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METABOLIC RESPONSES TO 4-CHLOROINDOLE ACETIC ACID IN" VfGNA RADIATA (L.) WILCZEK

ABSTRACT

THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF

Boctor of ${iilo£(op||p IN

BOTANY

BARKET ALI

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2006

ABSTRACT

Metabolic responses to 4-chloroindole acetic acid in Vigna

radiata L. Wilczek.

BARKET ALI

Abstract of the thesis, submitted to Aligarh IMuslim

University, Aligarh, India, for the degree of Doctor of Philosophy

in Botany, 2006.

Three pot experiments were conducted, during 2003-2005,

to study the response of mung bean {Vigna radiata L. Wilczek) cv.

T-44 to indole-3-acetic acid (lAA) or 4-chloroindole acetic acid

(4-CI-IAA). These auxins were supplemented through three

different modes, (a) pre-sowing seed soaking, (b) percolation

through root or (c) spray to the foliage, to work out the optimum

concentration of the hormone, the most suitable mode and the

plant age for its application, and to compare the degree of the

impact of these two forms of auxins (lAA and 4-CI-IAA). The

salient features in each of the three experiments are mentioned

below: -t^iQ.

Experiment 1

The surface sterilized, healthy seeds of mung bean {Vigna

radiata L. Wilczek) cv. T-44 were soaked in 0, lO'^'^M, lO'^M, or

lO'^M of lAA or 4-CI-IAA for 8 or 12 hours. These treated seeds

were washed with DDW, followed by coating with a uniform layer

of Rhizobium and were sown in earthen pots and were allowed to

germinate and grow in a net house, under natural conditions, in

the months of April to June, 2003. The plant samples were

collected at 30 and 45 days, after sowing (DAS) to assess their

growth (length, fresh and dry mass of shoot and root); nodule

bearing capacity (number, fresh and dry mass of nodules); the

level of nitrogen, total carbohydrates and leghemoglobin and the

activity of nitrogenase in the nodules; the contents of total

nitrogen, nitrate and chlorophyll pigment; the activities of nitrate

reductase and carbonic anhydrase; the rate of photosynthesis in

the leaves; and the quantity of ethylene released by the whole

plant. The rest of the plants, at 65 day stage, were harvested to

study the number of pods per plant, number of seeds per pod,

100 seed mass, seed yield per plant and per cent seed protein

content. All the parameters mentioned above, except nodule

nitrogen and per cent carbohydrate content, exhibited a positive

response to the auxin (lAA or 4-CI-IAA). Moreover, the number of

seeds per pod and per cent protein content in the seeds did not

give a significant response to the treatment. Chloroindole auxin

(4-CI-IAA) expressed superiority in its effect, over lAA. A medium

concentration (10"^M) of 4-CI-IAA generated maximum response.

Experiment 2

The healthy, surface sterilized and Rhizobium coated seeds

of mung bean {Vigna radiata L. Wilczek) cv. T-44 were sown in

earthen pots. The resulting seedlings at 7 and/or 14 day stage/s

were percolated with 0, 10"^°M, 10"^M, or 10"^M of lAA or 4-CI-IAA

at the rate of 20 cm- per seedling. The pots were placed in a

randomized manner in a net house, under natural conditions,

during the months of April to June, 2004. The analysis of plants,

sampled at 30 and 45 DAS, revealed that auxin (lAA or 4-CI-IAA)

significantly affected the growth (length, fresh and dry mass of

shoot and root), nodulation and fresh and dry mass of nodules;

the contents of nitrogen, carbohydrate and leghemoglobin and

the activity of nitrogenase, at the level of nodules; the content of

total nitrogen, nitrate and chlorophyll; the activities of nitrate

reductase and carbonic anhydrase and the net photosynthetic

rate, at the level of the leaf and the ethylene released by the

whole plant. However, the contents of nitrogen and carbohydrate

in the nodules declined, in response to the treatment. At harvest,

the number of pods per plant, 100 seed mass and seed yield per

plant increased significantly but the number of seeds and seed

protein (%) gave no significant response to the treatment.

Furthermore, the percolation of 20 cm- of 4-CI-IAA (10"^M) twice

(7 and 14 DAS) proved best.

Experiment 3

The healthy, surface sterilized and Rhizobium coated seeds

of mung bean (Vigna radiata L. Wilczek) cv. T-44 were sown in

earthen pots. The resulting seedlings, at 10 and/or 20 day

stage/s were sprayed with 3 cm^ of 0, 10"^° 1^, 10'^ M or 10"^ M of

lAA or 4-CI-IAA per plant. Pots were placed in a net house in a

simple randomized manner and the plants were allowed to grow

under natural conditions. The samples were picked at day 30 and

45 whereas the rest of the plants were allowed to grow to

maturity. The parameters studied were the same as in

Experiment 1. The application of the hormone to the foliage

improved all the characteristics, except the contents of nodule

nitrogen and carbohydrate. Chloroindole auxin (4-CI-IAA)

surpassed indole-3-acetic acid (lAA) in its effect where lO'^M and

lO'^M of 4-CI-IAA generated a comparable response. The values

were significantly higher than any other treatment. The

application of the hormone twice (10 and 20 DAS) was better

than single application (10 or 20 DAS).

Moreover, on comparing the three modes of auxin

application, feeding directly to the foliage proved superior than

being given seed-soaking treatment or percolated through the

roots of the standing plants.

METABOLIC RESPONSES TO 4-CHLOROINDOLE ACETIC ACID IN VIGIsJA RADIATA (L.) WILCZEK

THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF

Boctor of $i|tlos;o9l)p IN

BOTANY

BARKET ALI

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2006

/ f-r^ :i

, ^

Dedicated

To

My Parents

and

Teachers

PROF. AQIL AHMAD M.Phil., Ph.D.

PD.Res.Train. (Denmark)

PLANT PHYSIOLOGY SECTION DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202002, INDIA E.mail: [email protected]

Dated 2 3> • -^5 - X o^es^

^tttxixtnit

This is to certify that the thesis entitled "Metabolic responses to

4-chloroindole acetic acid in Vigna radiata (L.) Wilczel<" submitted for the

degree of Doctor of Philosophy in Botany, is a faithful record of the bonafide

research work carried out by Mr. Barlcet AM, at the Aligarh Muslim University,

Aligarh, India, under my guidance and supervision and that no part of it has

been submitted for any other degree or diploma.

(AQILAHMAD)

mailto:[email protected]

ACKNOWLDGEMENTS

/ express my sincere gratitude and indebtedness to my esteemed

supervisor, Prof. Aqil Ahmad for his able guidance and continued interest

in the completion oj this research work.

I am thankful to Prof Ainul Haq Khan, Chairman, Department of

Botany, for providing the requisite facilities during the research work.

My thanks are also due to my teachers, Prof Samiullah (Retd.),

Prof Arif Inam, Prof Firoz Mohammad, Prof. Nafees A. Khan and

Dr. M.M.A. Khan for their useful suggestions and help in the academic

persuits.

I have no words to express my heartiest thanks to Dr. Shamsul

Hayat, Reader, Department of Botany, for his help and constant motivation

throughout the research work.

I also express my sincere thanks to my seniors, colleagues and

friends. Dr. Qazi Fariduddin, Ms. Syed Aiman Hasan, Mr. Mohd. Shaikhul

Ashraf Mr. Naseem Ahmad, Mr. Tariq Hussain, Mr. Qaisar Hayat, Ms.

Sangeeta Yadav, Dr. Manzar H. Siddiqui, Mr. Mohd. Naeem, Ms. Bushra

Shehroz, Mr. Mohd. Nasir Khan, Mr. Mohd. Idrees Khan, Ms. Shafia Nasir,

Mrs. Shaheena Afroz, Ms. Minu Singh, Dr. Mohd. Azam Musharraf Ms.

Samee Kausar, Ms. Bushra Zameer, Ms. Kauser Parveen, Ms. Saba Azad,

Mr. Bagh Hussain, Mr. Zafar Hussain Magray, Dr. Athar Hussain, Mr.

Waseem Raja, Mr. .lahangir Iqbal, Mr. Khalil Ansari and Mr. Mushtaq

Ahmad.

Mere words foil to express my appreciation to my grand parents,

parents, brothers, sisters and all other relatives who were the real source of

motivation, inspiration and patience.

1 am also thankfd to Prof. KC. Engvild, Riso National Laboratory,

Denmark, for the generous supply of4-Cl-IAA.

Financial assistance provided by University Grants Commission,

New Delhi, India, is also gratefully acknowledged. j .

(Bj(rket AH)

fH .r ^

CONTENTS

Chapters Page No.

1. Introduction 1

2. Review of Literature 4

3. Materials and Methods 11

4. Experimental Results 29

5. Discussion 50

6. Summary 60

References 64

Appendix

Ontfoduction

Chapter- 1

INTRODUCTION

The application of optimal quantities of inorganic fertilizers

to the crops that resulted in "Green Revolution" in India is now

proving to be counter productive because of the repeated

cultivation of the same genome and also a shift in the

environmental conditions. This practice is also spoiling the soil

characteristics as is prominent from the accumulation of nitrate at

a toxic level in the soil and the drinking water, released from the

nitrogenous fertilizers (Thenabadue, 1989). To overcome such

problems, agrophysiologists while exploring additional ways to

break the present yield limits of existing and those of the newly

evolved cultivars to fill the gap between productivity and the

demand. The most feasible, and successfully adopted technique is

the application of dilute, aqueous solutions of phytohormones to

the standing plants at an appropriate stage of growth, that may

be together with the insecticide/pesticide which are applied in

routine, to cut short the cost of the spray.

It is well documented that natural (lower) concentrations

of phytohormones regulate various aspects of plant growth and

development starting from seed germination passing through

embryogenesis to the death. A larger recognized class of such

hormones include auxins, gibberellins, cytokinins, abscisic acid,

ethylene and brassinosteroids. However, salicylates and

jasmonates are gaining acceptance as additional classes of

phytohormones (Arteca, 1997). I have a firm opinion that the use

of these chemicals in agriculture has a great potential as they

regulate various physiological processes. One day, these

substances, in varied proportions, may be exploited for

commercial purposes under marketable names and crops

assigned to each of them. Among the auxins, indole-3-acetic acid

(lAA) is the first known ubiquitous chemical in plants (Davies,

2004). lAA has been implicated in an innumerable array of growth

responses which at the level of cell, are the expression of

division, elongation, expansion and differentiation (Davies, 2004).

Moreover, from the practical point of view root differentiation,

embryogenesis, fruit setting, ripening and senescence are the

most important phases that involve auxins (Macdonald, 1997).

At present a selected group of plants is recognized to be

enriched with other related auxins namely, indole-3-butyric acid,

phenyl acetic acid and 4-chloroindole acetic acid (4-CI-IAA)

(Normanly et al., 1995; Normanly, 1997). Out of them, 4-CI-IAA

has been identified in representatives, belonging to the family

Fabaceae (Engvild, 1994; Reinecke, 1999) and also in one species

of Pinus sylvestris (Ernstsen and Sandberg, 1986). This

halogenated auxin is characterized with an activity many times

more than that of the auxin, demonstrated in a number of

bioassays (Reinecke, 1999; All et al., 2006b). I t is further

expressed in the induced synthesis and/or activation of the

enzymes (Hirasawa, 1989; Ahmad and Hayat, 1999; Ahmad et

al., 2001 a,b), seed germination (Ahmad et al., 2001a), higher

rate of photosynthesis and production of seeds (Ahmad et al.

2001b) on being supplemented from out side.

Keeping in view higher auxin like activity in 4-CI-IAA at

the metabolic state, its potential in exploiting the genetic make

up of the plants to improve growth and productivity was tested.

Three experiments on Vigna radiata (L.) Wilczek, an economically

important crop, were planned, each corresponding to a different

mode of application of hormone (a) pre-sowing seed treatment

(b) percolation through roots and (c) to the foliage of the plants.

