ROOTING IN RELATION TO HORMONES AND VITAMINS

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

iWafiter of ^fiilofiiopljp IN

BOTANY

SUPHU RAFIQUE

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 1 9 9 5

^'-^'^•Z-&>5'^ ''I

DS2856

DP. AOIL AHMAD M. Sc. Ph. D. (Alig.)

PD.Rts, Train., DANIDA (Denmark)

BOTANY DEPARTMENT ALIGARH MUSLIM UNIVERSITY

ALIGARH-202002

CERTIFICATE

This is to certify that the dissertation entitled

"ROOTING IN RELATION TO HORMONES AND VITAMINS", is

a bonafied work carried out under my guidance and

supervision by Miss SUPHIA RAFIQUE and can be

submitted in partial fulfilment of the

requirements for the award of the degree of Master

of Philosophy in Botany.

(DR. AQIL AHMA15T

Dedicated To My Parents

ACKNOWLEDGEMENTS

I am profoundly thankful to Allah for blessing me

with the wisdom to complete the endeavour in a form of

dissertation.

I express my sincere gratitude to my respected

supervisor Dr. Aqil Ahmad, Reader, Department of Botany,

Aligarh Muslim University, Aligarh for constant help and

encouragements. This research work could not have been

accompalished without his suggestions, evaluation and

untiring cooperation.

Thanks are also due to Prof. Wazahat Hussain,

Chairman, Department of Botany, Aligarh Muslim University,

Aligarh for providing adequate facilities.

I am highly obliged to Prof. Samiullah, Dr. Arif

Inam, Dr. Feroz Mohainiaad, Dr. Nafees A. Khan and Dr. M.M.A.

Khan for their inspiration and valuable criticism from

time to time.

My lab colleagues and other friends have contributed

in achieving this goal, for which I am highly thankful.

My sincere and hearty thanks to my family members

whose moral support, and affection sustained me during the

period of this research work.

SUPHIA RAFIQUE

C O N T E N T S

Page No.

1. INTRODUCTION 1 - 2

2. REVIEW OF LITERATURE 3-62

3. MATERIAL AND METHODS 63- 78

4- REFERENCES 79- 97

5. APPENDIX (i)-(vi)

CHAPTER 1

JNTRODUCTION

INTRODUCTION

Agriculture is still the world's biggest business, to which

more people are devoted, and which yields a greater volume

of product, than any of man's other activity. In

agricultural research the application of certain sciences,

including plant physiology plays an important role, to the

specific problems of crop production. The contribution of

physiological researchers could made it possible to

increase both the amount of crop produced per unit of human

efforts invested and the amount of crop produced per unit

of land area utilized. Further advancement in agricultural

techniques had brought tremendous increase in crop

production, and it has reached to a plateau. To break the

present yield limit agriculturists and horticulturists are

using chemical growth regulators.

Plant growth and development largely depends on the

roots. Whose initiation and growth is very much regulated

by the balance of phytohormones which was recognized as

early as Thiamann and Went (1935). In addition to this,

roots also require secondary factors (Vitamins of B-group)

for their growth (Bonner and Bonner, 1948).

The use of Phytohormones and vitamins has largely

been confined to tissue culture or in inducing rooting in

cuttings. The due attention has not been paid to their

usage in improving the plant growth and productivity.

However, at Aligarh, a great deal of work has

already been done on B-group vitamins applied through the

seeds and leaves of various crops as reviewed by Samiullah

et al, 1988. The treatment stimulated growth and enhanced

proliferation of the roots at the seedling stage.

Therefore, such plants explored more soil for their better

establishment from the very early stages of growth. This

favourable effect was further reflected in terms of greater

biological yield, at harvest.

In the following studies to be undertaken

'TRITICALE' is proposed to be taken as a test crop. This

cultivar is still not able to compete with the new high

yielding varieties of wheat, due to shrivled grains and poor

germinability. The experiments wiH include :

(a) The grains will be pre-treated with hormones and/or

vitamins of equimolar concentration, before sowing in sand

culture. The resulting seedlings will be picked with

intact roots, at different stages of growth and studied for

various physiomorphological parameters both of root and

shoot..

(b) The seedlings grown in sand culture, will be watered

with the nutrient solution incorporated with hormone and/or

vitamin, at different intervals. They will be rooted out

at appropriate stages of growth and analysed for various

physiomorphological characteristics of root and shoot.

CHAPTER 2

REVIEW OF LITERATURE

REVIEW OF LITERATURE

C O N T E N T S

Page No .

2.0 Introduction .... 3

2.1 Vitamins as Plant Hormones .... 10

2.2 Vitamins in Tissue Culture .... 12

2.3 Vitamin in Rooting .... 15

2.4 Seed treatment with Vitamins .... 16

2.5 Vitamins assessment and application .... 23

to Intact Plants

2.6 Hormones in Tissue Culture _, .... 26

2.7 Hormones in Rooting .... 31

2.8 Seed Treatment with Hormones .... 52

2.9 Hormones supplied through the .... 56

Roots

2.10 Hormones supplied through the .... 59 Leaves

2.11 Conclusion .... 62

REVIEW OF LITERATURE

2.0 INTRODUCTION

French horticulturist, Duhamel du Monceau (1758) was

first to put forward the concept of plant growth regulators

when rooting from the tissues above the zone of girdling,

under the influence of descending sap, was noticed. The

observation recorded by Duhamel remained unexplored for

about a century. However, at that time the main interest

of the Botanists was in understanding that how plant

achieve their distinctive form. During the mid-1800s, the

famous German plant Physiologist Julius Sachs, found the

mechanism of organ formation in plants and stressed that

plants form organs in an organised pattern. This is due to

the action of specific 'organ forming' substances such as

'root and flower forming substances' etc. Sach's views

were not accepted because such substances vi?ere not isolated

and identified by that time.

Later on, the methods to isolate and identify other

plant constituents (such as starch, sucrose, glucose, amino

acids, proteins and nucleic acids) were developed. This

led to float the view that plants contains substances that

initiate biochemical processes, which ultimately lead to

organ formation and other aspects of regulated growth.

Finally, several distinct, different groups of compounds

(eg. Auxin, Gibberellins, Cytokinins, Abscissic acid and

ethylene) were characterized.

CHj^-COOH

Indole acetic acid (lAA)

CHa CDDH L CH ,

Gibberellic acid

Structures of various Hormones

CH20H

Z e a t i n

/CH3 HN-CH^-CH = C^

HOH^C—C H H

OH OH

Zeatin riboside or ribosyl Zeatin

HN-

n ^

k. ^ N - -

-CH2-

K i n e t i n

HC —CH

/ W 'C C

^ 0 /

— N \

CH

^ /

N H

CO OH 1

C==CH

Absicisic acid

H H

c- £

H •H

Ethylene

N ^

NH2 I C

H

C - - C H 2 — N ^

C — S

H3G C<>. ^V_JH N

C = C — C H ,

OH I

-CH2

CH.

Thiamine (Vitamin B )

NH, H

W X—CH2 — N //

C—S

\

o o

N G = G —CH2—CH2—O

CH,

-P-II O

-0—P—O

u

Thiamine Pyrophosphate

Structures of various B-group Vitamins

H I

H - C - O H

HO

H3C N

CH2OH

H

Pyridoxine

H 1 C=0

H I

H-C-NH2

Pyridoxal Pyridoxamine

-0-P-O

Pyridoxal phosphate

1 "* HOCH2- G - CHOH - CO - NH - C H 2 - C H 2 - COOIl

1 CH 3

Pantothenic acid

COOH

(CH2)A^H

/ C H - C — N H \ 5^ I /C = 0

XH2-C—NH H

B i o t i n

R H I i

S I /C=0 ^CH,-C —N

^ 1 I _ H COO

Where "R = NAD+ B i o c y t i n

10

The essentiality of another group of organic

substances (Vitamins), in addition to Phytohormones, was

noticed when incorporation of certain members of 'B-group'

vitamins to the culture medium was found to be necessary

for normal growth of fungi. The suggestion for including

the vitamins as plant growth substances was entertained

only when it was proved that isolated plant parts required

their exogenous application for normal growth under in

vitro conditions.

2.1 VITAMIN AS PLANT HORMONE

Bonner and Addicott (1937) reported that _in vitro

culture of excised pea root in a basic medium supplied with

amino acids, sugar (sucrose) and inorganic''salt, yielded

satisfactory results, in addition to this they substituted

yeast extract. Another accessory factor, required in small

amount, was Vitamin-B, for the growth of pea root, hence

considered phytohormone. However, vitamin-B^ alone was

inadequate to support the growth of excised roots.

Similarly, Day (1943; cultured excised tomato roots in

modified pfeffer's solution containing thiamine,

pyridoxine, nicotinamide, neo-peptone, glutamic acid and

glycine in various combinations. Thiamine and pyridoxine

enhanced root growth and roots possessed characteristic

hooks and curls.

Otakar (1969) studied the effect of vitamins B,, B„, B,, C, 1 2 o

P-aminobenzoic acid (PABA) and nicotinamide (.NA) on the

11

growth of pure culture of Suillus veriegatus. Vitamin B„,

B, and NA promoted growth whereas B, and PABA exhibited

comparatively an inhibitory effect.

Ramaiah et. ai (1984) studied the effect of B-group

vitamins (i.e. Thiamine, riboflavin, pyridoxine,

pantothenic, nicotinic and folic acids 100 mg/1) on the

growth of cluster bean (Cyamopsis tertragonaloba cultivar

PNB) plants. Among them, pyridoxine and folic acid were

most effective in increasing the level of GA-like

substances and growth. In 1987, they also studied the

influence of B-vitamins on the absorption of mineral

elements (e.g. Calcium, Sodium, Potassium and Phosphorus)

by the seedlings of green gram (Vigna radiata). Pyridoxine,

pantothenic acid and nicotinic acid had a profound

influence on the absorption of potassium and phosphorous.

Thiamine riboflavin and biotin slightly favoured the

absorption of these elements only at the late stages of

growth. They finally concluded that B-group vitamins act

as a plant hormone by inducing bio-chemical changes, even

at low concentrations.

Mozafar and Oertli (1992) reported that the

seedlings toth of barley and soyabean absorbed thiamine through the

root and transported it to the aerial parts. The uptake of

14

C-thiamine by soyabean roots is linear regarding the

concentration of thiamine but non-linear in terms of time.

An increase in root temperature from 5 to 35 degree

12

I 14

centigrade increased the uptake of C-thiamine and its

transport to the top. The aeration of the rooting media

v\7ith the nitrogen gas or the addition of 2,4-DNP in the 14 culture solution decreased the uptake of C-thiamine.

Thiamine concentration and the supply of oxygen affected

the partitioning of root absorbed thiamine between plant

roots and tops. They concluded that the uptake of thiamine

by plants does not require metabolic energy. The role of

thiamine in plants is not clear but is considered to be a

growth factor for roots.

2.2 VITAMINS IN TISSUE CULTURE

Almestrand (1950) supplemented the culture medium

with thiamine, pyridoxine and niacin for the culture of

isolated wheat roots. It was only pyridoxine (0.5 to 10

mg/1) which exhibited marked effect on the growth by

promoting cell division.

Lee and Whaley (1953) cultured excised tomato roots

in the medium with or without thiamine, pyridoxine and/or

niacin. The treatment did not induce any change in growth

rate during the first week but, in the third week, it was

significantly greater in the medium containing all the

three vitamins than in any other medium. The growth rate

was, however, markedly reduced in the fourth week by the

treatment. They proposed that the best period for

investigating the interaction between vitamins and root

13

metabolism in regulating its growth lies between second and

third week.

