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Bioassay for plant growth regulators

The document presents a comprehensive overview of plant growth regulators (PGRs), including auxins, gibberellins, cytokinins, ethylene, and abscisic acid, detailing their functions, characteristics, and bioassay methods for testing potency. It discusses the historical context of PGRs and the evolution from classical bioassays to modern analytical techniques like HPLC and GC-MS. The document also outlines various bioassay techniques for each PGR, illustrating how they influence plant growth and development.

Education◦
DR. VIBHA KHANNA Asso. Prof. (Botany) SPC GOVERNMENT COLLEGE AJMER (Rajasthan)
PLANT PHYSIOLOGY PLANT GROWTH REGULATORS PRESENTATION : BIOASSAYS FOR: AUXINS GIBBERELLINS CYTOKININS ETHYLENE ABCISSIC ACID
PLANT GROWTH REGULATORS [“In the early 1900 s F. W. Went made the profound statement :-without growth substances, no growth.”] • CHARACTERISTICS 1. Organic Molecules 2. Required in Very Less Quantity 3. Regulate Various Metabolic Processes 4. Generally Translocated From Site Of Synthesis To Site Of Action (Except Ethylene)
MAJOR GROUPS OF PLANT GROWTH REGULATORS PLANT GROWTH REGULATORS PLANT GROWTH PROMOTERS AUXINS GIBBERELLINS CYTOKININS PLANT GROWTH INHIBITORS ABCISSIC ACID ETHYLENE* *Ethylene can be grouped either into the promoters or into the plant inhibitors.
CONCEPT OF BIOASSAY • Biological assays are methods for the estimation of nature, constitution or potency of a material by means of the reaction that follows its application to living matter • An assay is a form of biological experiment. The response generated by the test solution is compared with that of the standard sample. • Bioassay is assessment of biological substance. It is essential in development of new drugs and plant growth regulators and in monitoring environmental pollutants • A typical bioassay involves applying a stimulus to a subject. Application of stimulus is followed by change in some measurable characteristic of the subject. The magnitude of the change depends upon the dose. • The intensity of stimulus is varied by using the various doses by the analyst
BIO ASSAY/ BIOLOGICAL ASSAY • DEFINITION – Bio assay is defined as the estimation or determination of concentration or potency of physical chemical or biological agents by means of measuring and comparing the magnitude of response of the test with that of standard, over a suitable biological system under standard set of conditions. – Bioassay/Biological assay are extremely sensitive and specific test that can determine the biological activity of a particular hormone by showing its measurable/detectable effect on a specific part of plant material.
1.AUXIN • Auxin is a plant phytohormone involved in practically every dimension of plant development, including responses to light and gravity, organ patterning, vascular development and regulating intrinsic growth and environmental responses in both shoot and root architecture. • BIOASSAY: I. Avena Coleoptile Curvature Test II. Split Pea Stem Curvature Test III. Avena Coleoptile Section Test IV. Cross Root Inhibition Test
“AVENA COLEOPTILE CURVATURE TEST” • Principle: • This test is based on the polar transport of the auxin in Avena coleoptile. • The auxin applied on one side of the cut coleoptile stump will diffuse down that side only and will cause that side to grow more resulting in curvature of the coleoptiles. • Within limits this curvature is directly proportional to the amount of auxin applied
“AVENA COLEOPTILE CURVATURE TEST”
“SPLIT PEA STEM CURVATURE TEST” • Principle: This test is based on the differential growth response of the epidermal cells and the cortical cells of the stem sections, to Auxins. • The curvature occurs mainly due to more growth of epidermal cells as compared to the cortical cells.
“SPLIT PEA STEM CURVATURE TEST”
2. GIBBERELLINS • The gibberellins (GAs) which is a large family of tetracyclic diterpinoid plant growth substances, are associated with various growth and development processes such as – seed germination, – stem and hypocotyls elongation, – leaf expansion, – floral initiation, – floral organ development, – fruit development, and – induction of some hydrolytic enzymes in the aleurone of cereal grains • BIOASSAY I. Barley endosperm bioassay II. Dwarf pea bioassay III. Lettuce hypocotyl IV. Dwarf corn
“BARLEY ENDOSPERM BIOASSAY” • PRINCIPLE: Based on the fact that during germination of grains such as barley, wheat and oats, gibberellin is released from the embryo which moves to the aleurone cells where it induces synthesis of the enzyme α-amylase This enzyme hydrolyses starch into simple reducing sugars.
