Some Changes and Transformation in Pea (Pisum Sativum L.) Infected With Mosaic Virus

Virus infection alters the entire metabolism of the host plant by changing the biochemical processes. Carbohydrate, proteinsamino acids and phenols have received considerable attention in relation to resistance in plants against diseases. Duringhost-pathogen interaction, amino acids may act as substrates for the pathogen (Fric, 1964, Titarenko et al., 1993). Increasein the carbohydrate constitution due to severity of the disease may serve as easily metabolized carbon source for the fungapathogen (Patil et al., 1985, Jeun and Hwang, 1991). These effects are brought-about; possibly, through the virus-inducedsynthesis of new proteins by the host, some of which are biologically active substances and can interfere with the normametabolism of the host. Effect of virus pathogen on vegetative growth on plant has been recorded by several workers(Bawden, 1959, John, 1963, Farakas and Solymosy, 1965, Srivastava, 1971, and Ram et al.1984). Based on the above informations, attempts have been made to study the changes in total carbohydrate, protein, amino acidand phenolics in pea plants infected with pea mosaic virus. The key argument against increasing the acreage for growing peas (which is desirable not only for economical but also forecological reasons) is the relatively low yield stability. This, in turn, reduces the grower's confidence in pea as a crop (Hebblethwaite et al. 1985). Being a multifactorial problem, strategies to improve yield stability necessarily have to bemanifold and include: 1. Improvement of harvesting characteristics: e.g., improved standing ability, concentration of the pods at the top of theplant, more pods per node, more seeds per pod and, most important, reduced pod abortion due to stress. 2. Introduction of or selection for resistance against diseases like pea wilt (Fusarium oxysporum and F. solani)powdery mildew (Erysiphe polygoni) and diseases like seedling and foot rot (Aschocyta ssp.), bacterial bligh(Pseudomonas syringae pv. pisi) and common root rot (Aphanomyces eutiches). Of particular and recent importanceare resistances against certain viral pathogens (PSbMV, PEV, PSV). 3. Problems concerning more sustainable techniques in controlling weeds and strategies to implement traits essentiafor surviving climatic stresses. Besides improving yield and yield stability, a second objective will be to optimize the protein and starch quality of peaObjectives in this field will include: 1. Improvement of storage protein composition by increasing the amount of essential amino acids (by addition ocodons, e.g. methionine, or the introduction of heterologous storage protein genes, e.g., 2S-albumin gene). 2. Increase the percentage of amylopectin in the starch. It is noteworthy to acknowledge that through conventional plant breeding more than 1000 varieties have been establishedbut only a few are of commercial importance. To solve some of the problems, genetic engineering may provide some new and promising methods which could speed upclassical breeding to further improve pea as an important crop species.

A major prerequisite for the application of the different methods available for introducing new or foreign genes into a givencrop or to select for desirable traits in vitro is the ability to regenerate fertile plants from complex tissue or isolatedprotoplasts. In a first report by Gamborg et al. (1974), intact plants were regenerated via organogenesis from a maceratedcell mass derived from apical meristems. Kartha et al. (1974) used the complete shoot apical meristems and obtained asomewhat higher efficiency. Malmberg (1979) was able to regenerate pea plants via organogenesis from calli derived fromepicotyl explants after a time-consuming tissue culture, however, with a low frequency. Only a few genotypes responded tothis protocol. Other tissues used for regeneration were immature leaflets (Mroginski and Kartha 1981), mature embryoderived callus (Hussey and Gunn 1984), node ex plants (Griga et al. 1986), immature embryos (Natali and Cavallini 1987)cotyledonary nodes (Jackson and Hobbs 1990), hypocotyl slices (Nielsen et al. 1991), and nodal thin layers (Nauerby et al1991). In contrast, reports on regeneration via somatic embryogenesis are rather limited (Kysely et al. 1987; Tetu et al. 1990and immature zygotic embryos or apical meristems seem to be the only useful explant. No convincing report is available onthe regeneration of pea plants from cell suspensions, although some embryo-like structures expressing storage proteinshave been obtained (Bohmer and Jacobsen, unpubl.).