The plant samples were assessed at specific stages of growth to

explore:

(a) Comparability of the degree of impact between indole-3-

acetic acid and 4-CI-IAA

(b) Select the stage of growth, giving maximum response,

(c) The best suited hormonal concentration and site of

application,

(d) The parameters that may be considered as the scale for

assessing the impact of hormone and to correlate them with

growth and productivity.

CRevietD of literature

CONTENTS

Page No.

2.1 Historical background 4

2.2 Occurrence 4

2.3 Metabolism 6

2.4 Bioactivity 7

Chapter - 2

REVIEW OF LITERATURE

2.1 . Historical background

The discovery of auxins is the outcome of Darwin's

experiments on phototropism and gravitropism. Although it

became clear in 1870 that transportable signals exist in plants but

solid evidences for the presence of specific hormones took

another half a century. It was Fitting (1909), who first introduced

the term 'hormone' into plant physiology and showed that orchid

pollinia contained some factor that causes swelling of orchid

ovaries. However, he failed to identify the active substance. The

breakthrough came in 1926 when Went isolated a crude

substance from the coleoptile tip that caused cell elongation and

was called as "auxin". Later on this substance was characterized

as indole-3-acetic acid (lAA) (Went, 1928). Moreover, nearly 40

years later chlorosubstituted auxin, 4-chloroindole acetic acid

(4-CI-IAA) was identified in the immature seeds of pea (Gander

and Nitsch, 1967; Marumoefa/., 1968a).

2.2. Occurrence

4-CI-IAA and its methyl ester were, for the first time,

noticed in the immature seeds of Pisum sativum (Gander and

Nitsch, 1967; Marumo et al., 1968a; Engvild et al., 1978) and

later on in mature pea seeds (Marumo et al 1968b; Schneider

et al 1985), leaves and both mature and immature seeds of Vicia

faba (Pless, et al., 1984; Hofinger and Bottger, 1979; Engvild et

al., 1980), immature seeds of Lens culinaris, Vicia sativa,

Lathyrus maritinus, Lathyrus sativus and Lathyrus orodatus

(Engvild et aL, 1981), Vicia amurensis (Katayama et ai., 1987)

and Lathy rus lotifolius (Engvild, 1980). The seeds of Pinus

syivestris also possessed 4-CI-IAA or its methyl ester (Ernstsen

and Sandberg, 1986). However, some other species of Vicae

tribe, namely Glycine max, Vigna catiang, Dolichos lablab, Glycine

soja and Cicer arietinum did not possess chlorolndole auxins

(Hofinger and Bottger, 1979; Katayama et al., 1987; Engvild,

1994). Moreover, Engvild (1975) screened 14 plant types

{Hordeum vulgare, Avena sativa, Triticum aestivum, Secale

cereale, Brassica napus, Linum usitatissimum, Nicotiana tabacum,

Lycopersicon esculentum, Zea mays, Allium schoenoprasum,

Phaseolus vulgaris, Glycine max, Lapidium sativum and

Halianthus annuus) and observed that none of these synthesized

detectable quantities of either 4-CI-IAA / its methyl ester but

incorporated chloride into unidentified compounds. However, the

identification of 4-CI-IAA in Pinus syivestris (Ernstsen and

Sandberg, 1986) provided a clue for a possible wider distribution

of 4-CI-IAA in the plants. A concrete conclusion is therefore,

lacking unless a more sensitive technique is employed in the

estimation of these halogenated auxins in various groups of

plants.

2.3 Metabolism

Metabolic pathways for the biosynthesis and catabolism of

4-CI-IAA in plants have been studied a little, due to the non

availability of labeled 4-CI-IAA and its precursors

(Reinecke,1999). Protocols for ^'^C-4-CI-IAA synthesis have been

described (Bald! et al., 1985; Engvild, 1994). However

radiolabled 4-CI-IAA has so far been employed only as a tracer in

the quantification of endogenous 4-CI-IAA (Pless ef al., 1984;

Magnus et al., 1997).

Watering of pea plants, at pre-flowering stage, with

radiolabled chlorine (^^Cl) resulted in the accumulation of

radiolabled 4-CI-IAA and its methyl ester in immature seeds

(Engvild, 1975). L-4-CI-Tryptophan was identified in two week old

plants of Vicia faba and these plants also incorporated deuterium

labeled indole to 4-chloroindole. Moreover, deuterium labeled L-

and D-4-CI-tryptophan were synthesized and supplied to Vicia

plants. L-4-CI-tryptophan was incorporated to 4-CI-IAA, but the

incorporation ratio was very low (Manabe et al., 1999). Existence

of 4-CI-tryptophan in plants has also been reported in Vicia faba

seeds and seedlings (Fock et al., 1992; Higashi et al., 1998).

Based on these observations it has been concluded that

L-4-CI-tryptophan is the possible precursor of 4-CI-IAA, against

the earlier belief where D-tryptophan was considered as precursor

of 4-CI-IAA (Marumo and Hattori, 1970).

Tryptophan is probably not the only source for auxin

synthesis, because tryptophan deficient mutants of maize (Wright

et ai, 1991) and Arabidopsis (Fink, 1991) synthesize a sufficient

quantity of lAA. Hence the question of biosynthesis of lAA and

that of 4-CI-IAA under a non-4-CI-tryptophan pathway may not

be ruled out (Reinecke, 1999).

The metabolism of 4-CI-IAA to 4-CI-dioxindole in

Bradyrhizobium japonicum (Jensen etal., 1995) is likely mediated

by 4-CI-dioxindole-3-acetic acid, since oxidases in B. japonicum

convert 5-CI-IAA to 5-CI-dioxindole-3-acetic acid and 5-CI-

dioxindole (Reinecke, 1999). This shows a synergism with the

catabolism of lAA (Bandurski et al, 1995). Similarly, methyl ester

and amide conjugates of 4-CI-IAA have been identified in pea

seeds and pericarp (Marumo et al., 1968a,b; Hattori and Marumo,

1972) and quantified by base hydrolysis (Magnus et al., 1997).

The methyl ester of 4-CI-IAA has also been identified in immature

seeds of Vicia faba (Hofinger and Bottger, 1979). Moreover, 4-CI-

lAA-aspartate has been reported to be highly active in Phaseolus

mungo hypocotyl swelling assay (Hattori and Marumo, 1972).

Apart from this, additional work is required to be done before the

emergence of a clear cut understanding of the metabolism of

chloroindole auxins.

2.4 Bioactivity

The biological activity of 4-CI-IAA has been tested in a

number of bioassays, where some times it is 50 fold more active

than lAA (Engvild, 1985). 4-CI-IAA is highly active in Avena

coleoptile curvature assay (Gerhold and Muir, 1989), the classical

auxin bioassay. Similarly, Avena coleoptile elongation (Marumo et

al., 1974; Bottger, et al, 1978; Hatano et al., 1987), wheat

coleoptile elongation (Katekar and Geissler, 1982), split pea

curvature (Porter and Thimann, 1965), mung bean hypocotyl

elongation (Marumo et al., 1974), mung bean hypocotyl swelling

(Marumo et al., 1968a,b, 1974; Hatano et al., 1987) exhibited

highly active response to 4-CI-IAA. Moreover, 4-CI-IAA also

induced lateral root initiation and their subsequent growth in pea

cuttings (Ahmad et al., 1987) and wheat and cucumber seedlings

(Stenlid and Engvild, 1987). 4-CI-IAA also enhanced the

formation of root primordia in pea, although, a few of them

developed into root proper (Wightman et al., 1980).

The maize, which is not a natural source of chlorinated

auxins (Hofinger and Bottger, 1979), the exogenous application of

4-CI-IAA to the plants activated its growth, over and above that

of lAA (Fischer et al., 1992; Karcz and Burdach, 1995; 2002;

Karcz et al., 1999). Similarly, 4-CI-IAA favoured growth in maize

coleoptile segments and that was 10 times faster, compared with

that of 1-naphthylacetic acid (NAA) but the binding affinities of 4-

CI-IAA to auxin binding site-I (ABS-I) and - I I (ABS-II) were 100

times lower, compared with NAA (Hertel, 1994; 1995; Stotz and

Hertel, 1994). This discrepancy, between the observed biological

activity of 4-CI-IAA and its binding efficiency to ABS-I and I I , was

argued by these authors by stating that auxin binding sites (I and

II) are not the true auxin receptor sites. However, at a later stage

(Rescher ef al., 1996) a correlation was observed between the

growth promoting activity of 4-CI-IAA and its binding affinity to

ABS-I. Furthermore, the values of membrane potential, in

response to 4-CI-IAA, was two times more than that of lAA,

suggesting specificity in signal pathway, generated by 4-CI-IAA,

in maize coleoptile (Karcz and Burdach, 2002).

Germinating seeds, in a natural course maintain GA

metabolism which is considered to be indispensable for pericarp

growth (Sponsel, 1982; Ozga et al., 1992). However, the growth

of pericarp, in deseeded ovaries of pea was restored by 4-CI-IAA

(Reinecke ef al., 1995) by favouring the conversion of GAig to

GA20 (Ozga and Brenner, 1992; Huizen et al., 1995).

4-CI-IAA favoured the production of ethylene in cucumber

and wheat seedlings (Stenlid and Engvild, 1987) and also in pea

leafy cuttings, where ethylene production was irreversible, for a

certain duration of time during which it caused the loss of apical

dominance and favoured the origin of laterals (Ahmad ef al.,

1987). Like auxin, in deseeded bean pods, 4-CI-IAA caused leaf

senescence (Tamas et a!., 1981). It is also known to inhibit the

level of the enzyme (9-cytokinin-alanin synthase) that metabolize

cytokinin (Parker et a/., 1986).

Out of the various chloroindole auxins tested, seed

germination gave maximum response to 4-CI-IAA (Ahmad et al.,

2001a) by possibly mediating the effect on Inydrolases (Hirasawa,

1989; Ahmad et al., 2001a). (Moreover, pre-sowing seed

treatment with 4-CI-IAA strongly favoured shoot and root length,

their fresh and dry mass and the activity of nitrate reductase

compared to lAA and also other chlorosubstituted auxins, in

Solanum melongena seedlings (Hayat et al., 2005).

The application of aqueous solution of 4-CI-IAA to the

foliage of 12-day old seedlings of pea increased the level of

activity of nitrate reductase in their leaves (Ahmad and Hayat,

1999). Similarly, Ahmad et al. (2001b) observed an elevated

activity of carbonic anhydrase and enhanced chlorophyll content

and photosynthetic rate, in 50 day old plants of Brassica juncea

and seed yield, at harvest.

At present, a measurable quantity of chloroindole auxin

and its methyl ester is reported in a limited number of plant

species (e.g. Pisum sativum, Vicia faba, Vicia sativa, Vicia

amurensis, Lathyrus lotifolius, Lathyrus maritinus, Lathyrus

orodatus, Lathyrus sativus, Lens culinaris and Pin us sylvestris).

However, in others it may be a matter of time when the adoption

of more sensitive methodologies may quantify their presence.

i^laterials and cMethods

CONTENTS

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.7.1

3.7.1.1

3.7.1.2

3.7.1.3

3.7.2

3.7.2.1

3.7.2.2

3.7.2.3

3.7.2.4

3.7.2.5

3.7.2.6

3.7.2.7

3.7.2.8

3.7.2.9

3.7.2.10

3.7.3

3.8

3.8.1

3.8.2

3.8.3

3.9

Seeds

Preparation of pots

Hormones and their preparation

Experiment 1

Experiment 2

Experiment 3

Parameters

Growth and nodulation

Shoot and root length

Fresh and dry mass of shoot and root

Nodule number and their fresh and dry mass

Biochemical analysis

Nodule/leaf nitrogen content

Nodule carbohydrate content

Nodule leghemoglobin content •

Nodule nitrogenase activity

Leaf nitrate content

Leaf nitrate reductase activity

Leaf carbonic anhydrase activity

Lcal'chlorophyll content

Net photosynthetic rate-

Seed protein content-'

lUhylenc estimation

Yield characteristics

Number of pods per plant

Number of seeds per pod

Seed yield per plant

Statistical analysis

Page No.

11

12

12

12

14

15

17

17

17

17

17

18

18

19

20

21

22

23

24

25

26

26

27

28

28

28

28

28

Chapter- 3

MATERIALS AND METHODS

Seeds of summer moong or mung bean {Vigna radiata L.

