Effects of nitrogen nutrition on salt stressed Nicotiana tabacum var. Samsum in vitro.

Abstract

The responses of Nicotiana tabacum L. var. Samsun to alterations in the nitrogen (N) supply under saline conditions in vitro were monitored. The aim was to test the hypothesis that nitrate-nitrogen supplementation to salt stressed plants alleviates the deleterious effects of salt on plant growth. Due to its capacity to be maintained under stringent environmental conditions, in vitro shoot cultures were chosen as the system of study. Nicotiana tabacum plantlets regenerated from callus in vitro were excised and rooted on solid MS culture medium containing a range of concentrations of NaCI (0 - 180 mM) and N (0 - 120 mM, as NO3--N, NH4+-N or a combination). A variety of parameters of root and shoot growth, nutrient utilisation and nitrogen metabolism were assessed over a 35 d period. Plant growth on 40 mM NO3--N + 20 mM NH4+-N (standard MS nutrients) was inhibited by the presence of salt, with root growth being more adversely affected by salt than stem growth. Root emergence was delayed from 6 d (0 mM NaCI) to 15 d (180 mM NaCI). Similar suppression of growth for all parameters, except root mass and leaf chlorophyll content, was observed when NaCI was replaced with mannitol at equivalent osmolalities. Root mass and leaf chlorophyll were significantly improved in plantlets supplied with mannitol. The time of root emergence was unaffected by mannitol supply, with all roots emerging after 10 d in culture. Plantlet growth on NH4+-N only (0 - 60 mM) was severely inhibited, even in the absence of NaCI, and was inferior to growth on NO3--N. Nitrate additions to salt stressed plantlets could not match growth in control (0 mM NaCI) plantlets. When plantlets were cultured on NO3--N only (0 mM, 30 mM, 60 mM, 120 mM), the increase in nitrate supply up to 60 mM resulted in a small improvement in growth on 90 mM NaCI, but had almost no effect on growth at 180 mM NaCl. A nitrate supply of 120 mM led to growth inhibition in all parameters, even in the absence of NaCl. Plantlet growth on isosmotic concentrations of mannitol in the presence of 0 - 120 mM NO3--N essentially mimicked that of NaCI, except for leaf chlorophyll content which was improved on mannitol at all NO3-·N levels. Nitrate uptake (measured as depletion from growth medium) by plantlets grown on 0 - 180 mM NaCI was positively correlated to availability of nitrate but negatively correlated to NaCI supply. Similar results were obtained for a mannitol supply except nitrate uptake was enhanced significantly on mannitol compared to NaCl. Sodium and chloride uptake appeared unaffected by nitrate concentration. Leaf protein content responded favourably to an increase in the NO3--N supply up to 60 mM and, in particular, appeared to be stimulated in the presence of 180 mM NaCl. Nitrate reductase (NR) activity was found to be inhibited drastically by salt and NO3--N supplementation to the salt medium had no effect on enzyme activity. A reduction in leaf total RNA content was recorded with an increase in NaCI concentration from 0 - 180 mM. A positive response to an increase in the NO3--N supply from 30 mM to 60 mM was detected in the presence of NaCl. Attempts were made to assess the levels of mRNA for NR in response to the various NaCl and N regimes. The plasmid pBMCI02010 containing a NR cDNA insert was isolated and purified and used in both radioactive and non-radioactive RNA slot blot hybridisation procedures. However, due to problems of non-specific binding of the probe, no quantification of the levels of NR mRNA in response to the various treatments could be made. Nitrate supplementation to plantlets of Nicotiana tabacum growing in vitro did not appear to ameliorate the effects of salinity stress, such that growth of plantlets in the presence of NaCI was always inferior to that in the absence of NaCl. As a large portion of growth inhibition was found in this study to be a result of osmotic rather than ionic effects of salt, it is questioned whether a nitrate supply would have an ameliorating effect on plant growth under field conditions.