1 Defence Agricultural Research Laboratory. Pithoragarh, 262 501, Uttaranchal, India. Email firstname.lastname@example.org
It is possible to sustain agriculture at high altitudes if farmers have plants tolerant, if not completely resistant, to low temperature. Genetic engineering is a method that permits induction of cold tolerance in plants where classical and conventional methods have failed. We are developing transgenic tomatoes with the osmotin gene introduced by Agrobacterium mediated genetic transformation and are assessing the feasibility of this method for induction of cold tolerance in plants. The osmotin gene is known to impart tolerance to salinity, drought and fungal attack. It is quite possible it will impart tolerance to cold also. Preliminary tests have revealed transgenics with the osmotin gene are more tolerant of cold than wild types.
Tomato plants have been introgressed with the osmotin gene. Preliminary data indicate that modified plants are more tolerant of cold than wild types.
Lycopersicon esculentum, transgenics, osmotin, cold tolerance.
Sustainable agriculture is today’s word. How is it possible to sustain the already existing agriculture and develop more of it? Land for agricultural production is available, but the current proportion under cultivation can no longer be substantially increased without taking massive ecological risks and the requirement for enormous capital investment, technical upgrading, innovation and energy. Also, the agriculturally usable land per capita is continuously decreasing. Global requirements for food, fuel and fodder now essentially need to be fulfilled by a comparable increase in yield, or by cultivating land not previously suitable for agricultural production such as high altitude regions. But nature has imposed certain limitations in these regions, namely abiotic stress factors like cold, frost, and limited radiation and water.
Temperature dependant changes in the structure of plant membranes are now held to be mainly responsible for the cold sensitive physiological function. In low temperature, conductivity of critical membranes decreases, viscosity of water increases and the hydroactive closure of stomata are inhibited, and thus water stress also occurs. In frost, the temperature falls below 0°C and water freezes in cells. This leads to loss of a range of essential physiological functions. Many abiotic stresses like drought, cold, salinity have a common consequence of osmotic stress or water deficit. The response of plants is the synthesis and accumulation of osmoprotectants to save them. Any change in the plant membrane structure, availability of osmoprotectants in cell plasma, molecules to tolerate stress can help make the plant tolerate, if not resist, abiotic stress.
A number of classical methods already exist that have helped agriculture by making plants tolerate stress. For example, the use of plant growth retardants can inhibit axial growth and delay senescence without damaging the plant, and even stimulate resistance to cold, heat, SO2, and fungal infection (Grossmann, 1989). However, the opportunities for such improvements are sometimes limited by a lack of naturally available genetic variability. Genetic engineering of plants is the new road to enhance, develop or modify tolerance to stress in high altitude regions. This, in turn, may enhance cropping intensity and crop yields of target crops.
Transgenic tobacco plants generated having P5CS enzyme (γ-pyrroline-5-carboxylate synthetase) showed enhanced expression of this enzyme and 10-18 fold more proline on giving salt and drought stress (Kishore et al. 1995). Tarczynski et al. (1993) showed that transgenic tobacco with bacterial mannitol –1-phosphate dehydrogenase gene was more resistant to osmotic stress. Research has been conducted in over expressing a fusion of DRE-containing promoter from a dehydration induced gene (rd29A) with a DREB gene in Arabidopsis that resulted in tolerance of plants to freezing, water stress and salinity (Kasuga et al. 1999). The ability of a plant to scavenge toxic oxygen species is considered to be critical for abiotic stress tolerance. Hence, the genes for superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase all contribute to tolerance of abiotic stresses. In short, there are numerous genes now available that can help tolerate cold. Zhang and Blumwald (2001) made transgenic tomatoes tolerant to salt using vacuolar Na+ / H+ antiport gene. Fan et al. (2002) cloned an antifreeze gene from carrot and successfully transferred it into tobacco.
Our laboratory is interested in the development of vegetables tolerant to cold stress that may grow at high altitudes. Of the many high altitude regions across the globe, Ladakh region in India is a cold desert consisting of Leh and Kargil districts, covering an area of 97,000 sq. km. This cold desert is characterized by hostile climatic conditions, high altitude and remoteness. The region is an elevated territory presenting a system of alternating valleys and mountains except in Eastern part, which is a plateau. These ranges are a barren, rocky surface largely devoid of vegetation cover. Human settlement and agriculture is confined between 2500 m and 4500 m above mean sea level. Poor precipitation and low temperatures are the most factors impeding crop cultivation. Although agriculture provides a range of staple foods including vegetables, fruit, milk, wool and meat, the yield is limited and dependant on trench cultivation methods. There is an increasing need to develop vegetables that are tolerant to cold.
The Defence Agricultural Research Laboratory, Pithoragarh initiated work in this direction. The strategy employed was Agrobacterium- mediated genetic transformation of tomato using osmotin gene. The research work includes assessment of osmotin as a potential gene for imparting cold tolerance to plants. To date, no work has been reported on the use of osmotin for cold tolerance.
Osmotin is a stress responsive protein adapted to NaCl and desiccation. Singh et al. (1985) studied the protein in tobacco var. Wisconsin 38 and gave the name to a basic 24 kDa protein that accumulates in cells on osmotic stress adaptation. Osmotin was seen to accumulate in salt adapted cells (Bressan et al. 1987). Woloshuk et al. (1991) and Vigers et al. (1991) demonstrated in vitro that osmotin has antifungal activity against a variety of fungi including P. infestans, Candida albicons, Neurospora crassa and Trichoderma reesi. Barthakur et al. (2001) gave evidence that over expression of osmotin induces proline accumulation and confers tolerance to osmotic stress in transgenic tobacco. It has been hypothesised that the synthesis of osmotin protein could induce synthesis and accumulation of certain solutes or could be involved in metabolic or structural changes. (Singh et al. 1987). Starting from here, we aim at developing transgenic tomatoes using osmotin gene and testing their efficacy for cold tolerance.