Wilczek) cv. T-44, commonly known as 'greengram' were used as

test material. This variety was selected, on the basis of our earlier

experience where it performed best compared with others, in the

prevailing climatic conditions. Three pot experiments were

conducted, during 2003-2005, to study the response of moong to

lAA or 4-CI-IAA, at selected stages of growth.

The comprehensive details of the material used and the

methodologies adopted, during the course of the present

investigation, are presented in this chapter.

3.1 Seeds

The authentic seeds of Vigna radiata L. Wilczek cv. T-44

were obtained from National Seed Corporation Ltd., Indian

Agricultural Research Institute, New Delhi, India. Before the start

of each experiment, the healthy seeds of uniform size were tested

for their per cent viability. Mercuric chloride solution (0.01%) was

used for surface sterilization of seeds. This was followed by

rinsing the seeds with double distilled water (DDW), at least

thrice, to remove the adhering solution of the mercuric chloride.

12

3.2 Preparation of Pots

The earthen pot (25x25 cm) were filled with uniform

quantity of sandy loam soil mixed with farmyard manure, in a

ratio of 9 :1 . Inorganic fertilizers (urea, single superphosphate and

muriate of potash) were added to each pot, at the rate of 40, 295

and 73 mg. The pots were arranged in a simple randomized

design, in a net house.

3.3 Hormones and their preparation

lAA was obtained from Sigma Chemicals Ltd., USA and

4-CI-IAA was gifted by Prof. K.C. Engvild, RISO National

Laboratory, Copenhagen, Denmark. The required quantities of the

hormones were dissolved in 0.5 cm^ of ethanol, separately in 100

cm- volumetric flasks. 0.5 cm- of surfactant Tween-20' was

added to each flask and the volume was maintained up to the

mark by using DDW.

3.4 Experiment 1

This experiment was laid down according to simple

randomized block design, during the summer season of 2003. The

surface sterilized dry seeds of mung bean (Vigna radiata L.

Wilczek) were treated in the following manner:

(A) The seeds were soaked in the solution of lAA or 4-CI-IAA

(10"^°M, lO'^M or lO'^M) for 8 hours.

(B) The seeds were soaked in the solution of lAA or 4-CI-IAA

(10'^°M, 10"^M or 10"^M) for 12 hours.

(C) The seeds were soaked in double distilled water (DDW) for 8

or 12 hours to be used as control for the above sets (A and

B), respectively.

The seeds, after being treated with the hornnone, were

washed with DDW to remove the adhering solution. These treated

seeds were coated with a uniform layer of specific Rhizobium sp.

and were sown in the pots. A uniform basal dose of N, P and K

was added at the time of sowing (refer to 3.2). Thinning was done

7 days after sowing (DAS), leaving three plants per pot, where 15

pots were maintained per treatment. Irrigation was done with tap

water as and when required. At each stage of sampling (30 and

45 DAS) plants were uprooted to assess the following

characteristics :

1. Length of root and shoot per plant.

2. Fresh and dry mass of root and shoot per plant.

3. Number of nodules per plant

4. Nodule fresh and dry mass per plant

5. Nodule nitrogen content

6. Nodule carbohydrate content

7. Nodule leghemogtobin content

8. Nodule nitrogenase activity

9. Leaf nitrogen content

14

10. Leaf nitrate content

11. Leaf nitrate reductase activity

12. Leaf carbonic anhydrase activity

13. Leaf chlorophyll content

14. Net photosynthetic rate

15. Ethylene production

The remaining plants were allowed to grow upto maturity

and were harvested after 65 days to study the following yield

characteristics.

1. Number of pods per plant

2. Number of seeds per pod

3. Seed yield per plant

4. 100 seed mass

5. Seed protein content

3.5 Experiment 2



surface sterilized and Rhizobium sp.coated seeds of moong {Vigna

radiata L. Wilczek) were sown in earthen pots, at the rate of 8

seeds per pot. A uniform basal dose of N, P and K was applied to

each pot, at the time of sowing (refer to 3.2). Three plants per

pot were maintained after thinning and all the pots were divided

into three sets.

15

(A) Seven-day-old seedlings were percolated with lAA or 4-CI-

lAA (10"^°M, lO'^M or lO'^M) through their roots, at a rate

of 20 cm- per seedling.

(B) Fourteen-day-old seedlings were percolated with lAA or

4-CI-IAA (10"^°M, 10" M or lO'^M) through their roots, at a

rate of 20 cm^ per seedling.

(C) The seedlings were percolated with lAA or 4-CI-IAA (10"^°M,

lO'^M or 10"^M) through their roots, at a rate of 20 cm^ per

seedlings, both at 7 and 14 day stages.

Control plants were maintained with each above set,

where the 7 and/or 14 day old seedlings were percolated with 20

cm^ of DDW, through roots, at each corresponding stage of

hormone treatment. Fifteen pots were maintained per treatment.

Irrigation was done, as and when required. The plants were

sampled at 30 and 45 DAS to study the parameters already listed

in experiment 1 (pages 13 and 14).

3.6 Experiment 3



surface sterilized and Rhizobium coated seeds of moong ( Vigna

radiata L. Wilczek ) were sown in earthen pots at a rate of 8

seeds per pot. A uniform basal dose of N, P and K was applied to

16

each pot, at the time of sowing (refer to 3.2). Three plants were

maintained in each pot that were divided into three sets

(A) The foliage of 10 day old seedlings was sprayed with lAA or

4-CI-IAA (10"^°!^, 10" M or lO' IM) at the rate of 3 cm^ per

seedling.

(B) The foliage of 20 day old seedlings was sprayed with lAA or

4-CI-IAA (10"^°M, lO'^M or 10"^M), at the rate of 3 cm^ per

seedling.

(C) The foliage of the seedlings was sprayed with lAA or 4-CI-

IAA (10"^°M, lO'^M or 10"^M) at the rate of 3 cm^ per

seedling, both at 10 and 20 day stages.

Control plants were maintained with each above set,

where the foliage of the seedlings was sprayed with DDW, at the

above corresponding stages of hormone treatment. The noozel of

the sprayer was adjusted in such a way that it pumped out 1 cm-

of water/hormonal solution per sprinkle. Each seedling was

sprinkled three times, therefore, each seedling received 3 cm ^ of

DDW/hormonal solution. The spraying was done carefully so that

the solutions did not fall in the soli or on the adjoining seedling.

Each treatment was represented by 15 pots. Irrigation was done

as and when required. The parameters, studied at each growth

stage (30 and 45 DAS) were the same as in experiment-l(pages

13-14).

17

3.7 Parameters

The methods applied to assess each parameter are

described below:

3.7.1Growth and nodulation

3.7.1.1 Shoot and root length

The whole mass of soil from each pot was pulled out and

dipped in a bucket filled with water to uproot the plants with

intact roots. The plants were gently moved to remove the soil

particles. This was followed by washing under running tap water.

The length of shoot and root was measured by using a meter

scale.

3.7.1.2 Fresh and dry mass of shoot and root

The shoot and root of each plant was weighed separately

to record their fresh mass. These were subsequently transferred

to an oven run at 80°C and left there for 24 hours. The shoot and

root were again weighed separately to record their dry mass.

3.7.1.3 Nodule number and their fresh and dry mass

The whole mass of soil was taken out of the pot and

placed in a bucket filled with tap water. The plants were moved

gently to get the intact root system with no damage to the

nodules. The roots were then washed under running tap water

and the number of nodules per plant was counted. The nodules

were picked and weighed to record their fresh mass.

Subsequently they were transferred to petri plates for overnight

drying in an oven, run at 80°C to record their dry mass.

3.7.2BiochemJcal analysis

3.7.2.1 Nodule/leaf nitrogen content

The nodule/leaf nitrogen content was estimated by

employing the method worked out by Lindner (1944).

50 mg of oven dried nodule/leaf powder was transferred to

a digestion tube to which 2 cm- sulphuric acid (AR grade) was

added. The tube was heated on a temperature controlled

digestion assembly for 2 h to allow the complete digestion of the

sample. After cooling the digestion tube for about 15 minutes, 0.5

cm- of 30 % H2O2 was added drop by drop and the solution was

heated again for about 30 minutes until the colour of the allequot

turned to light yellow. Additional 3-4 drops of 30 % H2O2 were

added, after cooling the tube followed by heating for about 15

minutes. This process was repeated till the contents of digestion

tube turned colourless. Digested material was transferred to a 50

cm^ volumetric flask, after 2 washings. The final volume of the

allequot was made up to the mark by using DDW.

10 cm- of the allequot was taken in a 50 cm^ volumetric

flask and neutralized by adding 2 cm^ of 2.5 N NaOH (Appendix

1.1) and 10 % sodium silicate (Appendix 1.2). Volume was made

up to the mark by using DDW. 5 cm- of this sample was pipetted

into a graduated test tube to which 0.5 cm^ Nessler's reagent was

added dropwise, with repeated shal<ings. The final volume was

made up to 10 cm^ with DDW. After waiting for 5 minutes, to get

optimum colour development, absorbance of the solution was

read at 525 nm on a spectrophotometer (Spectronic-20D, Milton

Roy, USA). A blank consisting of Nessler's reagent and DDW was

run simultaneously with each set of samples. Standard curve was

plotted by using known, graded dilutions of ammonium sulphate

solution. The absorbance of each sample was compared with that

of the calibration curve and per cent nitrogen, in each sample,

was computed on dry mass basis.

3.7.2.2 Nodule carbohydrate content

The carbohydrates were extracted from the samples

following the method of Yih and Clark (1965) and estimated by

adopting the procedure of Dubois et al. (1956).

50 mg of dried nodule powder was transferred to a glass

centrifuge tube containing 5 cm- of 1.5 N H2SO4 (Appendix 2.1).

The sample was centrifuged at 4,000 rpm for 10 minutes. The

supernatant was decanted into 25 cmi- volumetric flask with two

washings of the residue with DDW. The volume was made up to

the mark by using DDW. 1 cm^ of this extract was taken in a test

tube to which 1 cm^ of 5 % distilled phenol (Appendix 2.2) was

added. The test tube was placed in chilled water and 5 cm^ of

H2SO4 (AR grade) was added. The absorbance was read at 490

20

nm on a spectrophotometer. A blank was run simultaneously with

each set of samples. Standard curve was plotted by using known

graded dilutions of glucose solution. The absorbance of each

sample was compared with the calibration curve and per cent

carbohydrate content was calculated on dry mass basis.

3.7.2.3 Nodule leghemoglobin content

The leghemoglobin content, in fresh nodules was

estimated following the method described by Sadasivum and

Manickam (1992).

200 mg fresh nodules were mixed with 3 cm- of 0.1 M

phosphate buffer (Appendix 3.1) and macerated in a mixer

followed by filtration through two layers of cheese cloth. The

nodule debris was discarded. The turbid reddish brown filtrate

was centrifuged at 10,000 rpm for 15 minutes.

3 cm- of pyridine reagent (Appendix 3.2) was added to 3

cm^ of extract and mixed. The solution became greenish yellow

due to the formation of hemochrome.

The hemochrome was divided equally into two test tubes.

To the first test tube, 50 mg of potassium hexacyanoferrate was

added to oxidize the hemochrome and read at 539 nm on a

spectrophotometer. To the second test tube, 50 mg of sodium

dithionite was added to reduce the hemochrome. The absorbance

was read at 555 nm, after an interval of 5 minutes, against a

21

reagent blank. The leghemoglobin content (mM) was computed by

putting the values in the formula:

A556 - A539 Leghemoglobin concentration (nnM) =

23.4 X 2D

where D is the initial dilution.

A556 and A539 are the absorbances at 556 and 539 nm,

respectively.

The calculation is based upon the equation E = 23.4x10-^

mol"^ cm"^ The readings were converted to laM g'^ fresh mass.