Boll (1954) reported necessity of thiamine,

pyridoxine and niacin for the optimal growth of the

excised tomato roots in iji vitro culture. Pyridoxine was

replaceable whose order of activity was

pyridoxal>pyridoxine>pyridoxamine. Similarly, niacin was

replaceable by more active niacinamide. Pyridoxine could

also be replaced by glycine, but more effectively in the

presence than in the absence of niacine. The morphogenetic

changes induced by pyridoxine and glycine were comparable

although glycine appeared to exert an independent effect on

the initiation of the lateral. The determination of the

root morphology was, therefore, suggested to be regulated

by the growth factors supplied in the medium.

Fujiwara and Ojima (1954) observed an increase in

the growth rate of excised roots of rice in a liquid medium

supplemented with either thiamine or pyridoxine, whereas

wheat roots responded to pyridoxine oniy. Similarly, Das

and Das (1966) noted the necessity of thiamine and

pyridoxine (0.001 ppm to 100 ppm) in the culture medium for

promoting the growth of isolated root sections of pea.

Otilia (1968) observed a synergistic effect of

thiamine and lAA on the growth of excised roots, of

Lycopersicum esculentum. Root growth was most favourably

14

-7 -11

affected by a combination of 3 x 10 M thiamine and 10 M

of lAA. Similarly, an interaction effect of the vitamin

and hormones was reported by Mihaly (1969). It was noted

that the excised tobacco tissue removed lesser quantity of

thiamine from the solution in the presence of kinetin,

however, it could be nullified if lAA was incorporated. It

was concluded that proper growth of excised tobacco

tissues in culture medium could be achieved by

incorporating thiamine with lAA and kinetin.

Sandra and Spurr (1973) succeeded in inducing

greening response in albino mutant (al/al) by supplying

thiamine pyrophosphate (TPP) with the nutrients. Six week

old plants revealed that the mutant had 76% more free

thiamine than the normal plant but only 11% was

phosphorylated. It revealed that the mutant had a blockade

in the conversion of thiamine to thiamine pyrophosphate.

Inoue £t. a_l (1980) selected thiamine requiring and

non-requiring strains of rice to study the role of the

vitamin on the organogenesis in _in vitro culture. The

strain (not requiring thiamine) cultured on a medium

without vitamin,exhioited surface meristem, root and leaf

like structures but green regions were induced only in the

presence of thiamine and kinetin. The vitamin also

affected the surface architecture in the culture. It was,

therefore, evident that thiamine operated as a factor in

regulating organogenesis in rice culture.

15

2.3 VITAMINS IN ROOTING

Pastena (1974) left the cuttings of American vine in

water solution of vitamin A, B^/ B , B, (125 to 250 ppm) B,

(5 to 250 ppm) or C (150 to 25000 ppm) before planting them

in nursery, in the month of March. Vitamin B, and B,,

although had an inhibitory action on root formation but

enhanced the growth of cuttings. The other vitamins exhibited a

negative effect on rooting and growth.

Pyasyatskene (1974) treated annual cuttings of

Arctostaphylos uvaursi (L) spreng, with the mixture of

heteroauxin (150 mg/1) and ascorbic acid (1500 mg/1j. The

treatment not only increased the rooting but also the

length and weight of the roots. The rate of elongation of

the cutting and the number of shoots and leaves was also

favourably affected.

Hussein and Mansour (1978) supplied thiamine,

pyridoxine and cyanocobalamine continuously to the roots of

young seedlings of Vicia faba. It resulted in the death of

the roots within 48 hrs. However, the recovery could be

induced in water if the treatment was restricted to a

shorter duration (i.e. 4 to 24 hrs.). A higher

concentration of the vitamins or a treatment for longer

duration decreased the mitotic index. The frequency of

different mitotic stages exhibited that the treatment

reduced the number of prophases which was directly

proportional to the vitamin concentration. The number of

16

metaphases and anaphases increased but the telophase

decreased. The vitamins also induced ce r t a in abnormalities

(Stickeness of micronuclei, bridges and spindle

dis turbances) which increased with an increase in the

vitamins concent ra t ion .

Adinaryana and Vijayamma (1991) Studied the effects

of vitamins (thiamine, r ibof lavin , pyridoxine, b io t in ,

n iac in , pantothenic acid and fo l ic acid) and hormones (IBA

and GA ) to study the i r role in rhizogenesis of isolated

leaves of bean in nutr ient medium. IBA and niacin

increased the root number. Biotin and pyridoxine enhanced

l a t e r a l r o o t s , whereas, r iboflavin increase the root

length. Thiamine, b io t in and IBA recorded for maximum water

uptake. I t was concluded that vitamins mimic hormones and

also capable of increasing water uptake 3ust l ike a

hormones.

2 . 4 SEED TREATMENT WITH VITAMINS

The vitamin content exhibited a var ia t ion in plant

organs. The germinating seeds of some cerea ls and legumes

possessed more vitamins than dry seeds, possibly released

from the source on the hydration of the seeds (Tallarico,

1950).

Kulesha and Moshchinskii (1968) reported that the

wheat seed l ing i r r ad ia t ed by V-rad ia t ion for 1-20 h r s . .

17

synthesised more vitamin-B. than non-irradiated ones. The

vitamin synthesizing capability increased five fold if the

seedlings were provided with thiazol and pyrimidine, the

two fractions of thiamine included in the nutrient

solution.

Ovcharov and Kulieva (1968) soaked cotton seeds in

0.01% solution of vitamin B,, PP(vitamin B^) and nicotinic

acid amide (derivative of PP) for 1-3 hrs. , and supplied

with different forms of fertilizers. Vitamin PP and NAA

increased water absorption by the seeds and the weight of

their embryo. The germinability also improved. Hov;ever,

the effectiveness of the vitamin B, was largely determined

by the form of the fertilizer. The treatment increased

root length by 49% in the presence" of ammonium sulphate but

decreased it by 14% if calcium nitrate was present.

Zavenyagina and Bukin (1969) observed the effect of

pyridoxine and its antagonists, on the rate and power of

germination of seeds and the development of root and shoot

of pea and wheat seedlings in water culture. Pyridoxine

antagonists at the concentration ranging from 10~ to 10~

M decreased the germinability of the seeds and also

suppressed growth of root and shoot of both the cultivars.

However, the symptom of pyridoxine a-vitaminosis could

partially be avoided by the introduction of the vitamin to

the nutrient solution. The vitamin also stimulated the

18

growth of normal wheat and pea seedlings and increased the

chlorophyll content in the leaves.

Mullick and Chatterji (1971) reported that the

lettuce seedlings grown in the presence or absence of light

in 500 ppm of niacin solution exhibited an inhibition of

root growth. However, the growth of the hypocotyl was not

affected, in contrast to the observation made with the

monocots, where, germination and subsequent growth was

inhibited by the vitamin. It was suggested that niacin

might have disrupted the endogenous auxin level by

competing for tryptophan, in the substrate induced

biosynthesis.

Srivastava et al (1974) studied the effect of

various growth regulators like ascorbic acid, napthalene

acetic acid, lAA and GA on the improvement of seed

germination in Cichorium intybus L. (Chicory). It has been

found that GA and ascorbic acid at 200 and 400 ppm improved

germination upto 92% with the better seedling growth.

Asthana and Srivastava (1978) studied the effect of

pre-sowing treatment of maize seeds with ascorbic acid and

salicylic acid. It was observed that ascorbic acid

stimulated seed germination, whereas, salicylic acid either

alone or in combination with ascorbic acid was inhibitory.

Ascorbic acid and salicylic acid inhibited growth of root

and shoot. However, ascorbic acid increased the growth of

19

primary leaves though there was little effect on nitrate

reductase activity. While ascorbic acid alone or in

combination with salicylic acid enhanced the 80% ethanol

soluble and protein-N in the leaves. Both these acids,

either individually or in combination hastened the

flowering and ear emergence.

Vardjan (1978) germinated the dormant seed of

Gentiana lutea cv. symphyandra murb by chilling or the

application of GA^. Ascorbic acid as such did not make any

effect in association viyith GA^, the response of the seeds

was prominent. A similar, minor synergism was also

observed between GA-, and thiamine but not with riboflavin.

Ahmad et al (1981) studied the effect of

pre-treatment of grain with pyridoxine on the growth of

five varieties of barley, namely NP-13, NP-21, K-572/10.

K-572/28 and Clipper. Seeds sown in the field in a

factorial randomised trial. They observed a significant

effect of the treatment on tiller number, leaf number,

shoot length and fresh and dry weight at tillering, heading

and milky grain stages. Seeds soaked in 0.1% pyridoxine

solution proved optimum at all stages of growth.

Asfaque et aj (1983) conducted a field trial at

Aligarh, to study the effect of soaking the grain, for 12

hrs., in 0.0, 0.1, 0.2, 0.3 and 0.4% aqueous pyridoxine

20

solution on the yield characteristics of triticale variety

Branco-90. Most of the yield attributes studied were

significantly affected by the seed soaking treatment.

Maximum grain yield was obtained in 0.2% treatment, 37.7%

more than obtained in unsoaked controls.

Samiullah et a]^ (1983) also at Aligarh observed that

pre-soaking treatment with pyridoxine or by spraying at

appropriate stage on a standing crop of moong (Vigna radiata)

significantly enhanced leaf nitrate reductase activity

(NRA). Leaf NRA level significantly correlated with seed

yield can be utilized for predicting for predicting crop

productivity and for adopting corrective measures at an

early growth stages of the crop.

Khan and Ansari {19S4) soaked the seeds of Phaseolus

radiatus cv. T-9 in 0.03% aqueous pyridoxine solution for 10

hrs., the seedlings were supplied with phosphorous (0 to 60

ppm) on alternate days. The treatment with pyridoxine

increased the fresh weight and the number of lateral roots

in ten day old seedlings.

Ansari and Khan (1986) in a field trial on summer

moong var. K-851 found that pre-sowing seed treatment for 4

hrs. with graded aqueous pyridoxine solution, 0, 0.1, 0.2,

0.3, 0.4 and 0.5% significantly enhanced the plant length,

leaf number, fresh and dry weight at 30, 40 and 50 days

growth. They also observed significant effect on NRA at

20-30, 30-40 and 40-50 days growth and pod number/plant,

pod length and seed number/pod, at harvest. Among the

21

pyridoxine treatments 0.3% proved optimum. Seed yield was

positively correlated with plant length at 30 and 50 days;

with leaf number and dry weight at 30, 40 and 50 days; with

fresh weight 30 and 40 days with NRA, at all intervals.

Seed protein contents also showed positive correlation with

plant length at 30, 40, and 50 days; with fresh and dry

weight at 40 and 50 days, with NRA at 30-40 and 40-50 days

while pod number, pod length and seed/pod were correlated

with seed yield only.

Ahmad et a_l (1986a) conducted a field experiment

with five barley varieties 'NP-13', 'NP-21', •K-572/10',

'K-572/28' and Clipper. Seeds were pre-soaked in 0%, 0.2%

or 0.5% aqueous pyridoxine solution. Significant effect on

ear number and ear weight/plant, grain number/ear and grain

and straw yield was noted. The combination 0.01% x

K-572/10 proved optimum for ear weight/plant, length/ear,

spikelets/ear and grains/ear. Ahmad et al_ (1986b) also

studied the effect of pre-sowing treatment of seeds with

pyridoxine on the test weight, carbohydrate and protein

content on the same varieties (as discuss above) of barley.

It was reported that K-572/28 gave the heighest 1000 grain

weight, grain carbohydrate and protein percentage while

NP-13 was lowest yielder.

