the response of vigna radiata and …the response of vigna radiata and brassica jiincea to...

THE RESPONSE OF VIGNA RADIATA AND BRASSICA JUNCEA TO

28-HOMOBRASSINOLIDE AND KINETIN

ABSTRACT THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

Boctor of ${)tlo^ap{)p IN

BOTANY

QAZI FARIDUDDIN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNI\ ERSITY

ALIGARH (INDIA)

2002

The response of Vigna radiata and Brassica jiincea to

28-homobrassinolide and kinetin

QAZI FARIDUDDIN

Abstract of the thesis, submitted to the Aliga'"h M.'slim

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

Botany, 2002.

Four experiments were conducted with the aim to elucidate the

effect of 28-homobrassinolide and/or kinetin on the selected paramet2'"j

of the Vigna radiata L. Wilczek and Brassica juncea czern c'i coss. The

salient features in each of the experiment are mention'^d below :

Experiment 1

The surface sterilized, healthy seeds of moong (Vigna radicta L.

Wilczek) cv. T-44, were sown in sandy loam soil, filled in pots ( 25

cm^). The leaves of 25(A) or 30(B) day old plants were applied w'tn

water, aqueous solution of (KN) kinetin (lO-^M/lO-^M/Ur'^M) o- (HBR)

28-homobrassinolide (10-'°M/10-^M/10-^M). The plants v/ere sampled

26, 31, 35, 40 and 45 days, after sowing (DAS) from group-A an^ 31,

35, 40 and 45 DAS from group-B. These samples were analyzed for leaf

area; leaf dry mass; nitrate reductase activity; the contents of pr jte^i and

chlorophyll; carbonic anhydrase activity; net photosynthc' ate;

carboxylation efficiency; ethylene production; nodule nun^bcr; nciale

fresh and dry mass per plant and yield characteristics, at harvest.

All the above parameters, except nodule number; nodule fresh

and dry mass; seed number and 100 seed mass, gave positive response lO

the application of KN/HBR. HBR proved superior, than KN, in its effe-.t

where 10" M of HBR, applied at 25 DAS(A), generated best economical

response in the plants.

Experiment 2

The surface sterilized healthy seeds of mustard (B'-CSS'CJ

/iincea czcrn & coss) cv. Varuna, were sown in sandy loam soil, filled in

pots (25 cm^). The application of distilled water/aqu^ouj solutions of

(KN) Kinetin (10-*^M/I0-<' M/10- M) or (HBR) 28-homobrassinoiide (10"

"'M/IO^^M/10'^M) was done to the leaves of the plarus, oroe 29(A)/

44(B) days after sowing or twice (C) at both A and B. TA-^ letv^s were

sampled 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80 days Tffer sowing

(DAS) in group A or at 45, 50, 55, 60, 65, 70, 75, and 80 DAS m group

B and C. Leaf dry mass; nitrate reductase activity; the contents o. protein

and chlorophyll; carbonic anhydrase activity; net photosyiithttr- "xx and

carboxylation efficiency in first, second, third and founrh leaves were

significantly affected by the application of KN/HBR. The appl'cation of

lower concentrations of HBR (10'^° M/10'^ M) or higher conCwit'"atluns

of KN (lO'^M/lO'^'M) once at 29 DAS generated values mTdch highei than

other treatments and the control.

The biological yield, number of pods and seed yield per plant

were enhanced by the treatment. Moreover, the application of HBR, at A

or C, proved superior than KN, where lO' M of HBR proved best.

Experiment 3

The surface sterilized, healthy seeds of moor.g (Vigna radiala

L. Wilczek) cv. T-44, were sown in sandy loam soil, filled in pots (25

cm^). The leaves of the plants were applied with water or a mixture ( I I )

of aqueous solutions of lO' M of KN and lO' M of HBR at 2'; (A)/3C (B)

days after sowing, respectively. The plants were sampiea 26, 31, 35, 40

and 45 in group-A and 31, 35, 40 and 45 days after sowing in pro'';"B.

The treated plants possessed more leaf area; leaf dry mass; r.urate

reductase activity; the contents of protein and chlorophyll; carbonic

anhydrase activity; net photosynthetic rate; carboxylation efficiency and

biological yield; pod number; seed yield and seed protein consent, at

harvest.

Hormone application to the plants at 25 days afte'- s jvmg (DAS)

proved superior in its effect than 30 DAS.

Experiment 4

The surface sterilized, healthy seeds of mu^ta 'd {Brassica

jimcea czern & coss) cv. Varuna, were sown in sandy loam scii, i^Ved in

pots (25 cm^). The leaves were sprayed with water or a mixûie C :l) of

aqueous solutions of 10" M of HBR and 10" M of KN, on .p at 29/44

days after sowing (DAS) or twice at 29 and 44 DAS. Tl f-. leaves \/ere

sampled 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 DAS Tre êaves

receiving hormonal treatment twice both at 29 and 44 D. S ''x'îbited

maximum values of leaf dry mass; nitrate reductase aciivitv; tlie contents

of protein and chlorophyll; carbonic anhydrase a^'iîty; net

photosynthetic rate and carboxylation efficiency, l i e c e plants, at

harvest, exhibited higher biological yield, pod nuiribRi7%ad seed yield

per plant.

THE RESPONSE OF VIONA RADIATA AND BRASSICA JUNCEA TO

28-HOMOBRASSINOLIDE AND KINETIN

THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF

Boctor at ^Ul^eopl^p m

BOTANY

QAZI FARIDUDDIN

DEPARTMENT OF BOTANY AtlGARH MUSLIM UNI\ ERSiTY

ALIGARH (INDIA)

2002

DEDICATED

TO

MY PARENTS

DR. AQIL AHMAD M Phil ,Ph D PD Res Train (Denmark)

Plant Physiology Section Department of Botany Aligarh Muslim University AJigarh-202 002 U P ,(India) Phone 91-571-2408009 e-mail aqil_ahmad@lycos com

Dated 2 S . / ' 2 - - ^ 2 „

CERTIFICATE

This is to certify that the thesis entitled, "The response of Vigna

radiata and Brassica juncea to 28-homobrassinolide and kinetin",

submitted for the degree of Doctor of Philosophy in Botany, is a faithful

record of the bonafide research work carried out at the Aligarh Muslim

University, Aligarh, India by Mr. Qazi Fariduddin under my guidance

and supervision and that no part of it has been submitted for any other

degree or diploma.

(AQIt~?tmiABX

ACKNOWLEDGEMENTS

/ wish to express my sincere gratitude and indebtedness to my

esteemed supervisor, Dr Aqil Ahmad, Reader, Department of Botany, for

his able guidance and continued interest for the completion of this

research work.

J am thankful to Prof. SamiuUah, Chairman, Department of

Botany, Aligarh Muslim University, for providing the necessary facilities

during the research work.

I would like to place on record my profound thanks to respected

teachers, Drs. Ariflnam, Firoz Mohammad, MMA Khan, Nafees A. Khan

and Zaki A. Siddiqui for their useful comments and encouragement.

I have no words to express my heartiest thanks to Dr ShamsuI

Hayat for his help and constant motivation throughout the research

work.

Fortunately, 1 am blessed with a galaxy of sincere and adoring

friends and colleagues Mr M. Afzal Khan, Dr M. Azam Khan, Mr.

Rashid A. Faridi, Mr Devendra Kumar, Mr Ram Vilas, Mr. Shahid, Mr

Yousif, Mr. Ashfaque, Dr Arhsad Hussain, Dr. M. Mobin, Mr. Zake M.

Azam, Mr Ramzan Mir, Mr Fayaz, Mr Javid, Mr Shoukat, Mr Irfan,

Mr Manzer, Mr. Barket, Ms. Shahla Saeed and Ms. Naheed. Thanks are

also due to Mr H.K. Sharma for computer tying.

Mere words fail to express my appreciation lo my parents,

brothers and sisters who were the real source of motivation, inspiration

and patience for me to be at this stage.

1 thank Dr B.N. Vyas (General Manager, Godrej Agrovet Ltd.,

Mumbai, India) for the generous gift of 28-homobrassinolide.

Financial assistance rendered by Aligarh Muslim University,

Aligarh and Council of Scientific and Industrial Research, New Delhi in

the form of Junior Research Fellowship and Senior Research Fellowship,

respectively, are gratefully acknowledged.

(Qazi Fariduddin)

CONTENTS

CHAPTERS Page No.

1 INTRODUCTION I - 3

2 REVIEW OF LITERATURE 4 - 2 1

3 MATERIALS AND METHODS 22 - 34

4 EXPERIMENTAL RESULTS 35 - 55

5 DISCUSSION 56 - 62

6 SUMMARY 63 - 66

REFERENCES 67 - 95

APPENDIX 1 - ill

Chapter - 1

INTRODUCTION

INTRODUCTION

The success of "green revolution" in India, was the outcome of

the adoption of high yielding cultivars, supplemented with the optimal

level of inorganic fertilizers. However, the yielding capacity of most of

these crops has now attained a plateau, therefore, the application of

additional quantities of fertilizers is not only proving to be wasteful but

also counter productive. Moreover, the excessive use of inorganic

fertilizers is spoiling the soil characteristics as is evident from

nitrogenous fertilizers, releasing nitrate to a toxic level in the soil and

the drinking water (Thenabadue, 1989).

Agrophysiologists are, therefore, trying to evolve additional

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

evolved cultivars to fill the gap between productivity and 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.

These regulators are known to enhance the physiological efficiency of

the cells and thus exploit the genetic potential to a maximum extent.

Some of these hormones are known to enhance the rate of photosynthesis

and the nitrogen fixation (Arteca and Dong, 1981; Meena and Goswami,

1992; Hayat et al., 2000 and 2001a and b; Ahmad et al., 2001), delay

leaf senescence (Hopkins, 1995; Arteca, 1997), treated plants develop

higher stress resistance (Sairam, 1994) and finally exhibit better harvest

index (Ghosh and Biswas, 1991; Khan et al., 2002). The hormone treated

plants, therefore, have high seed/grain productivity (Bhatia and Kaur,

1997; Helmy et al., 1997; Khripach et al., 1997; Krishnan et al., 1999;

Khan et al., 2002).

Compared with auxins, gibberellins, cytokinins, abscisic acid

and ethylene.there is a new class of phytohormones that includes steroids

(brassinosteroids). These brassinosteroidl(BRs) not only regulate natural

growth and development (Clouse, 1996; Li and Chory, 1999) but have

higher potential in improving agricultural productivity by their

exogenous application (Ramraj et al., 1997; Khripach et al., 2000)

through an increase in the rate of cell elongation, activation of proton

pump, differentiation of vascular tissues (Mandava, 1988; Sakurai and

Fujioka, 1993; Yokota, 1997; Clouse and Sasse, 1998) and various other

metabolic activities (Khripach et al., 1999).

In certain aspects, kinetins are comparable with BRs (Arteca,

1997), therefore, in the present study, a comparative account has been

approached keeping in view the state of the plant because the plant

response is the result of the interaction between hormone/s and the

responsiveness of the tissue (Trewavas, 1982). Two crops {Vigna radiata

L. Wilczek and Brassica juncea czern and coss) were selected because

of their economical value and adoptibility by the local farmers.

Moreover, each crop is grown in different seasons of the year, covering

the two important climatic conditions (summer and winter). Kinetin (KN)

and/or 28-homobrassinolide (HBR) were applied, as aqueous solution,

to the plants at varied stages of growth to assess :

(a) the best concentration of the hormone/s

(b) the most suitable stage of growth having best interaction with the

applied hormone/s

(c) the metabolic and growth parameter showing maximum response

to the treatment and may be designated as a scale for forecasting

future growth and productivity..

Chapter - 2

REVIEW OF LITERATURE

CONTENTS

Page No.

2.1 Growth response 5 - 1 0

2.2 Metabolism 10 - 16

2.3 Plant protein content 16

2.4 Senescence 16 - 19

2.5 Yield parameters 1 9 - 2 1

2.6 Conclusion 21

REVIEW OF LITERATURE

Phytohormones are recognized as the regulators of plant

metabolism and thus integrate their developmental activities. These

characteristics of the hormones are being exploited, by their exogenous

application to plants, to regulate senescence, root differentiation,

abscission, fruit development, plant stature, weed control and in many

other economic gains (Arteca, 1997). Although, they are in use, in

agriculture, horticulture and floriculture since long but many of the

important aspects have remained unresolved in order to maximize the

true potential of the chemicals. This review, in the following pages,

includes the metabolic and morphological responses of the plants and

their parts, supplied with phytohormones.

Brassmosteroids may be listed among the recognized group of

phytohormones because of their involvement, as a regulator, in the

metabolism of plants. Brassinolide, as an active component of brassins,

was first extracted and identified in the pollen oi Brassica napus (Grove

el al., 1979). It has seven membered lactone ring, as part of a fused ring

system, and is the first natural plant growth substance shown to have a

steroidal structure. Moreover, the other sterol (Castasterone), a

biosynthetic precursor of brassinolide, highly active in, "rice-lamina

inclination test", was isolated from the insect galls of the chestnut

{Castanea sps) tree (Yokota et al., 1982). By this date, about 40 steroids,

structurally and functionally different from each other have been

characterized from various natural sources (Khripach et al., 1999). They

are widely distributed in plants (Kim, 1991). BRs are obligatory plant

constituents, largely concentrating in the reproductive organs and in the

actively growing tissues (Fujioka et al., 1998; Sasse et al., 1998).

The identification of certain mutants of Arabidopsis thaliana (Li

et al., 1996) and Pisum sativum (Nomura et al., 1997) capable to

synthesize brassinosteroids, have provided compelling evidences that

BRs are essential for plant growth and development. The analysis, at the

molecular level, proved their essentiality in diverse developmental

programmes including, cell division, cell expansion, vascular differen

tiation, etiolation and reproductive development (Clouse and Sasse,

1998). Therefore, brassinosteroids are now largely regarded as a new

class of plant hormone, in addition to auxins, gibberellins, cytokinins,

abscisic acid, ethylene and jasmonates. Moreover, the presence of BRs

also restores the normal response of plants to other phytohormones in

Arabidopsis mutants, by altering the hormonal sensitivity (Ephritikhine

el al., 1999).

