Dear Sir. Concerning your issue about the protocol for Ureide assay based quantification of BNF. The ureide assay represent the most useful techniques to assess biological N2 fixation. The 15N natural abundance technique and ureide analyses of legume xylem sap can provide on-farm measures of N2 fixation without perturbing the soil-crop system, or the need for special pre-crop treatments. The 15N enrichment technique is most suitable for use in pot or field experiments specifically designed and planned for its application. The ureide technique may provide an alternative to 15N technologies at least for the measurement of N2 fixation by a number of tropical and sub-tropical legumes species that transport ureides in their xylem sap. The ureide assay of N2 fixation based on vacuum extracted sap or aqueous extracts of plant stems would be used most in field situations with root-bleeding sap relevant in glasshouse studies or to plants in the humid tropics. I think the following below links may help you in your analysis:

https://www.researchgate.net/publication/279972188_Quantification_of_Nitrogen_Fixation https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742256/pdf/fpls-08-02111.pdf

https://www.jstor.org/stable/42947552?seq=1#page_scan_tab_contents

Thanks

Dear Prof. Isam,

Thank you very much, I heartily appreciated for your in depth explanation !

What I am still demanding is that, how practically do it in the field?, what equipment/tools needed ? i.e. a step by step procedure for it. Thank you so much !