Hague, I. (1988) studied the comparative response of

two triticales (Tiger 'S' and Muskox 'S') grains treated by

pyridoxine solution with various concentrations 0, 0.001 or

22

0.1% for 8 hrs., and raised such plants in sand culture

till the maturity. The leaf NPK status was examined at

tillering heading and milkey grain stages. While seeds were

screened for their total protein and carbohydrate contents,

at harvest. Pyridoxine concentration 0.1% proved optimum

followed by 0.01 and 0.001%. Cultivar Tigre 'S' and 0.1% of

the vitamin combination proved to be the best.

In a factorial randomized field trial, Samiullah et_

al (1992) studied the effect of soaking the seeds of lentil

(Lens culinaris L. Medic) var. T-36 for 12 hrs. in 0, 0.2,

0.3, and 0.4% aqueous pyridoxine solutions and 15, 30, 45

and 60 kg P/ha of their interaction on nitrate reductase

activity. (NRA) at 60-90 and 120 days; net assimilation

rate (NRA) at 60-90 and 90-120 days an; pods plants, length

pod, seed pod, 1000 seed weight, seed yield and seed

protein content at 140 days (harvest). The application of

0.3% pyridoxine and 30 Kg P/ha separately and the

interaction between 0.2% pyridoxine x 30 Kg P/ha proved

optimu' for most of the parameters studied, except the

interaction effect for 1000 seed weight. NRA and NAR, at

all sampling, showed a strong correlation (P < 0.01) with

seed yield and protein content, while yield attributes

showed a similar correlation with seed yield only. Thus

pre-sowing seed treatment with 0.3% pyridoxine in

combination with 30 Kg P/ha ensure high yield and quality

of lentil.

23

2.5 VITAMINS ASSESSMENT AND APPLICATION TO INTACT PLANTS

Bejishvili (1968) reported maximum contents of

pyridoxine and pantothenic acid (0.25 and 0.28 mg per cent

respectively) in root nodules of pea. Nicotinic acid

possessed maximum values in the roots (0.23 mg/%). While

lowest values were recorded in the leaves.

Pavel et_ a_l (1968) estimated thiamine and

riboflavin content in hydroponically cultivated barley

plants receiving lAA and GA once. Plants treated with lAA

had an increased thiamine level in shoot but decreased in

root while the riboflavin content decreased slightly.

Plants received GA had an increased thiamine level in the

root, riboflavin content showed a varying^ trends in

comparison with control, on the 1st and 4th day of illumi­

nation it was high, later it registered a mark decrease.

In control plants riboflavin level both in the root and

aerial parts was high throughout the study, but thiamine

content decreased in the above ground parts with time.

Rao and Sastry (1972) compared thiamine and

pyridoxine level in an early (TMV-2) and late (TMV-3)

varieties of Arachis hypogea L. The former had more

vitamins than the later and had a direct correlation with

the age. Epicotyl and roots had higher endogenous level

than the cotyledons. The late variety possessed more

24

vitamins probably due to higher Chlorophyll contents which

appeared to go hand in hand with vitamin synthesis.

Epachinov (1973) in a field experiment studied the

effect of mineral fertilizers on accumulation of nicotinic

acid, biotin, thiamine and pyridoxine in soil of root zone

of maize. It was noted that mineral fertilizers favoured

the accumulation of these vitamins and enhanced maize

yield. Thus, it was suggested that vitamins intensify the

growth of maize seedling and entry of nutrients into the

roots of the plants.

Gopala Rao (1983) working with Phaseolus radiatus

(green gram) noted that root elongation was inhibited by a

temperature of 4U° centigrade but thiamine and riboflavin

counteracted this effect. On the other hand, the

inhibition of shoot growth elongation could be counteracted

by niacin and pantothenic acid. These vitamins, therefore,

served as temperature lesions and protected the seedlings

from heat injury.

Oertli (1987) reviewed the work conducted on plant

responses to the exogenous application of vitamins as

regulators for growth and development of plants. Mode of

application of vitamins, function of vitamins in plants,

morphogenetic changes induced by exogenous vitamins appli­

cations, protective function of vitamin, effect of vitamins

on plants under stress and vitamins in the rhizosphere.

25

were thoroughly reviewed. In tabulated form function of

vitamins in higher plants, response of plants to spray with

vitamins, response of plants to soaking seeds with vitamins

and interaction of vitamins with other growth regulators

was also given. Vitamins have been applied by soaking seeds

or dipping cuttings; by spray or dusts or as drenches to

soils, exogenous vitamins application also proved helpful

in increasing substantial yield. It was also mentioned

that vitamin may cause morphogenetic response in plants,

the most pronounced among these responses are stimulation

of root formation and flowering under non-inductive

conditions. Interestingly role of vitamins as protector

against Ozone and Sulpher di-oxide, the two important

agents of air pollution wes also reviewed. Finally it was

concluded that some responses to vitamins are reproducible

and for others further experimental varification is

necessary only further research justify the use of

vitamins in agriculture but before this a number of

technological and econcmical problems may need to be solved.

Gopala Rao e^ aA (1987) working on cluster bean and

mustard observed that vitamins of B-group (thiamine,

riboflavin, pantothenic acid, pyridoxine and folic acid)

increased the uptake of zinc. Vitamin induced absorptive

capacity for zinc was higher in cluster bean than mustard.

At pH 9, uptake of zinc was maximum and minimum at pH 6. The

chelating agent (EDTA) was more effective in increasing

zinc uptake in mustard than in cluster bean, exhibiting a

26

possible interaction between the chelate and the vitamin.

Samiullah et_ al (1988) while reviewing the role of

the B-vitamins in relation to crop productivity mentioned

the role of B-vitamins inrelation to plant physiology,

nutrient uptake, seed germination, vegetative growth and

crop productivity. In their comprehensive review they also

reported the work related with foliar application of

B-vitamins with special reference to work conducted at

Aligarh. They concluded that B-vitamins involved in various

physiological process of plants as respiration, photosyn­

thesis, chlorophyll synthesis, protein synthesis and also

participate in improvement of growth, yield and quality

performance of vegetables, cereals,leguminous and oil crops

and also few aromatic and medicinal plants. However, they

affirmed that the role of vitamins in some areas of plant

physiology such as vernalization, photoperiodism, phyto-

chrome action, biological nitrogen fixation, has not been

studied so far. Therefore^ research on these neglected

aspects wer° also suggested. In addition, to this role of

vitamins in agriculture, particularly in dry land farming

has also been discussed by them.

2.6 HORMONES IN TISSUE CULTURE

This technique is used for the propagation of plants

on one hand and on the other hand it is also employed to

27

understand the mechanism of growth and development and role

of growth hormones in these processes.

Lavee and Galston (1968) reported the absence of

peroxidase activity in freshly excised pith tissues of

Pelargonium, however, it developed after 36 hrs., in

asceptic culture. The presence of lAA in the medium

initially (upto Ca. 50 hrs., in culture) inhibited the

activity of the enzyme but later (Ca. 100-150 hrs., in

culture) promoted. This dual effect of lAA resembled with

that reported earlier for specific iso-peroxidase, in

tobacco pith cells. The presence of kinetin alone also

inhibited peroxidase synthesis but in association with lAA,

a concentration which enhances growth also increased

peroxidase activity.

Mitra (1968) reported an enhanced rate of growth of

isolated roots of Rauwalfia serpentina on a medium

supplemented with 2-naphthalene acetic acid (0.001 mg/1)

and B-indole butyric acid (0.01 mg/1) but declined after 21

days. The enhanced rate could, however, be maintained if

successive transfer of 21 days old cultures was made

through 2 passages. In addition to this, kinetin (0.001

mg/1) and GA (0.01 mg/1) slightly enhanced the level and

duration of growth in the dark only.

Robbins and Annette (1969) successfully cultured

large quantities of inocula (dry weight 2.0 mg or more) of

excised root tips of Bryophyllum calycinum in 50 ml of a

28

mineral salt sucrose medium supplemented with vitamins.

However, the root tip in isolation failed to grow in the

absence of the exogeneously applied auxins ( °C -naphthalene

acetic acid, lAA and 2,4-D). This indicated the leaching

of auxins from the root tips in isolation.

Chadwick (197 0) proposed that the growth of excised

roots of Pisum sativum is not regulated only by the auxins

but its interaction with the ethylene.

Saniewski et aJ (1974) studied the effect of

different hormones on the regeneration of the bulblets from

a few month old bulbs of Hyacinthus orientalis. The emer­

gence of the roots and bulblets was dependent on the

quantity of the hormones incorporated with the basic

culture medium. NAA was noted to be essential for callus

formation and root differentiation but the best combination

was that of NAA (1.0 ppm) and BA (10 ppm) for the

initiation of bulblets.

Colijn £t. a_l (1979) cultured root meristem of

germinated seeds of Petunia hybrida on a medium added with

varying quantities of hormones. Medium, supplemented with

the combination of 6-benzylamino-purine (0.05 mg/1) and NAA

(0.05-2.0 mg/1) induced the formation of shoot. Whereas

the change in the quantity of these hormones to 0.25-2.0

mg/1 and 0.05-0.5 mg/1, respectively, resulted in the

development of complete plants.

29

Tanimoto and Hiroshi (1982) observed the

differentiation of adventitious roots in _in vitro cultured

cotyledons, stem and leaf explants of Rudbeckia bicclor by

the application of NAA but its replacement with BA resulted

in the formation of adventitious buds. If a lower

concentration of NAA was supplemented with BA, bud

development was promoted. The incorporation of GA induced

stem elongation of the adventitious buds and also occa­

sionally differentiation of floral buds at the apices of

adventitious shoots.

Ahmad and Nag (1985) reported an induction of root

formation on a Lac host Maghonia macrophylla by lAA, IBA

and NAA. Out of these three hormones, lAA was less

effective than IBA but more than NAA in rooting in host

plant.

Watanabe and Takahashi (1987) varied the relative

concentration of various hormones ( "^C-NAA, lAA, IBA, BA,

Kinetin and zeatin) in the culture medium to grow the shoot

of Actinida chinesis. They reported the growth of good

callus mass on a medium rich in lAA (0.1-10.0 mg/1) only

but it was better if lAA was replaced by NAA. A media con­

taining 0-0.01 mg/1 of NAA and 0-0.1 mg/1 of lAA promoted

shoot formation. Kinetin as compared with zeatin, was less

effective on the shoot differentiation from callus. IBA

(1.0 mg/1) favoured root development in cuttings.

30

Perez et al (1989) reported necessity of 6-benzyl-

amino-purine (BA), at a concentration of 1 mg/1 in the

medium for the induction of axillary, viable buds on

epicotyl-hypocotyl explants of Pinus canariensis chr.sm.ex

DC-culture. The addition of IBA with BA did not make any

significant difference in the bud formation, but 16% of the

buds at their base striked roots if auxin quantity was 0.1

mg/1. Which were not normal and the culture develop the

problem of organization.

Magnus et al (1992)reported stimulation in the develop­

ment of root and shoot on the xjci vitro culture of tomato

(Lycopersicon esculentum mill c.v. Monglobe) hypocotyl

tissue in a media supplemented with free lAA. In contrast,

lAA-aminoacid conjugates supported largely callus growth.

However, if free lAA and conjugates were supplied together

both organogenesis and callus growth resulted.

Bansal and Pandey (1993) cultured hypocotyl explants

of Sesbania aculeata in a Murashige and Skoog's basal

medium supplemented with IBA, NAA singly or in association

with BAP (Cytokinin). They observed budding directly from

the explant as well as from the callus. Both shoot and

root differentiated in a single step in the cultures

supplied either auxin alone or with cytokinin of the same

concentration.

31

Hu et al (19 93) cultured shoots, removed, from the

seedling and mature plants of Crategus scrabifolia and

observed the level of endogenous lAA depend upon each

individual (i.e. derived from seedling & mature plant).