Plant pathogens and insect herbivores can interact when they co-exist on the same host plant: they might compete directlyfor plant resources or interact indirectly via induced changes in plant morphology, physiology and the activation of plandefences. These „tripartite‟ plant-insect-virus interactions are further complicated when the pathogen is obligately dependenon the insect for its transmission. The overall interaction between the pair of species is now a combination of facilitation othe pathogen by the vector and the varying reciprocal response in the insect, and can lie anywhere along a continuumbetween mutualism (+, +), commensal (+, 0) and contramensal (+, -) (Hodge and Arthur 1996). It can be envisaged that therewould be evolutionary pressures on the pathogen not to be antagonostic towards its insect vector and that those pathogensthat modified plant biology so as to improve vector performance would subsequently be more successful in terms of theirown transmission. Various estimates suggest that aphids account for the transmission of between 25-50% of the plant viruses disseminated byinsects. A number of previous field and laboratory investigations have examined the responses of aphids to infected hosplants . Aphids developing on virus-infected plants have been demonstrated to show reduced, improved or no change in individual and/or population growth rates on infected plants, depending on the system examined. It is often found that thedistribution of aphids exhibits a bias towards virus-infected plants although this is not always the case (see Castle et al1998). There are also reports of increased production of winged alate-form progeny on infected plants, a factor liable toenhance subsequent dispersal of the plant pathogen. Pea enation mosaic virus (PEMV) is a widespread aphid-borne virus that infects a number of leguminous plants, causingstunting and deformation of the plant and mottling and curling of leaves, and the disease can result in severe crop losses (c50%) in beans and peas. PEMV consists of a symbiotic mutualism between an Enamovirus and Umbravirus and istransmitted by a number of aphid species in a circulative persistent (non-propagative) manner. The virus can be acquiredduring access feeding periods of only a few minutes, and after a latent period the aphids can inoculate new plants in bouts ostylet probing less than 30 seconds duration. The pea aphid, Acyrthosiphon pisum , is responsible for the transmission of a number of viruses affecting legume field cropsincluding PEMV. A. pisum has previously been found to show varying responses to single and multiple virus infections oclovers, the response often being dependent upon the stage of infection and severity of disease symptoms . Previously, weexamined the response of A. pisum to PEMV infection of Vicia faba L. and found that although the A. pisum showed cleapreferences for settling on the yellow foliage of virus-infected plants there were no effects on their growth, reproductiveoutput or production of winged progeny. The outcome of many non-trophic interactions between pairs of species can be dependent upon the biotic and abioticenvironmental conditions in which the interaction occurs. In particular, the occurrence of interspecific facilitation is oftenfound to be more prevalent when conditions are marginal for at least one of the species involved, and some abiotic or bioticstress is ameliorated by one species to the benefit of the other. It has been suggested that plant pathogen-induced facilitationof insect herbivores is more likely to occur when the uninfected host-plant possesses high resistance or is in some way aninferior resource to the insects. Vicia faba L. is considered one of the highest quality host plants for A. pisum due to its lowaphid resistance, and it is possible that virus-infection could not improve (or degrade) the resource sufficiently to induceobservable changes in aphid performance.

The effect of virus infection on certain aspects of changes of the host was studied. Ten days old seedlings of the test plantswere taken into two groups of 120 each. The first group of plants was left as healthy control after inoculation with only neutraphosphate buffer, while those of the second group were inoculated with pea mosaic virus. Twenty plants of each group wereharvested on 30,35,40 and 45 day of inoculation. One gram of fresh leaves was macerated with 5 ml of chloroform, methanol and water in ratio 1:1:1. The extract was filteredthrough Whatman filter paper No.1and the residue was extracted with 5ml. of extraction solvent. The extract was pooledtogether to obtain a volume of 10ml. extract. On keeping the mixture for some time the lower layer clearly settled down(chloroform layer) and upper layer (methanol & water layer) was separated. Then, the lower layer consisting of chloroformextract was further evaporated to dryness. The precipitate was redissolved in methanol, water (1:1) total phenolic contentTotal phenol was estimated spectrophotometric ally by Prussion blue method at 700nm as modified by Graham (1992). The upper layer was utilized for the estimation of total carbohydrate (Sadasivam and Manikam, 1996), total protein (Lowry eal., 1951 and Bergersen, 1980) and total amino acid (Yemm and Cocking, 1955). The solvent was evaporated to drynessunder vacuum and redissolved in 5ml. of 0.6mM phosphate buffer (pH 6.2). From this extract carbohydrate, amino acid andprotein were estimated as per the standard protocols (Danial, 1991).