Cytokinins, the well recognized group of phytohormones, are

adenine derivatives which regulate a number of activities in plants,

including cell division (Arteca, 1997). They are present in all the

members of plant community. At present, there are more than 200 natural

and synthetic cytokinins (Matsubara, 1990). They are largely synthesized

in the roots and transported to the shoot with the ascending sap but they

are in abundance in the young fruits and seeds.

2.1 Growth response

The desired orientation in plant growth, in response to various

phytohormones, is becoming a regular phenomenon for academic and/or

economic gains. The involvement of brassinosteroids in such cases is

very well supported by the observed deviation from normal development

in the mutant of Arabidopsis, lacking the capability to biosynthesize

BRs, but could be restored by their exogenous application (Altman,

1998, 1999; Choe et al., 1998, 1999a and b). However, the normal plants

of wheat and mustard, applied with BRs, achieved more fresh and dry

mass of the leaves and that of the whole shoot (Braun and Wild, 1984)

and an increase in length, width and fresh mass of the leaves of tobacco

(Diz et al., 1995). Likewise, the application of biobras-6 to tomato

plants (Nunez et al., 1996) and BRs to Vicia faba (Helmy et al., 1997)

favoured plant growth. BRs (brassinolide/epibrassinolide/

homobrassinolide) increased shoot length, dry mass production in Pinus

banksana (Rajasekaran and Blake, 1998), Arachis hypogea (Vardhini and

Rao, 1998), Brassica juncea (Hayat et al., 2000) and the length of

epicotyl of the soybean seedlings (Clouse et al.,1992). The growth

promoting capability was further corroborated by the observations on

the mutants of Arabidopsis thaliana, insensitive to BRs, showing

multiple growth and developmental defects (Clouse et al., 1996) which

were confirmed to be related with the genes, involved in BRs synthesis

(Clouse, 1996). The studies on Arabidopsis thaliana also demonstrated

the role of BRs alone, in cell elongation (Catterou et al., 2001a) by

rearranging the position of the tubules in the cell (Catterou et al., 2001b)

or in association with auxins (Mayumi and Shibaoka, 1995). This could

have also favoured the differentiation of trachary elements, in isolated

mesophyll cells of Zinnia elegans (Iwasaki and Shibaoka, 1991). The

other possible routes adopted by BRs might be the proton extrusion,

resulting in the hyperpolarization of the membrane (Cerana et al., 1983;

Romani et al., 1983; Dahse et al, 1990 and 1991; Cao and Chen, 1995;

Bajguz and Czerpak, 1996) and the induction of gene expression and

protein synthesis (Mussing and Altman, 1999). More specifically it has

been noted that BRs promote elongation by regulating gene expression

in the stem of Glycine max where the molecular cloning and the

characterization of epicotyl involved BRUl protein (Zurek and Clouse,

1994).

The effectivity of BRs, in regulating the cell elongation, very

much depended on the presence or absence of light (Mandava, 1988). It

was further proposed by Kamura and Inada (1991) that the elongation of

the epicotyl of Vigna radiata involved the phytochrome and it possibly

also mediated the BR action, in enhancing the growth of the epicotyl.

BRs (DA-6/epibrassinolide) in association with other hormones

(GA,) increased shoot mass and the length of spinach plants (Liang

et al., 1998). Similarly, synthetic brassinosteroid (22,23-S-homo-

brassinolide) interacted with lAA and GA,, exhibiting the capability to

enhance the auxin mediated elongation in Cucumis sativus L., but the

action did not depend on GA, (Katsumi, 1985). Similar conclusions with

auxins were also drawn by Yopp et al. (1981a) and Sala and Sala (1985).

Such a synergism was also observed in the bending responses of plants

(Yopp et al., 1981a and b). The BRs mediated elongation is proposed to

be mediated by auxins (Takeno and Pharis, 1982; Cohen and Meudt,

1983; Meudt, 1987) through the amplification of its action or by

sensitizing the tissue (Katsumi, 1985). However, Sasse (1990)

dissociated from the above suggestions and concluded that brassinolides

do not act through the auxins in the process of elongation. Moreover, in

tomato and soybean stem elongation promoted by BRs most likely did

not involve the auxin signal transduction pathways, even though both

the hormones effect the cell wall relaxation (Zurek et al., 1994).

The cell division was also favoured by the application of 24-

epibrassinolide to the protoplasts of cabbage (Nakajima e( al., 1996) and

brassinolide to that of Petunia hybrida (Oh and Clouse, 1998) in

association with auxin/cytokinin but failed to replace either of the

hormones.

Deviating from the above pattern of the application of the

hormone, it was supplied through the seeds by the pre-sowing soaking

treatment in the dilute aqueous solutions of the BRs. The seeds of Coffea

arabica treated with biobras-16 (Soto et al., 1997), Oryza sativa with

BRs (Wang, 1997), Cicer arietimim (Fariduddin et al., 2000) and

Triticum aestivum (Hayat et al., 2001b) with homobrassinolide

germinated to produce taller plants with higher fresh and dry mass than

the water soaked, controls.

BRs are also capable to strengthen the plants, under stress. The

water stressed plants of wheat, raised from the seeds, pre-treated with

homobrassinolide or its aqueous solution applied to the foliage of the

standing plants, increased leaf area and the total biomass (Sairam, 1994).

Similarly, pie-sowing seed treatment of Arachis hypogea with

brassinolide, 24-epibrassinolide or 28-homobrassinolide, supplemented

with 500 mM of NaCl solution, increased fresh and dry mass of the

seedlings (Vardhini and Rao, 1997).

The root growth, at the natural level of 24-epibrassinolide, in

the seedlings of wheat, mungbean and maize was inhibited but was

determined by the species, the age of the root and the site of application

of the hormone (Roddick and Ikekawa, 1992), Likewise, the response of

apical and basal regions of the excised roots of tomato (Roddick et al.,

1993) or that of Arabidopsis thaliana (Clouse el al., 1993) to

submicromolai concentrations of 24-epibrassinolide was negative. The

root length and its proliferation in soybean seedlings was also reduced

by epibrassinolide (Hunter, 2001). In contradiction to the above, the

treatment with BRs improved the rooting in the seedlings of Cucumis

sativiis (Ding et al., 1995) rice (Wang and Deng, 1992). Similarly, the

cuttings of Picea abies treated with 3, 15 or 60 ppm of (22S, 23S)-

homobrassinolide possessed more adventitious roots than the control

(Ronsch el al., 1993).

The special status extended to the legumes, over the other crops,

is because of their nitrogen fixing capability by forming nodules at the

level of their roots by symbiotic association. It is, however, a complex

phenomenon but involves external and internal factors, including the

phytohormones whose role is not very well defined. Moreover, not much

emphasis has been given in this direction, therefore, requires serious

efforts of the researchers. Among the various phytohormones tested, the

plants of Pisum sativum develop early nodules by kinetin treatment but

lAA delayed the process. These two hormones, together, induced

considerable increase in the mass of nodules (Nandwal et al., 1981).

Similarly, nodule number improved by the application of GA and kinetin

in Cicer arielinum (Garg et al., 1992) and NAA in Trifoliiim

alexendi'iainim (Garg et al., 1990). However, the nodulation in Glycine

max was inhibited by 24-epibrassinolide (Hunter, 2001).

2.2 Metabolism

The phytohormones are employed to improve the efficiency of

plants. They are known to affect the photosynthetic rate of the plants

and regulate the partitioning of the assimilates resulting in an increase

or decrease of the plant size (Arteca, 1997). However, the response in

terms of photosynthesis, varies form plant to plant and hormone to

hormone.

The leaf applied aqueous solution of homobrassinolide to wheat

and mustard (Sairam, 1994; Hayat et al., 2000, 2001a) and DA-6

(dialkylaminoethylalkanoate) or BR (epibrassinolide) in association with

gibberellic acid (GA^) to spinach enhanced the photosynthetic rate

(Liang et al., 1998).

The application of kinetin to potato plants (Borzenkova, 1976),

benzyladenine to mungbean (Abbas et al., 1987), 6-benzylaminopurine

and thidiazuron to Beta vulgaris, Pisum sativum, Festuca pralensis and

Festuca arundirtacea (Chernyad'ev, 1994) improved their net photo-

synthetic rate. It was also activated, at the level of flag leaf, if

benzyladenine (BA) was injected to the base of the mother shoot ear of

wheat and Triticale (ICumar and Nath, 2000). Moreover, cytokinin, N,N-

diphenylurea, N-phenyl-N-(2-chloro-4-pyridyl)-urea, thidiazuran and

atrazine, applied to the leaf disc from decapitated and non-decapitated

Fhaseohis vulgaris plants elevated the photosynthetic activity but

atrazine decreased it, in intact plants (Iliov et al., 1987).

The photosynthetic efficiency exhibited varied response to

gibberellins and auxins, depending on the plant type. Its rate was

enhanced by GA (Chatterjee et al., 1976; Khan et al., 1996) and auxins

(Borzenkova, 1976; Chatterjee et a/., 1976; Hayat et al., 2001a) but

remained unaffected by GA (Hayashi, 1961; Little and Loach, 1975) and

auxins (Robinson et al., 1978). In extreme cases, GA (Sanhla and Huber,

1974) or lAA (Erkan and Bangerth, 1980) induced a decrease. In a very

like manner, the effect of ethylene and ethylene releasing compounds on

photosynthesis was also diverse, promotive (Cliquet and Morot-Gaudry,

1989), no effect or partial inhibition (Pallas and Kays, 1982; Taylor and

Gunderson, 1986). Likewise, foliar application of aqueous solution of

ABA was inhibitory to photosynthesis (Fisher et al., 1985; Davies and

Jones, 1991; Rajasekaran and Blake, 1999; Hayat et al., 2001a) but

under water stress conditions, ABA/triadimefon increased the rate of

photosynthesis in wheat (Sairam et al., 1989). Similarly, photosynthetic

CO^ fixation in barley seedlings was inhibited by jasmonic acid (Popova

et al., 1988).

11

The other aspect of photosynthesis, CO^ assimilation, is also

variedly affected by the phytohormones. The foliar application of BR to

wheat and mustard (Braun and Wild, 1984), epibrassinolide to seedlings

of cucumber (Ding et al., 1995) and brassinolide to rice (Fujii, et al.,

1991) improved the rate of COj assimilation. However, the epicotyl of

cucumber, treated with epibrassinolide, did not respond but the transport

of the labelled ('"'C) glucose towards the epicotyl was favoured

(Nakajima and Toyama, 1995). Similarly, Hill activity was favourably

affected by the application of the aqueous solution of HBR or humicil to

the foliage of Vigna radiata (Bhatia and Kaur, 1997). Likewise,

6-benzylaminopurine (BAP) or N-2-chloro-4pyridyl-N -phenylurea

(4-PU-30) increased its activity in Phaseohis vulgaris (Metwally et al.,

1997).

The contents of chlorophyll a, b or total increased in the leaves

of Vigna radiala by HBR and humicil (Bhatia and Kaur, 1997) and that

of Brassica jimcea by homobrassinolide (Hayat ef al., 2001a) applied

directly to the plants or in the rice and gram plants raised from the seeds

pre-treated with BRs (Wang, 1997) or homobrassinolide (Fariduddin et

al., 2000). Moreover, the water stressed wheat plants, treated with

homobrassinolide, exhibited an increase in the chlorophyll level (Sairam,

1994). Similarly, rice (Ray et al., 1983) or potato plants (Ghosh and

Biswas, 1991), treated with kinetin possessed higher chlorophyll level in

their leaves. Benzyladenine was also equally effective in improving their

contents in the leaves of Vigna radiata (Abbas et al., 1987), Helianthus

anmiiis (Goswami and Srivastava, 1988) and wheat and Triticale (Kumar

12

and Nath, 2000). The application of BAP (6-benzylaminopurine) to

wheat stabilized the shape of the chloroplast (Sun et al., 1998). The

cytokinins efficiency was further confirmed by Kaminek et al. (1992)

and Mok and Mok (1994) where it favoured the transformation of

etioplast to chloroplast and chlorophyll production in the leaves and

cotyledons of etiolated plants. Moreover, the response of plants to

gibberellic acid is not very prominent. GA, application to rice (Oryza

saliva) leaves decreased the chlorophyll contents (Prakash and

Prathapasenang, 1990; Singh, 1996) but had mixed effect on plastid

development (Sundqvist et al., 1980; Bishnoi and Krishnamoorthy,

1992). However, the application of NAA to Arachis hypogea L.

significantly enhanced leaf chlorophyll content (Meyyappan et al.,

1991).

Carbonic anhydrase (CA, EC 4.2.1.1) is the second most

abundant soluble protein, other than RuBPCase, in C,-chloroplast (Reed

and Graham, 1981; Okabe et al., 1984). It is a zinc containing protein of

180 kDa (Lawlor, 1987) and is ubiquitous enzyme, among living

organisms. It catalyzes the reversible interconversion of bicarbonates

(HCOj") and CO.,, at physiological pH (Sultemeyer et al., 1993). The rate

of conversion of HCO^ to CO2 is normally slow in alkaline conditions.

However, CA activates the use of HCO^' and the production of COj

(Lawlor, 1987). In C, plants, CA has a close association with RuBPCase

and elevates the level of CO^ at the active site of the RuBPCase (Badger

and Price, 1994). An increase in the activity of CA in the leaves could

be attained by the application of cytokinin (Sugiharto et al., 1992).GA,

13

(Khan et al., 1996) or homobrassinolide (Hayat et al., 2000, 2001a) to

the shoot of the plants. Similarly, the seedlings of wheat raised from the

grains, pre-treated with homobrassinolide, possessed high CA activity in

leaf (Hayat el al, 2001b).