However the balance of lAA and Z + ZR during the course of

development from seedling to mature plant was multiform. An

increase in lAA level and decrease in Z + ZR level induced

rooting in cultural shoot derived from adult plant. The

endogenous ABA content stood at a lower level and no

remarkable difference was observed between the shoots from

the two different sources.

Roddick et_ a]^ (1993) reported that 10 pM or lower

concentration (1 uM - 10 uM) of 24-epibrassinolide applied

basally or to the apex region of excised roots of tomato

(Lycopersicon esculantum) inhibited growth in apical region

but basal region remained unaffected. Roots, therefore,

appeared to transport epibrassinolide acropetally. The

lack of response by tomato roots to epibrassinolide by this

method is probably due to larger inoculum used rather than

physiological age of the root.

2.7 HORMONES IN ROOTING

It was interesting to note that Dutch gardners had a

centuries old practice of embeddling grain seeds into

cuttings of trees to promote root formation. Before the

32

discovery of auxins, many chemical compounds were reported

to increase the rooting of cuttings (e.g. permanganate and

carbon monooxide) but auxin found to be most effective and

this property of auxin is highly useful in the vegetative

propagation of plants.

Nanda et al (1968) demonstrated that the segments

of Populus nigra, shorter than 3.5 cm in length, rooted

only when exposed to dark for 3 or more days and the number

of roots were directly proportional to the number of dark

days. However, this requirement could be fulfilled if the

cuttings were treated with lAA or IBA. The effect

increased in the presence of sucrose. The inability of

control cuttings to root in continuous light was because of

low auxin level which might have been due to lack of its

synthesis or photo-oxidation. It could be overcome either

by the exogenous supply of the auxin or the increase in the

size of the segments. GA^, under favourable conditions,

inhibited rooting but stimulated bud emergence and

subsequent elongation.

Ryuzo (1968) found that stem cuttings from Pinus

densiflora (1 or 2 year old seedlings) produced more number

of roots if treated with 100-500 ppm of lAA, whereas

kinetin inhibited rooting at concentration above 10 ppm but

promoted rooting at 1 ppm, Gibberellin (1-50 ppm)

inhibited rooting if used alone. In a mixture of lAA and

kinetin, higher concentration of kinetin inhibited rooting

33

while lower concentration of kinetin increased rooting to a

higher per cent than that produced by lAA and kinetin alone.

The inhibition was more prominent if all the three hormones

were provided together.

Basu et_ aj^ (1969) dipped the leafy cuttings of Eranthemum

tricolor in the solution of tannic acid, gallic acid,

p-hydroxybenzoic acid and salicylic acid (1000, 100, 10 and

1 mg/1) for 24 hrs. These treated cuttings were again

dipped in a 1000 mg/1 solution of lAA, IBA and NAA for 10

seconds. It was noted that phenolics did not promote

rooting. However, tannic acid in combination with NAA and

IBA enhanced rooting. Similarly, gallic acid markedly

increased the number of roots per cutting in combination

with NAA and IBA. Synergism was also recorded betv een

p-hydrobenzoic acid and lAA or NAA, but not with IBA.

Salicylic acid greatly promoted rooting in association with

lAA, IBA and NAA.

Chin et. BX_ (1969) observed that ABA (abscissic acid)

stimulated rooting in stem cuttings of mung bean and

English ivy. It also overcame the inhibitory effects of GA

on root formation in mung bean cuttings but at the same

concentration. It failed to do so in the cuttings treated

with kinetin.

Katsumi and Machi (1969) working with the cucumber

seedling observed that the removal of the apex produced no

34

effect on the rooting of the hypocotyl but it decreased on

the loss of cotyledons. lAA treatment promoted rooting.

The presence of cotyledons resulted in the requirement of

lower concentration of the auxin (1 mg/1) for promotion of

rooting but in the absence of major part of the cotyledons,

the effective lAA concentration was higher (i.e. 10 mg/1).

It was, therefore, concluded that cotyledons play a major

role in the differentiation of roots in cucumber hypocotyl

cuttings probably by supplying the auxin to the hypocotyl

region.

Chailakhyan and Sarkisova (1970) noted that

principal factor in the formation of roots in stem cuttings

of fruit trees is their ability to regulate the relative

level of endogenous growth Regulator (i.e. auxin) and its

inhibitors. Those varieties, whose stem cuttings can not

be rooted showed a constant inhibitor composition,

therefore, did not respond to the exogenous application of

growth regulators. This was proposed to be major point of

difference between easy and hard-to-root varieties.

Samananda et aj (1972) treated the stem cuttings of

Chrysanthemum moriflouim with aqueous solutions of Ethernai

(2-chloroethyl phosphonic acid) '" <* IBA. Etheral (1 mg/1)

applied as a dip or as spray to the leaves promoted root

length and branching only in hard-to-root (Mrs. Roy and

Clipper) but it had no effect on easy-to-root "improved

35

mefo" cultivar. However , IBA increased root number in

"Clipper" as well as in "improved mefo". It was suggested

that IBA and Etheral might be acting at different processes

of root differentiation. IBA promoting initiation and

etheral stimulating elongation and branching. It was

accompanied by a decrease in the level of soluble

carbohydrates particularly at the bases of the stem.

Sharma et aJ (1975) reported that air layering of

guava (Psidium guajava L.) by growth regulators (IBA and

NAA) showed the maximum rooting percentage and better

survival rate in the month of March. Survival was nil in

the July air-layering. While if the full ring of bark is

removed then treatment with IBA and NAA mixture showed

higher survival and rooting rate.

Similarly in bean hypocotyl the lAA supplied either

acropetally or basipetally increased the number of roots

without altering the pattern of their emergence. The

radioactive lAA ( C-IAA) was translocated mainly through

the vascular -tissues but a smaller quantity also moved

radially towards the cortex and pith which was proposed to

be largely responsible for the promoting effect of the

auxin on rooting (Friedrrian et al 1979).

Haissing (1979) compared the influence of arylesters

of lAA and IBA with simple lAA on adventitious root

primodium initiation and development in bean and jack pine.

36

Phenyl-IAA (P-IAA) induced 95-154% more macroscopically

visible root priraordium in two days old cuttings of bean as

compared with lAA. Even though, the treatment with

3-hydroxyphenyl {3-HP) lAA decreased the number of root

primordia as compared with P-IAA but the effect of both

these esters of lAA was 10 or more times as effective as

lAA in bean cuttings Methyl esters of lAA was ineffective

in this regard. P-IBA enhanced the number of rooted

jack-pine seedling cuttings by 11-12% while higher

concentration of IBA enhanced rooting upto 27%. The number

elongated roots (2 mm or more) after S-days increased

165-276% for P-IAA than lAA, treated bean cuttings, 3HP-IAA

showed similar but less increase as P-IAA. Lastly, it was

suggested that these esters were, required by. the cuttings

of the initial stages for inducing the differentiation of

root primordia.

Bandzaitene (1981) treated the cowberry cuttings

-3 -4 -5 with the solution of lAA (10 , 10 , 10 M) and a mixture

of lAA (10~ M) and succinic acid (10"" M) , before their

plantation. The treatment promoted rooting and the

cuttings developed well. However, the cuttings treated with

-3 the solutJ.on of kinetin (10 M) and lAA resulted in

inhibition. The treatment v/ith Pyruvic acid, succinic acid, citric

_3 acid, gibberellic acid (10 M) alone or in combination with

lAA (10 M) has no significant effect, as compared with

the control.

37

Balasimha and Subramoniam (1983) used 0-and

p-hydroxybenzoic acid (0- and p-HBA) as synergists of IBA.

Rooting and rhizogenesis were enhanced by a combined

application of p-HBA and IBA. Peroxidase and

iso-peroxidase activity were higher before rooting compared

with the onset of rooting in cuttings. Treated cuttings

also possessed higher enzymes activity than control.

Ellasson and Karin (1984) succeeded in getting the

stem cuttings of pea rooted in a nutrient solution in the

presence of light. The number of roots, hov/ever, decreased

if the solution was also supplemented with lAA (10 M) for

24 hrs. The response in terms of rooting was also poor if

the treatment duration was shortened. But lAA (100 uM)

usually increased the number of roots. The number of roots

exhibited a strong increase if lAA was replaced by

indolebutyric acid (IBA), or 2,4-dichlorophenoxy acetic

acid (2,4-D). They suggested that the decrease in the

number of roots per cutting was due to the sub-optimal

concentration of the auxins. In addition to this the

experiment with 10 ^M, IBA showed that stimulation of

rooting was obtained only if the auxin was present in the

rooting solution for several days.

Kundal and Bindra (1984) studied the effect of

washing of cuttings of Peach and Almond for 0, 24, 48 and

72 hrs. followed by their treatment with 500, 1000 and 2000

38

ppm each of NAA and IBA on rooting. Washing was not

effective in improving the rooting because it lowered the

endogenous level of starch, total sugars, phenolics, total

nitrogen, C/N ratio and HCN contents. However, washing

for 72 hrs. followed by their treatment with 1000 and 2000

ppm of IBA and tAA induced rooting in the ratio of 12:30 and 11:70%

cuttings. Little rooting occured in unwashed and untreated

cuttings where the endogenous level of HCN (265.7 ug/gm)

was supposed to be the main inhibitory factor.

Massel and Valio (1984) studied the effect of lAA,

IBA, CEPA (2-chloroethyl phosphonic acid), AVG (amino

ethoxy vinylglycin) and AgNo, on the initiation of

adventitious roots on the stem of tomato (Lycopercicum

esculentum cv. eaqui) under an air-layer. Auxins, in

general did not improved rooting. CEPA (a source of

ethylene) promoted rooting and this was reversed by AVG and

AgNO-, (anti-ethylene agent). Therefore, in this case ethy­

lene was proposed to play regulatory role on rooting.

Le et al (1984) found that the cytokinin activity in

Kidney bean leafy cuttings increased in the course of root

formation, stimulated by lAA. They, therefore, suggested

that the increase in the level of endogenous cytokinin may

be one of the mechanism by which lAA stimulated rooting in

cuttings.

39

Weigel and Bertold (1984) reported a basipetal

decrease of free auxin in the cuttings of Chrysanthemum

morifolium cv. yellow Galaxy but the middle part had the

maximum quantity of the esters of lAA. Both the forms of

auxins increased as the cuttings developed the first root.

Prolonged irradiance of stock plants to higher intensities

of light (40 Wm ) delayed the increase of both free and

esters of lAA in the cuttings concomitantly with the lower

number of roots, compared to the control stock plants grown

_2 at 4.5 Wm . A distinct relationship was found between lAA

contents of stock plants at the time of taking the cuttings

and the number of adventitious roots formed by the cuttings,

20 dyas later.

Zeadan and Macleod (1984) exposed the attached and

excised roots of Pisum sativum to lAA to study the

initiation of root primordia and their subsequent emergence.

In attached and excised roots, the presence of auxin

stimulated the process under study. This effect of lAA on

the development of lateral roots was probably due to changes

in the proliferative activity in the apical meristems of

primary roots.

Jarvis and Shaheed (1986) applied triodobenzoic acid

(TIBA) or morphactin (Chlorofluoronel) alone or in

14 combination with C-IAA to the base of mung bean hypocotyl

cuttings where they inhibited rooting. It was due to the

fact that both the chemicals inhibited the accumulation of

14 C-IAA at the base of the cuttings.

40

Mato and Vieitez (1986) during rhizogenesis of the

IBA treated shoots of Castanea sativa Mill. reported an

increase in auxin protectors in the basal parts and a

decrease in the upper parts. However, lAA-oxidase activity

declined in the basal part while it increased in the upper

parts. Whereas in control cuttings the enzyme level was low

in the upper end and high in the lower parts. Therefore,

rooting regions of the shoot had more lAA in the

pre-treated cuttings. It was proposed, at last that the

pre-treatment of the shoot with IBA could have the

controlling effect on the endogenous auxin level through

lAA-oxidase activity or by their transport.