3.7.2.4 Nodule nitrogenase activity

The nitrogenase activity was assayed by adopting the

procedure of Hardy et al. (1958). Assay was carried out in fresh

samples, immediately after harvesting the plants. Nodulated roots

were separated and shaken slowly to remove attached soil

particles. Samples were incubated in 30 cm- special glass tubes

sealed with a rubber subseal and the perforated lid to allow it to

be pierced by a hypodermic needle. 10 cm^ of air was withdrawn

from the incubation tube by using a syringe and that was replaced

by the same volume of pure acetylene gas. After Ih of the

incubation, at room temperature, 0.5 cm^ of the gas drawn from

the tube was injected into gas chromatograph (GC 5700, Nucon,

New Delhi), equipped with 1.8 m porapack N (80/100 mesh)

column, a flame ionization detector and an integrator. The

acetylene injected into the tube was converted to ethylene by the

22

catalytic activity of nitrogenase. The quantity of the ethylene so

produced was estimated by comparing the values with that of the

standard curve, drawn by using pure ethylene. The results were

expressed in terms of nM of ethylene formed / g nodule fresh

mass / hour.

3.7.2.5 Leaf nitrate content

The leaf nitrate content was estimated following the

method of Johnson and Ulrich (1950).

50 mg of the oven dried leaf powder was transferred to a

dried centrifuge tube (25 cm- ) with the addition of 400 mg of

calcium sulphate and 12.5 cm- of DDW. The sample was

centrifuged at 6,000 rpm for 10 minutes. The supernatant was

transferred to a 50 cm- conical flask containing 1 cm^ of 0.5 %

calcium carbonate solution. The excess solution was evaporated

on a water bath leaving the final volume to about 5 cm- . To the

above solution, 0.5 cm^ of hydrogen peroxide (30 %) was added

and the flask was plugged with a lid. After 5 minutes, the lid was

removed and the solution in the flask was heated further to

dryness on a water bath, in order to remove the peroxide. The

flask was cooled and 1.25 cm^ of phenol disulphonic acid

(Appendix 4.1) was rapidly added with continuous stirring, 35 cm-

of DDW was also added to this solution. Lastly, 3 cm^ of 50 %

ammonium hydroxide solution was pipetted into it. Yellow colour

developed. The absorbance of this solution was read, after 15

23

minutes, at 400 nm, using a spectropliotometer and was

compared with that of a standard curve, plotted by taking known

graded concentrations of potassium nirtate.

3.7.2.6 Leaf nitrate reductase activity

The activity of nitrate reductase was measured following

the method laid down by Jaworski (1971), in fresh leaf samples.

The leaves were cut into small pieces (1 cm-^). 200 mg of

these chopped leaves was weighed and transferred to plastic

vials. To each vial 2.5 cm- of O.IM phosphate buffer (pH 7.5)

(Appendix 5.1) and 0.5 cm- of 0.2M potassium nitrate solution

(Appendix 5.2) was added followed by the addition of 2.5 cm- of

5 % isopropanol (Appendix 5.3). These vials were incubated in a

BOD incubator for 2 h at 30±2°C, In dark. 0.4 cm^ of the

incubated mixture was taken in a test tube to which 0.3 cm- each

of 1 % sulphanilamide solution (Appendix 5.4) and 0.02% N-1-

nepthyl-ethylendiamin hydrochloride (NED-HCI) (Appendix 5.5)

were added. The test tube was left for 20 minutes, for maximum

colour development. The mixture was diluted to 5 cm- by using

DDW. The absorbance was read at 540 nm on spectrophotometer.

A blank was run simultaneously with each sample. Standard curve

was plotted by using known graded concentrations of NaN02

(sodium nitrite) solution. The absorbance of each sample was

compared with that of the calibration curve and nitrate reductase

(NR) activity (nM NO2 g"^ fresh mass h" ) was calculated.

24

3.8.2.7 Leaf carbonic anhydrase activity

The carbonic anhydrase (CA) activity in fresh leaf samples

was measured by following the method described by Dwivedi and

Randhava (1974).

The fresh leaf samples were cut into small pieces at a

temperature below 25°C. 200 mg of these leaf pieces was

weighed and transferred to petriplate. The leaf pieces were

further cut into fine pieces in 10 cm^ of 0.2 M cystein

hydrochloride (Appendix 6.1) and were left at 4°C for 20 minutes.

The leaf pieces were blotted and transferred to a test tube

containing 4 cm^ of phosphate buffer of pH 6.8 (Appendix 6.2). To

this test tube, 4 cm- of 0.2 M sodium bicarbonate (Appendix 6.3)

solution and 0.2 cm^ of 0.002 % bromothymol blue (Appendix

6.4) was added. The test tube was shaken gently and left at 4°C

for 20 minutes. CO2 liberated by the catalytic action of carbonic

anhydrase on NaHCOs was estimated by titrating the reaction

mixture against 0.05 N HCI (Appendix 6.5) using methyl red as

indicator (Appendix 6.6). The volume of HCI used to develop light

purple colour, persisting for at least five seconds, was noted. A

blank consisting of all the above components of the reaction

mixture, except the leaf sample, was run simultaneously with

each set of the samples. The activity of enzyme was calculated by

putting the values in the formula :

25

V X 22 X N CA activity = [mol (CO2) kg'^ (leaf fresh mass) s" ]

W

V = Difference in volume (cm^ of HCI used, in control and test

sample titrations)

22 = Equivalent weight of CO2

N = Normality of HCi used

W = Fresh mass of tissue, used.

3.7.2.8 Leaf chlorophyll content

The chlorophyll content in fresh leaf samples was

estimated following the method of Mackinney (1941).

One gram of finely cut fresh leaves was ground to a fine

pulp by using a mortar and pestle after pouring 20 cm- of 80 %

acetone. The mixture was centrifuged at 5,000 rpm for 5 minutes.

The supernatant was collected in a 100 cm- volumetric flask. The

residue was washed three times, using 80 % acetone (Appendix

7). Each washing was collected in the same volumetric flask and

the final volume was made upto the mark by using 80 % acetone.

The absorbance was read at 645 and 663 nm against a blank

(80 % acetone only), on a spectrophotometer. The chlorophyll

content (mg g~ fresh tissue) was calculated using the following

equation. ^

V Total chlorophyll = [2.02 (A645)]+[8,02 (Agea)] x (mg g'^ fresh mass) content 1000 x W

26

A = absorbance at specific wave length

V = final volume of chlorophyll extract in 80 % acetone

W = fresh mass of tissue, used for extraction.

3.7.2.9 Net photosynthetic rate

The photosynthetic rate, at each selected stage, was

measured in fully expanded leaves of the sample plants by using

LI COR-6400, portable photosynthesis system (Nebraska, USA).

All the measurements were made on cloudless clear days between

11.00 to 13.00, solar time.

3.7.2.10 Seed protein content

The total protein content in the dry seeds, at harvest, was

estimated by adopting the methodology of Lowry et al. (1951).

50 mg of the oven dried seed powder was transferred to a

mortar. The sample was ground with the addition of 1 cm- of 5 %

trichloroacetic acid (Appendix 8.1). The pulp was transferred to a

glass centrifuge tube with repeated washings and the final volume

was made up to 5 cm^. The mixture was centrifuged at 4,000 rpm

for 15 minutes and the supernatant was discarded. 5 cm- of IN

NaOH (Appendix 8.2) was added to the residue. The tube was left

in a water bath at 60°C for 30 minutes. After cooling for 15

minutes, the mixture was centrifuged at 4,000 rpm for 15

minutes. The supernatant was collected in 25 cm^ volumetric flask

27

with repeated washings. Volume was made up to the mark by

using IN NaOH that was used to estimate the protein content.

1 cm^ of the above extract was transferred to a test tube

and 5 cm^ of Reagent C (Appendix 8.3) was added to it. The

solution was shaken well and allowed to stand at room

temperature for 15 minutes. 0.5 cm- of Folin phenol reagent

(Appendix 8.4) was added rapidly with immediate mixing. The

blue colour developed whose intensity was read at 660 nm using

spectrophotometer. A blank, consisting of all reagents of reaction

mixture, except protein extract, was run simultaneously with each

set of samples. The total protein content was calculated by

comparing the absorbance of the samples with those of a

calibration curve, plotted by using known graded concentrations

of bovin albumin.

3.7.3 Ethylene estimation

The ethylene released by the plants was measured by the

procedure adopted by Ahmad et al. (1987).

The whole plant sample was incubated for an hour in a

cor)\ca\ flask (250 cm^) and sealed with rubber septum, at 25°C. 5

cm- of the gas from the flask was withdrawn by using an air tight

hypodermal syringe and was injected into a gas chromatograph

(GC 5700, Nucon, New Delhi) equipped with 1.8 m porapack N

(80/100 mesh) column, a flame ionization detector and an

integrator. Nitrogen was used as carrier gas at 3 kg/cm^ The

column temperature was set at 65°C and that of detector at

28

150°C. The retention time of ethylene gas was 70-80 seconds.

The standard curve was obtained by using pure standard

ethylene, diluted with air to get desired concentrations. Ethylene

production was expressed as nL g"^ fresh mass (F.M.) h"^

3.8 Yield characteristics

3.8.1 Number of pods per plant

At harvest (65 DAS), 9 plants (3 from three pots)

representing each treatment were randomly sampled and counted

for the number of pods per plant.

3.8.2 Number of seeds per pod

25 pods from each treatment were randomly selected and

computed for the number of seeds per pod.

3.8.3 Seed yield per plant

The pods from each replicate were crushed, cleaned and

computed to assess seed yield per plant. 100 seeds were

subsequently randomly picked and weighed to record 100 seed

mass.

3.9 Statistical analysis

The experimental data was analysed following the standard

procedures described by Gomez and Gomez (1984). Each treatment

was represented by fifteen pots where each pot was considered as

a replicate. Three observations (from three different pots) were

recorded per treatment. The 'F' test was applied to assess the

significance of the data at 5 % level of probability. Least significant

difference (LSD) was calculated to compare the effect of various

treatments.

Experimental Results

CONTENTS

Page No.

4.1 Experiment 1 29

4.1.1 Shoot and root length 2 9

4.1.2 Fresh and dry mass of shoot 30

4.1.3 Fresh and dry mass of root 30

4.1.4 Number of nodules 30

4.1.5 Fresh and dry mass of nodules 31

4.1.6 Nodule nitrogen content 31

4.1.7 Nodule carbohydrate content 31

4.1.8 Nodule leghemoglobin content 32

4.1.9 Nodule nitrogenase activity 32

4.1.10 Leaf nitrogen content 33

4.1.11 Leaf nitrate content 33

4.1.12 Leaf nitrate reductase activity 33

4.1.13 Leafcarbonicanhydrase activity 34

4.1.14 Leaf chlorophyll content 34

4.1.15 Net photosynthetic rate 35

4.1.16 Ethylene production 35

4.1.17 Yield characteristics and seed protein content 35

4.2 Experiment 2 36

4.2.1 Shoot and root length 36

4.2.2 Fresh and diy mass of shoot 37





4.2.7 Nodule carbohydrate content 39




4.2.11 I,eaf nitrate content 40

4.2.12 Leafnitrate reductase activity 41

4.2.13 Leaf carbonic anhydrase activity 41

4.2.14 Lcafchlorophyllconlcnl 41




4.3 Experiments 43

4.3.1 Shoot and root length 43

4.3.2 Fresh and dry mass of shoot 44





4.3.7 Nodu 1 e carbohydrate content 4 6




4.3.11 Leaf nitrate content 47

4.3.12 Leafnitrate reductase activity 47

4.3.13 Leafcarbonicanhydrase activity 48

4.3.14 Leaf chlorophyll content 48




Chapter- 4

EXPERIMENTAL RESULTS

4.1 Experiment 1

The surface sterilized, healtiiy seeds of mung bean {Vigna

radiata L. Wllczek cv. T-44) were soaked in 0.0 (control), 10"^°M,

lO'^M or 10"^M aqueous solution of lAA or 4-CI-IAA for 8 or 12

hours. These treated seeds were sown in earthen pots (25 cm

diameter), filled with soil and farmyard manure, in a ratio of 9 :1 .