The reduction of nitrate, absorbed by the roots, is initially

catalyzed by nitrate reductase (EC 1.6.6.1), located in the cytosol of the

cell, which reduces nitrate to nitrite. The produce is then carried to

chloroplast where nitrite reductase (EC 1.7.7.1) catalyzes its further

reduction to ammonia, the state in which it is metabolized. The level of

nitrate reductase is the known rate limiting step of the whole reduction

process and is regulated by a number of external and internal factors.

One of these is the concentration of phytohormones whose elevation, by

exogenous application, may induce a shift in NR level (Elliot and

Peirson, 1980; Prakash and Kapoor, 1984; Ahmad, 1988 and 1994;

Ahmad and Hayat, 1999; Ahmad et al, 2001; Hayat el al, 2001b). The

application of auxin and/or gibberellin to the plants of Pisum sativum

elevated the level of nitrate where the former was more effective

(Zholoback, 1985) and even within auxins, IBA proved to be a better

promoter than lAA (Ahmad, 1988). This high substrate (Nitrate) level,

could have been an additional reason to explain the observed increase in

the leaf nitrate reductase activity, reported in wheat raised from the seeds

pre-treated with aqueous solution of GA, (Haque et al, 1989). Similarly,

GA, in association with ABA, improved the activity of the enzyme in the

roots of Cichorinm intybus (Goupil et al, 1998), all plant parts of

Solarium tuberosum (Palmer, 1985) and the plants of Zea mays and

14

Gussypiiim, grown under saline/non-saline conditions (Munjal and

Goswami, 1995; Khan and Srivastava, 1998). Moreover, the other

hormones, particularly BRs increased the NR activity in rice (Mai et al.,

1989), maize (Shen el al., 1990), water stressed wheat (Sairam, 1994)

and kinetin in the plants of Leiicanea leiicocephala (Pandey and

Srivastava, 1995), Brassica jimcea (Chanda ei al., 1998). In contra

diction to above, Desouky el al. (1989) noted an inhibition in NR

activity in the root and the shoot of the plants of Fisum sativum, treated

with lAA, GA,, BA, chloroethylphosphoric acid or paclobutrazole but

failed to assign any reasonable explanation to their findings.

The level of gaseous phytohormone, ethylene, is enhanced by

BRs in the etiolated mungbean hypocotyl segments (Arteca el al., 1983)

and that of ACC in tomato plants, supplied through the roots

(Schlagnhaufer and Arteca, 1985a). Similarly, in rice lamina BR alone

or in combination with auxin induced the synthesis of ethylene (Cao and

Chen, 1995). A synergism was also recorded between the ethylene and

ACC production in the etiolated mungbean hypocotyls, treated with BR

and/or lAA (Arteca et al, 1988) but was sensitive to the inhibitors of

ethylene synthesis (Schlagnhaufer et al., 1984a; Schlagnhaufer and

Arteca, 1985b). BR, in association with kinetin, also generated elevated

level of ethylene in mungbean etiolated segments (Schlagnhaufer el al.,

1984b). Moreover, the water stressed plants of jack pine, treated with

homobrassinolide, evolved more ethylene (Rajasekaran and Blake,

1999). However, the auxin antagonists, 2,3,5-triiodobenzoic acid (50

fiM) or 2-(p-chlorophenoxy)-2-methylpropionic acid (10 p.M) acted as

15

antagonists of BRs in ethylene production in mungbean hypocotyl

segments (Arteca et al., 1991). With regard to the site of action of BRs

and auxin in the ethylene production, Yi et al. (1999) are of the opinion

that the two hormones act at different gene levels for the expression of

three members of the 1-aminocyclopropane-l-carboxylate synthase in

Vigna radiata.

2.3 Plant protein content

The plants treated with phytohormones in general, exhibited

high protein content (Schuster and Davies, 1983; Prishchepa, 1997). In

specific cases, BRs, applied to the plants of Phaseolus vulgaris and

Phaseohis aureus (Kalinich el a!., 1985) and the algal cells of Chlorella

vulgaris (Bajguz, 2000) had high protein content. The tomato seedlings

also responded to biobras-6 in a similar way (Nunez et al., 1996).

Likewise, Hordeum vulgare (Callebaut et al., 1983; Nissen, 1988) and

Pisum sativum (Maksyutova et al., 1987) synthesized more protein, in

response to GA^ application. Moreover, supplementing GA, in

association with NAA, cytokinin and chloromequat to pea (Shende et

al., 1987) or with 2-chloro-4-pyridyl/phenylurea to Zea mays (Stefano et

al., 1998) improved their protein content. The leaves of Cassia

angustifolia supplied with benzyladenine or ascorbic acid through the

roots elevated the leaf protein content (Singh et al, 1999).

2.4 Senescence

It is the process which refers to, endogenously regulated

deteriorative, changes that become the natural cause of death of cells,

tissues, organs or organisms (Leopold, 1975; Nooden and Leopold,

1978). Buchanan-Wollaston (1997) isolated and characterized a large

range of cDNA clones representing genes that show increased expression

in senescing leaves. A number of parameters, like changes in the level of

chlorophyll, total protein, nucleic acid, photosynthetic rate (decrease in

photosynthetic enzymes) variation in plant growth substances, increased

membrane permeability and abscission which may be used as the markers

of the state of senescence. However, the factors, such as, development

of metabolic sinks (e.g. fruits), unfavourable photoperiods, lengthy

exposure to shade or excessive light, insufficient nutrients or water,

extremes in the temperature or other stresses affect the crop yield (Reddy

et al., 1993).

The senescing leaves of Triticum aeslivum L. lost chloroplasts

per cell (Peoples et al., 1980) and that could be because of their

degradation (Wittenbach et al., 1982). Moreover, the level of stomatal

enzymes exhibited a decline prior to photochemical activities in wheat

(Camp et a!., 1982). The protein for RuBPCase decreased and had a

close relationship with photosynthetic activities in wheat (Wittenbach,

1979) but this relation could not be observed by Hall et al. (1978).

However, in monocarpic senescence of rice plants, the process is

initiated as a result of mobilization and consequent depletion of

metabolites from leaves to grain (Biswas and Choudhuri, 1980; Ray and

Choudhuri, 1981a and b).

The senescence of the leaves, which are the major source of the

metabolites, may be delayed by the application of selected chemicals so

that the devehaping seeds/fruits may get the supply for an extended

period. The plants give varied response to phytohorrnones, although

cytokinins, auxins and gibberellins, in general, retard the process of

senescence (Arteca, 1997) while ethylene, ABA and methyl Jasmonate

promote it (Abeles el al., 1992).

Therefore, many of the plants, exogenously supplied with

cytokinins. exhibited delayed senescence (Van Staden el al., 1988),

Similarly, kinetin application delayed the monocarpic senescence in

Oryza saliva leaves but ABA promoted (Ray el a/., 1983). Moreover,

Solanum hiherosvm plants applied with kinetin or urea remained

vegetative for a longer period than the control (Ghosh and Biswas,

1991). Contrary to above, lAA enhanced senescence in the isolated

petals of DIanlhus carophyllus (Wulster el al., 1982). However, BRs

retarded the abscission of leaves and fruits of citrus (Iwahori, 1990),

fruit drop in oranges (Sugiyama and Kuraishi, 1989) and persimmons

(Suzuki el al., 1988). Likewise in maize, BRs enhanced greening of

etiolated leaves, at low temperature in the presence of light (He el al.,

1991) whereas 24-epibrassinolide accelerated senescence of the isolated

leaf/cotyledon tissues but cytokinin delayed (He et al., 1996).

Nooden el al. (1990) have observed a decline in the cytokinin

contents in the xylem sap of the senescing plants which is assigned to be

the reason for the leaf senescence. The hormones are recognized to delay

many of the processes that contribute to plant senescence (Nooden and

Leopold, 1978; Mok and Mok, 1994; Smart, 1994).

The gaseous hormone, ethylene, promotes the ripening process

of the fruits (Theologis, 1993) and the rate of senescence of flowers

(Borochov and Woodson, 1989) by co-ordinately inducing the expression

of various genes encoding enzymes. However, in young Arabidopsis

leaves ethylene has no impact on the expression of senescence-related

genes (Grbic and Bleeker, 1995), therefore, senescence oiHemerocallis

flowers (Smart, 1994) and monocot leaves (Valpuesta et ai, 1995) does

not require ethylene signalling.

2.5 Yield parameters

The biological yield of the plants may be improved by the

exogenous application of the phytohormones, either directly through its

impact on the characteristics determining it or indirectly as an expression

of the healthy vegetative growth, generated by the treatment. The

application of homobrassinolide to the water stressed wheat (Sairam,

1994), Vi^na radiata (Bhatia and Kaur. 1997), Tnliciim aeslivvm, Oryza

safiva, Brassica juncea, Gossypium hirsiilum, Arachis hypogea (Ramraj

el ai, 1997), Brassica juncea (Hayat et a/., 2000 and 2001a) or pre-

sowing seed treatment of Cicer arietimim with homobrassinolide

(Fariduddin el al., 2000) improved the yield characteristics (expressed

as pod or ear number per plant). Similarly other hormones, 6-

benzyladenine applied to the foliage oi Glycine max (Crosby el al., 1981;

Carlson el al., 1987) and GA,, lAA or cytokinin to Brassica juncea

(Hayat ei al., 2001a) enhanced pod/spikelet number per plant. Moreover,

the seed number per pod/per plant in bean and rapeseed by the spray of

4-chlorophenoxy acetic acid or auxin (Morgan, 1980), Glycine max by

tricontanol (Shende el al.. 1987) and Brassica jvncea by chlorocholine

chloride/ethrel (Grewal et al., 1993) could successfully be increased.

However, Moigan et al. (1983) did not observe a significant increase in

the pod number per plant in rapeseed plants, supplemented with

benzyladcnine.

The values for the other important yield determining

characteristic (i.e. seed mass) was improved in Vigna radiata (Bhatia

and Kaur. 1997), irrigated and rainfed wheat (Sairam, 1994) by the

application of homobrassinolide to their foliage. Likewise, BRs with or

without BAP. GA,, KN enhanced 1,000 seed mass in rice (Krishnan et

al., 1999). Among the other hormones, benzyladcnine alone (50 and 100

ppm) was effective in enhancing seed mass in Helianthiis ai^nutis

(Goswami and Srixastava. 1988) and in rice (Ray and Choudhuri,

1981a). However, 1,000 seed mass, in mustard plants, failed to respond

to homobrassinolide treatment (Hayat el al., 2000).

Finally, the seed yield, in various groups of plants, could be

improved by the exogenous addition of the phytohormones both in the

pot and the field conditions. The brassinosteroids tested, either as foliar

spray or pre-sowing seed treatment, included BRs on Vicia faba (Helmy

el al., 1997), Bras.sica jimcea (Kumawat et al., 1997), epibrassinolide on

potato (Khripach el al., 1997) and homobrassinolide on wheat, rice,

mustard, groundnut, potato, cotton (Ramraj et al., 1997), Vigna radiata

(Bhatia and Kaur, 1997), Brassica jiincea (Hayat el al., 2000, 2001a),

Cicer arieiinum (Fariduddin el al., 2000), In all the above cases and in

20

water stressed wheat (Sairam, 1994) tlie yield of the plants significantly

increased by the hormonal treatment. The response of the plants to the

other hormones was not uniform. The spray of aqueous solutions of lAA,

IBA and/or NAA enhanced the biological yield in Loliiim speciosum

(Shafi and Khan, 1992; Park, 1996), (jossypnim harhaden.se (Sawan el

al., 1988). Lens culmans (Setia el al., 1993) and egyptian cotton (Sawan

el al., 1989). The application of kinetin (Ray el al., 1983) and that of

GA, (Yoshida, 1981; Singh, 1996) to the plants of rice favoured grain

production, however, ABA (Ray el al., 1983) decreased it. Moreover,

ethylene generated a varied response where it enlianced the values for

seed yield in wheat (Dahnous et al., 1982; Leary and Oplinger, 1983;

Wiersma el al., 1986) but Nafziger el al. (1986) noted a decrease.

2.6 Conclusion

The available literature, reviewed above, reveals that due

attention has not been given to plant growth regulators particularly 28-

homobrassinolide and kinetin with regard to their impact on growth,

metabolism, senescence and yield of the plant. Moreover, the results in

many of the cases are inconsistent. Therefore, this study was planned to

observe the effect of 28-homobrassinolide and/or kinetin on the selected

parameters of the Vigna radiata L. wilczek and Brassica juncea czern &

coss.

21

http://harhaden.se

Chapter - 3

MATERIALS AND METHODS

CONTENTS

3.1

3.2

J.J

3.4

3.5

3.6

3.7

3.8

3.8.1

3.8.2

3.8.3

3.8.4

3.8.5

3.8.6

3.8.7

3.8.8

3.8.9

3.9

3.9.1

3.9.1.1

3.9.1.2

3.9.1.3

3.9.2

3.9.2.1

3.9.2.2

3.9.3

Seeds

Preparation of pots

Experiment 1

Experiment 2

Experiment 3

Experiment 4

Senescence

Parameters

Leaf area and dry mass

Number of nodules/plant

Nodule fresh and dry mass

Biological yield/plant

Number of pods/plant

Number of seeds/pod

Seed yield/plant

Net photosynthetic rate

Carboxylation efficiency

Chemical analyses

Chlorophyll content

Extraction

Chlorophyll estimation

Calculation of chlorophyll

Ethylene estimation

Moong

Mustard

5/plant

content

Estimation of carbonic anhydrase (CA) activity

Page No

22

22 - 23

23 - 24

24 - 25

26

26 - 27

27

27

27

28

28

28

28

28

28

28 - 29

29

29

29

29

29

30

30

30

30 - 31

31 - 32

3.9.4. Estimation of nitrate reductase activity 32

3.9.4.1 Development of colour 32 - 33

3.9.5 Estimation of proteins 33

3.9.5.1 Extraction 33

3.9.5.2 Colour development of protein 34

3.10 Statistical analyses 34

MATERIALS AND METHODS

Seeds of summer moong (Vigna radiala L.Wilczek) cv. T-44,

commonly known as 'greengram', and those of mustard (Brassica juncea

czern & coss) cv. Varuna were used as test materials. These varieties

were selected, on the basis of a preliminary screening where they

performed best in the local climatic conditions. Four pot experiments

were conducted, during 1999-2001, to study the response of moong and

mustard lo the leaf applied aqueous solution of 28-homobrassinolide

(HBR) and/or kmetin (KN) at selected stages of growth.