Primary and secondary metabolites viz.carbohydrates, proteins, amino acids and phenols have received considerableattention in relation to resistance in plants against diseases. Total carbohydrate, amino acid and protein in different parts oinfected plants by pea mosaic virus and healthy plants were carried out. Root nodulation and root abnormality were alsoobserved. Carbohydrate amount of healthy and infected leaves, stem and root increased, with the increase in growth oplants. Amino acid amount in different counter parts of diseased plants were higher as compared to healthy. Protein contenwas always higher in infected plant parts (leaf, stem and root) than their healthy counterparts, but maximum protein contenwas found in diseased leaves followed by root and stem) The results revealed, that pea mosaic virus infection was alsofound to reduce the number, size and fresh weight of root nodules. The number of secondary roots and nodules decreasedsignificantly in diseased plants. The phenolic content decreased with the increase in infection in plants. Some prior investigations into plant virus-aphid interactions have suggested that increased alate production on diseasedplants is caused by physiological changes in the host plant, such as modification of nitrogen metabolism and changes inamino acid profile of the phloem sap. Poor nutrition seems an unlikely stimulus for alate production in the system used in thisexperiment, as the results of the aphid performance assays suggested that PEMV-infected peas were, if anything, superiohosts compared to control plants. Also, there was no increase in alate progeny when using a single founding A. pisum indicating that infection of the plants per se (and any associated nutritional differences) did not directly induce production owinged forms. When multiple founding aphids were housed in clip cages the proportion of alate progeny on infected plantswas almost double that observed on the controls. In terms of numbers of aphids, levels of crowding within the clip cageswould be very similar in the control and PEMV-treated plants: the density of founding adults was equal, overall nymphproduction was not affected and any virus-induced increases in aphid size would only be slight within the short duration othe assay. Thus it appears that a combination of factors is required to produce the high numbers of alate progeny observedon the PEMV-infected plants, the effects of maternal crowding being somehow heightened when present in conjunction withhost plant infection. The effects of crowding can be accentuated by higher contact rates resulting from increasedrestlessness of aphids, although this behavioural response was not examined explicitly