The plants were sampled at 30 and 45 days after sowing (DAS) to

assess the following characteristics. The rest of the plants were

allowed to grow to maturity to study the yield characteristics. The

observations are summarized in tables 1-12 and are briefly

described below.

4.1.1 Shoot and root length

The length of shoot and that of root increased with the

advancement of the age of the plant and was significantly

affected by the treatment (Table 1). The plants obtained from the

seeds pre-treated with 4-CI-IAA possessed the shoot and root

longer than those receiving lAA treatment. 4-CI-IAA (lO'^M)

generated the best response where the values for shoot and root

length, at 30 and 45 days, increased by 45 %, 27 % and 29 %,

24 %, over the control, respectively. However, the extension in

the duration of seed soaking, from 8 to 12 hours made no

significant impact.

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4.1.2 Fresh and dry mass of shoot

The data depicted in Table 2 indicates tliat the fresh and

dry mass of the shoot exhibited an increase as the growth

progressed. I^oreover, the pre-sowing seed treatment with either

of the auxins (lAA or 4-CI-IAA) caused a significant improvement

in shoot mass, where 10"^M of 4-CJ-IAA proved best. The increase

in fresh mass was of the order of 37 % and 29 % and dry mass of

52 % and 49 %, over the control, at 30 and 45 DAS, respectively.

Furthermore, the values that closely followed were generated by

the highest concentration (10"^M) of 4-CI-IAA.

4.1.3 Fresh and dry mass of root

Fresh and dry mass of the root (Table 3) followed a

pattern similar to that of the shoot. The mass increased with the

age of the plant and that too more significantly in the plants

raised from auxin treated seeds, irrespective of the duration of

soaking. 4-CI-IAA (10"^M) proved best where the values for fresh

and dry mass increased by 49 % and 26 % at day 30 and 34 %

and 23 % at day 45.

4.1.4 Number of nodules

The nodule number increased during the observation from

day 30 to 45 (Table 4). The plants resulting from the seeds pre-

treated with either of the auxin (lAA or 4-CI-IAA) had more

nodules per plant than the control. A concentration of 10"^M of

4-CI-IAA induced maximum number of nodules, 34 % and 38 %

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day) but was statistically equal to that of the other concentrations

(10'^°M and lO'^M) of 4-CI-IAA, at day 45 stage.

4.1.5 Fresh and dry mass of nodules

Both fresh and dry mass of the nodules increased with the

age of the plant and followed a pattern of response to the auxins,

very much comparable with nodule number (Tables 4 and 5).

Among the concentrations (10"^°M, lO'^M or lO'^M) of 4-CI-IAA

used, the medium (10"^M) one, induced the best response but the

values were statistically equal to the other concentrations (10'^°M

and 10"^M) of 4-CI-IAA, at 45 DAS.

4.1.6 Nodule nitrogen content

The nitrogen content of the nodules was higher at day 45

than at day 30 (Table 5). Control plants possessed maximum

values but decreased in the plants, generated from auxin treated

seeds. The nodules developed on the plant roots raised from the

seeds pre-treated with 4-CI-IAA had lower concentrations and

that too the lowest (32 % and 28 %, less than control) were with

lO'^M but were statistically equal to that of lO'^M.

4.1.7 Nodule carbohydrate content

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that of nodule nitrogen content, during the growth period (Table

6). The values in the nodules of the plants resulting from auxin

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32

treated seeds, were significantly lower than the control.

Compared with lAA, 4-CI-IAA was more effective in declining the

nodule carbohydrate level. Among the three concentrations of

4-CI-IAA employed, lO'^M was most effective where the values

decreased by 26 % and 28 % at 30 and 45 days of sampling, over

the control.

4.1.8 Nodule leghemoglobin content

The leghemoglobin content in the nodules increased as the

plant growth progressed (Table 6). Pre-sowing seed treatment

with auxin resulted in the plants whose nodules had more

leghemoglobin than that of the control. Among the auxins, 4-CI-

IAA was more effective where 10" M generated a response that

was 39 % and 41 % higher at 30 and 45 day stages, than the

control, respectively. However, at 45 day stage the effect was

statistically equal to that of 10"^M.

4.1.9 Noduie nitrogenase activity

The values increased as the age of the plants and that of

the nodules advanced from 30 to 45 days (Table 7) and increased

further in the plants raised from the seeds pre-treated with

auxins (lAA or 4-CI-IAA). Nevertheless, 4-CI-IAA proved better

than lAA. lO'^M of 4-CI-IAA generated maximum values at 30 and

45 day stages that were 36 % and 37 % more than the control,

respectively.

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4.1.10 Leaf nitrogen content

Pre-sowing seed treatment with auxins improved the leaf

nitrogen content in the resulting plants that increased further with

the age of plant (Table 7). Chloroindole auxin proved superior

than indole-3-acetic acid. Among the three concentrations of

4-CI-IAA, 10"^M proved superior but the values generated were

statistically equal to that of 10"^M. A maximum increase of 36 %

and 32 % was recorded, at 30 and 45 day stages, under the

influence of lO'^M of 4-CI-IAA.

4.1.11 Leaf nitrate content

As the growth progressed from day 30 to 45, the leaf

nitrate content decreased significantly (Table 8)'. However, the

treatment of the seeds with lAA or 4-CI-IAA increased its level in

the leaves of resulting plants. Out of various auxin treatments, 12

hours soaking in 4-CI-IAA (10"^M) generated the best response

where the values were 22 % and 23 % higher, over the water

soaked control, at 30 and 45 DAS, respectively. These values

were statistically equal to that resulting from the treatment with

lO'^M of 4-CI-IAA.

4.1.12 Leaf nitrate reductase (NR) activity

Comparing the values at 30 and 45 day stages, a slight

decrease in NR activity was observed at the latter stage of growth

(Table 8). However, the treatment with auxin generated

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34

significantly liigher values, irrespective of the concentration of the

hormone and the duration of soaking. Maximum activity was

noted in the leaves of the plants resulting from the seed

treatment with 10"®M of 4-CI-IAA that resulted in an increase of

36 % and 32 %, over the control, at 30 and 45 DAS, respectively.

Moreover, soaking for an extended period of 12 hours proved

superior to 8 hours.

4.1.13 Leaf carbonic anhydrase (CA) activity

The activity of CA, like that of NR decreased from day 30

to 45 (Table 9). Pre-sowing seed treatment with auxin caused a

significant improvement in the activity of the enzyme in the

foliage of the resulting plants. The response was better to 4-CI-

IAA than lAA, whose medium concentration (lO'^M) generated

values that were 39 % and 33 % higher over the control, at 30

and 45 DAS, respectively. However, these values were

statistically equal to those generated by the rest of its

concentrations (10"^°M and lO'^M).

4.1.14 Leaf chlorophyll content

Compared with 30 day stage, the leaf chlorophyll content

decreased at day 45 (Table 9). Irrespective of the duration of

soaking, auxin treatment elevated the level of leaf chlorophyll. A

maximum increase of 24 % and 22 %, at day 30 and 45, was

recorded with lO'^M of 4-CI-IAA that was significantly higher over

all the treatments and the control.

— < •O Q C -—'

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35

4.1.15 Net photosynthetic rate

The data presented in table 10 expresses that the plants

raised fronn the seeds given pre-sowing treatment with auxin (lAA

or 4-CI-IAA), irrespective of' their concentration and duration of

soaking, photosynthesized more efficiently than the control

plants, at both the stages (30 and 45 DAS) of growth. However,

at the advance stage of growth (45 DAS) the plants

photosynthesized at a slower pace. Maximum values 32 % and

25 % higher over the control, were recorded in 30 and 45 day old

plants, raised from the seeds pre-treated with lO'^M of 4-CI-IAA.

4.1.16 Ethylene production

The plants released more ethylene (Table 10) as they aged

and also with the type and the level of the auxin (lAA or 4-CI-

IAA). Chloroindole auxin generated more ethylene than indole-3-

acetic acid. However, increase in the concentration of either of

the auxins increased its level. 4-CI-IAA (lO'^M) was the strongest

stimulator of ethylene production, among all the treatments that

generated 56 % and 58 % more ethylene than the control, at 30

and 45 DAS, respectively.

4.1.17 Yield characteristics and seed protein content

At harvest (65 DAS), the number of pods per plant, 100

seed mass and seed yield per plant were significantly enhanced

by the treatment (Tables 11 and 12). Out of the two auxins (lAA

0) c QJ > x: •i-i



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36

and 4-CI-IAA), the latter, irrespective of its concentration,

generated better response in the plants. A nnaximunn, respective

increase of 29 %, 30 % and 23 % in the number of pods, 100

seed mass and seed yield per plant, over the control was

recorded with lO'^M of 4-CI-IAA. A comparable response was also

generated by the higher concentration (10"^M) of 4-CI-IAA in

certain aspects. The seeds produced did not differ significantly in

their protein percentage.

4.2 Experiment 2

The surface sterilized, healthy seeds of moong {Vigna

radiata L. Wilczek cv. T-44) were sown in earthen pots, filled with

sandy loam soil and farmyard manure (9:1). The resulting 7

and/or 14 day old seedlings were percolated with 0, 10'^°M, 10"^M

or lO'^M of lAA or 4-CI-IAA, through their roots. Plants, at the

age of 30 and 45 days, were sampled to assess the

characteristics, briefly explained below. The rest of the plants

were allowed to grow to maturity (65 days) to study the yield

characteristics. The observations are summarized in tables 13-34.


The data presented in tables 13 and 14 gives an

impression that the length both of root and shoot increased up to

day 45. The auxins, irrespective of their concentration and time of

application favoured the elongation. However, the seedlings gave

a better response to the treatment at the early stage of growth

c

CL

0) N U

c ro

rsi ro ro

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ro LD

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37

(7 or 7 and 14 day stage/s). Chloroindole auxin generated a

better response than indole-3-acetic acid and that too with lO'^l^

which induced maximum values but were statistically equal to

that of 10"^M.


The plants, with the advancement of the age and the

application of either of the auxins exhibited higher fresh and dry

mass of the shoot (Tables 15 and 16). 4-CI-IAA proved better

than lAA, applied at any stage of growth where the maximum

values were recorded with 10" M which Increased shoot fresh and

dry mass by 78 % and 138 % at day 30 and 38 % and 89 % at

day 45. A statistically equal response of 10"^M was observed with

10"^M of 4-CI-IAA, at day 30.


Like that of shoot mass, the fresh and dry mass of root

also increased in response to the auxin treatment and the age of

the plants (Tables 17 and 18). The seedlings gave maximum

response to 4-CI-IAA whose 10" M concentration proved best

enhancing the values by 52 %, 48 % and 73 %, 59 % of fresh

and dry mass of the roots at day 30 and 45, respectively, over

the control. Moreover, 10" M of 4-CI-IAA generated values


to 4-1

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0) N U

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cn VI 0) to E x: 1/1 0) L.

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4.2.4 Number of nodule

The number of nodules increased up to day 45 (Table 19).

This increase got a boost from the exogenous application of

auxins (lAA or 4-CI-IAA). Out of these two auxins, 4-CI-IAA

excelled in its effect over lAA. A maximum 42 % and 47 %

increase at 30 and 45 DAS, over the control, was recorded with

lO'^M of 4-CI-IAA fed to the seedlings, both at 7 and 14 day

stages. However, these values were comparable with those

expressed by the plants supplied with 4-CI-IAA (lO'^M) at day 7

only.


The fresh and dry mass of nodules exhibited an increasing

trend with the advancement of the plant age (Tables 20 and 21).

Moreover, the seedlings supplemented with either of the auxins

(lAA or 4-CI-IAA) had more mass of nodules at both the stages

of sampling (30 and 45 day). Out of the treatments, 4-CI-IAA

(10"^M) had maximum impact which improved fresh and dry mass

by 68 % and 60 % at day 30 and 64 % and 55 % at day 45, over

the control, respectively. These values were followed by those

generated by 10"^M of 4-CI-IAA. Furthermore, the application of

the hormones twice (at 7 and 14 DAS) generated a better

response than fed at either of the two stages of growth.

