טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDarbow Workenash
SubjectAmino Acid Overproduction - a New approach for Broomrape
Control
DepartmentDepartment of Biology
Supervisors Professor Emeritus Shimon Gepstein
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Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The broomrape (Orobanche and Phelipanche spp.) is an absolute root-parasite plant which causes great damage to various agricultural crops in Israel and worldwide. It is very difficult to eradicate broomrape . Chemical control, solar disinfection and other measures give only a partial solution. In such circumstances in which it is very difficult to eradicate the pest, the use of biological control alone or as part of a comprehensive interface should be considered. It is known, in general, that excessive production or application of one amino acid can cause negative feedback which inhibits a key allosteric enzyme, thus blocking the biosynthesis of other amino acids produced by the same enzyme. The tested hypothesis is that the parasite, obtaining its nutrition from the host rather than synthesizing all its metabolites as most plants do, would be more sensitive than the host to feedback inhibition .

In this study, a tissue culture system was developed to directly test the hypothesis that excess amino acids can inhibit broomrape growth.

Work objectives were:

A.    Prove that one can prevent the spread of broomrape on host plants that have been engineered to produce certain amino acids in surplus in their roots.

B.     Identify the amino acids most effective in inhibiting growth of sunflower broomrape and Egyptian broomrape and determining the minimum lethal concentration.

We examined the net effect of the amino acids on the tissue culture of sunflower broomrape and Egyptian broomrape without the host affecting the process. The amino acids Glu, Asp, Cys-HCl, Arg, Phe, Thr, and Try completely inhibited the growth of cultures at a concentration of 2mM, and others at higher concentrations .

To confirm that death was caused by an excess of amino acids, we examined their contents in tissue cultures of broomrape grown in the presence of 5mM of amino acids Asp, Glu, Ile, Phe. In these cultures, the rate of growth was slowed. At this stage of the two cultures we performed a mini amino acid analysis. A concentration of 5mM of isoleucine resulted in isoleucine accumulation of 7-fold of cultures in sunflower broomrape, while in Egyptian broomrape there was almost no accumulation. Unexpectedly, however, Leucine and Valine concentration was low. When the cultures were grown in excess of phenylalanine (a single aromatic amino acid) the result was an accumulation of phenylalanine, 9 times the control level in sunflower broomrape cultures, while in Egyptian broomrape cultures there was no accumulation. Here, too, the concentrations of tyrosine and tryptophan were similar to the controls. The feedback model was not supported by the results, because addition of a single amino acid did not reduce the levels of other amino acids belonging to the same biosynthetic pathway, but seem to have a more general effect . Nevertheless, the cultures did die, providing an exciting possibility for parasite control strategies. It is possible that cultures are dying from nonspecific toxicity of amino acids, perhaps by their detection through various cell receptors that signal for cell death. This eventually leads to cumulative damage and cell culture mortality.