Alorda et a_l (1987) removed hardwood cuttings of

Ceratonia siuilqua L. in the months between February and

May which exhibited better rooting on being treated with

4000, 8000 and 10,000 mg/1 of IBA singly or in association

with kinetin (10,000 and 20,000 mg/1) respectively. Highest

percentage of rooting was reported at the end of March.

This difference in rooting in various months was due to the

development of sclerenchymtous bands in the cortex.

Lucas and Valio (1987) examined that in the leafy

cuttings of Phaseolus vulgaris, auxin was the only growth

regulator which was capable of inducing root formation.

These roots were usually formed in the petiole above the

41

pulvinus. Other growth regulators like NAA and lAA,

stimulated root initiation, while IBA was more effective

among them. However, ABA did not effect rooting because

its level was higher in the lower part of petiole than in

pulvinus. The transport of these labelled material (lAA

and ABA) did not help in understadning the inhibitory

effect of the -palvinus. But the presence of substance

similar to oestrogens in the extract of pulvinus suggests

the possibility of its involvement in the inhibitory effect

of the pulvinus on the rooting process.

Thimba and Italya (1987) studied the effect of three

concentrations of IBA (0, 1000, 3000 and 8000 ppm) on the

endogenous level of soluble carbohydrates in the cuttings

of Passiflora edulis f. sins removed from regions of the

mother plant and their relationship with the rooting. It

was noted that 3000 ppm proved best for all types of the

cuttings, except the terminal decapitated ones, therefore,

was termed as optimal level. The cuttings treated with

this optimal concentration of IBA had the lowest

carbohydrate level whereas those treated with other

concentrations exhibited higher level of carbohydrates.

Carbohydrate level in the cuttings was supposed to be

related to the percentage of rooting, number of roots and

root dry weight per cutting.

42

Kakkar and Rai (1988) reported that the treatment of

hypocotyl cuttings of Phaseolus vulgaris L. by Actinomycin-D _7

and cycloheximide (10 M) for 6 to 24 hrs. increased root

primordia, root number and their length, as compared with

controls.

Wiesman et al (1988) reported that IBA was much more

effective in inducing adventitious root formation in mung

bean (Vigna radiata) cuttings than lAA. The effect was

more prominent if the duration of the treatment was

extended from 24 to 96 hrs. Both the labelled auxins were

transported acropetally from the base, the site of their

application, but larger part was retained in the rooting

zone. Relatively, more JBA was found in the upper sections

of the cuttings. Rate of metabolism,of both the auxins was

similar and within 24 hrs. only a small fraction of free

auxin was left. It was, therefore, suggested that higher

root promoting activity of IBA was not due to a difference

in the rate of transport or metabolism but the IBA

conjugates being the better source of free auxin than

those of lAA.

Berthon et aj (1989) observed first a rise followed

by a fall in soluble peroxidase activity in Sequoiadendron

giganteum cuttings during rooting. The level of the free

lAA exhibited an opposite trend i.e. first a fall then

rise. The contents of absci'-sic acid, zeatin amd zeatin-

43

riboside increased upto day 6, passed through a minimum day

13, and then again increased slightly. They proposed a

scheme explaining the possible events connecting rooting

and the variation of endogenous plant hormones.

Haissing (1989a) observed lesser adventitious roots

in jack pine (Pinus baniksiana) and bean (Phaseolus

vulgaris) cuttings when treated with H-carbomethoxy

vinylene phenyl indole-3-acetic acid, (CMVP-IBA), whereas

treatment with 2-4^ dichlorophenoxy indole-3—butyrate

(DCP-IBA) induced comparable number of roots in both

genera. Bean cutting treated with phenyl indole-3 butyric

.acid (P-IBA) possessed more number of roots than IBA. It

was concluded that phenyl ester of indole auxins have equal

or greater root inducing capability as compared with the

parent auxin, only if their phenolic moieties do not

contain a substituent (e.g. P-IBA) or contain only a simple

substituent such as -OH (e.g. 3-hydroxyphenyl indole-3

acetate) or -CI (eg. DCP-IBA), rather than more complex

susbtituent like -CH=CH-C00CH2 (eg. CMVP-IBA). In addition

to this, the studies were also extended to the effect of

the removal of the apes? of the cuttings of Pinus

baniksiana and on the partitioning of the carbohydrates.

It was observed that presence of the stem apex (control)

and their treatment with IBA increased rooting, associated

with an increase in the fresh and dry weight of the basal

44

part of the cutting. Removal of the stem apex affected

partitioning of the carbohydrates, however, the pattern

changed if the cuttings were supplied with IBA either at

the base or apex but it was not comparable with control.

IBA treatment of decapitated cuttings had lesser rooting

potential, as compared with controls, probably due to

increased reducing sugar-starch ratio in their basal parts

(Haissing, 1989b).

Pluss et a_l (1989) observed a positive correlation

between the number of new roots and the duration of the

application of lAA (for first 5 to 6 hrs. ) , to the basal

cut surface of the cuttings of J opulus tremula. It was due

to the induction of a metabolic system that transform lAA

to a compound as 2-indolene-3-acetyl aspartic acid (OXIA-

asp) (which is unable to provok new roots).

Riov and Yang (1989) investigated the role of

ethylene and its interaction with auxin in adventitious

root formation in cuttings of mung bean (Vigna radiata L.).

Indole-3 acetic acid (30 ^M) inhibited rooting but a

similar concentration of IBA promoted, significantly.

Ethylene, released from ACC (1-aminocyclopropane-l-

carboxylic acid) enhanced the number of roots but inhibited

their emergence and elongation. The cuttings treated with

lAA possessed larger quantities of ethylene, ACC and

malonyl ACC, but later on it was observed that IBA produced

45

higher levels of ethylene, ACC and M-ACC during most of the

rooting process, and this is the reason IBA had more

stimulating effect on rooting.

Kanwar (1990) noted that the propagation of

sugarcane was favoured with kinetin (1 ppm) treatment by

facilitating rooting and germination. These processess

were also enhanced by a treatment with lower concentration

of lAA, ethrel and TIBA (triiodo benzoic acid).

14 Berthon et_ a]^ (1991) supplied radio active ( C),

2,4-D to the shoot cuttings of Sequioadendron gigantum

from juvenile (12 months old seedlings) and mature (30

years old tree) during rooting. Juvenile (easy -to-root)

clones accumulated larger quantity of the hormones than the

mature (hard-to-root) clones of which larger part was

confined to their bases. However, the basal and apical

parts of both types of clones exhibited unequal

distribution of free 2,4-D.

Nagy et. al_ (19 91, a) observed that the amount of lAA

in paclobutrazol treated hypocotyls of Phaseolus vulgaris

was higher than the control. More lAA accumulated in the

basal region correlating with the increased root system of

intact plants and more intensive rooting of stem cuttings.

Leaves also had higher lAA-level than control. It was

proposed that distribution of the hormone between the

petiole and leaf-blade was not favourable for rooting

46

process. In another study (Nagy et a_l, 1991b), they

reported that paclobutrazol besides retarding expansion

growth, stimulated rooting process both in intact plants

and stem cuttings. Differentiation of root primordia was

inhibited in petioles, which v/as overcome by IBA, ABA and

ethylene.

Vazquez and Maria (1991) examined the degree of

rooting and lAA-oxidase activity in the cuttings of

Phaseolus vulgaris treated with 2,3 or 4-hydroxybenzal-

dehyde. 2-hydroxybenzaldehyde was most effective and acted

synergistically with 10 uM of lAA at 0.4 mM. 3- and 4-OH-

Bal also stimulated rooting and acted additively with lAA.

It appeared that 2-OH-Bal inhibited lAA-Oxidase activity.

All the aldehydes, after 4 hrs of the treatment, were

converted to the corresponding alcohol or acid. The

alcohol/acid ratio was 10 for 3- and 4-OH-Bal and 20 for

2-OH-Bal. It was, therefore, concluded that oxidative

effect of the aldehyde induced beneficial response in

rooting, at early stages.

Kakkar and Rai (1992) noted that polyamines

(spermine and spermidine; cyclic AMP and indole-3 acetic

acid stimulated rooting in hypocotyl cuttings of Phaseolus

vulgaris L. cv. white panchraary. Spermine, C-AMP and lAA

added together exhibited synergistic effect on rooting. lAA

alone or in combination with polyamines and cyclic- AMP was

47

less effective than their individual sets. It was

concluded that C-AMP and polyamine triggers the effect of

lAA and stimulated rhizogenesis.

Liu and Reid (1992) reported an acropetally

decreasing gradient of the level of free lAA in the

hypocotyls of Helianthus annus, but the level of conjugated

lAA increased acropetally (i.e. free to total lAA ratio was

highest in the basal portion of the hypocotyls, where most

of root primordia were found ) . Some primordia were seen in

this region within 24 hrs. after the roots were excised.

In the middle portion of the hypocotyl the quantity of free

lAA increased upto 15 hrs after excision and then

decreased. In this portion there were fewer root primordia

and they could not be seen until 72 hrs. In the apical

portion the amount of free lAA steadily increased and no

root primordia were seen by 72 hrs. Surgical removal of

various parts of the hypocotyl tissues caused adventitious

root formation due to the accumulation of basipetally

transported lAA. N-1-naphthylphthalmic acid and 2,3,5-

triiodobenzoic-acid inhibited basipetal flow of auxin thus

resulting in fewer adventitious roots. Only few root

primordia were seen if the major source of endogenous auxin

(apical bud and cotyledons) were removed. The endogenous

auxin not only promoted rooting but also overcame the

inhibitory effect of 2,3,5-triiodobenzoic acid.

48

Vander ejt al (1992a) reported that the stem

cuttings of apple incubated in the dark on a medium

containing 3.2 or 10 ;aM of IBA plus riboflavin horned

maximum number of roots. Here, about 95% of the IBA taken

up by the cuttings was conjugated to inactivate it, about

4% was in the free (active) form IBAH and only 1% as lAAH.

The incubation of cuttings in light with or without

riboflavin, on a medium containing 10 ;JM of IBA possessed a

level of IBAH and lAAH comparable with the cuttings grown

in dark on a medium containing 3.2 pM IBA plus riboflavin,

however, former had lesser number of roots.

Vander et_ al (1992b} correlated the contents of free

IBA and that of IBA-derived lAA with adventitious root

formation on the stem cuttings of apple. An incubation of

the cuttings on a medium supplemented with 32 M of IBA for

3 days or for 5-days in 3.2 or 10 ^M of the hormone

produced maximum roots. Larger part of the observed IBA

remained confined to the rooting zone (1 mm base of the

stem). As regards the metabolism of the hormones, about

60% of the IBA in the medium from 6 hrs to 3-days of

T n +•

incubation was in the free from (IBA ) whereas IBA

derived lAA was about 16%. A shorter period required a

higher IBA and lAA content to produce same number of

roots in an incubation period of 2 and 3-days. This gave

an impression that mode of action of IBA in root formation

resembled a dose effect of the active auxin components, in

49

the stem base. It was, therefore, concluded that root

formation was not related to the specific type of auxin

metabolism.

Selby et_ £]^ (1992) grew the seedlings of Picea

sitchensis (Bong carr.) at low light intensities (15-20

-2 -1 ^Mol m s ) so that tissues become responsive to root

inducing treatments. Spontaneous rooting occured in

untreated cuttings, and at a low frequency, depended upon

the supply of auxin. A continuous treatment with indole-3

butyric acid (IBA) was more effective than either indole-3

acetic acid (lAA) or 1-napthalene acetic acid (NAA) at

— fi — s concentrations ranging between 10 and 10 M. The effect

of IBA on rooting was inhibited by other growth regulators

such as 6-benzylaminopurine (BA) whose most effective

concentration was 3 x 10 M. Gibberillic acid (GA^)

abscissic acid (ABA) and 2-chloroethyl phosphoric acid

(etheral) were less potent inhibiters of rootings.