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Over all nitrogen level in the nodules increased as their

age advanced from 30 to 45 days (Table 22). However, the

nodules developed at the control plants possessed maximum

nitrogen content compared with those fed with either of the

auxins at any stage of growth. The plants fed at 7 and/or 14 day

stage/s with 4-CI-IAA (10"^M) possessed least nitrogen in their

nodules, at both the stages of sampling.


Like nitrogen content, the carbohydrate level in the

nodules improved with the age of the plant and was maximum in

the control plants (Table 23). Auxin treatment generally

decreased nodule carbohydrate level and the most by 4-CI-IAA

(lO'^M), irrespective of the time of application.


The treatment of the plants with either of the auxins

generated a significant response in the leghemoglobin content of

the nodules (Table 24). While comparing the effect of two auxins

with each other, chloroindole auxin had a better effect than the

indole-3-acetic acid and that too with 10'^ M which was

statistically equal to that of lO'^M. The application of the auxins

twice (7 and 14 DAS) proved superior to single application (7 or

14 DAS). Moreover, the values were higher as the age advanced

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from 30 to 45 DAS. The best combination was observed to be the

application of 10" M of 4-CI-IAA twice (7 and 14 DAS).

4.2.9 Nodule nitrogenase activity

The activity of nodule nitrogenase increased as the growth

progressed from 30 to 45 DAS (Table 25). The values were

significantly higher in the nodules of the plants fed with auxin.

The application of the auxin twice (7 and 14 DAS) proved superior

than single application (7 or 14 DAS). Maximum values at either

of the stages of sampling (30 or 45 DAS) were recorded with

10"^M of 4-CI-IAA, supplied at 7 and 14 DAS but was statistically

equal to that of 10"^M of 4-CI-IAA.


The content of nitrogen in the leaves increased with the

age of the plants (Table 26). Moreover, the leaves exhibited a

larger quantity of nitrogen in the plants supplied with auxin.

Chloroindole auxin excelled in its effect over indole-3-acetic acid.

A medium concentration (10"^ M) of 4-CI-IAA proved best but the

values were statistically equal to that of 10'^ M.


The nitrate content in the leaves decreased as the growth

advanced, but increased to a significant level by auxin treatment,

at both the stages of sampling (Table 27). The application twice

(7 and 14 DAS) proved better than single application (7 or 14

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DAS). 4-CI-IAA, irrespective of its concentration, excelled over

lAA, wliere 10'^ M concentration was most effective.


The activity of NR was significantly accelerated by the

application of either of the hormone (lAA or 4-CI-IAA), but

decreased as the plant growth progressed from day 30 to 45

(Table 28). 4-CI-IAA proved superior to indole-3-acetic acid.

Compared with single application (7 or 14 DAS), two applications

(7 and 14 DAS) were better where maximum values were

generated by 10"^ M of 4-CI-IAA which were statistically equal to

that of the maximum concentration (10"^ M) of chloroindole auxin.


The activity of CA decreased with the age of the plant

(Table 29). The treatment had a significant impact on the enzyme

activity, where 4-CI-IAA generated a better response than lAA.

The plants that were fed hormone twice (7 and 14 DAS)

possessed higher CA activity than single application (7 or 14

DAS). Highest values 53 % and 49 % more than the control were

recorded in the plants supplemented with 10'^ M of 4-CI-IAA at 7

and 14 DAS, recorded at the two growth stages (30 and 45 DAS),

respectively.


The leaves possessed more chlorophyll at early stage (30

DAS) of plant growth than at the later stage (45 DAS) (Table 30).

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However, the plants that received auxin treatment exhibited a

larger quantity of the pigment than the control, at the two stages

of growth (30 and 45 DAS). The plants were more responsive to

4-CI-IAA than lAA where 10" M of the auxin proved best.


The plants at 30 day stage photosynthesized more

efficiently than at day 45 (Table 31). The values improved further

if the plants were supplied with either of the auxins. Out of the

two auxins, 4-CI-IAA proved better than lAA whose medium

concentration (10"^M) generated maximum values 44 % and 59%

more than the control at 30 and 45 day stages, respectively.


The production of ethylene from the plants increased as

the growth progressed from day 30 to 45 (Table 32). The

application of auxins further stimulated its release (synthesis) and

that too to a maximum level with 4-CI-IAA. The plants receiving

single application (7 or 14 DAS) produced lesser quantity than

applied twice (7 and 14 DAS). The maximum values 144 %

and 59 % higher, over the control, were recorded with 4-CI-IAA

(lO'^M) at day 30 and 45, respectively.


The seedlings that received auxin treatment possessed

significantly larger member of pods per plant and seed yield per

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plant and 100 seed mass, at harvest (Tables 33 and 34). The

application at 7 and 14 DAS was more effective than once at

either of the two stages (7 or 14 DAS). Maximum values for the

above parameters 43 %, 44 % and 17 %, over the control, were

recorded with IC'^M of 4-CI-IAA, supplied twice (7 and 14 DAS).

Number of seeds per pod and per cent protein content of the seed

did not show any significant response.

4.3 Experiment 3

The surface sterilized, healthy seeds of moong {Vigna

radiata L. Wilczek) cv. T-44 were sown in earthen pots filled with

sandy loam soil and farmyard manure (9:1). The resulting

seedlings were sprayed with 0, 10"^°M', 10" M or lO'^M of lAA or

4-CI-IAA, on their foliage at 10 and/or 20 day stage/s of growth.

The plants were sampled at 30 and 45 DAS to assess the

characteristics briefly explained below and summarized In tables

35-56. The rest of the plants were allowed to grow to maturity to

study the yield characteristics, at harvest (65 days after sowing).


The values for both shoot and root increased as the growth

of the plants progressed from day 30 to 45 and improved further on

being sprayed with the auxins, irrespective of the time of

application (Tables 35 and 36). Chloroindole auxin (10"^M) proved

best for both shoot and root growth where the values increased by

45 %, 28 % and 52 %, 34 % at day 30 and 45, respectively.

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However, in case of the former the values were statistically equal to

that of lO'^M, at both the stages of sampling (30 and 45 DAS).


As the age progressed, both shoot fresh and dry mass

increased (Tables 37 and 38). Keeping aside the auxin

concentration and the time of application, the treatment

significantly enhanced both fresh and dry mass of the shoot where

4-CI-IAA excelled in its effect. Maximum values were generated by

10" M of 4-CI-IAA that were 84 %, 46 % and 148 %, 86 % more

than the control at 30 and 45 day stages, respectively but were



Root fresh and dry mass (Tables 39 and 40) followed a

pattern similar to that of shoot. Auxin application favoured fresh

and dry mass to improve where 4-CI-IAA excelled over lAA and that

too with higher concentrations (10"^M and lO'^M) which were

statistically equal in their effect. A maximum increase of 45 %,

42 % and 85 %, 42 % in their respective values, at 30 and 45 day

stages, over the control, was recorded with 10'^ M of 4-CI-IAA.

4.3.4 Number of nodules

The nodule number increased with the age of the plants (30

to 45 days) and exhibited a positive response to the auxin,

irrespective of the application pattern (Table 41). At the early stage

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of growth (30 DAS) the higher concentrations (10"^M and lO'^M) of

both the auxins generated statistically equal values but at a later

stage (45 DAS) 4-CI-IAA surpassed the others and increased it by

38 % and 21 %, over the control, at the respective stages of

sampling, and was closely followed by 10" M of 4-CI-IAA.


The values for fresh and dry mass of nodules increased with

the age of the plants (30 to 45 days) and were significantly higher,

over the control, in those where the foliage of the plants was

supplemented with the auxins, irrespective of the age of the plant

(Tables 42 and 43). Higher concentrations (lO'^M and 10"^M) of

4-CI-IAA proved best generating maximum values at both the

stages of sampling (30 and 45 DAS). However lAA (10"^M and

lO'^M) did not lag far behind in making its impact on the nodule dry

mass at 45 day stage and was statistically equal to that of the other

auxin (4-CI-IAA).


Nitrogen content increased from day 30 to 45 (Table 44).

However, it exhibited a significant decrease in the plants sprayed

with either of the auxins, compared with the control, irrespective of

the pattern of application. This decrease was more prominent in

those treated with 4-CI-IAA than lAA. The maximum values were,

therefore, recorded in the control.

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46


Carbohydrate content (Table 45) followed a trend

comparable with that of nodule nitrogen. Nodules borned at the

control plants had maximum carbohydrate that increased further

with the advancement of the age from 30 to 45 day but decreased if

the plants, irrespective of their age, were sprayed with auxin. Loss

was more prominent if the auxin concentration was increased from

10" °M to lO'^M.


The extended plant growth (30 to 45 day) favoured an

increase in the leghemoglobin content (Table 46). Moreover, the

plants applied with auxin to their foliage exhibited higher

leghemoglobin content than the control. At both the stages of

sampling (30 and 45 DAS), 4-CI-IAA generated significantly higher

values compared with lAA. A maximum increase of 22 % and 21 %,

at 30 and 45 day stages, was recorded with lO'^M of 4-CI-IAA,

compared with control, but was statistically equal to that of 10"^M.

4.3.9 Nodule nitrogenase activity

Nitrogenase activity increased both with the advancement of

age and the application of the auxin (Table 47). Out of the two

auxins tested, 4-Cl-IAA proved superior in its effect than lAA. The

plants supplied with auxin at 10 and 20 DAS possessed higher

values at both the stages (30 and 45 day) of growth than being

supplemented once either at 10 or 20 DAS. A maximum increase of

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51 % and 32 %, over the control, was recorded with lO'^M of 4-0-

lAA sprayed twice at 10 and 20 DAS, estinnated at 30 and 45 DAS,

respectively. This effect was statistically equal to that of lO'^M at

day 30 but lagged behind at the later stage of sampling (45 DAS).


Nitrogen content increased with the age of the leaves (Table

48). Moreover, its level improved further In the plants that were

treated with auxin and in particular with 4-CI-IAA. At both the

samplings (30 and 45 DAS), the higher concentration (lO'^M and

lO'^M) was most effective that increased the values by 39 % and

40 % over the control, at the respective stages of growth.


It is evident from table 49 that nitrate content decreases as

the leaves attain an age but exhibited a significant improvement if

they are applied with auxin at 10, 20 or 10 and 20 DAS where the

latter proved most effective at the two stages of sampling.

Chloroindole axuin, compared with indole-3-acetic acid, proved

superior. Maximum increase, over the control, was of the order of

28 % and 30 % with lO'^M of 4-CI-IAA, sprayed twice (10 and 20

DAS), sampled at day 30 and 45, respectively.


As the leaves aged (30 to 45 DAS), the NR activity

decreased but improved to a significant level on being fed with

auxin (Table 50). Single application (10 or 20 DAS) did not prove as

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48

effective as twice (10 and 20 DAS). 4-CI-IAA excelled in its effect

over lAA and produced maximunn values that were 69 % and 40 %

more than the control, at the two stages of sampling if sprayed both

at day 10 and 20 stages.


The activity of the enzyme was less at day 45 than at day 30

(Table 51). However, the level increased in plants sprayed with

auxin, at the two stages of sampling, compared with their

respective controls. 4-CI-IAA surpassed lAA in its effect whose

higher concentration (lO'^M or lO'^M) produced maximum values

that were significantly different from each other at 30 day stage but

at 45 day stage, the effect was comparable.


The content of the pigment exhibited a trend comparable to

CA activity (Table 52). The increase generated by the auxin was

more prominent with 4-CI-IAA than lAA. The higher concentration

(lO'^M or 10"^M) of 4-CI-IAA produced maximum values that were

statistically equal to each other at both the stages of sampling (30

and 45 DAS). The increase was 46 % and 45 %, over their

respective controls.


Like that of the earlier parameters (Tables 50, 51, 52) that

declined with age, the ageing leaves also photosynthesized at a

slower pace (Table 53), but their treatment with auxin improved the

efficiency at both the stages of sampling (30 and 45 DAS). 4-CI-IAA

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49

was more effective than lAA whose medium concentration (lO'^M)

improved the rate by 37 % and 41 %, at 30 and 45 day stages,

over the control, respectively. However, a comparable response was

generated by 10" M of 4-CI-IAA.


