A smaller amount of N was taken up by the plant when irrigated with 32.3 as compared to 16.8 cm of water for each N application rate.This is presumably because the extra water leached some of the N below the root zone. On the other hand, higher head weight was achieved under 32.3 as compared to 16.8 em of water under the two highest N application rates. Apparently a maximum of 13.0 T/ha of broccoli could be produced with 16.8 em of water at the experimental site regardless of the fertilizer application.Nitrogen was applied as NH4N03 by continuous injection with the irrigation water at constant concentrations of 25, 50, and 75 mg Nil. The two irrigation treatments allowed the soil water suction at a 25 em depth to drop to approximately 10 or 30 cb at time of irrigation. Pragmatically the two irrigation treatments resulted in irrigation of approximately every day compared to every other day applications. There was no effect of the irrigation treatment on the yields. During the period of peak production, the lowest yields occurred under the lowest N application treatments. However, this yield deficit appears to have been partially offset by greater production of these low N treatments during the earlier part of the season. The total yields of the lowest N treatment were 8~ to 90 per cent of the maximum and yields were not consistently improved by applying N at higher rates. As the rate of N application increased, an increasingly greater proportion of the N taken up by the crop was partitioned into the vines and foliage. In contrast,chicken fodder system an increase in N application rate from 120 to 585 kg/ha increased the a~ount of N removed in the fruit by only 40kg/ha. Thus, the total amount of N actually removed from the field depended very little on the rate of N fertilizer application.
It is apparent from the fruit yield and N uptake data that although total N removal continued to increase with increasing N application, the additional N assimilated in the two higher treatments did little to improve fruit yield but served primarily to elaborate vegetative material and increase the storage of N in the vines. Therefore, N fertilizer efficiency expressed in terms of the per cent recovery of applied N, the amount of N used by the plant to increase yield, or the economic return for unit of N applied, decreased substantially at the higher N rates. It should be noted, however, that since almost all of the excess N taken up was partitioned into the vines, a large amount of N could be returned to the soil and should serve as an important N source for subsequent crops. The results of the tomato experiment as well as some of the sprinkler-celery experiments illustrate that relatively high N concentration during the initial stages of growth is important. Even though the plant removal is small in total quantity, the plant has a very small root system and must, therefore, be provided with nitrogen in relatively high concentration to prevent nitrogen deficiency at the early stages of growth. On the other hand, as the plant grows larger and the root system becomes more extensive, supplying nitrogen in the amount necessary for uptake appears to be adequate. Nitrogen applied in excess of this amount may potentially be lost through denitrification or leaching depending upon water-soil interactions.Furrow-irrigated sweet corn was grown on the UCR Experiment Station to test the effects of a nitrification inhibitor and three rates of irrigation water application . On a sandy soil, the nitrification inhibitor significantly increased the average weight of the corn stalks. On a sandy loam soil, the nitrification inhibitor increased average N concentrations per stalk at low rates of water application but had little effect at higher application rates. Generally soil N levels were maintained higher with the nitrification inhibitor, and the effect was more pronounced at higher rates of water application .To assess the involvement of the MsLEC1 and MsLEC2 genes in nodulation of alfalfa, we examined the responses of rooted cuttings of transgenic vector control plants, plants expressing the antisense transgene for MsLEC2 , and plants expressing the antisense transgene for MsLEC1to inoculation with -glucuronidase – or green fluorescent protein -marked strains of S. meliloti Rm1021. Because legume lectins have been associated with facilitation of nodulation, reduced nodulation of lectin loss-offunction plants was predicted.
However, contrary to our expectations, all the transgenic plants, including the controls, were nodulated 7 days post inoculation . By this time, the LEC1AS plant lines had already developed abnormally large numbers of nodules . The colonized nodules, as evidenced by the presence of GUS- or GFP-marked rhizobia, were frequently adjacent to each other or directly opposite one another on the root. Infection thread development in root hairs, as viewed by fluorescent microscopy of GFP-marked Rm1021, was not impaired in the LEC1AS roots, although some of the nodules appeared to be uninfected . Occasionally, uninfected nodules also developed on the roots of LEC2AS plants , but generally, the LEC2AS root nodules contained the marked strains . With one exception, line 49b, the LEC2AS roots developed markedly separate rather than clustered nodules. By 12 to 14 dpi, many of the LEC1AS nodules were already beginning to show signs of senescence, as indicated by the reduction in overall staining in a nodule 13 dpi with a GUS marked strain and by the decrease in Rm1021 GFP fluorescence in the center part of a 2-week-old nodule . In the Rm1021 GFP nodules, there was a concomitant accumulation of auto fluorescent compounds, presumably flavonoids, in the central and proximal parts of the nodule . Sections of senescent nodules demonstrated that the rhizobia had senesced from the inside outward . In contrast, the vector control and LEC2AS nodules as well as the plant lines containing the cognate sense transgenes did not show any symptoms of senescence until many weeks after inoculation. All nodules were colonized by rhizobia . Potting soil and Turface. Nodules formed on vector control plants grown in potting soil appeared identical, i.e. pink and elongated, to nodules formed by nontransgenic, wild type alfalfa . Similarly, nodules formed on the LEC2AS plants were elongated and pink in color . In contrast, although some LEC1AS root nodules appeared morphologically normal, many LEC1AS nodules were large, multilobed structures that showed signs of senescence, including loss of pink color due to breakdown of leghemoglobin, whereas others showed arrested development at early stages of nodule formation . Nodulation was also examined in an inert root medium . The Turfacegrown plants exhibited an identical nodulation phenotype . The LEC1AS lines displaying the most severe developmental and reproductive abnormalities had the highest proportion of abnormal nodules. We also examined plants that expressed the sense transgene for MsLEC1 and found that these roots also developed some large, albeit pink, multi-lobed nodules , suggesting that cosuppression might be the cause. We did not pursue this analysis further. In contrast, the LEC2ST plants produced nodules that were identical to those of the vector control . Hydroponic conditions. When grown hydroponically,fodder systems for cattle the differences between inoculated versus uninoculated plants became very obvious.
