Represented by - Komal Purohit B.Sc. Biotechnology II year

EVALUATION IN PLANT UNDER SODIUM

CHLORIDE STRESS

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WHAT IS STRESS ?

•Stress can be defind as any change in environmental condition that reduce the plant growth.

TYPES OF STRESS

•Biotic stress- The stress in plant is caused by any living ;disease causing organism, is called abiotic stress.

•Abiotic stress- The stress in plant is caused by non living factors, is called abiotic stress.

ABIOTIC FACTORS

SALINITY STRESS

•Salinity is defind as the occurrence various concentration of salts in soil or water that may interfere the normal growth of the plant.

•Presence of salts in lower concentration results in relatively lower growth rate, however presence in higher amounts may result in complete death of the plant.

•The problem of salinity is present in the high temperature area ( arid and semi arid region).

BENEFITS OF ABIOTIC FACTORS

•Stress as a positive point in plant’s life , it is a situation where abiotic stress plays a constructive role in ecosystem.

•Stress resistance mechanism can be grouped into two general categories-

•Avoidance mechanism

•Tolerance mechanism

OBJECTIVE

•In the desert area of Rajasthan, the two major environmental factors that currently reduce the plant growth that are drought and salinity.

•So our aim was to study the influence of salinity on germination and growth of Subabul seeds (Leucaena leucocephala ) and onion bulb (Aillum cepa) at the different concentration of sodium chloride (NaCl).

PLANT MATERIAL

•Seeds of subabul (Leucaena leucocephala )

•Root tips of onion bulb (Aillum cepa)

MORPHOLOGICAL PARAMETERS

•In the morphological parameters, we analyzed the morphological growth of subabul seeds under different salt concentration.

1.SEED GERMINATION

2.ROOT SHOOT LENGTH

RESULTS OF MORPHOLOGICAL

PARAMETERS

control (normal condition) Seeds grown in 50mM NaCl solution

CONTINUE…

seeds grown in 100mM NaCl solution seeds grown in

200mM NaCl solution

BIOCHEMICAL PARAMETERS

•In the biochemical parameters, analyzed the protein estimation using Bradford’s dye binding method.

•The Bradford method is a colorimetric protein binding assay, it is based on absorbance in the dye commassie brillent blue G- 250 (CBBG- 250).

•In this method, the commassie dye is previously in red color but when it binds to protein it changes its color to blue.

RESULT OF QUANTITATIVE ESTIMATION OF PROTEIN

•The graph above shows the protein activity of the sample at different salt concentration (sodium chloride) that is at 50mM, 100 mM, 200 mM.

ISOENZYME ACTIVITY

•ESTIMATION OF PEROXIDASE ACTIVITY-

Peroxidase plays important role in plant metabolism

In the stress condition, peroxidase response in lignification, cross linking of cell wall component, wound healing in the plants.

RESULT OF PEROXIDASE ACTIVITY

•In the presence of NaCl, the activity of peroxidase gradually increases from lower concentration (50mM) to higher concentration (200mM).

•This indicates that during abiotic stress, the plant cell synthesis specific isoenzyme in higher concentration.

RESULT OF PEROXIDASE ACTIVITY

•The graph above shows the peroxidase activity of the sample at different salt concentration that is at 50mM, 100 mM, 200 mM.

ISOENZYME ACTIVITY

•ESTIMATION OF ACID PHOSPHATASE -

Acid phosphatase is widely distributed in plants and animals. These enzymes catalytically break down the wide variety of phosphate esters.

In plant roots the main function of this enzyme is to provide inorganic phosphate to the growing plant during germination.

RESULT OF ACIDPHOSPHOTASE

ACTIVITY

•The activity of acid phosphatase gradually increases in the presence of NaCl .

•Its indicating that the activity of acid phosphatase is increased during abiotic stress.

RESULT OF ACIDPHOSPHOTASE

ACTIVITY

The graph above shows the acid phosphate activity of the sample

at different salt concentration that is at 50mM, 100 mM, 200 mM.

ESTIMATION OF PROLINE

•Proline is a component of salt stress response of plants.

•Proline, instead, produces an orange/yellow color during stress condition.

•It acts as a osmolyte for enzyme and membrane against salt inhibition.

RESULT OF PROLINE

•The activity of proline gradually increases in the presence of NaCl .

•Its indicating that the activity of proline is increased during abiotic stress.

RESULT OF PROLINE

•The graph above shows the proline activity of the sample at different salt concentration (sodium chloride) that is at 50mM, 100 mM, 200 mM.

QUALITATIVE ESTIMATION OF PROTEIN

•QUALITATIVE ESTIMATION OF PROTEIN: BY POLYACRYLAMIDE GELS

SDS-PAGE: Sodium dodecyl sulphate- polyacryl amide gel electrophoresis

SDS-PAGE is the most widely used method for analyzing protein mixtures qualitatively.

QUANTITATIVE ESTIMATION BY SDS-PAGE

•Estimation of protein by SDS.

QUANTITATIVE ESTIMATION BY SDS-PAGE

•Estimation of acid phosphatase by SDS

CYTOLOGICAL STUDY

•CYTOLOGICAL STUDY OF ONION ROOT TIP (Alium cepa)

1.It was discovered by Rudolf Virchow that new cells can only arise from previously existing cells.

2.This process of cell division is called as mitosis.

3.The cells of onion undergo mitosis once in 24 hours.

GERMINATION OF ROOT TIPS AT VARIOUS SALT CONCENTRATION

PROCEDURE

onion bulb root tips were cut

placed in fixative

washed with distilled water

placed in glass slide

cover slip was put

with the help of a needle it was squashed properly

observe stages of mitosis

PHASES O MITOSIS- AT CONTROL

Control (Prophase) Control (Metaphase)

Control (Anaphase) Control (Telophase)

PHASES OF MITOSIS- AT 50MM

50mM (Prophase) 50mM (Early Metaphase)

50mM (Late Metaphase)

CONCLUSION

•From the above experiments it is concluded that in the presence of NaCl, the length of seedling gradually decreases after 5 days of treatment.

•With this the concentration of protein & amount of acid phosphatase is increases.

•In the stress conditions the plant system synthesizes the peroxidase enzyme which gradually increases from 2nd day to 5th day.

CONTINUE..

•Thus peroxidase and proline act as stress markers in the plant.

• NaCl is toxic to plants so they do not survive in the higher concentration .

FUTURE WORK

•Conventional plant breeding have been using the genetic variability and to select the wild type strain or mutant strain of plant species.

•Other techniques have also been used to increase the salt tolerance through in vitro selection, somaclonal variation, mutation breeding, recombinant DNA techniques.