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• Phytotoxicity Bioassay

•  

• Importance:
  Plants have proven useful in curing the human disease and also constitute as an important source of compounds against herbs. Members of the family of Lamnaceae are suitable plants to investigate physiological processes and effect of different chemical substances. Lemna gibba and lemna minor are used in testing the Phytotoxity of pesticides and other environmental chemicals to higher plants. Lemna plants are also used for detection of herbicidal drugs and used for the removal of undesirable herbs from crops.

   

• Method: Modified Protocol of Prof. Mc Laughlin et al. (1991)

  For the phytotoxicity assay, Lemna plant is used.  Three different concentration of test compound are exposed to the plant for seven day in Essential medium. Percent growth regulation is calculated. Paraquat is used as the standard drug.

• Nature of extracts tested:
  Crude plant extracts and fractions.

   

• Requirements:

  • • Lemna minor

  • • E-Medium

  • • Micropipettes (10,100, and 1000 µl)

  • • Conical flasks (50 ml) 3 per test compound.

  • • Growth Cabinet, at 30 °C, 56 +10 °rh (relative humidity), 9000 lux light intensity and 12 day length for seven days.

  Composition of E-medium:

  Chemical Name /  Constitutes                          g/L

  • • Potassium dihydrogen phosphate (KH2PO4)    0.68

  • • Potassium nitrate(KNO3 )                             1.515

  • • Calcium nitrate (Ca(NO2)2.4H2O)                  1.180

  • • Magnesium sulfate (MgSO4.7H2O)                0.492

  • • Boric acid (H3BO3  )                                    0.00286

  • • Manganous chloride (MnCl2.4H2O)               0.00362

  • • Ferric chloride (FeCl2.4H2O)                        0.00540

  • • Zinc sulfate (ZnSO4.5H2O)                          0.00022

  • • Copper Sulfate (CuSO4.5H2O)                     0.00022

  • • Sodium Molybdate (Na2MO4.2H2O)              0.00012

  • • Ethylene diamino tetra acetic acid (EDTA)    0.01120

   

  Preservation and cultivation of Lemna plants:
  For cultivation of Lemnaceae generally includes

  • • Cleaning of the new clone in water and cultivation under optimal conditions for one or two days. In this way a good number of healthy fronds are obtained.

  • • Short washing in H2O and transferring to nutrient solution.

    
    Procedure
    

  • • E-Medium is prepared by mixing various constituents in 1000 ml distilled water and pH is adjusted between 6.0 to 7.0 by adding KOH pellets (Stock solution).

  • • Working E-medium is prepared by mixing 100 ml of stock solution and 900 ml of distills water.

  • • 30 mg for crude extract/ compound is dissolved in 1.5 ml of solvent (Methanol/ Ethanol etc.) serving as stock solution.

  • • Three flasks are inoculated with 10, 100 and 1000 µl of solution pipette from the stock solution for 10, 100 and 1000µg/ml.

  • • Allow solvent to evaporate overnight.

  • • Add 20 ml of working E. medium and then plant of Lemna minor, each containing a rosette of two to three fronds, to each flask. (total 20 fronds).
  • • Other flasks supplemented with E-medium and reference (standard drug) plant growth inhibitors and promoters serving as negative and positive controls, respectively.

  • • Place the flaks in growth cabinet for seven days.

  • • Plants should be examined daily during incubation.
  • • Count and record number of fronds per flasks on day 7.
  • • Results are analyzed as growth regulation in %age, calculated with reference to the negative control.

   

• Calculation

  %Regulation = 100 -   No. of fronds in test            X   100
                                   No. of fronds in –ve control

  Criteria:
              0-39% inhibition            Low activity
             40-59% inhibition           Moderate activity
             60-69% inhibition           Good activity
             Above 70%                    Significant activity