The comprehensive details of the material used and the

methodologies adopted, durmg the course of the present investigation,

are presented in this chapter.

3.1 Seeds

The authentic seeds of Vigna radiala L. Wilczek and Brassica

jiincca czern & coss cultivars were procured 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 sterilized, double distilled water, at least thrice, to

remove the adhering solution.

3.2 Preparation of Pots

Earthen pots of 25 x 25 cm size were filled with equal 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 applied, to each pot, at the rate of 40, 295 and 73 mg for moong

and 359, 295 and 63 mg of N, P and K for mustard, respectively. The

pots were properly arranged in the net house.

3.3 Experiment 1

This experiment was laid down according to simple randomized

block design, during the summer season of 1999. The surface sterilized

seeds of moong {Vigna radiata L.Wilczek) were sown in the pots of 25

cm^, at the rate of 8 seeds per pot. A uniform basal dose of N, P and K,

as urea, single superphosphate and muriate of potash, was applied at the

time of sowing. These pots were divided into two groups.

(A) An aqueous solution of lO'io / lO' / lO"* M of HBR or lO'^ /lO"' /

10"" M of KN was applied to the leaves of moong, 25 days after

sowing (DAS).

(B) An aqueous solution of 10"'" / 10" / 10" M of HBR or 10-^10"^/

10"" M of KN was applied to the leaves of moong, 30 days after

sowing (DAS).

Control plants were sprayed with double distilled water only.

Each treatment was replicated thrice. Irrigation was done with tap water

as and when required. The plants were sampled at 26, 31, 35, 40 and 45

DAS, from group-A and 31, 35, 40 and 45 DAS, from group-B to study

the following parameters :

1. Leaf area

2. Leaf dry mass

3. Leaf nitrate reductase activity

23

4. Leaf protein content

5. Leaf chlorophyll content

6. Leaf carbonic anhydrase activity

7. Net photosynthetic rate

8. Carboxylation efficiency

9. Ethylene production

10. Number of nodules/plant

11. Nodule fresh and dry mass/plant

The rest of the plants were allowed to grow and harvested at the

maturity of the seeds, to study the following :

1. Biological yield/plant

2. Number of pods/plant

3. Number of seeds/pod

4. 100 seed mass

5. Seed yield/plant

6. Seed protein content

3.4 Experiment 2

This experiment was laid down according to simple randomized

block design, during the winter season of 1999-2000. The surface

sterilized seeds of mustard {Brassica juncea c'zern & coss) were sown in

the earthen pots (25 cm^) filled with the mixture of the soil and farmyard

manure (9:1). A uniform basal dose of N, P and K was applied at the

time of sowing (page 23). These pots were divided into three groups.

(A) An aqueous solution of W^^IW'^IW^ M of HBR or lO'VlO-^/lO-^M

of KN was sprayed on the leaves of 29 day old plants.

24

(B) An aqueous solution of lO-'O/lO-^lO-^M of HBR or lO-VlO-^/lQ-^M

of KN was sprayed on the leaves of 44 day old plants.

(C) The leaves were applied with HBR (IQ-^'^/lO-^/lO-^ M) or KN(10-V

10"' /10- M) at both the stages of growth (29 and 44 day old plants).

The leaves of control plants were sprayed with double distilled

water only. Each treatment was replicated thrice. Irrigation, with tap

water, was done as and when required. The plants were assessed for the

following chaiacteristics at 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80

DAS.

1. Leaf dry mass

2. Leaf nitrate reductase activity

3. Leaf protein content

4. Leaf chlorophyll content

5. Leaf carbonic anhydrase activity

6. Net photosynthetic rate

7. Carboxylation efficiency

8. Ethylene production, at the level of each leaf

The following parameters were recorded, at harvest ;

1. Biological yield/plant

2. Number of pods/plant

3. Number of seeds/pod

4. 1,000 seed mass

5. Seed yield/plant

25

3,5 Experiment 3

This experiment was conducted according to simple randomized

block design during summer season of 2000. The surface sterilized seeds

of moong {Vigna radiata L. Wilczek) were sown in the earthen pots (25

cm^) at the rate of 8 seeds per pot, filled with the mixture of soil and

farmyard manure (9:1). A uniform basal dose of N, P and K as urea,

single superphosphate and muriate of potash (page 23) was applied at

the time of sowing. These pots were divided into two groups.

(A) The leaves of 25 day old plants were applied with a mixture (1:1)

of the aqueous solutions of HBR (lO'^M) and KN (IQ-^M).

(B) The leaves of 30 day old plants were applied with a mixture (1:1)

of the aqueous solutions of HBR (lO' ^M) and KN (lO-^^M).

The leaves of the control plants were sprayed with double

distilled water only. Each treatment was replicated thrice. Irrigation was

done with tap water as and when required. The plants were sampled at

26, 31, 35, 40 and 45 DAS, from group-A and 31, 35, 40 and 45 DAS,

from group-B and studied for the characteristics, mentioned in

Experiment 1.

3.6 Experiment 4

This experiment was laid down in a simple randomized block

design during the winter season of 2000-2001. The surface sterilized

seeds of mustard {Brassica juncea czern & coss) were sown in the pots

(25 cm^) filled with sandy loam soil, mixed with farmyard manure in 9:1

ratio. A uniform basal dose of N, P and K (page 23) was given at the

time of sowing. These pots were segregated into three sets.

26

(A) The leaves of 29 day old plants were sprayed with aqueous solution

of HBR (lO-^M) and KN (IQ-' M), mixed in a ratio of 1:1.

(B) The leaves of 44 day old plants were sprayed with aqueous solution

of HBR (10-^M) and KN (IQ-^M) mixed in a ratio of 1:1.

(C) The leaves of the plants were sprayed with aqueous solution of

HBR (10-* M) and Kx (IQ- ^M) mixed in a ratio of 1:1, at both the

stages of growth (ie. 29 and 44 days, after sowing).

The leaves of the plants applied with double distilled water were

used as control. Each treatment was replicated thrice. The plants were

irrigated with tap water as and when required. The sampling was done at

30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 days, after sowing and the

characteristics studied were same as in Experiment 2.

3.7 Senescence

The schedule of sampling, in all the experiments, was planned

in such a way that the pattern of leaf senescence may be studied and

visual observations may be recorded.

3.8 Parameters

The methods applied to assess various growth and yield

parameters are described below :

3.8.1 Leaf area and dry mass

It was determined by gravimetric method where the leaf area of

randomly selected leaves, from each treatment, was determined by

tracing its outline on the graph sheet and the leaves dried (at 80°C) to

determine their dry mass.

27

3.8.2 Number of noduies/plant

Whole plant was uprooted with the precaution that the roots may

not be damaged. They were washed under the tap water and the number

of nodules per plant was counted.

3.8.3 Nodules fresh and dry mass/plant

The nodules from each plant were weighed followed by their

transfer to petriplates for drying overnight in hot air oven, run at 80°C.

The dried material was weighed and was recorded as fresh and dry mass

per plant, respectively.

3.8.4 Biological yield/plant

The plants with the pods, at harvest, were dried under sun light

and weighed.

3.8.5 Number of pods/plant

At harvest, 9 plants (3 from each replicate) from each treatment

were randomly sampled and counted for the number of pods per plant.

3.8.6 Number of seeds/pod

25 pods from each treatment were randomly selected and

counted for the seeds per pod.

3.8.7 Seed yield/plant

The pods from each replicate were crushed, cleaned and

computed to assess the seed yield per plant.

3.8.8 Net photosynthetic rate

The photosynthetic rate at each selected stage was measured in

fully expanded leaves, of somewhat the same age in all the replicates of

28

plants by using LICOR-6200, portable photosynthetic system (Nebraska,

USA). Each observation was replicated thrice. All the measurements

were made on cloudless clear days betweenll.OO and 13.00, solar time.

3.8.9 Carboxylation efficiency

The formula used by Tiwari el al. (1998) was adopted for this

purpose where the obser\cd \alues were put to compute the rate of

carboxylation reaction.

Net photosynthetic rate Carboxylation efficiency (CE) = (mol m'^s"')

Internal CO2

3.9 Chemical analyses

The details for the estimation of vairous components are

described below :

3.9.1 Chlorophyll content

Total chlorophyll content was estimated following the method

of Mackinney (1941).

3.9.1.1 Extraction

Fresh leaves (100 mg) were homogenized in a mortar in the

presence of sufficient quantity of 80% acetone. The extract was filtered

and supernatant collected in the volumetric flask. The process was

repeated thrice and each time the supernatant was collected in the same

flask. Finally, the volume was made up to 10 cm^, with 80% acetone.

3.9.1.2 Chlorophyll estimation

5 cm^ sample of chlorophyll extract was transferred to a cuvette

and the absorbance was read at 645 and 663 nm on spectrophotometer

(Spectronic 20D, Milton Roy, USA).

29

3.9.1.3 Calculation of chlorophyll content

The following equation, given by Arnon (1949), was adopted to

calculate the chlorophyll content.

V Total chlorophyll mg/g = 2.02 (D645)+ 8.02(D663) x

1000 X W

where. V = Total volume of the solution (cm- )

W = Weight of the tissue (g). used for the extraction of the

pigment.

3.9.2 Ethylene Estimation

Ethylene was estimated following the method described by

Sadasivam and Manickam (1992).

3.9.2.1 Moong

In case of moong {Vigna radiaia L. Wilczek), whole plant was

enclosed in a polythene bag of known volume and made air tight, for a

period of two hours. The gas sample of 5 cm- was drawn in a hypodermal

airtight s\ringe. The analysis was done on a Nucon series-5500 gas

chromatograph fitted with a glass column packed with porapack T, 80-

100 mesh and a flame ionization detector. Nitrogen was used as the

carrier gas al 3 kg/cm^. The column temperature was set at 65°C,

injection 100°C and detector 150°C. The retention time of ethylene was

70 to 80 seconds. The standard curve was obtained by using pure

standard ethylene, diluted with air for desired concentrations. Ethylene

production was expressed on fresh weight basis.

3.9.2.2 Mustard

The ethylene production in mustard {Brassica juncea czern &

coss) was estimated at the level of each leaf. The whole leaf, attached to

the mother axis, was enclosed in polythene bag of known volume and

made air tight. It was incubated for 2 hours after which the gas sample

was injected out in an air tight hypodermal syringe of 5 cm- . The sample

was run on GLC in the same way as for moong.

3.9.3 Estimation of carbonic anhydrase (CA) activity

CA was assayed by adopting the method of Dwivedi and

Randhawa (1974).

The fresh leaf samples were cut into small pieces at a

temperature, below 25°C. 200 mg, of these pieces, was weighed and cut

further into small pieces in 10 cm"* of 0.2M cystein hydrochloride

(Appendix ].l) and left for 20 minutes, at 4"C. The leaf pieces were

taken out from the petriplate and the adhering solution was soaked with

the help of blotting paper. These leaf samples were then transferred to a

test tube containing 4 cm^ of phosphate buffer of pH 6.8 (Appendix 1.2).

To this test tube, 4 cm'' of 0.2M sodium bicarbonate (NaHCOj) in 0.2M

sodium hydroxide (NaOH) solution (Appendix 1.3) and 0.2 cm"* of

bromothymol blue (0.002%) indicator (Appendix 1.4) were added. The

tube was shaken gently and left for 20 minutes, at 4°C.

CO2 liberated by the catalytic action of CA on NaHCOj, was

estimated by titrating the reaction mixture against 0.0 IN hydrochloric

acid (Appendix 1.5), using methyl red as an indicator. The reaction

mixture of the control was also titrated against O.OIN hydrochloric acid.

In each case, the quantity of hydrochloric acid was used to neutralize the

sample and blank and the difference was calculated.

The activity of the enzyme was calculated by putting the values

in the formula :

V X 22 X N [mol (CO,)kg-i (leaf f.m)S-i]

W

wjiere, V = Difference in volume (cm'' of hydrochloric acid in

control and the test sample)

22 = Equivalent weight of CO^

N = Normality of HCl

W = Fresh mass of tissue used

3.9.4 Estimation of nitrate reductase activity

Nitrate reductase activity (NRA) was estimated by following the

method of Jaworski (1971). The fresh biological sample was cut into

small pieces, out of which 200 mg was weighed and transferred to plastic

vial (25 cm') containing 2.5 cm' of phosphate buffer (Appendix 2.1),

0.5 cm' of 0.2M potassium nitrate solution (Appendix 2.2) and 2.5 cm'

of 5% isopropanol (Appendix 2.3). Two drops of chloramphenicol were

also added to check the bacterial growth. These vials were incubated in

the dark in a BOD incubator run at 27±2"C, for two hours.

3,9.4.1 Development of colour

To the test tube, 0.4 cm^ of test extract and 0.3 cm^ each of

naphthyl ethylene diamine dihydrochloric acid (NED-HCl) and

sulphanilamide (Appendix 2.4 and 2.5) were pipetted. The colour

changed to pink which was left for 30 minutes for the development of

maximum intensity. It was diluted to 5 cm- with double distilled water

(DDW). The optical density of the sample was read at 540 nm, using

32

spectrophotometer which was ah^eady caliberated for 100% transmittance

by using a blank containing 4.4 cm' water and 0.3 cm^ each of

sulphanilamide and NED-HCl.

A standard curve was plotted by using known graded

concentrations of sodium nitrite. Optical density of the test extract was

compared with that of tiie caliberated curve. NRA was calcuiated in

terms of n moles NO^ h''g''f.m.

3.9.5 Estimation of proteins

The method of Lowry et al. (1951) was followed for the

estimation of total protein in the samples.