Dubeikovsky et. al (1993) inoculated soft wood

cuttings of black currant by using recombinant strains

(isogenic) Pseudomonas fluorescens) (differing in plant

hormone production). Indole-3-acetic acid i.IAA) produced

by these bacteria had stimulatory effect on the root

development of black currant soft wood cuttings, but was

inhibitory for sour cherry. The size of the population of

the inoculated strain on the root surface of the cuttings

50

exhibited a direct correlation with the degree of the

observed effect.

Epstein and Ackerman (1993) while studying the IBA

transport and its metabolism in early and late flowering

varieties of Protea and Lucadendron discolor, found

enhanced rooting in the cuttings of an early variety

treated with IBA. In easy-to-root variety more radioactive

IBA was transported to the leaves (35-40%) than difficult

to root (10%). IBA was metabolized rapidly by both the

varieties and afte 24 hrs., most of the label^ was in the /

new metabolites, most probably an ester conjugate of

glucose. However, 10% of free IBA was present in the

cuttings during the whole period of rooting (i.e. 4 weeksj.

As regards the distribution of free IBA, most of it

accumulated at base of easy-to-root cuttings but in diffi­

cult to root cuttings it was found in larger quantities in

the leaves. It was suggested that it was the level of free

IBA at the rooting zone of the cuttings which made the

differenceof easy and difficult to root varieties.

Flygh et. aj_ (1993) divided epicotyl cuttings of

Pinus sylvestris into two groups i.e. those where roots

differentiated within 6 weeks after removing the cuttings

and those which required more than 6 weeks. Auxin

stimulated rooting and number of roots per cutting;

significantly, irrespective of the physiological state of

51

the plant and their cuttings. Anatomical studies showed

that the roots developed from the wound tissues in both the

group of cuttings.

Hausman (1993) observed that the shoot of (Populus

tremula X Populus tremloides) did not strike roots in the

absence of auxin in the medium. Soluble peroxidase

activity increased sharply corresponding with a fall in the

level of free lAA in the shoot. These early changes were

followed by a peak of free lAA level finally initiating the

phase of rooting. It was followed by an increase in

ethylene production both in the rooting zone and the shoot

but in the former the level was higher and earlier. The

use of ACC, suggested that the application of NAA might

have increased the level of ACC-synthase activity.

Nordstrom and Eliasson (1993) noted an increase in

ethylene production and the reduction in root number in the

cuttings of Pisum sativum L. cv. Marma treated with

1-aminocyclopropane-l-carboxylic acid (ACC). The treatment

with ACC (0.1 mM) had little effect on the lAA level at the

base of the cuttings after 24 hrs, but decreased during the

3 following days.Indole-3-acetylaspartic acid, at the

base, increased in the first 3 days and then declined and

remained unaffected by ACC treatment in the first 2 days of

the treatment but later this conjugate decreased more

rapidly than in the control. They proposed that the

inhibitory effect of ethylene on rooting in these pea

52

cuttings was due to a decrease in the level of lAA in the

rooting zone, however, the exogenous supply after 24 hrs,

may compensate this loss and stimulate rooting. Ethylene

was said to mediate no inhibitory effect of applied lAA on

rooting.

2-8 SEED TREATMENT WITH HORMONES

The seed is the representative of the next

generation and is equipped with sufficient quantity of food

reserves, minerals, vitamins and hormones, which sustain

life till the emerging seedling becomes self-sufficient. In

some seeds the insufficient quantity of hormones has

profound influence on the germination and growth. This

physiological barrier jsan be removed by sowing the seeds

pre-treated with hormones.

Brown et_ al (1968) studied the effect produced in

tomato (Lycopersicum esculantum) by seed or root treatment

with various concentrations of gibberellic acid and indole-

3-acetic acid. It was noted that growth, in length, of

stem and leaves was accelerated by 5.0 ^g GA-s applied to

the seed or 5.0, 0.5 and 0.05 p.g given to the roots.

Higher concentration of GA^ (5.0 ;jg) also increased the

number of buds and lengthen the time between bud appearence

and fruit formation on the first truss by 1-8 days. This

time was further shortened to 1-4 days if smaller amounts

53

of GA^ are applied to roots. lAA (0.5 ug) alone or in

combination with GA^ applied to the roots proved

ineffective. However, lower concentration of GA^ applied

through seed or roots enhanced growth rate of both stem and

leaves.

Dunlap and Morgan (1977) observed a lower rate of

ethylene production in the first 12 hrs of the germination

of Lactuca sativa L. cv. primier great lakes, seeds at

20°C. However, it increased by 100 fold between 12th and

16th hrs, Synchronizing with the protrusion of the radicle .

Exposure to a supra-optimal temperature, i.e. 32°C, was

inversely proportional to the per cent germination but

ethylene production and growth not affected by incubating

visibly germinated seeds at 32°C. The germination at 32°C

was partially restored by exogenous application of

ethylene, in the presence of light only. The requirement

of light could be substituted by GA. They concluded that

an increase in temperature raised the threshold concentra­

tion of ethylene required for germination which was

satisfied by its exogenous supply. The low rate of

ethylene production, at the initial stages of seed

germination, probably regulates subsequent germination.

Post germination ethylene production did not break thermo

dormancy but stimulated the emergence of radicle.

Khryanin et_ a]^ (1978), subjected the seeds of hemp

and spinach to pre-sowing treatment with solutions of GA,

54

lAA and 6-BAP at concentrations of 25 and 50 mg/1 and ABA

10 and 20 mg/1. The treatment with GA increased growth to

a larger extent than lAA but 6-BAP and ABA inhibited both

growth and flowering. GA induced maleness in hemp plants,

under long day conditions. However, sexualization in

spinach was not affected by hormones in spinach. They

suggested that the sex of the plants may be manipulated by

using proper concentrations of selected hormones even from

the seed stage.

Hans-Jorg (1978) supplied 2,4-D to the germinating

seeds of pea at the begining of the irtibibiticai or 12 hrs

after being imbibed in water. The treatment reduced the

fresh weight increase-^nd enhanced the dry weight decrease.

In the 30,000 x g - aapernatant of the homogenate 2,4-D

induced no significant effect on acidic cytoplasmic RNAase

from 12 to 72 hrs but from 72 to 144 hrs the treatment

decreased the activity, as compared with the control. The

dialyzed and concentrated homogenates of 30,000 g

supernatants, the activity increased from 12 to 72 hrs but

exhibited a parallel decreased from 72 to 144 hrs both in

control and treated seeds.

Tudor (1984) observed that at 18 and 25°C, in the

dark, 50% of the seeds of celery soaked in polyethylene

glycol (PEG) germinated after 3-days whereas those treated

with gibberellin (GA. + GA^) plus ethylene reguired 7 days.

55

The control (untreated) seeds did not germinate, at all.

The cytokinin activity was little in dry and dried-back

seeds, irrespective of the treatment.

Setia and Shelly (1985) studied the interaction

effect of salanity and hormones and noted a declined in the

per cent germination, length and fresh and dry weight of

the seedlings in Pisum sativum with an increase in the

salanity. The effect of salanity stress on seed

germination was accelerated by GA^, kinetin and morphactin

(5 mg/ml each). The growth of shoot and root was restored

partially by GA, and kinetin.

Sisler and Carmen (1986) observed that the compound _3

2,5-norbornadiane at 8000 1/1, silver nitrate at 5 x 10 _3

M, or amino oxyacetic acid at 3 x 10 M which block

ethylene synthesis in the seeds of Nicotiana tabacum L. cv.

Mc Nair 944 inhibited their germination but was overcome by

exogenous application of ethylene. However, another -3

inhibitor of ethylene biosynthesis, CoCl„ at 5 x 10 M,

did not affect the germination. They proposed that in

tabacco a low level of endogenous ethylene was essential

for germination.

Grabisic and Neskovic (1988) induced the germination

of the seeds of Paulownia tomentosa either by red light or

GA. The red light effect was completely neutralized by

56

far-red, ABA, ancymidole, teteyelasis, paclobutrazol but

not by AMO-1618, CCC. However, GA^ over come the inhibitory

effect of far-red light or that of growth retardants, while

that of ABA was reversed by fusicocein. It was further

noted that the germination of light insensitive wheat,

corn, alfa alfa and mung bean seeds was not inhibited by

growth retardants.

Patel and Chatterji (1988) soaked the seeds of

Carica papaya L. for 24 hrs in dilute solutions of NAA,

lAA, IBA, p-NAA and GA. The treatment stimulated the rate

of germination and growth of the seedlings. They finally

recommended the soaking of papaya seeds for 24 hrs, in 100

ppm solution of lAA for better germination and 100 ppm

solution of GA for enhanced rate of growth of the

seedlings.

2-9 HORMONES SUPPLIED THROUGH THE ROOTS

Here the plants are allowed to grow either in the

inert material (sand), nutrient solution or even in the

soil. Except in the last condition, it becomes easier to

manipulate the supply of the substances, whose effect has

to be studied, particularly, those of minerals, vitamins

and hormones which may be added in required quantities with

the nutrient solution for a definite period.

Brouwer and Kleinendorst (1967) cultured bean plants

in solution at varying temperatures with or without

57

aeration. Gibberellic acid supplied with the solution

increased stem weight by enlarging leaf and produced more

dry matter but decreased root weight which did not depend

on either of the above two factors (Temperatures and

aeration) but on the plant age and the duration of the

supply of the hormone.

Rauser et. al (1975) compared the effect of lAA and

ethylene on the growth of intact pea roots. Both lAA and

ethylene inhibited growth within 20 minutes/ however, this

effect was lost within 60 minutes of removal of these

growth regulators. A supramaximal concentration of

ethylene inhibited root growth less, than did, 5 to 20 M

of lAA indicating that, the inhibition of root growth by

auxin was not due to lAA induced ethylene production.

By using gas chromatography-mass spectrometry

technique Pilet and Martial (1985) estimated the endogenous

level of lAA in the elongation zone of the intact primary

roots of Zea mays and noted, its linear correlation with the

growth rate. However, treatment with lAA decreased the

elongation of the roots, probably due to the supra—optimal

concentration of the auxin. On the other hand the growth of some

rapidly extending roots were enhanced by such treatments.

Hurren eib £1 (1988) noted that in the intact roots

°- ^^^ i ys lAA induced stimulation of primordium

initiation which was followed by a similar increase in

58

lateral emergence. However, excised roots did not give

any response to auxin. The proliferative activity of the

meristem at the apex, did not show any correlation with

number of primordia initiated or the number of laterals

produced or the rate of root elongation either.

Macisaac et al (1989) reported 600% increase in the

development of laterals in Lettuce (Lactuca sativa L. cv.

Grand Rapids) seedling roots, treated with 10 M of NAA.

For maximum stimulation of these laterals the auxin was

required by them for only the first 20 hrs of the 72 hrs

period of treatment. On the other hand, Kinetin (2 x 10 M)

not only restricted the spontaneous formation of lateral

root primordia but also acted as antagonists of NAA in the J-

production, in this regard the sensitivity to the Kinetin,

by the roots, was maximum in the first 20 hrs.

Wu and Yu (1991) markedly increased the growth and

auxin content of epicotyl of one week old seedlings of

Phaseolus mungo by their treatment with epibrcLSsinolide

(0.5 ppm) for 48 hrs, this effect was lost either by the

removal of some of the leaves or by their treatment with

TIBA (inhibitor of polar auxin transport). A simultaneous

application of lAA counteracted the inhibition of TIBA on

the epicotyl growth induced by epibrassinolide. It was

suggested that epibrassinolide promoted growth through the

enhancement of endogenous lAA by inhibiting the activity of

oxidase and peroxidase.

