promoters from heterologous species such as Avicennia marina, Porteresia coarctata and Prosopis juliflora.

Promoter sequences upstream of PcNHX (Na+/H+ antiporter) from P. coarctata; PR244, a salt inducible gene from the mangrove A. marina; MYB1 transcription factor from A. marina; Ascorbate peroxidase from A. marina; Lipid Transfer Proteins from P. juliflora; atypical LEA protein from P. juliflora; Photosystem II PsbR from P. juliflora; Metallothioneins from P. juliflora are being characterized.

Identification of Genes that are Uniquely Regulated during Oil Biosynthesis in Jatropha curcas seeds

Fossil fuel resources are reaching finite limits and a sharp increase in oil prices all over the world has resulted in an unprecedented impetus for research on alternative sources of fuel, especially from that of plants. Many plants have the property of accumulating oil in the seeds. Jatropha curcas is one such oil yielding plant that has gained widespread attention in recent times as a source of biodiesel. In addition to many factors that influence seed oil yield; regulation of genes that control the expression of proteins that bring about oil accumulation in seeds is one very basic factor that differentiates a high oil yielding plant from that of the average.

In order to identify genes that control active oil accumulation, J. curcas seeds were grouped into four maturation stages as follows: Early maturation stage, mid-maturation stage, late-maturation stage and full-maturation stage. Since protein expression will be more active when the oil accumulation is still progressing, mid- and late-maturation stages of seeds was used for the identification of genes. The experimental design essentially involved ‘subtraction’ of genes which are common to the stages mentioned above wherein genetic information pertaining to late stages of oil biosynthetic pathway would only be left out as ‘unique’, for further analyses. In this regard, a cDNA subtraction library consisting of ‘unique’ information that relate to genes involved in seed oil biosynthesis in J. curcas was constructed last year. To identify and characterize unique information, annotation of the same was performed through sequencing of a small portion of the ‘expressed’ genetic information called, Expressed Sequence Tag (EST). Information annotation of about 300 ESTs have been performed till date, of which, a few show homology to genes that have already been reported to be involved in the regulation of oil biosynthesis in other plants.

Standardization of tissue culture methodologies for transforming J. curcas plants through gene transfer has been initiated using emerging cotyledons as starting plant material. Tissue culture methodology development will aid in transferring genes controlling active oil accumulation into J. curcas to obtain high and uniform oil yield.

Flower and sex-specific genes

In an effort to identify flowering as well as putative sex-specific genes, a cDNA library was constructed by isolating good quality, high concentration total RNA from a fresh flower of Pandanus using a modified LiCl method. A total of 979 flower-specific ESTs were thus obtained. CAP3 analysis performed on the above data set grouped them into 82 contigs (549 ESTs) and 430 singlets. Approximately 512 unigenes were identified. Important genes like Dehydrin, metallothionein, LEA, GST, Serine protease inhibitor, ATPases, chaperons, transcription factors, Zinc finger proteins, cellular transport proteins, ribosomal proteins, proteins involved in carbohydrate metabolism, lipid metabolism, proteolysis etc. have been isolated from the library. In addition to these several putative sex-specific genes like pollen specific protein, Arabinogalactan, MADS-box transcription factor, LIM transcription factor, profilin etc were also isolated. Expression studies (Northern) and Southern hybridizations are currently being performed to identify putative sex-specific genes in this plant.

Development of Transgenics

Salt and drought stress treatment: Seeds collected from the transgenic plants were germinated on MS medium and grown for a period of 2 weeks in 16 h light/8 h dark cycle. Then the plants were transferred to Yoshida medium maintained at pH 5.0 and grown for a period of three weeks in a hydroponic solution, in a green house with 12 h dark/light photoperiod. After three weeks of hydroponic growth the nutrient solution was replaced with fresh solution consisting of 150 mM NaCl to study the effect of salt stress on the transgenic plants.