3.9.5.1 Extraction

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

mortar. Grinding was done with the addition of 1 cm^ of trichloroacetic

acid (5%). It was transferred to a centrifuge tube with repeated washings

and the final volume was made up to 5 cm' with trichloroacetic acid

Complete precipitation of the proteins was allowed to take place by

leaving the sample for about 1 hour. The material was centrifuged at

4,000 rpm for 15 minutes and the supernatant was discarded. 5 cm- of

sodium hydroxide (IN) was added to the residue and mixed well by

shaking. It was left for 30 minutes in a waterbath at 60°C so that all the

precipitated proteins may completely get dissolved. After cooling for 15

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

the supernatant, containing protein fraction, was collected in 25 cm-'

volumetric flask and volume was made up to the mark with IN sodium

hydroxide and used for the estimation of protein.

3.9.5.2 Colour development of protein

One cm'' of sodium hydroxide extract was transferred to a test

tube and 5 cm^ of Reagent D (Appendix 3.4) was added to it. The

solution was mixed well and allowed to stand, at room temperature, for

10 minutes. 0.5 cm'' of Folin-phenol reagent was added rapidly with

immediate mixing. The blue colour developed. It was left for 30 minutes

for maximum colour development. The per cent transmittance of this

solution was read at 660 nm, using spectrophotometer. A blank was

simultaneously run with each sample. The protein content was calculated

by comparing the optical density of each sample with a caliberation

curve plotted by taking known graded dilutions of a standard solution of

bovine serum albumine.

3.10 Statistical analyses

The experimental data was analyzed following the standard

procedures described by Gomez and Gomez (1984). The 'F ' test was

applied to assess the significance of the data at 5% level of probability.

The error due to replication was also determined. Critical difference

(CD) was calculated to compare the effect of various components by

putting the values in the following formula :

Standard Error x 2 C D . - J x t (value) 5%

Replicates

Chapter - 4

EXPERIMENTAL RESULTS

CONTENTS

4.1

4.].1

4.1.2

4.1.3

4.1.4

4.1.5

4.1.6

4.1.7

4.1.8

4.1.9

4.1.10

4.1.11

4.1.12

4.1.13

4.2

4.2.1

4.2.2

4.2.3

4.2.4

4.2.5

4.2.6

4.2.7

4.2.8

4.2.9

4.2.10

Experiment 1

Leaf area

Leaf dry mass

Leaf nitrate reductase activity

Leaf protein content

Leaf chlorophyll content

Leaf carbonic anhydrase



Ethylene production

Nodule number/plant

(CA)

(NRA)

activity

Nodule fresh and dry mass/plant

Yield characteristics

Senescence

Experiment 2

Leaf dry mass

Leaf nitrate reductase activity (NRA)

Leaf protein content

Leaf chlorophyll content

Leaf carbonic anhydrase



Ethylene production

Yield characteristics

Senescence

(CA) activity

Page No

35

35

35 - 36

36

36

36 - 37

37

37

37 - 38

38

38

38

38 - 39

39

39 - 40

40

40

41

41

42

42

42 - 43

43

43 - 44

44 - 45

4.3 Experiment 3 45 . 46

4.3.1 Leaf area 46

4.3.2 Leaf dry mass 46

4.3.3 Leaf nitrate reductase activity (NRA) 4 6 - 4 7

4.3.4 Leaf protein content 47

4.3.5 Leaf chlorophyll content 47

Leaf carbonic anhydrase (CA) activity 47

Net photosynthetic rate 48

Carboxylation efficiency 48

Ethylene production 48

Nodule number/plant 49

Nodule fresh and dry mass/plant 49

Yield characteristics 49

Seed protein content 49

Senescence 49 - 50

Experiment 4 50

Leaf dry mass 5 0 - 5 1

Leaf nitrate reductase activity (NRA) 51

Leaf protein content 5 1 - 5 2

Leaf chlorophyll content 52

Leaf carbonic anhydrase (CA) activity 52 - 53

Net photosynthetic rate 53

Carboxylation efficiency 53 - 54

Ethylene production 54

Yield characteristics 54

Senesence 54 - 55

4.3.6

4.3.7

4.3.8

4.3.9

4.3.10

4.3.11

4.3.12

4.3.13

4.3.14

4.4

4.4.1

4.4.2

4.4.3

4.4.4

4.4.5

4,4.6

4.4.7

4.4.8

4.4.9

4.4.10

EXPERIMENTAL RESULTS

4.1 Experiment 1

The surface sterilized, healthy seeds of moong (Vigna radiala L.

Wilczek cv. T-44) were sown in soil, filled in pots (25 cm^). The leaves

of 25(A) or 30(B) day old plants were applied with water (T,), aqueous

solution of kinetin (10-**M,T2 /IQ-^'MJ, /10-^M,T^) or 28-hoino

brassmolide (10-IOM,T3 IIO'^UJ^^ l\Q-%^J^). The plants were sampled

26, 31, 35, 40 and 45 days, after sowing (DAS) from group-A and 31,

35, 40 and 45 DAS from group-B and analyzed for 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 to 14 and are briefly described below :

4,1.L Leaf area

The leaf area of the plants, in both the sets (A and B), increased

with the advancement of the plant age. The effect of the treatment was

noticed only after about a week of the treatment where all the

concentrations of both the hormones generated a significant response

(Table 1). However, 28-homobrassinolide (HBR) proved better than

kinetin (KN) in its effect. The plants supplied with lO'^M of HBR (T^) at

either 25 or 30 day stage possessed maximum values, at all samplings. It

was closely followed by the next lower concentration 10'^°M of HBR

(Tj) in its values.

4.L2 Leaf dry mass

The leaf dry mass increased up to 40 days and the effect of the

treatment was noticed only after about a week of the treatment (Table 2).

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The treatment increased the values in both the sets (A and B) and the

effect of HBR excelled over that of KN. Among the treatments, Tg (10"^

M of HBR) applied at 25/30 DAS proved best generating a significant

increase of about 12% and 7%, over the control, at 40 day stage of

growth.

4.1.3 Leaf nitrate reductase activity (NRA)

The enzyme activity increased up to 35 day stage but decreased

as the age of the leaf progressed (Table 3). The treatment significantly

increased the values, irrespective of the hormone, the concentration and

the time of application. However, early treatment (25 DAS) was more

effective than at later stage (30 DAS). T^ (lO'^M of HBR) proved best

enhancing the values by 44% and 31%, over the control, at 35 DAS, in

sets A and B, respectively and differed significantly from the rest of the

values.

4.1.4 Leaf protein content

It is evident (Table 4) that the per cent protein content had a

linear increase up to 35 DAS but declined, thereafter. The application of

K N / H B R improved the level of total protein, irrespective of the time of

application but the treatment at early stage(A) proved better than at later

stage (B) of growth. The leaves sprayed with 10" M of HBR at 25/30

DAS, had values 21%) and 12% more than the control, at 35 DAS.

4.1.5 Leaf chforophyll content

Leaf chlorophyll content increased as the growth progressed up

to 35 DAS (Table 5). The leaves sprayed with KN or HBR possessed

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concentration of 10''°M was more effective than the others, in set A.

However, in set B the effectivity of the higher concentration (10"^M)

was more prominent, at 35 DAS.

4.1.6 Leaf carbonic anhydrase (CA) activity

The activity of the enzyme increased up to 35 day stage and the

values improved further by the application of KN or HBR, at either of the

stage (A/B) of the treatment. The values declined as the age of the leaf

progressed (Table 6). The application of the hormones at the early stage

of growth (25 DAS) proved better than at the later stage (30 DAS). The

leaves supplied with lO' M of HBR, at 25/30 DAS, had maximum

enzyme activity throughout the growth period and the values being 43%

and 33% more, respectively than the control, at 35 DAS.


It is evident from Table 7 that the leaves of moong, sprayed with

K\ or HBR photosynthesized more efficiently than the control, at all

stages of sampling. Its rate increased as the growth progressed, up to day

35 but exhibited a linear decline, thereafter. HBR excelled in its effect

over KN. Treating the plants, 25 DAS proved more fruitful than at the

later stage (30 DAS) of growth. In both the sets (A and B) maximum

values were recorded in the plants sprayed with 10" M of HBR where

the respective increase was 31% and 22%, at 35 DAS, over the control.


The values exhibited a progressive increase up to day 35 (Table

8). However, the application of K N / H B R to the leaves improved its

37

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values, over the control, and among the two regulators, latter proved

better. A maximum increase of 31% and 23%, over the control, was

recorded at day 35, in the plants applied with HBR (lO'^M) at 25 and 30

DAS, respectively.

4.1.9 Ethylene production

The production of ethylene increased significantly from day 26

to 35 in the plants sprayed with KN/HBR, at either 25/30 DAS (Table 9).

Lower concentration of KN (TJ) or medium concentration of HBR (T^)

sprayed at either of the stages of growth (25/30 DAS) enhanced the

production of ethylene by 10% & 8% and 8% & 7% respectively, over

the control, at day 35.

4.1.10 Nodule number/plant

The number of nodules increased up to 40 day stage and

decreased, thereafter. The plants sprayed with aqueous solution of KN or

HBR had no impact on the number of nodules (Table 10).

4.1.11 Nodule fresh and dry mass/plant

The values increased up to day 40 and decreased, thereafter

(Tables 11 and 12). The treatment had no impact on either of these

parameters.

4.1.12 Yield characteristics

Biological yield increased significantly by the spray of hormone

(KN/HBR) at 25/30 DAS. HBR (lO'^M) proved best by enhancing its

values by 24% and 23%, over the control, in sets A and B, respectively

(Table 13). The pod number increased significantly (Table 13). The seed

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yield was enhanced to a maximum of 26% and 19%, over the control, in

the plants supplemented with HBR (lO'^M) at 25/30 DAS, respectively.

Moreover, the seeds so produced possessed 27 and 24% more proteins,

than the control (Table 14).

4.1.13 Senescence

From day 25 to 30, irrespective of the treatment, all the plants

looked healthy and green. At day 35, water sprayed plants had 25%

visual senescence symptoms in the lower leaves, whereas, the plants

treated with KN/HBR, at day 25/30, did not show any visual symptoms of

senescence. The middle leaves of the control plants also started

developing the symptoms at day 45. However, the plants sprayed with

KN (10-6/10-^ M) or HBR (lO-i^/lO-^ M) at either of the two stages (25/30

DAS) developed 10-20%) visual symptoms at the basal (older) leaves

only at 45 DAS. At each successive stage of growth the rate of

senescence in control plants was much faster than the plants treated with

KN (10-6/10-4M) or HBR (lO-'O/lQ-^M). Moreover, the early treatment (25

DAS) proved better than at 30 DAS.

4.2 Experiment 2

The surface sterilized, healthy seeds of Brassica jnncea cv.

Varuna, were sown in pots (25 cm^) filled with soil. The application of

distilled water (Tj)/aqueous solution of kinetin (10"^M,T2 /10"^M,T3

l\0-^M,T^) or 28-homobrassinolide {lQ-^ÛJÎ\0-Û,i:^/\Q-^M,i:^) was

done to the leaves of the plants 29 (A)/44 (B) days after sowing or at

both the stages (C). The leaves were sampled 30, 35, 40, 45, 50, 55, 60,

65, 70, 75 and 80 days after sowing (DAS) in group A or at 45, 50, 55,

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60, 65, 70, 75 and 80 DAS in B and C. Sampled leaves were analyzed for

the following parameters. The remaining plants were allowed to grow to

maturity to study the yield characteristics. The observations are

summarized in tables 15 to 48 and are briefly described below :

4.2.1 Leaf dry mass

The mass of the first, second, third and fourth leaves increased

up to day 35, 50, 55 and 55, respectively and was significantly affected

by the application of KN/HBR (Tables 15-18). Treating the leaves once

(29 DAS) or twice (29+44 DAS) proved best and possessed maximum

values at the above samplings. The application of lower concentration of

HBR or higher concentration of KN once (29 DAS) generated values

much higher than other treatments and the control. The leaves sprayed

with 10'^ M of HBR (T^) at stage A or C possessed maximum mass.


The activity of the enzyme decreased as the age of the first leaf

increased (Table 19). However, the values increased with age up to day

40 in second leaf (Table 20) and day 50 in the third (Table 21) and fourth

(Table 22) leaves. Moreover, the level of NR was much higher at the

level of the 1st leaf and exhibited a linear decrease up to leaf four.

Treating the leaves 29 DAS (A) proved superior over late (44 DAS)

application. The application of lower concentration of HBR or higher

concentration of Kwonce (29 DAS) generated values much higher than

other treatments and the control. The leaves sprayed with 10"^M of HBR

(T^) at stage A possessed maximum activity and was followed by T3

(lO-^M of KN).

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a


The per cent protein content was significantly affected by the

treatment (Tables 23-26). The effect was quite prominent if the

hormones were applied at 29 DAS (A) than being applied at late stage

(B) of growth. The values in the first leaf were maximum at 30 day

sampling and then decreased as the age advanced. However, in the

second, third and fourth leaves the per cent content increased from day

30 to 40, 50 and 55, respectively and then exhibited a decreasing trend.

HBR (10-VlO-'"M) or KN (IQ-^/lO-^M) generated values significantly

higher than the other treatments. Maximum values were recorded with

the medium concentration (lO'^M) of HBR followed by its lower

concentration (10'"^M) and the two higher concentrations (lO'^/lO'^'M)

of the KN at the level of each leaf, in all the samplings.

4.2.4 Leaf chlorophyll content

At the first leaf (Table 27), the content decreased as the age

advanced from day 30 onwards. However, in the second (Table 28), third

(Table 29) and fourth (Table 30) leaves its values exhibited a progressive

increase up to day 40, 50 and 55, respectively. The treatment

significantly improved the chlorophyll content, over the control, at all

the stages of growth. Treating the leaves, at early growth stage (29 DAS)

with 10-"Vl0-^M of HBR or lO- /lO^^M of KN proved superior, among

the treatments. However, the maximum values were recorded in the

leaves sprayed with 10" M of HBR (T^) once (29 DAS) and the values

were comaprable with that of C (ie. sprayed both at 29 and 44 DAS).