59

Lloret and Pulgarin (1992) reported promotion of

lateral root formation and inhibition of the elongation of

the main root by NAA (oC-Naphthalene acetic acid) supplied

to the adventitious roots of Allium cepea L. They finally

suggested that the basal part was less sensitive to the

auxin than the apical portion of onion root. It

rejuvenated the cells of the pericycle enabling them to

differentiate into root primordia which was not possible in

the absence of NAA.

Atzmon and Staden (1993) treated Pinus pinea seedlings

growing in half-strength Hoagland's nutrient solution with

zeatin and isopentanyladenie (iP), for 24 hrs. Root length

did not show any response but the development of lateral

roots was inhibited by Zeatin and stimulated by iP. Root/

shoot ratio and root dry weight increased by a treatment

— 8 with lower concentration of zeatin (10 M ) . A concentration

of 10"^ M of both (i.e. Z & iP) proved to be most effective

in enhancing (40%) most of the root characteristic

studied.

2-10 HORMONE SUPPLIED THROUGH LEAVES

The dilute solution of commercial fertilizers are

sprayed on the leaves of standing crop which has proved to

be more beneficial than the traditional way of supplying

all nutrients at the time of sowing or top dressing. In

foliar application the nutrients are readily available at

the site of its metabolism. However, the application of

60

other chemicals, particularly hormones spray on the leaves

has been paid less attention and is confined only to some

fruit plants.

Coleman and Richard (1977) noted that GA^ stimulated

the rooting in tomato leaf disc, cultured in a nutrient

medium in continuous darkness. It was found that GA^ at

— 8 —7

concentration 1 x 10 M to 1 x 10 M significantly

enhanced root initiation (in presence of tryptamine, 2 x 10"

M) . Effect of GA- could be replaced by GK..^ but not by

kinetin. Indole-3-lactic acid (a indole derivative) highly

active in the induction of rooting in the tomato leaf

discs, further this effect was enhanced by GA^.

Bosselaers (1984) tf-eated the primary leaves of bean

plants (Phaseolus vulgaris L. cv. limberrgse vroege) with

bezyladenin (BA) or kinetin, at concentration 0.5 mM, for

5-consecutive days resulting in thicker leaves and a

decrease in intercellular air space volume, as compared

with control. Stomata of treated plants were not fully

closed in the dark and they did not open in the middle of

the light period as in control suggesting that treatment

resulted in impared stomatal action. Thus changes in leaf

structure and function have no important effect on total

leaf diffusion resistance to C0_ during the light period.

Mingo et_ £l (1984) found that benzyladenine and GA

sprays (100 mg/1) on Euphorbia lathyris L. plants induced

61

L

a change in its normal decussate phyllotaxes. Benzyladenine

produced a change to tetracussata, tricussata and bijugate

and GA to spiral phyllotaxis. Benzyladenine and GA

treatment resulted in a significant increase in apex

diameter (72.8% and 19.1%, respectively). CCC

{2-chloroethyltrimethyl ammonium chloride) Ancymidol, Alar

and Glyphosine did not alter decussate phyllotaxes.

Mardanov, (1985) grew pumkin plants (Cucurbita pepo

L.) for 16 days in Knop's medium lacking nitrogen. Half of

the plants of each treatment were sprayed daily with water

and the rest were sprayed with kinetin (30 mg/1). In

nitrogen-deficient watered plants, the root/shoot dry mass,

protein/non-protein nitrogen and K/Ca contents in roots

increased. While chlorophyll content in cotyledons

decreased considerably. In kinetin treated plants the

nitrogen starvation features were less pronounced, roots

showed greater content kinetin due to utilization of essential

nutrients.

Mardanov (1987) observed in nitrogen deficient

plants whose shoots were processed with water, that ratio

of dry mass increased in root and shoot, ratio of protein

and non-protein nitrogen (K/Ca in roots) also increased,

however, chlorophyll content decreased in the leaves

especially in seed lobes. Nitrogen-deficiency was not

pronounced when the shoots were sprayed with kinetin

62

solution,. Auxin showed three responses and outer surface of

plasmalemma which blocks H pumping; a protective or

restorative effect on the H -ATPase; an increased capacity

for K"*" influx during the developmental phase of washing,

which is augmented by the presence of GA^

Middleton et al (1980) noted that IBA promoted

rooting in the cuttings of Phaseolus aureus only in the

presence of leaves. IBA had direct action on root

initiation but it enhanced free sugars in the leaves.

Light grown cuttings were also affected by exogenous boron

for root development, before root initiation IBA and boron

14 promoted the accumulation of C in hypocotyls when leaves

were treated with sucrose. Illuminated leaves proved

beneficial for rooting (IBA for one day; followed by boron

for 6 days) while, leaves in darkness showed lack of

response to IBA and Boron.

2.11 CONCLUSION

The above review of literature broadly establishes

that vitamins and hormones have profound effect on the

growth and development of the plants, especially rooting.

However, the information regarding the application of

vitamins and hormones on Triticales is very less. Thus it

is desirable to carry out a detailed scientific study by

soaking the seeds of new selected varieties of Triticales

in various concentrations of thiamine and lAA.

CHAPTER 3

MATERIALS AND METHODS

MATERIAL AND METHODS

C O N T E N T S

3 . 1 Seeds

3.2 Sand Purification

3.3 Preparation of pots

3.4 Experiment 1

3.5 Experiment 2

3.6 Experiment 3

3.7 Experiment 4

3.8 Experiment 5

3.9 Nutrient Solution

3.10 Morphological characteristics

3.11 Chemical Analysis

3.11.1 Estimation of reducing and non-reducing Sugars

3.11.1.1 Preparation of Extract

3.11.1.2 Estimation of Reducing Sugar

3.11.1.3 Estimation of Non-reducing Sugar

3.11.2 Estimation of Carbohydrates

3.11.2.1 Preparation of Powder

3.11.2.2 Extraction:* Estimation

3.11.3 Estimation of Proteins

3.11.3.1 Extraction of Soluble and Insoluble Protein

3.11.3.2 Estimation of Soluble Protein

Page No.

63

63

63

64

65

65

66

66

66-

68

69-

69-

69

70

70

-68

-78

-71

71-72

71

72

72-74

72

73

3.11.3.3 Estimation of Insoluble ... 74 Protein

3.11.4 Estimation of Nitrate ... 74-75

3.11.4.1 Preparation of Powder ... 74

3.11.4.2 Extraction and colour ... 75 development

3.11.5 Estimation of Nitrate ... 75-76 reductase activity (NRA)

3.11.5.1 Colour development ... 76

3.11.6 Estimation of Thiamine ... 76-78 content

3.12 Statistical Analysis ... 78

Appendix

63

MATERIAL AND METHODS

To achieve the objectives mentioned in chapter-1, it

is proposed to conduct five pot experiments on 1*riticale

during the 'rabi' (winter) season. The details of

materials and methods to be used are given in the following

pages :

3.1 SEEDS

The grains of a local variety TL40190, released by

PAU Ludhiana, will be used in the following experiments.

The healthy grains of uniform size will be tested for their

per cent viability. Mercuric chloride solution (5%) will

be used for surface sterilization of the grains.

3.2 SAND PURIFICATION

Sand will be washed once with tap water and then

left in Hydrochloric acid (18%) for 24 hrs.« in plastic

buckets. The next day, it will be thoroughly leached by

using tap water which will be allowed to percolate

upwards, in the buckets, by means of a glass tube. Final

washing will be done with demineralized water before

transferring the sand to pots.

3.3 PREPARATION OF POTS

Earthen pots of 10 inch diameter will be used in

these experiments. The inner wall of each pot will be

64

lined with polythene sleeves whose other end will be

allowed to pass through the opening at the base of each

pot. It will facilitate the drainage and aeration. Each

pot will be filled with an equal amount of the sand and

lined properly in the net house.

3.4 EXPERIMENT-1

The effect of pre-sowing grain treatment with

thiamine on the physiomorphological changes of seedlings

will be studied. The grains of Triticale will be soaked in

10~ M and 10 M aqueous solution of thiamine hydrochloride

for 6 and 12 hrs. Ten pre-treated grains will be sown in

each pot(filled with acid washed sand). All the pots will

be watered with 250 ml of full nutrient solution, every day

in the morning. The thinning will be done after

germination and five plants per pot will be left. Sampling

will be done 20, 40 and 60 days, after sowing. The

following characteristics will be studied both in the root

and shoot at each sampling :

3.4.1 (A) MORPHOLOGICAL CHARACTERISTICS

1. Root number per plant

2. Leaf number per plant

3. Fresh and dry weight of roots

4. Fresh and dry weight of shoots

65

3.4.2 (B) CHEMICAL ANALYSIS

1. Reducing and non-reducing sugars

2. Total carbohydrate content

3. Soluble and insoluble proteins

4. Nitrate content

5. Nitrate reductase activity and

6. Thiamine content

3.5 EXPERIMENT 2

Ten surface sterilized, grains of Triticale will be

sown in each pot filled with acid washed sand. The

seedling will be provided with 10 ml of 10 M or 10 M of

thiamine solution only once after 5, 10 or 15 days, after

sowing. Thinning, other cultural practices, sampling

techniques, the stages and the characteristics to be

studied will be the same as in experiment 1.

3.6 EXPERIMENT-3

The surface sterilized grains of the same cultivar

-3 of Triticale will be soaked for 6 and 12 hrs. in 10 M or — fi

10 M of indole-3-acetic acid. Ten grains will be sown in

each pot filled with acid washed sand. Thinning will be

done on germination, leaving five seedlings in each pot.

The sampling stages and the parameters to be studied will

be same as in experiment 1.

66

3.7 EXPERIMENT 4

Ten, surface sterilized, grains of the same cultivar

of Triticale will be sown in each pot. Out of which five

seedlings will be left to grow in the same way as in

experiment-1. These seedlings will be supplied once with 10

ml of indole-3-acetic acid (10~ M or 10~ M) 5, 10 or 15

days, after sowing. Sampling schedule and the analysis of

the seedlings will be done in the same way as in experiment

1.

3.8 EXPERIMENT 5

In this experiment, a combined effect of vitamin and

hormone will be studied. The surface sterilized, grains of

Triticale will be soaked ijj 10~ M or 10~ of thiamine

solution for 3 or 6 hrs. followed by their respective

additional soaking for 3 and 6 hrs. in 10 M or 10 M of

lAA, before sowing in the pots. The seedlings will be raised

in sand culture and physiomorphological studies at different

stages of growth will be done in the same way as in

experiment 1.

3.9 NUTRIENT SOLUTION

Stock solutions of the required essential macro and

micro-untrients (Table-1) will be prepared according to

Hewitt (1966). The stock solutions will then be diluted as

per Table-2 for watering the plants.

67

Table 1

Concentration of the standard stock solution.

gg-j ts Quantity (g) in 100 ml stock solution

( a ) M a c r o n u t r i e n t s

C a ( N 0 2 ) 2 ( A n h y d r o u s ) 3 2 . 8

KNO^ 2 0 . 2

MgSo .7H 0 1 8 . 4

NaH2P0^ .2H20 2 0 . 8

FeCgH^0^ .3H20 ( F e r r i c C i t r a t e ) 5 . 9 8

(b) M i c r o n u t r i e n t s

M n S 0 . . 4 H „ 0 2 . 2 3 0 4 2

C U S 0 . . 4 H 0 0 . 2 5 0

Z n S 0 . . 7 H „ 0 0 . 2 9 0 4 2

H ,BO. 1 . 8 6 0 3 4

( N H 4 ) g . M 0 ^ 0 2 4 . 4 H 2 0 0 . 0 8 8

68

Table 2

Nutrient solution will be prepared from stock solution.