“BARLEY ENDOSPERM BIOASSAY”
“DWARF CORN TEST” PRINCIPLE: This bioassay is based on the fact that gibberellins cause elongation of the internodes, and in case of corn they also increase the length of the leaf sheaths which enclose the internodes. PROCEDURE:
3. CYTOKININ • Cytokinins are a class of purine-type phytohormones that promote – cell division, – shoot and root morphogenesis, – chloroplast maturation, – cell enlargement, – axillary bud release, and – senescence. • Bioassay for Cytokinin I. Carrot root phloem bioassay II. Chlorophyll retention test III. Tobacco pith culture IV. Cell enlargement test V. Differentiation test
“CARROT ROOT PHLOEM BIOASSAY” • The cell division tests which are based on the induction of cell division in cytokinin-requiring tissue cultures . • Tobbaco stem pith may also be used
“CHLOROPHYLL RETENTION TEST” • Principle: The senescence delaying effect of cytokinin is expressed as chlorophyll retention
4. ETHYLENE (C2H4) : A gaseous hormone • BIOASSAY OF ETHYLENE I. Triple Response II. Tomato Leaf Epinasty Test III. Pea Stem Swelling Test
“TRIPLE RESPONSE” ASSAY • Pratt and Biale (1944) developed this method for bioassay of ethylene • Principle: it is based on the physiological effect of ethylene to cause – Subapical thickening of stem. – Reduction in the rate of elongation. – Horizontal nutation (transverse geotropism) of stem in etiolated seedlings.
“TRIPLE RESPONSE” ASSAY • Dark-grown (2-4 days ) seedlings germinated on agar medium in the presence of exogenous ethylene display the triple response phenotype: – Horizontal growth, reduced longitudinal growth and increased thickness of hypocotyl, due to • Swelling of nodes (Due to altered pattern of microfibrils) • Inhibition of elongation of internodes of the stem. • Induction of horizontal growth of stem against gravity. – an exaggerated apical hook and – a short root
“Epinastic Response Of Tomato Petioles” • Epinasty is observed as a response to the flooding of roots of plants (Stress condition). • Leaf epinasty is the downward bending of leaves as a result of disturbance in their growth. More growth on adaxial surface as compared to that at abaxial surface.
Leaf Epinasty Bioassay •Young healthy tomato plants are made use of •They are exposed to the test substance in a closed environment for 2 to 3 days. •Epinastic growth is then measured.
“PEA STEM SWELLING TEST” • Cherry (1973) used pea seedlings to measure ethylene concentration by marked increase of stem swelling expressed as a ratio of weight to length. • In one ppm of ethylene the ratio is about 4.0.
5. ABSCISIC ACID • Abscisic acid (ABA) is a stress hormone and helps a plant adapt to stress by regulating the stomatal movements, bud development and seed dormancy. • It exhibits antagonistic effect. ABA can decrease, overcome, reverse, counteract and inhibit the responses of various growth-promoting hormones, particularly Gibberellins • BIOASSAY OF ABA: I. Inhibition of Cucumber hypocotyl elongation II. Rice Seedling Growth Inhibition Test III. Inhibition of α-amylase Synthesis in Barley Endosperm Test IV. Stomatal closure of mung bean hypocotyls V. Acceleration of abscission in petiole explants
“INHIBITION OF CUCUMBER HYPOCOTYL ELONGATION TEST” (Lin et al., 1988) The test is based on studying or measuring the inhibition of elongation of cucumber hypocotyls.
PROCEDURE FOR THE “INHIBITION OF CUCUMBER HYPOCOTYL ELONGATION TEST”  Hypocotyls from the seedlings of 0-5 mm below the cotyledons are excised in green, safe light.  Excised hypocotyls are placed in a 50 ml flask containing 1 ml of test solution (the test solution consists of 2 mM (milimolar) sodium phosphate (pH 5.8), 40 mM Potassium Chloride and absicic acid standard to be assayed  The bottles containing that test hypocotyls are placed on a Shaker under darkness at 28 degree Celsius and shaken at 10 RPM for conditioning
PROCEDURE FOR THE “INHIBITION OF CUCUMBER HYPOCOTYL ELONGATION TEST” (Contd.) After 20 hours of incubation the samples are exposed to continuous rotation on a roller wheel up set at 1 RPM The length of hypocotyl is measured on a dissecting microscope using a micrometre after 24 hours of exposure to light. Under these conditions the inhibition of hypocotyl elongation is proportional to the concentration of the basic acid applied
“INHIBITION OF α-AMYLASE SYNTHESIS IN BARLEY ENDOSPERM TEST” • ABA inhibits the synthesis of α amylase in the aleurone layers which is triggered by gibberellins. • Goldschmidt and Monselise (1968) developed the bioassay method to estimate ABA activity by determining the extent of inhibition of α- amylase synthesis, induced by treating barley seed endosperm with Gibberellic Acid.
“RICE SEEDLING GROWTH INHIBITION TEST” • Mohanty, Anjaneyulu and Sridhar (1979) used rice growth inhibition method to measure ABA like activity. • The length of second leaf sheath after six days growth is measured.
CURRENT STATUS • The classical methods for bioassays of plant growth regulators have now been replaced with modern methods of separation and quantification such as – high performance liquid chromatography (HPLC) and – gas chromatography (GC) followed by mass spectrometry (MS) to provide proof of structure. • Immunoassay methods are also used which are more rapid and thousand times more sensitive than bioassays
REFERENCES: • Plant Growth Substances : Principles and Applications. By Arteca • Fundamentals of Plant Physiology By V.K.Jain • Growth and Development By Lalit Srivastava