41

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The activity of the enzyme, at the level of first leaf decreased

with the age (Table 3 1). However, in the second leaf (Table 32) its value

increased up to day 40 and that in the third (Table 33) and fourth (Table

34) up to day 50. The treatment significantly enhanced the values, over

the control, throughout the study period. The leaves fed with HBR

(10"^M) at 29 DAS possessed maximum values and were closely

N. followed by the values generated by 10-'"M of HBR/IO"^'or lO- M of K


At the first leaf (Table 35), the photosynthetic rate decreased as

the age advanced from day 30 onwards. However, in the second (Table

36), third (Table 37) and fourth (Table 38) leaves its values exhibited a

progressive increase up to day 40, 50 and 50, respectively. The treatment

significantly enhanced photosynthetic rate, over the control, noted at all

the stages of growth. Treating the leaves, at early stage (29 DAS) with

two lower concentrations (T- and T^) of HBR or the two higher

concentrations (T^ and T ) of KN proved superior, among the treatments.

However, 10"**M of HBR proved best, applied as A or C.


The treatment significantly improved the carboxylation

efficiency of all the four leaves studied (Tables 39-42). The values were

higher, over the control, in the leaves treated with either of the two

hormones, irrespective of the time of application (29/44 DAS) and the

stage of sampling. However, in the first leaf the values started

decreasing from the day 35. On the other hand, in the second, third and

42

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fourth leaves its values increased up to day 40, 50 and 50, respectively

and then declined. The application of the hormones at early stage (A) of

the growth proved superior than B/C. Among the hormones 10"* M of

HBR proved best and was followed by 10-'"M of HBR and IQ-^IO-^M of

K\ which had a comparable effect.


It is evident from Tables 43 to 46 that the ethylene production

increases as the age of leaf advances. The effect of the treatment is

distinct only in the first two/three samplings at the level of each leaf

after the application of the hormone, irrespective of the time of treatment

(A, B or C) and then increases at the same rate as the control. However,

the values are not highly significant. On a comparative basis, it may be

said that the first leaf produces larger quantities of ethylene and the level

shows a successive decrease as one moves towards second, third and

fourth leaves.


The biological yield improved in most of the cases by the

treatment, except T2 (lO'^M, KN) and T-, (IQ-^M, HBR) where the values

were statistically equal to that of the control (Table 47). The application

of HBR at A (29 DAS) or C (29 and 44 DAS) proved better than KN.

Among the treatments, T^ (10"^M of HBR), irrespective of the stage at

which the treatment was done, proved best and the values increased by

26%, 12% and 28%, over the control, at A, B or C, respectively and

were closely followed by T5, T3 and T^.

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The application of HBR/K\ incieased the nuinbei of pods pei

plant (Table 47) The values weie maximum and significantly highei

than the contiol, m the plants supplied with hoimones twice (C) followed

by A and B, m that oidei Out of the tieatments, T^ (lO'^'M of HER)

geneiated maximum values at A, B and C which weie 27%, 14% and

31% highei, ovei the contiol Seed yield mcieased significantly by the

spiay of hoimone (KN/HBR) at 29, 44 oi 29+44 DAS HBR (lO'^M)

pioved best by enhancing its values by 21%, 11% and 25%, ovei the

contiol in sets A, B and C, lespectively (Table 48) The numbei of seeds

pel pod and 1 000 seed mass lemained unaffected (Tables 47 and 48)

4.2.10 Senescence

Senescence of the fust, second, thud and fouith leaves weie

assessed visually thioughout the giowth peiiod The fust, second, thud

and fouith leaves looked healthy and gieen up to 35, 50, 55 and 65 days,

lespectively, niespective of the tieatment Howevei, as the age of leaves

advanced diffeiences oiigmated Earliest state of 50% senescence

appealed m the fust leaves of the contiol plants and those tieated with

lO'^M of KN (T2) at day 45, that was followed with abscission within

few days A similai phenomenon was noticed m the fust leaves, supplied

with lowei concentiation of KN (T2) 01 a higher concentration (T^) of

HBR at late stage (C) of giowth Howevei, the plants applied with KN

(lO-^M) 01 10-'0/10-8 of HBR at A/C (1 e 29 DAS 01 both at 29 and 44

DAS) achieved the same state of senescence, after 50 day stage and did

not abscise at this stage Moreovei, m the second leaves, visual

observation of senescence could be detected only in the plants of the age

44

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of 55 days and water sprayed control plants exhibited more than 60%

senescence at the level of this leaf which was lost after attaining the age

of 60 days. The treated plants, at this stage of growth, started with about

25% senescence and retained the second leaf up to day 65 (T2/T7) or 70

(T,, T^, T- and T - ) in set A or C with more than 50% senescence. Late

(44 DAS) application did not pro\c to be as fruitful as early application

because the second leaf uas retained up to 65 days with more than 50%

senescence in T , T- and T^ only. The third leaf of the control plants

attained 50%) senescence at day 65 in all the sets (A,B and C) and

abscised within few days. Treating these leaves once (29 DAS)

prolonged their life span to 70 days with 60%o senesence. However, third

leaf may be kept intact up to 75 days with 50% senesence if it was fed

with Kx (10-'' M) or HBR (]0-"Vl0-^M) twice (29 and 44 DAS).

Moreover, at 70 day stage, the fourth leaf treated with KN (lO'^M) or

HBR (lO'^M) exhibited symptoms comparable with that of control (ie

about 50%o senesence) and abscised before attaining 75 days. Higher

concentrations of KN (10"^/10"' M) and lower concentrations of HBR

(10"''^/10"^M) enhanced the life span up to 75 days and 80 days

respectively, with about 50% senescence, if applied once (A).

4.3 Experiment 3

The surface sterilized, healthy seeds of moong (Vigna radiala,

cv. T-44) were sown in pots (25 cm^) filled with soil. The leaves of the

plants were applied with water (Sj/Sj) or a mixture (1:1) of aqueous

solutions of lO- M of KN and lO' M of HBR (S2/S4) at 25/30 days after

sowing (DAS), respectively. A specific number of plants was uprooted

45

at 26, 31, 35, 40 and 45 DAS to make the following observations and the

rest of the plants were harvested at maturity to study the yield

characteristics The observations are summarized in tables 49 to 56 and

are briefly described below :

4.3.1 Leaf area

The treatment of the leaves significantly increased their area and

that improved further with the age (Table 49a). At each stage oi' sampling

the values in the treated plants (Sj/S^) were higher than those fed with

water (S|/S,). Hormonal application at early stage (Sj) of growth proved

superior than at late (S^) stage. At 40 day stage, a maximum increase of

12% was recorded in S2, over its control (S,).

4.3.2 Leaf dry mass

The values increased up to day 40 (Table 49b). The treatment

generated a significant response and that too more prominent if applied

at early stage of growth (Sj). A comparative study revealed that at each

stage of sampling the values in S2 are higher than in S . At 40 day stage

the values were maximum, where S2 exhibited 19% increase over S,

(control).


The activity of NR improved as the age of the leaf advanced up

to day 35, followed with a decline which was sharper in the controls (S,/

S3) than the hormone treated leaves (S^ and S^). The application of the

hormones at 25 DAS (Sj) proved superior than at 30 DAS (S^). The

highest value was recorded in Sj at 35 DAS which was 22% more than

46

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the control (S|). Moreover, the decrease, at the subsequent growth

stages, in its values was not as sharp as in the control, therefore, at the

last stage of sampling (45 DAS) it was 38% higher, than S, (Table 50).


The effect of the treatment was significant on the per cent

protein content of the leaves (Table 51a). Its value increased up to day

35 with a decrease as the age advanced further but this decrease was

highly prominent in controls than those treated with hormones.

Significance of the treatment was more prominent in Sj (over S,) than S

(over S,).


The contents show an increasing trend up to day 35 (Table 51b).

The leaves treated, at either of the stage of growth (25/30 DAS) gave a

significant response to the treatment, however, Sj proved better than S .

Its values were maximum at 35 DAS but in terms of per cent increase, it

was highest (24% over Sj) at 31 DAS in S2 treatment, over S,.


The activity of the enzyme increased up to day 35 (Table 52a)

and declined, thereafter. The leaves treated with the hormones (82/8^)

possessed an elevated level of the enzyme activity, over their respective

controls, at each stage of growth. Sj proved better than S , where the

highest value was recorded at 35 DAS which was 30%) higher over S,

(control).

47

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It is evident from Table 52b that the leaves sprayed, at either of

the stage of growth (25/30 DAS), with the aqueous mixture (1:1) of KN

and HBR (S2/S_ ) photosynthesized more efficiently than the water

sprayed, respective controls (Sj/S,). The photosynthetic rate increased

as the plant growth progressed, up to 35 DAS followed with a linear

decline at the rest of the samplings. A maximum increase of 20%, over

the corresponding control (S,) was recorded, at 35 day stage, in S2 and

was also 8% higher over S . Therefore, early application of the hormones

favoured the process more efficiently than late application.


It is evident from Table 53a that the carboxylation efficiency of

the leaves was significantly affected by the treatment where the values

increased up to day 35 and declined, thereafter but with a very slow pace

than the controls. Early application (Sj) proved better than late

application (S^) where the maximum value was recorded at 35 DAS and

was 27% more than S, and also 19% higher over S .


The observed level of ethylene production was significantly

affected by the treatment at early stages of growth (Table 53b). However,

the values increased with the age of the leaves. It seems that the ethylene

was generated just after the applicaiton of the hormones, irrespective of

time of hormones application, within the first week only followed by a

decrease.

48

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4.3.10 Nodule number/plant

The nodule number increased with the age of the plants, up to

day 40 but remained unaffected by the treatment (Table 54a).

4.3.11 Nodule fresh and dry mass/plant

The fresh and dr}- mass of the nodules per plant exhibited a

pattern comparable with its number and did not give significant response

to the treatment (Tables 54b and 55).


The response of the treatment was significant in determining the

biological yield, number of pods and seed yield per plant (Table 56).

Treating the plants with hormones at early stage of growth (S2) proved

superior than being applied at late (S^) stage of growth. The values in

Sj, for the above characteristics, increased by 16%, 21% and 20% over

the control (S,), respectively.

4.3.13 Seed protein content

Like the other parameters, the per cent protein content of the

seed also improved significantly by the spray of the hormones at 25/30

DAS (Table 56). However, early application (25 DAS) proved better than

at late (30 DAS) stage of growth. The respective increase, over the

controls (Sj/Sj) in Sj and S was 15% and 8%).

4.3.14 Senescence

The rate of senescence of the leaves was assessed visually

throughout the growth period. The leaves of all the plants were green

49

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and Iieaitliy up to day 30 but at 35 day stage the lower leaves in control

plants showed the early symptoms of senescence which advanced further

to about 30% leaf senescence at 45 day stage. The process of senescence

got completed at day 60, in control plants. However, in the leaves treated

with hormones (Sj/SJ at either of the stage of growth (25/30, days after

sowing) the rate of senescence was delayed by 25-30%. Therefore, at the

last stage of sampling (60 DAS) treated leaves remained positioned with

75% senescence whereas the controls lost them.

4.4 E.xperiment 4

The surface sterilized, healthy seeds of Brassica jimcea cv.

Varuna, were sown in pots (25 cm^) filled with soil. The leaves were

applied with water or a mixture (1:1) of aqueous solutions of 10"* M of

HBRand lO' 'M of KN. once at 29 (H,/H2)/44 (H^/H J or twice at 29+44

DAS (H-/H^^). The sampling of the leaves, from the plants, was done at

30, 35, 40, 45, 50, 55. 60. 65. 70 and 75 days after sowing. The sampled

leaves were analyzed chemically for the following parameters. The

remaining plants were allowed to grow to maturity to assess the yield

characteristics. The observations are summarized in Tables 57 to 73 and

are briefly described below :

4.4.1 Leaf dry mass

The mass of the first, second, third and fourth leaves increased

up to day 35, 45, 50 and 55, respectively and was significantly affected

by the application of the hormones (Tables 57 and 58). Treating the

leaves once (29 DAS) or twice (29+44 DAS) proved best where

maximum values were recorded, at the above samplings. The per cent

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increase in tlie mass of first leaf at 3 5 days was 5% by H2 and that of

second, third and fourth leaves at 45/50/55 DAS bv H, and H. was 12%,

14%; 11%), 12%o and 9%, 12% respectively, over their controls (H1/H5).

Moreover, these treated leaves, exhibited a slower rate of weight loss up

to the last stage of sampling.


Irrespective of the treatment pattern (Hj/H^/H^^), the activity of

NR was significantly affected (Tables 59 and 60) and the values, at each

sampling, were significantly higher than the water sprayed, controls

(H1/H3/H3). Among the treatments, Hg proved best and was followed, in

their effect, by H^ and H . At the level of first, second and third leaves,

the values for the enzyme level increased with age up to day 35, 40 and

50, respectively. However, in the fourth leaf, the increase was noted in

Hj and H^ up to day 55 but in the rest of the treatments it was up to day

50. H(3 generated maximum values in the first and second leaves at 45

DAS, third leaf at 50 DAS and fourth leaf at 55 DAS which were 31%),

31%o, 26%o and 25% higher over the control (H^), respectively.


The plants sprayed with hormones, under any treatment pattern

(H^/H^/Hg), possessed significantly higher leaf protein content than their

respective controls (Hj/Hj/H^) at all stages of sampling (Tables 61 and

62). Hg (hormones sprayed both at day 29 and 44), in general, proved

best in generating maximum values in all the four leaves. The first leaf

exhibited a significant increase by the treatment which was maximum in

Hg (14%) more than H^, the control) at 45 DAS, however, the values

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leaves possessed highest values at 40/45, 50 and 55 day stages where

maximum increase of 11%, 10% and 13% was noted in H, , over H^,

respectively. Treating the leaves once (29 DAS) closely followed Hg, in

its effect.


