|Keywords (Extracted from title, table of contents and abstract of thesis)
SUGARCANE, MOSAIC VIRUS, SACCHARUM OFFICINARUM, INVITRO MERISTEM, CALLUS CULTURE, mosaic, streak, sereh, Fiji, ratoon stunting, organogenesis, embryogenesis
More than 50 viruses and mycoplasma are known to infect cereals. Sugarcane alone is affected by at least five major virus diseases, mosaic, streak, sereh, Fiji, and ratoon stunting. High susceptibility of sugarcane to mosaic virus causes huge loss (30-40%) in net productivity of this crop in Pakistan. Novel biological techniques like tissue culture offer tremendous prospects in the field of agricultural research. The present study deals with the application of in vitro methods for sugarcane improvement. In vitro techniques ware successfully utilised for the efficient regeneration of sugarcane mosaic virus free stock through apical meristem, organogenesis and embryogenesis in one of the locally cultivated mosaic virus susceptible variety Col-54. Amongst the leaves, apical meristem and pith, the leaves proved to be the best explant source for callus induction and subsequent regeneration. The optimum temperature for callus induction and multiplication was worked out to be 28±1oC for all explants. A significant increase in callus induction and proliferation was noticed in dark incubated cultures than the cultures grown in light. Among the auxins, 2,4-D was most effective for callus formation. The optimum callus formation occurred at 3 mg/l 2,4-D. Addition of cytokinin alone or in combination with auxin did not cause any promotive effect. Two prominent callus types, white compact and friable developed from the cultured explants which exhibited different morphogenic response.
In vitro regeneration from sugarcane callus was obtained via organogenesis and embryogenesis. Shoot and differentiation through organogenesis was obtained both under hormone-free and kinetin-mediated media. Shoot induction was also dependent on incubation period both for hormone-free and hormone-mediated conditions. Organogenic callus developed numerous meristemoids and bud primordial which subsequently differentiated into plantlet s. Vigorous rooting of plantlets was induced on MS basal medium. Somatic embryogenesis was induced in all explants either directly or indirectly via intervening callus. Direct embryos appeared after 8-10 days of incubation on MS basal medium with 1.5-2.0 mg/l 2,4-D. Initially transparent proembryoids .appeared which transformed into globular proembryoids. These ultimately developed into the plantlets. Direct embryogenesis was exclusively dependent on 2,4-0 concentration and incubation period. Highest frequency of regeneration was observed at 1.5-2.0 mg/l 2,4-0 after 4 week of incubation. Some of the explants incubated for embryogenesis also initiated callus formation. This callus was composed of embryogenic and non-embryogenic tissues. Maximum embryogenic callus induction and proliferation was observed at 3.0 mg/l 2,4-0. Highest response of embryo germination was noticed at 16 weeks. No positive effect of kinetin or kinetin-auxin interaction was observed on embryogenesis.
Along with organogenesis and embryogenesis micropropagation of sugarcane apical meristem was also carried out. Apical meristem was found not only a viable and an efficient source of clonal propagation but also a rapid and reliable mode of virus elimination . In present study, highest number of virus-free plants were obtained by apical meristem culture. Of the three medium M1, M2, M3, used for apical meristem culture, M3 showed highest propensity for regeneration, as well as, multiple shoot formation. Regeneration capability and success of virus elimination was highly dependent on the Size of cultured
SCMV indexation of in vitro regenerated plants was done by infectivity assay, serology test and by transmission electron microscopy. In vitro grown plants, indexed negative by infectivity assay also showed negative results in precipitin test and electron microscopy.
Another important aspect of virus-free stock is that these are useful experimental material for studying the effect of specific viruses on host plants. More importantly a biochemical comparison of infected clones with healthy stock could generate outstanding pool of information. In present study, biochemical investigation was undertaken to find out the qualitative and qualitative changes in total phenols, phenolic enzymes, soluble proteins esterases and acid phosphatases both in rejuvenated healthy and SCMV infected plants. The activity of total phenols and phenolic enzyme was found much higher in diseased plants compared with the healthy ones. Contrary to the above findings, soluble proteins and acid phosphatases content were found to be higher in the leaves of healthy plants in comparison with infected ones. The electrophoretic patterns of peroxidase, soluble proteins and acid phosphatases also reflected conformity with the total activities of the molecules.
From the present study rapid clonal propagation of sugarcane was achieved through apical meristem, organogenesis and embryogenesis. Degree of success for eliminating virus in cultured tissues appeared very high. Although meristem was an excellent source for virus elimination but reasonable success was also found in embryogenic and organogenic plants. In vitro grown healthy plants were further used as an experimental material to investigate the effect of virus infection on host plants in comparison with healthy ones. A clear cut biochemical alterations were recorded in virus-infected stock which confirmed the physiological and metabolic interference of virus particles on host plants.