Tomato cultivars nationally recommended for cultivation in high altitude regions were chosen. Of these, one with moderate inbuilt resistance to cold was chosen for research work so that the impact of integrated desired gene could be assessed in transformed plants. This was done deliberately because if an already good, cold tolerant cultivar had been chosen, then small, valuable, positive effects of the transferred gene would not be observed. The cultivar chosen was Pusa Ruby.
Fig.1. The osmotin gene construct
Initially the protocols for callus formation, regeneration and hardening in the tomato cultivar was standardized using cotyledonary leaf as the explant. A number of plant growth hormones were tested, with reference to available literature. Jia et al. (2002) used IAA, BAP and zeatin in combination for callus induction and shoot regeneration; Chesnokov et.al. (1995) used 2,4-D and isopententyl adenosine for the same; Davis et al. (1991) used zeatin alone, Barg et. al. (2001) and Hamza et al. (1993) used zeatin in combination with IAA while Frary et al. (1996) used only kinetin. Hormones were checked individually or in combination throughout a range of concentrations following the checkerboard method. Thereafter, antibiotic dosage sensitivity tests were done to assess the effect of antibiotics on calli formation and regeneration of explants. The antibiotic cephotaxime, used to prevent overgrowth of Agrobacterium in infected explants and kanamycin, the selectable marker antibiotic were tested for within a given dosage range.
A literature survey revealed immense variation in the method for Agrobacterium- mediated genetic transformation in tomato itself (Jia et al. 2002; Davis et al. 1991; Barg et al. 2001; Hamza et al. 1993 and Frary et al. 1996) and adopted the following protocol for Agrobacterium mediated genetic transformation of tomato.
Cotyledonary leaves were used as explants and infected with Agrobacterium culture. The bacterial culture was grown overnight, cells harvested, and volume made up so that the optical density (O.D.) of infection media was in the range of 0.1-0.6. Different batches of explants were infected with different O.D. of bacterial culture. Infected explants were co-cultivated and then transferred to selection media having required antibiotic dosages. The media was changed at least once a month. Fully regenerated tomato plantlets that had survived primary screening were hardened in hardening mixture and kept in a containment facility.
Molecular analysis of regenerating transformed tomato plants was tested using a polymerase chain reaction (PCR) method using gene specific primers, here osmotin and others like nptII and promoter, CaMV 35S.
Morphological characters like plant growth habit, leaf type, color, pubescence and foliage cover, stem type, thickness, pubescence and pigmentation, flower size and color, style position, fruit size, shape, color, surface, weight, yield per plant, pulpiness, firmness, locule number per fruit, days to flowering, days to first fruit set, first fruit harvesting, number of clusters per plants, flowers per cluster, plant height and, number of primary branches were studied in both wild as well as T0 plants.
Total protein was extracted using standard Bradford method (Bradford, 1976). Extracted protein was used for dot blot analysis using antibodies against the osmotin protein. Proline was extracted from normal and stress induced plants using the method of Bates (Bates et al. 1973). Physiological parameters were analyzed using the Leaf Chamber Analyzer (AC Bioscientific Ltd.). Electrolyte leakage tests were done using the Digital portable kit CK-704, India.
In the laboratory, callus induction and regeneration of tomato cv. Pusa Ruby was best in zeatin alone at 1.0 ppm, which supports the results of Davis et al. (1991). Agrobacterium mediated genetic transformation successfully gave transformed tomato plants that survived primary screening of antibiotics and regenerated nicely.
Molecular analysis by PCR of the regenerating transformed tomato plants (T0 generation) revealed presence of osmotin gene (Fig 2), nptII gene and CaMV 35S gene. Though ideally all genes between the right and left border of the T-DNA are transferred, we found recombinants in developed regenerated plants. Some plants analysed had all the three genes while some had one or more of them missing. PCR analysis has been done using genomic DNA extracted from leaves being bulked from all sides of the plant in order to reduce the possibility of mosaic transgenics. Results from Southern analysis and RT-PCR analysis are pending.
Fig.2. PCR of Osmotin gene in transformed plants
Morphological analyses of regenerating T0 tomato plants show remarkable variation with wild ones in some cases while others reveal similar traits. Fruiting pattern and fruit morphology has been under observation giving expected results, some similar and some dissimilar to wild.
Biochemical analysis has been done for osmotin protein and proline content. Varied expression of osmotin protein was observed on dot blot analysis. Proline content was higher in transformed plants under normal and stress conditions. Salt stress given to leaf discs from regenerated tomato plants gave a range of results. Enzyme analysis of transformed plants is planned.
Physiological analysis by electrolyte leakage tests, leaf chamber analyser parameters, stomata number and morphology, reveal relevant results on transformed tomato plants given cold stress.
In short, the transgenic approach embarked up on to assess the function of osmotin gene under cold environment conditions by developing transformed tomato plants developed using osmotin gene are responding to cold stress in a positive manner. Further analysis of the progenies of transformed tomato plants for molecular, physiological, biochemical and morphological parameters will provide further evidence of the effectiveness of the osmotin gene in cold stress conditions.
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