The values summarized in Tables 63 and 64, show a significant

improvement, over the controls, in the chlorophyll contents of the leaves,

supplemented with the hormones, at all stages of growth. However, the

first leaf had a decreasing trend with the advancement of its age from

day 30 but at each sampling the treated (H^/H^/H^) leaves possessed

more chlorophyll than their respective controls (Hj/H^/Hj). Moreover,

the values in the second leaf increased up to day 40 and that in third and

fourth leaves up to day 50. Treating the leaves once (H2) or twice (H^)

proved best and the generated values very close to one another. The

maximum values recorded at 50 day sampling in second and third leaves

were 13%), 19% and 13%), 10% higher in H^ and H^ over the controls (H,

and H3), respectively.


The treatment significantly increased the activity of the enzyme

at the level of all the leaves (Tables 65 and 66). However, in the first

leaf the values decreased from day 30 onwards but in the second, third

and fourth leaves the values increased up to day 45, 50 and 55,

respectively. Moreover, at each sampling, the treated leaves (Rj^U^/U^)

always had higher enzyme activity than the corresponding controls (H,/

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HJHj). Application of the hormones 29 DAS (H^) or 29+44 DAS (H^ )

proved best generating maximum values at each stage of growth. These

vlaues were 16%, 21%; 21%, 23% and 20%, 25% higher in H2 and H^ at

day 45, 50 and 55, in second, third and fourth leaves, respectively, over

the controls (Hj/Hj).


The photosynthetic rate as evident from Tables 67 and 68,

was influenced favourably by the treatment (Hj/H^/H . ). At each

sampling, the leaves treated with the hormones possessed significantly

higher vlaues than their respective controls. As the age advanced, the

first leaf exhibited a decrease from day 30 but in the second leaf the rate

increased up to day 45 whereas in the third and fourth leaves this

increase was further extended up to 50 DAS. A maximum increase of

15%) and 19%) in the first and second leaves was noted at 45 DAS and

23%) and 23% in the third and fourth leaves was noticed at 50 DAS in

Hg, over its control (H^).


The hormonal treatment significantly icnreased the

carboxylation efficiency of the leaves where, at each stage of sampling,

the values were higher than the controls, moreover, to a significant level

in H2 and Hg (Tables 69 and 70). The first leaf exhibited a decreasing

trend from day 30 to 35 but the maximum 17% increase was recorded

with Hg at 45 DAS. However, the efficiency of the second leaf increased

up to day 45 and that of third and fourth leaves up to day 50. In the latter

leaves, highest values were recorded in Hg which got enhanced by 24%),

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33% and 17% over H (the control), respectively, ll is to be pointed out

that the second, third and fourth leaves, even at the last sampling had the

differences in the carboxylation efficiency of the order of 39%, 32%;

61%. 50% and 35%, 30% by H^ and Hj, respectively, over the controls

(H,/H,).


The release of ethylene from the leaves increased as their age

advanced from day 30 onwards. The first leaf produced more ethylene

than the others (Tables 71 and 72) throughout the growth period.

Significantly more ethylene was released from all the four leaves if the

hormonal treatment was given once (H^) but that too in the first few

days (35/40 DAS) of the treatment.


Out of the parameters determining the productivity of the

plants, the biological yield, number of pods and seed yield per plant

were significantly affected by the treatment (Table 73). The values for

these characteristics increased most by H^ which were 17%, 25% and

21% more than its control (H^) and were followed by Hj, in its effect.

4.4.10 Senescence

The first, second, third and fourth leaves looked healthy and

green up to 35, 45, 55 and 60 days respectively, irrespective of the time

of application of hormones. However, as the age of first leaves

progressed, earliest state of 60% senescence appeared in the control

plants and those treated at later stage (H^) of growth at day 45, that was

54

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followed with abscission with in few days Hormones treated leaves (H2/

H ) abscised late where 50% senescence was noticed at day 50. However,

in the second leaf of the control plants and those treated with H , 40%

senesence was noticed at day 50. Hormone tieated second leaf (Hj/H^^)

expressed only 25% senescence at day 55. As the age of second leaf

progressed, control plants attained 80"/o senesence at 60 day stage but

those applied with hormones (Hj/H^ ) showed 50% state of senescence.

At day 65, control plants and those treated with H , could not retain the

second leaf but those treated with H /H ^ were 10% senesced and still

attached to the mother body. Moreover, third leaf of the control plants

attained 60% senescence at day 65 and abscised within few days. But the

hormones application (Uj^H^) prolonged the life span of the third leaf up

to 70 days with 65%) senescence. However, the fourth leaf of the control

plants, attained only 40% senescence at day 65. But, the leaves, treated

with hormones once (Hj) or twice (H^) abscised at a slower rate and only

20%) senesced at day 65. However, at day 75, hormone treated (Hj/H^J

leaves were 70%o senesced but water sprayed and those treated at later

stage (H^) of growth could not be retained up to this stage of growth.

'^

55

Chapter - 5

DISCUSSION

DISCUSSION

The expression of the genetic potential of the plants is very

much determined by the recognized group of chemicals called

phytohonnones (auxins; gibberellins; cytokinins; abscisic acid; ethylene;

jasmonatcs and brassinosteroids). They get involved through the

modificailon of transcription, translation and/or differential sensitivity

of the tissue. Firn (1986) has, therefore, suggested that the hormone

induced response largely depends on the physiological state of the plant.

Out of the above hormones, cytokinins and brassinosteroids were

selected for the present study as they not only get involved in growth by

activating cell division, elongation and their proliferation (Arteca, 1997)

but BRs also elicit a positive shift in the responsiveness of the plants

(Mandava el ai. 1981; Yovp el ai. 1981a; Mandava, 1988; Cutler, 1991

and Khripach el al.. 2000).

The nitrate reducing power of the plants is one of the important

factors determining growth. However, the process of nitrate reduction

depends on three important factors (a) substrate (NO,) level in the

cytoplasm (b) the level of functional nitrate reductase (NR) and/or (c)

the activity level of functional NR. Each of these processes is, directly

or indirectly dependent on the metabolic sensors and/or signal

transducters (Campbell, 1999). Moreover, the major rate limiting step in

the whole process of nitrate reduction is the reduction of nitrate to nitrite

(Salisbury and Ross, 1992) which is catalyzed by nitrate reductase (NR).

The level of the enzyme is dependent on a number of factors, borned

within or outside the plants. One of the major regulatory factor.

determining tiie activity of NR is the level of phytohoiniones per se or

added from outside (Prakash and Kapoor, 1984; Ahmad, 1988, 1994;

Khan el a/., 1996; Ahmad and Hayat. 1999; Ahmad et al., 2001).

Brassinosteroids, like other hormones, also act as inducers of NR

activity (Mai ef al., 1989; Shen et al.. 1990; Singh et a!., 1993; Hayat el

a/., 2001b). Present observations (Tables 3, 19-22, 50, 59, 60) are very

much comparable with the earlier findings. Here the leaves both of

moong and mustard, treated with K\ and/or HBR, possessed high NR

activity, compared with control and the latter hormone was a better

inducer than the former. Saroop el al. (1998) suggested hormone induced

de novo synthesis of proteins as a possible reason for the elevated level

of NR. It may be true in the present case also where the protein (per

cent) in the treated leaves was high (Tables 4, 23-26, 51a, 61,62) and

exhibited a positive correlation with the activity of the enzyme (Figs.

1-3).

The other enzyme, carbonic anhydrase (CA) which catalyzes the

reversible hydration of carbon dioxide and maintains its constant supply

to RuBPCase, at the level of the grana of the chloroplast (Majeau and

Coleman, 1994; Price et al., 1994). Otherwise, at ambient concentration

of inorganic carbon, the activity of RuBPCase is restricted (Majeau and

Coleman, 1994). Moreover, CA is also hypothesized to be involved in

photosynthetic electron transport system (Stemler, 1997) and in

maintaining chloroplast pH during rapid changes in light intensity (Reed

and Graham, 1981). The reported elevation in the activity of CA (Tables

6, 31-34, 52a, 65, 66) by KN and/or HBR is in agreement with others

(Sugiharto el ai, 1992; Khan el al., 1996; Hayat el al., 2000, 2001a and

57

20

B c 8

Q.

19 -

18

17

Y = 7421 + 0017X r = 0 936

--

• •

(a)

16

15 450 500 550 600 650

NR actvity

700 750

12

Y = 6 635 +0.012 X

r 2 r 0 5 2 0

c • 4 — '

O o c •5

a 10 1

280 300 320 340

N R activity

(b)

360 380

Fig. 1 Correlation coefficient values between nitrate reductase (NR) activity and protein content in the (a) moong and (b) first leaf of mustard plants (Experiment 2).

Y = -416 7 0 6 + 65 023 X

r = 0 926

B 15 c: o t) c: B •g

a. 14

(a)

13 , -420 440 460 480 500 520 540 560 580

NR activity

16

Y = -559 652+ 73 55 X

r2 = 0934

15

I c •

'B * S 14 , ' (b)

13 ' 1 - - 1- 1 i

420 4 4 0 4 6 0 4 8 0 5 0 0 520 5 4 0 5 6 0 5 8 0

NR activity

Fig.2. Correlation coefficient values between nitrate reductase (NR) activity and protein content in the (a) second and (b) third leaf of mustard plants in Experiment 2.

16 -

15

c -2 "c o o

c

"o 14

13

Y = 7.247 +0.013 X r = 0.897

J9

• / ^

•

^ •

•

• ^

440 460 480 500 520 540 560 580 600

NR activity

Fig. 3. Correlation coefficient values between nitrate reductase (NR) activity and protein content in the fourth leaf of mustard plants in Experiment 2.

b; Ahmad cl al., 2001). Sugiharto et al. (1992) noted cytokinin induced

elevation in CA-mRNA by acting either at the level of the transcription

and/or the stabilization of the transcripts. It was, therefore, suggested

that the enhanced level of mRNA could be a possible reason for an

increase in the activity of the enzyme. Similarly, HBR also involves

transcription and/or translation in generating an impact on the activity of

CA (Okabe ef al., 1980).

Higher activity of nitrate reductase (NR) and carbonic anhydrase

(CA) in the plants treated with KN and/or HBR may partly be an

expression of the observed increase in the protein content, both in moong

and mustard (Tables 4, 23-26, 51a, 61, 62). Moreover, both the enzymes

(NR and CA) exhibited a positive correlation with the protein content

(Figs. 1-6) of the leaves. This increase in protein content is suggested to

be the impact of the hormones on transcription and/or translation

(Kalinich el a/., 1985; Mandava, 1988; Moore, 1989; Nunez et al., 1996;

Bajguz, 2000).

Van Staden et al. (1988) proposed that cytokinins boost the

general metabolism of the chloroplast through their action on the related

processes operative at the level of the nucleus and/or cytoplasm,

facilitating an increase in chloroplast DNA, rate of protein synthesis in

the chloroplast, maintaining sufficient pigment level and promotes grana

formation. Theiefore, leaves supplemented with cytokinins (Tables 5,

27-30, 51b, 63, 64 and Ray et al., 1981a; Caers et al., 1985; Ghosh and

Biswas, 1991) possessed a larger quantity of chlorophyll than those

sprayed with water only (control). The application of HBR has generated

a level of leaf chlorophyll even more than that of the cytokinin (Tables

58

20

19

Y = 7 037 + 4 586 X

( = 0 955

m 18

c 0 o 17

DI

16

15

y"

20 22 24

CA actvity

26

(a)

28

17

Y = 8716 + 3 025X

r = 0 992

Fig 4 Correlation coefficient values between carbonic anhydrase (CA) activity and protein content in (a) Experiment 1 and (b) Experiment 3 of moong plants

_, c

r n o

0>

a

i : 0

11 9 -

11 8 -

11 7

11 6

11 5

, • Y = 7.650+1.632 X 1 r = 0 886

• (a)

11 2 - ) -

2 2 24 25

CA activrty

27

16 Y = 7.092 +2.305 X

r = 0.975

2.8 3.0 3.2

CA activity

3.4 3.6

Fig. 5. Correlation coefficient values between carbonic anhydrase (CA) activity and protein content in the (a) first and (b) second leaf of mustard plants in Experiment 2.

Y = 9 735 + 1 641 X

1 = 0 851

15

(a)

2S 30 32

CA activity

34 36

16

Y = 8913 + 1 701 X r = 0 942

Fig 6 Correlation coefFicient values between carbonic anhydrase (CA) activity and protein content in the (a) third and (b) fourth leaf of mustard plants in Experiment 2

5, 27-30, 51b, 63, 64). Similar observations of the effect of HBR have

also been reported earlier (Sairam, 1994; Bhatia and Kaur, 1997; Hayat

el a/., 2000). It may be suggested that both the hormones (KN and HBR)

have generated a comparative response in terms of protein content and

the activity of the enzymes (NR and CA) which may be regarded as an

expression of high metabolic state of the cells of the leaves. Moreover,

HBR has also enhanced the leaf chlorophyll content which could be

assigned to the above fact and/or acted in a way comparable with that of

the cytokinins.

This healthy metabolic state of the treated leaves was further

reflected in the observed increase in the net photosynthetic rate (PN) of

the leaves (Tables 7, 35-38, 52b, 67, 68). Moreover, it could also be an

expression of higher chlorophyll content (pages 36-37, 41, 47, 52) and

BR induced RuBPCase activity (Braun and Wild, 1984). High RuBPCase

activity, in the treated leaves, might have naturally been attained by the

observed increase in CA activity (pages 37, 42, 47, 52-53). Further, the

activity of CA was positively correlated with PN (Figs. 7-9). The rate of

CO, fixation was, therefore, maintained at a higher level, in the treated

leaves. The other crops have also been reported to possess high rate of

photosynthesis in response to KN (Meidner, 1969; Borzenkova, 1976)

and HBR (Sairam, 1994; Bhatia and Kaur, 1997).

The visual observations related with senescence may be

regarded as a scale to visualize the balance between metabolic and

catabolic processes, at the level of the leaf. Both in moong and mustard

the treatment with KN and/or HBR has delayed the appearance of visual

symptoms of senescence (pages 39, 44-45, 49-50, 54-55). Cytokinins are

59

Y = 1.666+8.448 X r = 0 783

18 20 22 24

CA activity

(a)

2'1

Y = 6.951 + 6.572 X P = 0.997

Fig. 7. Correlation coefficient values between carbonic anhydrase (CA) activity and net photosynthetic rate (PN), in (a) Experiment 1 and (b) Experiment 3 of moong plants.