The leaves released more ethylene as their age advanced

from day 30 to 45 (Table 54) and it increased further in those

plants which were treated with auxin. The ethylene released from

auxin treated plants, was in proportion to the concentration of the

hormone-applied, at both the stages of sampling. The plants

receiving auxin at two stages (10 and 20 DAS) of growth released

more ethylene than supplied once (10 or 20 DAS).


Out of the parameters determining the biological yield, the

number of pods per plant, mass of 100 seed and seed yield per

plant was significantly affected by the treatment (Tables 55 and

56). Over all effect of 4-CI-IAA was better than lAA. Higher

concentrations (10"^M or 10"^M) was quite prominent in its effect,

and generated values statistically equal to each other in terms of

number of pods, seed mass and seed yield. A maximum increase in

the values of these parameters was 49 %, 51 % and 56 % over the

control, that was obtained by 10" M of 4-CI-IAA. Single application

(10 or 20 DAS) of 4-CI-IAA was less effective than applied twice (10

and 20 DAS) in improving number of pods per plant and seed yield

per plant.

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Discussion

Chapter- 5

DISCUSSION

Out of the genomic information stored in a cell for the

synthesis of the proteins, needed in the whole life span of the

plant to regulate various activities, only a limited portion of it is

expressed at any given time of plant growth and development.

Each set of these proteins (enzymes) is specifically recognized to

regulate a definite process of physiochemical expressions, going

on in the cell. However, there are forces that play a significant

role in selecting the pattern of the repression/derepression of

these genes. The important ones are light (Thompson and White,

1991), poliutants/elicitors/phytotoxins (Royals et al., 1992),

phytohormones (Cleland, 1999) and also the electrical signals and

small biomolecules generated while establishing communication

between the cells (Robards and Lucas, 1990; Lucas et al., 1995).

Among the phytohormones, the very first recognized group is

classified as auxin, whose physiological functions are quite

diverse. The most recent one included in this group of chemicals

is 4-chloroindole acetic acid (4-CI-IAA) identified in various

groups of plants and demonstrated to be many times more active,

in a number of bioassays, than lndole-3-acetic acid (Engvild,

1994; Reinecke, 1999). Some of these that may be mentioned

here are cell division and elongation; apical dominance; fruit

setting and their ripening and abscission of plant organs by

51

regulating plant metabolism through the involvement of genes

(Davies, 2004).

In the present study the auxin (lAA or 4-CI-IAA) fed

through seeds, roots or leaves significantly increased the number

of nodules (Tables 4, 19 and 41), nodule fresh and dry mass

(Tables 4, 5, 20, 21, 42 and 43) and the activity of the enzyme

nitrogenase (Tables 7, 25 and 47) in mung bean plants, observed

at two stages of growth (30 and 45 DAS). The sequence that

leads to the establishment of nitrogen fixing mature nodules

begins with the infection thread formed by the joint action of

bacteria and the host that is some how activated by

phytohormones (Dart, 1977; Hopkins, 1995). The observation is

further supported by the reported increase in nodulation and

nodule mass, in lAA treated alfalfa (Gruodien and Zvironaite,

1971) and groundnut supplemented with NAA (Srinivasan and

Gopal, 1977). Similarly, pea (Nandwal and Bharti, 1982) and

groundnut (Bishoi and Krishna, 1970) exhibited higher nitrogen

fixing ability in response to cytokinin and groundnut (Vardhini and

Rao, 1999) and chickpea (Ali et al., 2006a) to brassinosteroids. It

gives an impression that chemicals belonging to various groups of

phytohormones have an inherent characteristic to activate the

process of symbiotic association between the host and the

bacteria by favouring an increase in the number of nodules and

their mass. Moreover, the volume of the atmospheric nitrogen

reduced to ammonia in the nodules of the auxin-treated plants is

52

further elevated by the observed increase in the activity of the

enzyme nitrogenase (Tables 7, 25 and 47) because of the

regulatory action of auxin on transcription and/or translation

(Macdonald, 1997).

The nitrogen fixing potential of each nodule is determined

by three main factors (Marschener, 2002). (a) Availability of

photosynthates to the bacteroids : It is evident from Tables 10,

31 and 53 that the host plants photosynthesized at a faster pace

on being treated with the auxin (lAA or 4-CI-IAA) than the

control. This should have facilitated the availability of a large

share of carbohydrates to the nodules, (b) Effective maintenance

of low oxygen level in the interior of the nodule so that the

enzyme nitrogenase may be maintained active at a maximum

degree to metabolize carbon and nitrogen (Vance and Gantt,

1992). This ideal interior atmosphere is achieved naturally by the

presence of two operations (i) existence of cortex borned

diffusion barrier around the nodule and (ii) high rate of oxygen

consumption in the bacteroid to generate respiratory energy.

Moreover, the oxygen supply is further controlled by the

mediation of leghemoglobin (Marschener, 2002) whose quantity

has increased in the nodules borned on the plants that received

auxin treatment (Tables 6, 24, 46). Therefore, a limited oxygen

supply may have further elevated the activity of nitrogenase. A

reasonable fraction of oxygen is also used in the cytosol of host

cells to hydrolyze carbohydrates by operating glycolysis (oxidative

53

pathway) to make available fumarate and succinate, preferred

respiratory substrate (Kim and Copeland, 1996) that are carried

to bacteroids for generating energy used by the enzyme

nitrogenase (McKay et al., 1988). (c) Rapid export/metabolism of

ammonia, produced after the reduction of nitrogen. Out of the

carbohydrates transported, a significant quantum serves as

receptor of ammonia, in the cytosol of the host cells. The

carbohydrate consumption in the nodules may be touching 50%

mark, at certain stages of growth, in cowpea (Pate and Herridge,

1978). Lower carbohydrate level (Tables 6, 23 and 45) in the

nodules, borned on the auxin treated plants, is possibly an

expression of increased bacterial activity that is self evident from

elevated level of the activity of nitrogenase. The enzymes that

catalyse the reduction of nitrogen to ammonia which diffuses

across the peribacterial membrane by simple diffusion (Udvardi

and Day, 1993) into the cytosol of host cells. This is the site

where ammonia is metabolized to glutamine. However, for

maintaining higher efficiency of nitrogenase both ammonia and

the metabolic product/s are to be exported at faster pace that is

very much regulated by glutamine synthetase (GS) and glutamate

synthase (GOGAT) system (Vance and Gantt, 1992). The activity

of GS and GOGAT enzymes, like those of others (Tables 7-9, 25,

28, 29, 47, 50 and 51, Ahmad and Hayat, 1999; Hussain, 2000;

Ahmad et al., 2001a,b; Hayat et al., 2005) could have been

favourably affected by the applied auxin. This will naturally result

in a decrease of nitrogen level in the nodules (Tables 5, 22 and

54

44) and an increase in tine leaves (Tables 7, 26 and 48). Nandwal

and Bharti (1982) have also reached to a similar conclusion in the

auxin treated pea plants.

Besides ammonia being incorporated into organic

compounds, a significant quantity of nitrate (NO3) is removed by

the roots from the soil to fulfill the nitrogen requirement of the

plant, out of these two, latter is the major source of inorganic

nitrogen (Marschener, 2002). The level of nitrate in the leaves of

auxin (lAA or 4-CI-IAA) treated plants was higher than the control

(Tables 8, 27 and 49). This increase in the level of NO3 may be

attributed to the elongation and/or proliferation of the roots

(Tables 1, 14, 36) that could have resulted in an increase in the

area of the soil, being explored by the roots, for the uptake of

NO3. Moreover, the auxins also modify the activity of membrane

bound ATPase and ionic channels, at the level of the roots,

affecting the solute uptake and the cellular turgor (Macdonald,

1997) which could have facilitated the influx of NO3 from the soil.

Ahmad and Hayat (1999) and Hussain (2000) have also reported

an increase in nitrate content, both in the leaves and the roots of

pea, in response to auxin (lAA, IBA or 4-CI-IAA). At different

levels of the plant, this nitrate is initially reduced to nitrite by the

enzyme nitrate reductase (NR) whose activity is determined by

the concentration of the substrate, its rate of synthesis,

degradation and reversible inactivation (Solomonson and Barber,

1990), the level of metabolic sensors and signal transducers

">,_ L<S ->

(Campbell, 1999) and the relative concentration of,-various

phytohormones e.g. auxins (Ahmad, 1994, 1998; Ahmad and

Hayat, 1999; Hussain, 2000; Ahmad et al., 2001b; Hayat et al.,

2005), gibberelins (Prakash and Kapoor, 1984; Haque e^ al.,

1989; Hayat et al., 2001; Fariduddin, 2002) and brassinosteroids

(Singh et al., 1993; Hayat et a!., 2003; Ali et al., 2006a,c). The

observed increase in NR activity (Tables 8, 28 and 50) is either an

impact of the auxin on the transcription and/or translation (Key,

1969) or a cumulative effect of the factors mentioned above as

the auxin is recognized to act at multiple points.

The plants supplemented with auxin exhibited high

carbonic anhydrase (CA) activity (Tables 9, 29 and 51 ; Ahmad et

al., 2001b), the enzyme that catalyses interconversion of CO2 and

HCO3 (Okabe et al., 1984). The level of CA is very much

determined by the photon flux density, CO2 concentration, the

availability of Zn (Tiwari et al., 2005) and the transcriptional state

of the genes that encode CA protein (Kim et al., 1994). Auxin

could have acted at specific genes to modify their state of

expression because these chemicals are involved in transcriptional

and/or translational processes (Key, 1969) hence increase the

level of required enzyme proteins. Moreover, this act of the auxin

may have also been the basic cause of auxin-induced

enhancement of chlorophyll pigments (Tables 9, 30 and 52).

Therefore, higher CA activity, larger quantity of the

photosynthetic pigments, lower stomatal resistance (Arteca and

56

Dong, 1981) coupled with higher rate of phosphorylation

(Chatterjii et al., 1975) induced by auxins, finally culminated into

improved rate of photosynthesis (Tables 10, 31 and 53).

Moreover, a positive correlation between CA and photosynthesis

reported by Edwards and Mohammed (1973), Okhi (1978) and

Ahmad etal. (2001b) further strengthened the above belief.

Plants regulate the production of ethylene at multiple

points (Arteca, 1997). However, from the metabolic point of view,

it is the endogenous level of 1-aminocyclopropane-l-carboxylic

acid (ACC) synthase and ACC oxidase (Imaseki, 1999) that

determine ethylene synthesis, in plants. Auxins have a positive

impact on the activity of ACC synthase (Arteca et al., 1993),

therefore, elevate the level of ethylene and its release from the

plants (Tables 10, 32 and 54; Ahmad etal., 1987).

It is quite evident from the earlier discussion that most of

the parameters studied have responded favourably to the auxin

treatment, irrespective of the auxin type and the methodology

adopted to feed the chemical. Moreover, auxin shifts the electrical

properties of the plasma membrane by influencing the membrane

bound proton pump, that favours the transfer of the protons into

the cell wall. This acidification of the cell wall generates a pH

most suited for the activation of hydrolases, involved in the

loosening of cell wall (Hopkins, 1995). Simultaneously, auxin also

bring about changes in the behaviour of ionic channels, resulting

in the readjustment of the direction of the movement of the ions

57

and solutes succeeding to a shift in the cell turgor (Macdonald,

1997). Specific signals so generated repress/derepress a selected

set of genes that alter the activity, level and/or the type of the

protein (Sitbon and Parrot-Rechenmann, 1997). These sequential

happenings will naturally develop a healthy plant as expressed by

length, fresh and dry mass of shoot and root (Tables 1-3, 13-18,

35-40). Moreover, auxin induced delay in the process of

senescence of plant organs, in particular that of leaves and

flowers (Hopkins, 1995; Davies, 2004) will automatically help the

plant in extending the life span of photosynthetically active sites

and also prevent the premature loss of flowers and fruits. This

consequently resulted in the observed increase in the number of

pods per plant (Tables 11, 33 and 55). The belief gets additional

support from Marschener (2002) that auxin increases the degree

of sink at the level of seeds, directing the flow of metabolites to

the developing seeds consequent to an improvement in the seed

mass (Tables 12, 34 and 56) and seed yield per plant, at harvest

(Tables 11, 34 and 56). A similar increase in seed yield in

mustard has also been reported by Ahmad et al. (2001b).