For pot experiments both transgenic and control plants were used for the study. Plants of untransformed control and homozygous transgenic plants of both the lines were used for the pot experiments of salinity and drought stress experiments respectively. For pot experiments 40 day old plants, grown in pots were watered with salt water (100 mM NaCl) every four days. For drought stress treatment, the transgenic plants were grown in pots for a period of fifty days with normal watering everyday. Drought stress treatment was provided by withholding irrigation. Two days prior to the drought stress treatment the irrigation was stopped to ensure the soil was dry and no moisture content was present in the soil. The drought treatment was done for two cycles of 100 h of drought stress in each cycle and the plants were irrigated once between the two cycles.

Three limited field trials were also conducted with the approval of the regulatory authorities of Govt. of India. This is the first ever report on the increased tolerance to salinity stress and drought stress by over expressing a cytosolic SOD in rice. Our study reconfirms the importance of cytosolic Cu/ZnSOD isoform in conferring abiotic stress tolerance in plants SOD genes exist as multiple gene families in mangroves. Isolating the genes coding for different isoforms of SOD from A. marina and other mangroves and studying the gene regulation of these isoforms would provide more input on the role of SOD enzyme in providing protection to the mangrove plants against abiotic stresses. These studies would in turn throw light on how these genes can be utilized for transforming to crop plants for enhancing their abiotic stress tolerance. This happens to be the first report on the transfer of a gene isolated from a mangrove to rice, which is a crop plant. The study revealed that mangroves could be potential sources for isolating novel genes for abiotic stress tolerance given the fact that they are halophytes and is capable of tolerating a high degree of salinity and abiotic stress.

Introgression to Local varieties: Once the trangenics are generated they could be crossed using breeding technologies, introgressed into any related varieties to create a diverse background for the transgenics. From the population of 200 BC2F1 plants in each variety of rice, the positive plants (50%) which possess the transgene were identified initially by PCR analysis and among them those plants with a higher resemblance to the respective local variety were identified by phenotypic selection and tagged, and the presence of the gene was confirmed by Southern blot analysis. The expression of the gene was confirmed through the isozyme analysis in the case of AmSOD. Such plants were used as the female parent; pollen from the respective local variety was dusted and BC3F1 seeds were obtained. The BC3F1 plants were screened as per the same procedure followed for BC2F1 plants, crossing was done and BC4F1 seeds were obtained. BC4F1 plants are being screened through PCR analysis for identification and selection of positive plants to serve as female parents for crossing. In addition, a few panicles in the BC3F1 plants, used as female parents for crossing, were selfed and the seeds were collected. These seeds were sown to get the BC3F2 generation. Twenty plants are maintained in the BC3F2 generation and the expected Mendelian ratio of 3:1 was observed with respect to the corresponding transgene. Homozygous plants will be identified in the next generation and subjected to stringent screening for salinity and drought stress tolerance.

Co-expression of Active Oxygen Species (AOS) scavenging genes in rice and evaluation of the same for increased salinity tolerance: In Agrobacterium mediated transformation of pCAM Am-APX + Am-MDAR in rice, two lines regenerated under hygromycin selection. PCR analysis for Am-APX and Am-MDAR revealed that line DC1 was positive for both genes, the 1.1kb and 1.9kb fragment specific to Am-APX and Am-MDAR respectively but in DC2 only MDAR was amplified but not APX. This might be due to partial removal of T-DNA during infection. Genomic DNA was isolated from DC1, DC2 and untransformed control and digested with Sac I (linearises the T-DNA) to determine the copy number of integration of Am-APX and Am-MDAR in the rice genome.

Twenty-four seeds (T0 generation) were sown from the DC1 lines. DNA was isolated from all the lines and PCR analysis revealed that among the 24 lines, 18 lines were positive for both APX and MDAR. The results revealed that the segregation of DC1 in T1 generation was in 3:1 Mendelian ratio.