The shoots of the uninoculated plants were paler than the nodulated plants and, of the three types of transgenic plants, the LEC1AS plants were the most chlorotic . The differences between the vector control, LEC2AS, and LEC1AS plants 35 dpi, were striking . The LEC1AS plants were much less robust than either the vector control or the LEC2AS plants; the plants were consistently small and chlorotic, with a poorly developed root system. Nevertheless, a number of large, prominent nodules were observed on the roots of the LEC1AS plants , as well as small, senescent nodules . The abnormal nodulation phenotype in LEC1AS plants was evident 15 to 20 dpi under hydroponic conditions. In contrast, nodulated vector control and LEC2AS plants appeared normal, and many fewer nodules formed on the root system . To express these findings in a quantitative way, the nodules were removed from the roots of the hydroponically grown plants and were separated into pink and senescent categories. Table 1 shows that the mean number of pink nodules was significantly lower for the LEC1AS plants than for the vector control and the LEC2AS plants. In contrast, the mean number of senescent nodules was significantly higher for the LEC1AS plants compared with the vector control andLEC2AS plants . The mean for the total number of nodules for the three plant groups, which included pink and senescent nodules, did not differ significantly . However, when the mean total nodule number was normalized to grams of root dry weight, the value was significantly higher for LEC1AS plants compared with vector control and LEC2AS plants; the vector control and LEC2AS plants did not differ significantly from each other . These results demonstrate that the LEC1AS plants produced more nodules, even though their overall root mass was less than the vector control and LEC2AS plants. MsLEC2 RNA accumulated to very low levels in the nodules of vector control plants, which is consistent with what we previously reported for wild-type, nontransgenic alfalfa . In contrast, RNA isolated from nodules formed on roots of the LEC1AS plants showed a highly variable level of MsLEC2 mRNA that depended on the plant line . Although the signal was detectable in all of the lanes, it was very intense in some , suggesting that introduction of the antisense-MsLEC1 transgene affected MsLEC2 mRNA accumulation in a plant line-dependent way . The most abundant MsLEC2 mRNA in the LEC1AS nodule samples was found in RNA isolated from one very large nodule. Nodule RNA from LEC2AS plants contained highly variable levels of both endogenous and antisense MsLEC2 mRNAs . RNA from LEC1AS roots with nodules contained detectable levels of the antisense-MsLEC1 transgene ; no endogenous sense mRNA was observed.
The nodulated roots from the different LEC1AS plants showed considerable variability in the amount of accumulation of this transgenic RNA. We had found earlier that there was a correlation between plants that demonstrated moderate to severe developmental and reproductive abnormalities and those with low accumulation of MsLEC1-antisense RNA in nodulated roots . Similarly, low levels of MsLEC1-antisense RNA were detected in nodules using Northern analysis. The transgenic plants were stably transformed, and different lines contained varying numbers and positions of transgene insertions , which may have contributed to variability in transgenic and endogenous lectin mRNA expression . Transcripts hybridizing to MsLEC1-sense mRNA were not detected in RNA isolated from nodules of LEC1AS plants . In situ hybridization analysis. Because of the difficulty in detecting MsLEC1 transcripts in nodules using Northern blot analysis, we performed in situ hybridization experiments on nontransgenic alfalfa nodules to get a better idea of the spatial expression pattern of this gene. Transcripts hybridizing to MsLEC1 were detected in alfalfa nodule meristems and adjacent cells of the invasion zone , whereas no transcripts were observed in the comparable cells of the sense controls . We also examined MsLEC1 expression in alfalfa roots and found that this lectin gene was expressed in the root apical meristem and also in cells of the elongation zone ; no transcripts were observed in the sense controls . It was difficult to evaluate the difference in the extent of MsLEC1 expression in individual uninoculated versus inoculated roots. The two sets of roots looked almost identical. For MsLEC2, essentially the same pattern of transcript localization was observed. Figure 6J illustrates an entire alfalfa nodule primordium 7 dpi; MsLEC2 mRNA was detected throughout the developing nodule using the WISH method. As the nodule matured, the signal became more concentrated in the cells of zones I and II, the nodule meristem and the invasion zone, respectively . More mature nodules showed the same pattern of MsLEC2 mRNA localization . MsLEC2 transcripts were also detected in the root meristems and adjacent regions and lateral root tips . Similar to the MsLEC1 results, there was no obvious difference in the amount of transcript observed in inoculated versus uninoculated roots. There was no signal detected in the nodule hybridized with the sense probe or in the comparable control for the root .