A trial was conducted in a private orchard at Matiyana (Shimla), India (during December, 2014 to December, 2015) to find out the alternate to calcium ammonium nitrate for nitrogen application to apple trees. Different nitrogen containing fertilizers (sources of nitrogen) alone or in combination were used as treatments and applied in split doses to the experimental trees. Different nitrogen sources significantly influenced fruiting, yield, fruit quality and leaf nutrient contents of apple. Trees subjected to calcium nitrate+urea treatment exhibited significantly higher fruit set (23.96%), yield (5.20 kg tree-1) as well as yield efficiency (0.183 kg cm-2 trunk cross sectional area (TCSA) and lower fruit drop (13.06%). The treatment (calcium nitrate+urea) significantly improved fruit weight (179.67 g), total soluble solids content (13.10 °B), total sugars (9.99%) and reducing sugars content (7.15%). Similarly, significantly higher leaf N (2.86%), K (1.93%) and Mg (0.44%) contents were recorded in trees subjected to calcium nitrate+urea treatment, whereas, leaf P (0.28%) and Ca (1.58%) were observed highest in trees treated with combination of di-ammonium phosphate and calcium nitrate and calcium nitrate, respectively. With respect to various observations recorded, calcium nitrate+urea treatment was either superior or statistically at par with calcium ammonium nitrate (control), thus calcium nitrate+urea was proved to be most suitable alternate to calcium ammonium nitrate for supplying nitrogen to the apple trees.

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The primary result of our experiment revealed that the germination percentage of N. oleander mature seeds is only 30%. From this observation, the concept of protocol standardization for zygotic embryo culture of this plant was originated. Zygotic embryo culture was proved an efficient in vitro multiplication system of N. oleander. The maximum germination percentage (96%) of zygotic embryos was observed on ¼ MS medium with 15 gm/L sucrose, whereas the best growth medium was optimized as ½ B5 with same sucrose concentration. The second part of this study was aimed to find out the cardiac glycoside accumulation pattern in both in vitro and acclimatized plants. For this purpose, one-month-old in vitro plantlets and acclimatized plants were subjected to LC-MS analysis and 09 cardiac glycosides were detected and quantified in both the systems. Most of the cardiac glycosides including odoroside A (32.71 mg/gm DW), odoroside H (4.69 mg/gm DW) and oleandrin (0.52 mg/gm DW) were found to be accumulated at maximum level within in vitro plantlets. CG 840b (1.89 mg/gm DW) is the only cardiac glycoside, which was maximally accumulated in acclimatized plants. From this study, it can be concluded that, zygotic embryo culture is a better choice for in vitro multiplication of N. oleander when compared to matured seeds and in vitro grown plantlets of this species favor cardiac glycosides biosynthesis in comparison to acclimatized plants. Therefore, all future research on the enrichment of cardiac glycosides from this plant may be conducted on zygotic embryos derived in vitro grown plantlets or cultures.