Throughout this study it has been observed that a

moderate concentration (10"^M) of the auxin has been most

effective possibly because of its being an optimal dose for

generating maximum response and a concentration above that

(lO'^M) being supraoptimal. Out of the two types of the auxins,

lAA and 4-CI-IAA, employed in this study the latter proved

58

superior in improving the values for most of the vegetative and

reproductive characteristics. Similar observations have also been

reported earlier in Avena coleoptile elongation (Marumo et al.,

1974), rooting and ethylene synthesis (Ahmad et al., 1987)

induction of NR synthesis (Ahmad and Hayat, 1999), a-amylase

activity (Hirasawa, 1989; Ahmad et al., 2001a), activity of CA and

rate of photosynthesis (Ahmad et al., 2001b) and

hyperpolarization of plasma membrane (Karcz and Burdach,

2002). This high auxm-like activity of 4-CI-IAA is proposed to be

because of its higher degree of resistance to metabolization

(Karcz and Burdach, 2002). Further, while testing various indole-

3-cetic acids, halogenated at different positions of the ring (4-CI-

IAA, 5-CI-IAA, 6-CI-IAA, 7-CI-IAA) it was resolved by Engvild

(1994) that the varied degree of degradation and/or conjugation

or other related processes is/are the basic reason/s for generating

diverse response m plants. Moreover, Reinecke et al. (1998) has

gone one step further and stated that the difference in their

activity lies at the level of the impact of 4-CI-IAA on the receptor

of signal transduction pathway.

Conclusion

The present study reveals that

(i) The efficiency of the site of application of the auxin

followed a trend; foliar spray > root application > seed

soaking.

59

•

(ii) The treatment of the plant roots/foliage twice (early + a

bit late) generated the best response, compared with the

single application, at either of these stages of growth.

Extended duration of soaking (12 h) of seeds proved

superior over 8 hours.

(iii) 4-CI-IAA excelled, over indole-3-acetic acid, in its effect.

(iv) Out of the three concentrations of the two auxins used,

10"^M generated the best response.

(v) Activity of the enzymes, viz. nitrogenase at' the level of

nodule; nitrate reductase and carbonic anhydrase, at the

level of leaf increased by the application of either of the

auxins (lAA or 4-CI-IAA), irrespective of the mode of

application.

(vi) The content of chlorophyll pigment and the rate of

photosynthesis, in the leaves of plants treated with the

auxin (lAA or 4-CI-IAA) was higher than that of the

control.

(vii) The yield of the plants was significantly affected by the

auxin (lAA or 4-CI-IAA), where the application of hormone

twice was more effective than single dose.

3ummart(

Chapter - 6

SUMMARY

This thesis is based on the following five chapters

Chapter 1 : It explains the significance of the problem entitled,

"Metabolic responses to 4-chloroindole acetic acid in Vigna radiata

(L.) Wilczek".

Chapter 2 : It represents a comprehensive review of available

literature, related with the above problem, pertaining to growth,

metabolism and yield characteristics of the plants.

Chapter 3 : It explains all the details of the materials used and

the methods employed in conducting the experiments,

assessment of vegetative characteristics and chemical analysis of

the biological material.

Chapter 4 : It is comprised of the tabulated data, recorded

during the study, and a brief description.

Chapter 5 : It attends to the possible explanation/s for the

observations, in the light of the earlier findings.

The salient features of the observations, recorded in each

of the three experiments, are summarized below :

Experiment 1

The surface sterilized, healthy seeds of mung bean {Vigna

radiata L. Wilczek) cv. T-44 were soaked in water (control), lAA

or 4-CI-IAA (10"^°M, lO'^M, or lO'^M) for 8 or 12 hours, in

sterilized petriplates. Treated seeds were washed with DDW and

subsequently coated with a uniform layer of Rhizobium sp.,

followed by sowing in earthen pots, filled with sandy loam soil.

The resulting plants were sampled at day 30 and 45 and analyzed

for the following characteristics :

(i) growth (length, fresh and dry mass of shoot and root);

(ii) nodule number, their fresh and dry mass;

(iii) leghemoglobin content and nitrogenase activity in fresh

nodules;

(iv) per cent nitrogen and carbohydrate in dried nodules;

(v) nitrogen and nitrate content in dried leaves;

(vi) activity of nitrate reductase (NR) and carbonic anhydrase

(CA);

(vii) the content of chlorophyll pigments and photosynthetic

rate,

(viii) ethylene production by the whole plant; and

(ix) the yield characteristics, at harvest.

The values for most of the parameters, referred above,

increased in response to the auxin (lAA or 4-CI-IAA). However,

nodule nitrogen and carbohydrate contents decreased. 4-CI-IAA

excelled in its effect over lAA. Soaking the seeds for 12 hours, in

lO'^M of 4-CI-IAA generated the best economical response in the

plants.

62

Experiment 2

The surface sterilized, healthy and Rhizobium coated seeds

of mung bean {Vigna radiata L. Wilczek) cv. T-44, were sown in

earthen pots, filled with sandy loam soil. The resulting seedlings

at 7 and/or 14 day stage/s were percolated with aqueous solution

of lAA or 4-CI-IAA (10'^°M, lO'^M, or lO'^M) at the rate of 20 cm^

per seedling, through their roots. The plants were sampled at day

30 and 45 to study the characteristics as stated in Experiment-1.

The overall response of the plant to the auxin (lAA or 4-CI-IAA)

was somewhat the same as in the earlier experiment. However,

two applications at 7 and 14 day stages proved superior than

single application at either of these stages.

Experiment 3

The seeds were sown in the same way as in Experiment 2.

The resulting seedlings, at 10 and/or 20 day stage/s were

sprayed with aqueous solution of lAA or 4-CI-IAA (10"^°M, lO'^M,

or lO'^M) at the rate of 3 cm- per seedling to their foliage. The

plants were sampled at day 30 and 45 to assess the

characteristics, referred in Experiment 1. The plants sprayed with

auxin exhibited higher values for most of the characteristics and

4-CI-IAA surpassed in its effect, over lAA. The application of the

hormone (lAA or 4-CI-IAA) twice at 10 and 20 day stages proved

superior than at any one of them. However, nodule nitrogen and

carbohydrates declined in response to the treatment and number

63

of seeds per pod and per cent seed protein did not give any

response.

Comparing the plant response to auxin, fed by three

varied techniques (Experiment 1, 2 and 3) the leaf applied proved

most effective in shaping the growth of plants for maximum

economical gains.

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K- '

* ^ i S " >

JAppandioc

APPENDIX

1. Preparation of reagents for nitrogen estimation.

1.2 2.5N NaOH

5 g of NaOH was dissolved in sufficient DDW and final volume

was made up to 100 cm^

1.2 1 0 % sodium silicate

10 g of sodium silicate was dissolved in sufficient DDW, in a

100 cm' volumetric flask and final volume was made up to

ti^e mark by using DDW.

2. Preparation of reagents for carbohydrate estimation

2.1 1.5 N H2SO4

40.8 cm^ of concentrated H2SO4 (AR) was pipetted into

sufficient DDW and final volume was made up to 1000 cm^

by using DDW.

2.2 5 % phenol

50 cm^ of distilled phenol was mixed with 950 cm- of DDW,

to get 1000 cm^ solution.

3. Preparation of reagents for leghemoglobin estimation

3.1 Sodium phosphate buffer (pH 7.4)

It was prepared by separately dissolving 13.9 g of NaH?P04

and 26.82g of Na2HP04 in sufficient DDW to make the final

volume of each solution to 1000 cm^ These solutions were

mixed in the ratio of 19:81, respectively.

3.2 Alkaline pyridine reagent

It was prepared by dissolving 0.8 g of NaOH in 50 cm- of

DDW and allowed to cool. 33.8 cm^ of pyridine was added to

it and diluted to 100 cm^ with DDW. This produced 4.2 M

pyridine in 0.2 M NaOH.

4. Preparation of reagents for nitrate estimation

4.1 Phenol disulphonic acid

25 g pure distilled phenol (AR) was dissolved in

concentrated sulphuric acid to which fuming sulphuric acid

was added (13 % SO3). The solution was heated for 2 hours

at 100°C, in a water bath. This solution was stored in a dark

bottle, in a refrigerator.

5. Preparation of reagents for nitrate reductase activity

5.1 O.IM Phosphate buffer (pH 7.4)

27.2 g of KH2PO4 and 45.63 g of K2HPO4.7H2O were

dissolved separately in 1000 cm^ of DDW. The above

solutions of KH2PO4 and K2HPO4.7H2O were mixed in the

ratio of 16:84.

5.2 0.2M KNO4

20.2 g of KNO4 was dissolved in sufficient DDW and final

volume was made up to 1000 cm"*, by using DDW.

Ill

5.3 5 % isopropanol

5 cm- of isopropanol was pipetted into sufficient DDW and

final volume was made up to 100 cm^, by using DDW.

5.4 1 % Sulphanilamide

Ig of sulphanilamide was dissolved in 100 cm^ of 3N HCI.

3N HCI was prepared by dissolving 25.86 cm- of HCI in

sufficient DDW and final volume was maintained to 100 c m \

by using DDW.

5.5 0.02 % N-1-nephthyl-ethylenediamine hydrochloride

(NED-HCI)

20 mg of NED-HCI was dissolved in sufficient DDW and final

volume was made up to 100 cm- , by using DDW.

6. Preparation of reagents for the estimation of carbonic

anhydrase activity

6.1 Cystein hydrochloride solution (0.2M)

48 g cystein hydrochloride was dissolved in sufficient DDW

and final volume was made up to 1000 cm" , by using DDW.

6.2 Sodium Phosphate buffer (pH 6.8)

27.8 g NaH2P04 and 53.65g Na2HP04 was dissolved each

separately in sufficient DDW and final volume was made

1000 c m l 51 cm^ of NaH2P04 and 49 cm^ of Na2HP04 were

then mixed to get the required solution.

IV

6.3 Alkaline sodium bicarbonate solution

16.8 g sodium bicarbonate (NaHCOs) was dissolved in

aqueous 0.2M NaOH solution [0.8g NaOH (1000 cm^)'^] and

final volume was made up to 1000 cm^, by using DDW.

6.4 0.002 % bromothymol blue

0.002 g of bromothymol blue was dissolved in sufficient

DDW and final volume was made up to 1000 cm^ by using

DDW.

6.5 0.05 N HCI

4.3 cm- of pure HCI was pipetted in sufficient DDW and final

volume was made up to 1000 cm^, by using DDW.

6.6 Methyl red indicator

5 mg of methyl red was dissolved in sufficient ethanol and

final volume was made 100 cm- , by using ethanol.

7. 80 % Acetone for chlorophyll estimation

80 cm- of pure acetone was mixed with 20 cm" of DDW.

8. Preparation of reagents for protein estimation

8.1 5 % trichloro acetic acid (TCA)

5 cm^ of TCA was mixed with 95 cm- of DDW.

8.2 I N NaOH

40g of NaOH was dissolved in sufficient DDW and final

volume was made up to 1000 cm^, by using DDW.

8.3 Reagent A : 2 % sodium carbonate (2 g dissolved in 100

cm^ DDW) and O.IN NaOH (4 g NaOH dissolved in 1000 cm^

DDW) were mixed in the ratio of 1:1.

Reagent B : 0.5 % copper sulphate (500 mg CUSO4

dissolved in 100 cm^ DDW) and 1 % sodium tartarate ( Ig

sodium tartarate dissolved in 100 cm' DDW), were mixed in

the ratio of 1:1.

Reagent C : 50 cm- of reagent A was mixed with Icm-^ of

reagent B, except omission of sodium hydroxide.

8.4 Folin's Phenol reagent

The reagent obtained from SRL Sisco Research Laboratory

Pvt. Ltd, Mumbai, India was diluted by DDW in the ratio

1:2.

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