Atkinson R.G., Gardner R.C. (1991). Agrobacterium-mediated transformation of pepino and regeneration of transgenicplants. Plant Cell Rep 10: pp. 208–212. Beachy R. (1990). Coat protein mediated resistance against virus infection. Annu Rev Phytopathol 28: pp. 451–474. Binns A.N., Thomashaw M.F. (1988). Cell biology of Agrobacterium infection and transformation of plants. Annu RevMicrobiol 42: pp. 575–606. Broer I., Arnold W., Wohlleben W., Pühler (1989). The phosphinotricin N-acetyltransferase gene as a selectable marker foplant genetic engineering. In: Galling G (ed) Proc Braunschweig Symp on Applied molecular biology. Zentralstelle füWeiterbildung der TU Braunschweig, Germany, pp. 240–246. Byrne M.C., McDonell R.E., Wright M.S., Carnes M.G. (1987) Strain and cultivar specificity in the Agrobacteriumsoybean interaction. Plant Cell Tissue Organ Cult 8: pp. 3–15. Chilton M.D., Drummond M.H., Merlo J.M., Sciaky D., Montoya A.I., Gordon M.P., Nester E.W. (1977) Stable incorporation oplasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11: pp. 263–271. Davies D.R., Hamilton J., Mullineaux P.M. (1992). A progress report on pea transformation. Proc 1st Eur Conf on Grainlegumes 1992, Angers. AEP, Paris, pp. 123–124. Eapen S., Köhler F., Gerdemann M., Schieder O. (1987) Cultivar dependance of transformation rates in moth bean after cocultivation of protoplasts with Agrobacterium tumefaciens. Theor Appl Genet 75: pp. 207–210CrossRef. Gadani F., Mansky L.M., Medici R., Miller W.A., Hill J.H. (1990) Genetic engineering of plants for virus resistance. Arch Viro115: pp. 1–21 Gamborg O.L., Constabel F., Shyluk J.P. (1974) Organogenesis in callus from shoot apices of Pisum sativum L. PhysioPlant 30: pp. 125–128. Griga M., Tejklova E., Novak F.J, Kubalakova M. (1986). In vitro clonal propagation of Pisum sativum L. Plant Cell TissueOrgan Cult 6: pp. 95–104. Hebblethwaite P.D., Heath M.C., Dawkins T.C.K. (1985). The pea crop. Butterworth, London. Hobbs S.L.A., Jackson J.A., Baliski D.S., DeLong C.M.O., Mahon J.D. (1990). Genotype and promoter induced variability intransient β-glucuronidase expression in pea protoplasts. Plant Cell( Rep 9: pp. 17–20. Hood E.E., Fraley R.T., Chilton M.D. (1987). Virulence of Agrobacterium tumefaciens strain A281 on legumes. Plant Physio83: pp. 529–534. Hussey G., Gunn H.V. (1984). Plant production in pea (Pisum sativum L. cvs. Puget and Upton) from longterm callus withsuperficial meristems. Plant Sei Lett 37: pp. 143–148. Janssen BJ, Gardner RC (1989) Localized transient expression of GUS in leaf discs following cocultivationwith Agrobacterium. Plant Mol Biol 14: pp. 61–72. Kartha K.K., Gamborg O.L., Constabel F. (1974). Regeneration of pea (Pisum sativum L.) plants from shoot apicameristems. Z Pflanzenphysiol 72: pp. 172–176 Khetarpal R.K., Maury Y. (1987). Pea seed-borne mosaic virus: a review. Agronomie 7(4): pp. 215–224 Krens F.A., Molendijk L., Wullems G.J., Schilperoort R.A. (1985). The role of bacterial attachment in the transformation ocell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens. Planta 166: pp. 300–308 Lulsdorf M.M., Rempel H., Jackson J., Baliski D.S., Hobbs S.L.A. (1991). Optimizing the production of transformed pea(Pisum sativum L.) callus using disarmed Agrobacterium tumefaciensstrains. Plant Cell Rep 9: pp. 479–483. Malmberg R. (1979). Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L. Planta146: pp. 243–244 Mroginski L.A., Kartha K.K. (1981). Regeneration of pea (Pisum sativum L. cv. Century) plants by in vitro culture of immatureleaflets. Plant Cell Rep 1: pp. 64–66. Natali L., Cavallini A. (1987). Regeneration of pea (Pisum sativum L.) plantlets by in vitro culture of immature embryos. PlanBreed 99: pp. 172–176. Nauerby B., Madsen J., Christiansen J., Wyndaele R. (1991). A rapid and efficient regeneration system for pea (Pisumsativum L.), suitable for transformation. Plant Cell Rep 9: pp. 676–679. Nielsen S.V.A., Poulsen G.B., Larsen M.E. (1991) Regeneration of shoots from pea hypocotyl explants. Physiol Plant 82: pp99–102. Penza R., Lurquin P.F., Fillipone E. (1991). Gene transfer by cocultivation of mature embryos with Agrobacteriumtumefaciens: application to cowpea (Vigna unguiculata Walp). J Plant Physiol 138: pp. 39–43. Potrykus I. (1990) Gene transfer to plants: assessment and perspectives. Physiol Plant 79: pp. 125–134. Pounti-Kaerlas J., Stabel P., Eriksson T. (1989). Transformation of pea (Pisum sativum L.) by Agrobacterium tumefaciensPlant Cell Rep 8: pp. 321–324. Puonti-Kaerlas J., Eriksson T. (1988). Improved protoplast culture and regeneration of shoots in pea (Pisum sativum L.)Plant Cell Rep 7: pp. 242–245. Puonti-Kaerlas J., Ottosson A., Eriksson T. (1992). Survival and growth of pea protoplasts after transformation byelectroporation. Plant Cell Tissue Organ Cult 30: pp. 141–148

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