15

13

Y = -0015 + 0178X r = 0 939

• •

(a)

12 22 23 24 25

CA activity

26 27

21 1

Y = 4.621+4.695 X

r = 0 969

CL

:io

19

18 (b)

17

16 2.4 2.6 2.8 3 0 3.2 3.4

CA activity

3.6

Fig. 8. Correlation coefficient values between carbonic anhydrase (CA) activity and net photosynthetic rate (PN) in the (a) first and (b) second leaf of mustard plants in Experiment 2.

21

20

Y = 5308+4 315X 1^=0 916

19 1 • - •

17

(a)

16 2 6 2 8 30 32

CA activity

3 4 3 6

23 1

'j Y = 5829+ 4 501 X 1 = 0 927

Fig 9 Correlation coefficient values between carbonic anhydrase (CA) activity and net photosynthetic rate (PN) in the (a) third and (b) fourth leaf of mustard plants in Experiment 2

already recognized to inhibit the deteriorative activities in the leaves by

elevating the activity of a-aminolevulinic acid synthase (Van Staden el

a/., 1988) thus maintain chlorophyll level. BRs have also been employed

to slow down the process of senescence in leaves and fruits of citrus

(Sugiyama and Kuraishi, 1989; Iwahori, 1990) and those of hypocotyl

segments of mungbean seedlings (Zhao el at., 1987), Both the hormones

have possibly acted, in delaying senescence through the stabilization of

protein content (pages 36, 41, 47, 51-52) and the photosynthetic

pigment, clilorophyll (pages 36-37, 41, 47, 52) which otherwise may

undergo hydrolysis (Ray and Choudhuri, 1983) .

The leaves of the treated plants possessed more surface area

(Tables 1,49a) which could mainly be an expresison of activated cell

division and their enlargement induced by the application of KN or BRs

(Nakajima el ai, 1996; Arteca, 1997; Clouse and Sasse, 1998). It is

further supported by Sairam (1994), Diz el al. (1995), Khan el al.,

(1996). Pipaltanawong el al. (1996) and Singh (1996) where hormonal

treatment increased leaf area. Moreover, the leaves also exhibited a

higher state of metabolic activity, induced by the treatment, which is

evident from the elevated level of net photosynthetic rate (pages

37,42,48, 53) and protein percentage (pages 36, 41, 47, 51-52) which

should have made a positive contribution to the leaf area and leaf dry

mass (Tables 1, 2, 15-18, 49a, 49b, 57, 58). Similarly, cytokinins (Hayat

el al., 2001a) or BRs (Braun and Wild, 1984; Nunez el al., 1996; Wang,

1997) improved net photosynthetic rate and accumulation of drymass in

other plants.

60

The abortion of flowers and pods is one of the important

determining factor of plant productivity where cytokinins are implicated

to rescue them from pre-mature fall (Reese el al., 1995; Nagel el ai,

2001). The hormone is suggested to provide strength to the sink by

redirecting movement of assimilate to the site of their application

(tissue), subsequently enhance their growth rate and prevent the loss of

the developing flowers and pods (Huff and Dybing, 1980; Dybing, 1994;

Reese ei al., 1995). The hormone induced effect is, therefore, expected

to increase pod number per plant as evident from the observed values

(Tables 13, 47, 56, 73) and those of others (Peterson el al., 1990; Khan

el a!., 1996; Kumawat et al., 1997; Khan et al., 2002). The effect

generated by HBR in improving the number of pods (Tables 13, 47, 56,

73; Nakaseko and Yoshida, 1989; Kadyrov el al., 1995; Hayat et al.,

2000) could have been on comparable lines with those of cytokinins

because both the hormones have also delayed leaf senesence (pages 39,

44-45, 49-50, 54-55). Moreover, higher rate of photosynthesis (pages

37, 42, 48, 53) in the treated leaves ensured regular supply of

photosynthates, at an elevated level and availability of more and more

organic nitrogen and high protein level (page 36, 41,47, 51-52) provided

additional support to the belief that the metabolic state of the leaf is one

of the important parameters to evaluate the biological yield (Sairam,

1994). The increase in seed yield (Tables 14, 48, 56, 73) in KN and/or

HBR treated moong and mustard plants is, therefore, an expression of

improved number of pods per plant and is in support of Takahashi el al.

(1994).

It may not be an exaggeration of the facts that there is complete

uniformity in both the crops (moong and mustard) in their response to

kinetin and 28-hoinobrassinolide. However, the superiority of early

application is well established from the data. Moreover, there seems to

be a limit to the concentration of the hormones to which the plants give

maximum response. These results clearly indicate that 10"''M of kinetin

and lO' ^M of 28-homobrassinolide proved best in improving most of the

parameters studied. The other concentrations were probably either sub-

optimal or supra-optimal for these crops.

CONCLUSIONS

The present study revealed :

(i) The activity, both of nitrate reductase (NR) and carbonic anhydrase

(CA), at the level of the leaf, increased by the application of

aqueous solutions of KN and/or HBR, to the shoot.

(ii) The leaves of the KN and/or HBR treated plants possessed a larger

quantity of chlorophyll and protein. Moreover, exhibited higher

rate of photosynthesis.

(iii) The plants, applied with either of the hormones, were more

responsive, at early stage of growth.

(iv) 28-homobrassinolide (HBR) proved superior in its effect than

kinetin (KN).

(v) Out of the concentrations, medium concentrations 10" M of HBR

and 10" M of KN generated maximum response both in raoong and

mustard.

62

Chapter - 6

SUMMARY

SUMMARY

This thesis is based on the following five chapters.

Chapter 1, includes the significance of the problem entitled,

"The response of Vigna radiata and Brassica Jiincea to 28-

hoinobrassinolidc and kinetin".

Chapter 2, represents a comprehensive review of the available

literature, related with the above problem, jiertaining to growth,

metabolism, senescence and yield characteristics of the plants.

Chapter 3, explains the details of the materials and methods

employed in conducting the experiments and chemical analysis of the

biological material.

Chapter 4, is comprised of the tabulated data, recorded during

this study, and its brief description.

Chaptei 5, deals with the possible explanations for the

observations, in the light of the earlier findings.

The salient features of the observations, recorded in each of the

four experiments, are summarized below :

Experiment 1

The surface sterilized, healthy seeds of moong {Vigna radiata L.

Wilczek) cv. T-44, were sown in sandy loam soil, filled in pots ( 25

cm^). The leaves of 25(A) or 30(B) day old plants were applied with

water, aqueous solution of (KN) kinetin (lO-^M/lO-^^M/lO- 'M) or (HBR)

28-homobrassinolide (10-IOM/10-^M/10-<^M). The plants were sampled

26, 31, 35, 40 and 45 days, after sowing (DAS) from group-A and 31,

35. 40 and 45 DAS from group-B. These samples were analyzed for leaf

area; leaf diy mass; nitrate reductase activity; the contents of protein and

chlorophyll; carbonic anhydrase activity; net photosynthetic rate;

carboxylation efficiency; ethylene production; nodule number; nodule

fresh and dry mass per plant and yield characteristics, at harvest.

All the above parameters, except nodule number; nodule fresh

and dry mass; seed number and 100 seed mass, gave positive response to

the application of K\/HBR HBR proved superior, than KN, in its effect

where 10" M of HBR, applied at 25 DAS(A), generated best economical

response in the plants.

Experiment 2

The surface sterilized healthy seeds of mustard {Brassica

jiincea czern & coss) cv. Varuna, were sown in sandy loam soil, filled in

pots (25 cm^). The application of distilled water/aqueous solutions of

(KN) Kinetin (IQ-^M/IO- 'M/IO-^M) or (HBR) 28-homobrassmolide (10"

'"M/10-^M/lO-^M) was done to the leaves of the plants, once 29(A)/

44(B) days after sowing or twice (C) at both A and B. The leaves were

sampled 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80 days after sowing

(DAS) in group A or at 45, 50, 55, 60, 65, 70, 75, and 80 DAS m group

B and C. Leaf dry mass; nitrate reductase activity; the contents of protein

and chlorophyll., carbonic anhydrase activity; net photosynthetic rate and

carboxylation efficiency in first, second, third and fourth leaves were

significantly affected by the application of KN/HBR. The application of

lower concentrations of HBR (10"'" M/10"* M) or higher concentrations

64

of KN (10"^'M/10""^M) once at 29 DAS generated values much higher than

other treatments and the control.

The biological yield, number of pods and seed yield per plant

were enhanced by the treatment. Moreover, the application of HBR, at A

or C, proved superior than KN, where lO' M of HBR proved best.

Experiment 3

The surface sterilized, healthy seeds of moong (Vigna radiala

L. Wilczek) cv T-44, were sown in sandy loam soil, filled in pots (25

cm^). The leaves of the plants were applied with water or a mixture (1:1)

of aqueous solutions of IQ-' M of KN and lO' M of HBR at 25 (A)/30 (B)

days after sowing, respectively. The plants were sampled 26, 31, 35, 40

and 45 in group-A and 31, 35, 40 and 45 days after sowing in group-B.

The treated plants possessed more leaf area; leaf dry mass; nitrate

reductase activity; the contents of protein and chlorophyll; carbonic

anhydrase activity; net photosynthetic rate; carboxylation efficiency and

biological yield; pod number; seed yield and seed protein content, at

harvest.

Hormone application to the plants at 25 days after sowing (DAS)

proved superior in its effect than 30 DAS.

Experiment 4

The surface sterilized, healthy seeds of mustard {Brassica

jiincea czern & coss) cv. Varuna, were sown in sandy loam soil, filled in

pots (25 cm^). The leaves were sprayed with water or a mixture (1:1) of

aqueous solutions of 10" M of HBR and 10"' M of KN, once at 29/44

65

days after sowing (DAS) or twice at 29 and 44 DAS. The leaves were

sampled 30, 35, 40, 45, 50, 55, 60, 65. 70 and 75 DAS. The leaves

receiving hormonal treatment twice both at 29 and 44 DAS exhibited

maximum values of leaf dry mass; nitrate reductase activity; the contents

of protein and chlorophyll; carbonic anhydrase activity; net

photosynthetic rate and carboxylation efficiency. These plants, at

harvest, exhibited higher biological yield, pod number and seed yield

per plant.

66

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APPENDIX

PREPARATION OF REAGENTS

The various chemicals/reagents used for biochemical analyses

were prepared by adopting the following procedures.

1. Reagents for the estimation of carbonic anhydrase activity

1.1 Cystein hydrochloride solution (0.2 M)

48 g cystein hydrocholoride was dissolved in sufficient

quantities of double distilled water (DDW) and final volume was

made up to 1000 cm-\

1.2 Phosphate buffer (pH 6.8)

(a) 27.8 g sodium dihydrogen orthophosphate (NaH2P04) was

disoslved in sufficient quantities of DDW and the final

volume was made up to 1000 cm-\

(b) 53.65 g di-sodium hydrogen orthophosphate (NajHPO^) was

dissolved in sufficient quantities of DDW and the final

volume was made up to 1000 cm-\

(c) 51 cm^ of solution (a) and 49 cm^ of solution (b) was mixed

and final volume was made up to 200 cm^ with DDW in

order to get the required pH 6.8.

1.3 0.2 M Sodium bicarbonate solution in 0.02 M sodium

hydroxide solution

16.8 g sodium bicarbonate was dissolved in aqueous sodium

hydroxide solution (0.8 g NaOH/1) and final volume was made up

to 1000 cm^ with sodium hydroxide solution.

1.4 Bromothymol blue (0.002%)

0 002 g biomothymol blue was dissolved in sufficient

quantities of ethanol and the final volume was made up to 100

cm"'

1.5 H>di-ochloi-ic acid (0.01 N)

0 86 cm' puie hydiochloiic acid was added to enough DDW

and final volume was made up to 1000 cm"*

2. Reagents for the estimation of nitrate reductase activity (NRA)

2.1 Phosphate buffer (pH 7.5)

(a) 13 6 g potasisum dihydiogen oithophosphate (KHjPO^) was

dissolved in sufficient quantities of double distilled watei (DDW)

and the final volume was made up to 1000 cm'

(b) 17 4 g dipotassium monohydiogen oithophosphate (K^HPO^)

was dissolved in adequate amount of double distilled wa+ei ^md

the final volume was made up to 1000 cm"*

(c) 160 cm"* of solution (a) and 840 cm"* of solution (b) was mixed

in 01 del to get the lequiied pH 7 5

2.2 Potassium nitrate (0.2 M)

2 0 g potassium nitiate was disoslved in required quantity c

DDW and the final volume was made up to 100 cm^

2.3 Isopropanol solution (5%)

5 cm' isopiopanol was added to 95 cm- of DDW

2.4 NED-HCl solution (0.02%)

20 mg N-l-(naphthyl)-ethyIene diamine dihydiochloiic acid

(NED-HCl) was dissolved in enough DDW and the volume v/as

made up to 100 cm"*

2.5 Sulphanilamide solution (1%)

Ig sulphanilamide powdei was dissolved m 100 cm' of 3N

hydiochloric acid

2.5.1 Hydrochloric acid (3N)

26 2 cm^ puie hydiochloiic acid was added to enough DDW

and the final volume was made up to 100 cm'

3.0 Reagents for the estimation of proteins

3.1 Reagent A

2% sodium carbonate was mixed with 0 IN sodium hydroxide

(1 1)

3.2 Reagent B

0 5% coppei sulphate was added to 1% sodium tartrate ( I D

3.3 Reagent C (alkaline copper sulphate solution)

It was prepared by mixing 50 cm- reagent 'A' with 1 cra^

reagent 'B '

3.4 Reagent D (Carbonate copper sulphate solution)

It was piepaied m the same way as reagent ' C except the

omission of sodium hydroxide from leagent 'A'.

ill

the response of vigna radiata and …the response of vigna radiata and brassica jiincea to...

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