Standard Stock Solution Vol.(ml) in 1 litre of Nutrient Solution

(a) Macronutrients

Ca(N02)2 (Anhydrous)

KNO.

MgS0^.7H20

NaH2P0^.2H20

Fer r ic C i t r a t e

2.0

2.0

2.0

1.0

0.58

(b) Micronutrients

Deionised Water

0.1 (From each Micro-nutrient stock solution

992.4

Total volume (ml) of the nutrient solution for watering

1000.0 ml

3.10 MORPHOLOGICAL CHARACTERISTICS

At each sampling, the polythene sleeve with the whole

mass of the sand and roots will be pulled out of the pet and

69

washed in running water to get the intact mass of roots with

aerial parts. Each plant will be analysed for :

i) Total number of roots per plant

ii) Leaf number per plant

iii) Fresh weight of the root and shoot.

The root and shoot of each plant will be dried

separately in an hot air oven run at 80 degree centigrade

for two days. Each sample will then be determined for its

dry weight.

3.11 CHEMICAL ANALYSIS

3.11-1 ESTIMATION OF REDUCING AND NON REDUCING SUGARS

3.11.1.1 Preparation of extract

Plant material will be dried overnight in an oven at

80 degree centigrade. Each sample will be ground in an

electric grinder and passed the powder through a 72 m*sh

screen.

5.675 gm powder will be weighed and placed in 100 or

125 ml erlenmeyer. Flask will be tipped so that all powder

may come at one side; powder than will be wet by adding 5 ml

alcohol. Further flask will be tipped so that wet powder is

at upper side and 50 ml acetate buffer solution will be

added. Then flask will be shaken to bring powder into

suspension. Immediately 2 ml Na-Tungstate solution will be

70

added and mixed thoroughly. Then solution will be filtered

at once with Whatman No-4 filter paper discarding first 8-10

drops of filtrate.

3.11.1.2 Estimation of reducing sugar

5 ml powder extract will be pipetted into Ca 75 ml

test tube. Then 10 ml KoFe(CN)^ will be added in the test

tube, mixed and then test tube will be immersed in boiling

water bath so that liquid in tube remains 3-4 cms below the

surface of boiling water. After boiling the tube for 20

mts, the tube will be cooled in running water and its

content will be poured at once into 100 or 125 ml erlenmeyer.

Test tube will be rinsed with 25 ml HoAc-salts solution and

mixed thoroughly. Th^n 1 ml starch-KI solution will be

added. The solution will be titrated with 0.1 N Na-,S„On

solution until blue colour completely disappears. The

consumed ml 0.1 N Na^S^O^ during titration v;ill be

subtracted from 10.00. This difference represents definite

quantity of reducing sugars/10 gm powder, will be calculated

as maltose from Table-3.

3.11.1.3 Estimation of non-reducing sugar

5 ml powder extract will be piptted into 8 inch test

tube and will be immersed in boiling water bath. Then test

tube will be cooled under running water and 10 ml K-,Fe (CN)^ J b

solution will be added. Test tube will be rinsed with 25 ml

HOAc- salt solution and will be mixed thoroughly. 1 mi

71

starch-KI solution will be added and than titrated with 0.1

N Na_S„0, solution until blue colour disappears completely,

ml 0.1 Na S-O-, used in titration will be subtracted from

10.00- K^Fe(CN)g reduced after hydrolysis and K, FetCN)g

reduced by maltose in powder will be equal to non reducing

sugars.. It will be calculated as sucrose and determined

from the Table-3 given brxcR.

3.11.2 ESTIMATION OF CARBOHYDRATES

Carbohydrate content in the plant material will be

estimated as follows :

3. 11.2-1 Preparation of powder

Plant material will be dried overnight in an oven run

at 80 degree centigrade. Each sample will be ground to a

fine powder in an electric grinder and passed through a 72

mesh screen.

3-11.2.1.1 Extraction and estimation

The extraction and estimation of carbohydrates, will

be done by adopting methodologies followed by Yih and Clark

(1965) and Dubois et a]^ (1956), respectively.

On a clean sheet of paper sufficient amount of the

powder will be spread and dried overnight in an oven at 80

degree centigrade. These dried Samples will be cooled in a

dessicator for about 10 mts.

50 mg of each sample will be weighed and transferred

to a 20 ml centifuge tube. To each tube will be added 5 ml

Table - 3 ; Reducing arid non-reducing sugars w i l l be c a l c u l a t e d as mal tose from Tab le -3 , given below

14.025 O.IN

0.1/V Ferricyar Reduced

ml

o.io' 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4,00 4.10 4.20 4.30 4.40

Ferricyanide Maltose-Sucrose Conversion Table'

ide Maltose per 10 g Flour-

mg 5

10 15 20 25 31 36 41 46 51 56 60 65 71 76 80 85 90 96

101 106 111 116

^121 126 130 135 140 145 151 156 161 166 171 176 182 188 195 201 207 213 218 225 231

Sucrose per 10 g Flour

rtig 5

10 15 19 24 29 34 38 43 48 52 57 62 67 71 76 81 86 91 95

100 104 109 114 119 123 128 133 138 143 148 152 157 161 166 171 176 181 185 190 195 200 204 209

0.1/V Ferricyanide Reduced

ml 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 (.00 8.10 8.20 8.30 8.40 8.50 8.60 8.70 S.80

Maltose per 10 g Flour

mg 237 244 251 257 264 270 276 282 288 295 302 308 315 322 328 334 341 347 353 360 367 373 379 385 392 398 406 412 418 425 431 438 445 451 458 465 472 478 485 492 499 505 512 519

Sucrose per 10 g Flour

V 7

mg 214 218 223 228 233 238 242 247 251 256 261 266 270 275 280 285 290 294 299 304 309 313 318 323 328 333 337 342 347 352 357 362 367 372 377 382 387 392 397 402 407

These values are arbitrarily given for 10 g flour altho detn is made on only 0.5 g flour

72

of 1.5 N Sulphuric acid. Digestion of the powder will be

completed by keeping the tube in a water bath for two hrs at

90-95 degree centigrade. The digested sample will be

centrifuged at 4000 rpm and the supernatant collected in a

25 ml volumetric flask with two washings and the final

volume made up with distilled water, 1 ml of this extract

will be diluted to a required level. 1 ml of this diluted

sample, 1 ml of water and 1 ml of phenol (5%) will be poured

in each test tube and placed in a beaker containing chilled

water, and 5 ml of concentrated Sulphuric acid will be

pipetted quickly into each test tube. After 1/2 an hr, the

pale yellow coloured solution will be transferred to a

colorimetric tube and the transmittance noted at 490 nm. On

a specific 'Spectronic-20' colorimeter. A blank will be

run simultaneously. The reading of each sample will be

compared with a calibration curve, obtained by using known

graded dilutions of standard glucose solution.

3.11.3 ESTIMATION OF PROTEINS

The methodology adopted by Lowry et al_ (1957) will be

followed for the estimation of proteins.

3.11.3.1 Estraction of soluble and insoluble proteins

Sufficient amount of powder will be spread over a

sheet of paper and dried over night in an oven at 80 degree

centigrade. The dried samples will be cooled in a dessicator

for about 10 mts, before weighing.

73

50 mg of each sample will be taken and transferred

into a mortar and pestle. It will be ground with 5 ml of

distilled water and collected in a centrifuged tube with

atleast two washings. The sample will be centrifuged at

4,000 rpm for 10 mts. The supernatant will be collected in

a 25 ml volumetric flask, together with two washings of the

residue with distilled water and this will be preserved for

estimation of soluble protein.

To the residue, 5 ml of 5% trichloroacetic acid will

be added. It will be left for 30 mts to allow complete

precipitation of proteins. The sample will be centrifuged

at 4,000 rpm for 10 mts and the supernatant will be

discarded. To the residue 5 ml of 1 N sodium hydroxJ.de

will be mixed well and allowed to stand in a water bath at

80 degree centigrade for 30 mts. The solution will be then,

allowed to cool and centrifuged at 4,000 rpm. The

supernatant together will three washings with 1 N sodium

hydroxide and will be collected in 25 ml volumetric flask.

The volume will be made upto mark with 1 N sodium hydroxide

and used for the estimation of insoluble protein.

3.11.3.2 Estimation of soluble protein

1 ml of the above water extract will be transferred

into a 10 ml test tube and 5 ml of reagent 'C (Appendixiii)

will be added to it. The solution will be mixed well and

74

allowed to stand for 10 mts at room temperature and 0.5 ml

of reagent 'E' will be added rapidly with immediate mixing.

After 30 mts, the blue colour will be developed in a

solution. The solution will be transferred to a colorimetric

tube and the percent transmittance will be read at 660 nm on

a 'Spectronic-20' colorimeter. A blank will be run

simultaneously with each sample. The optical density of

each sample with a calibration curve plotted by taking known

dilutions of a standard solution of egg-albumin.

3.11.3.3 Estimation of Insoluble protein

1 ml of sodium hydroxide extract will be transferred

to a 10 ml test tube and 5 ml of reagent 'D' will be added

to it (Appendix iii) . The solution wi^l be mixed well and

allowed to stand for 10 mts at room temperature. Rapidly

0.5 ml of reagent 'E' will be added. After a lag of about

30 mts the intensity of the blue coloured solution will be

measured on a "spectronic-20" colorimeter at 660 nm.

3.11.4 ESTIMATION OF NITRATE

The nitrate content will be estimated by following

the method of Johnson and Ulrich (1950),

3.11-4.1 Preparation of Powder

Dried sample will be ground in an electric grinder

and passed through a 72 mesh screen, and stored in polythene

bags for the anlysis of nitrate.

75

3.11.4.2 Extraction and colour development

A sufficient amount of the powder will be dried

overnight on a clean sheet of paper in an oven at 80 degree

centigrade, out of, 50 mg of the powder will be transferred

to a dried 25 ml tube. Calcium Sulphate (400 mg) and 12 ml

of double distilled water will be poured into each tube. The

sample will be centrifuged at 6000 rpm for 10 mts. The

supernatant will be transferred to a 50 ml conical flask

containing 1 ml of 0.5% calcium carbonate suspension. The

solution will be heated at water bath, leaving the final

volume to about 5 m l . 0.5 ml of H2O2 (30%) will be added

to the above solution and closed the flask with the lid. The

flask will be heated on a water bath to dryness in order to

remove the peroxidase. It will than be cooled and 1.25 ml

of phenol-di-sulphonic acid (11.11%) will be added quickly

with a continuous stirring, 3.5 ml of distilled water will

be added to this solution finally, 7.5 ml of 50% ammonium

hydroxide will be added. Yellow colour will be developed

transmittance after 15 mts will be noted at 397 nm using a

"spectronic-20" colorimeter. A standard curve will be

plotted using the graded concentration of standard nitrate

solution.

3.11.5 ESTIMATION OF NITRATE REDUCTASE ACTIVITY (NRA)

Nitrate reductase activity will be estimated, in the

fresh samples, according to the method of Jaworski (1971).

76

In polythene vials 500 mg of the sample, 2.5 ml of

phosphate buffer (Appendix iv), 0,5 ml of 0.2 M potassium

nitrate solution (Appendix iv) and 2.5 ml of 5% Isopropanol

will be poured atleast, two drops of the aqueous solution of

chloramphenicol will also be poured to avoid bacterial

growth in the test medium. The vials will be incubated for

2 hrs. in dark at 30 degree centigrade in a BOD incubator.

3.11.5.1 Colour development

Test extract (0.4 ml) will be taken in a test tube

to which 0.3 ml each of Sulphanil amide and NED-HCl

(Appendix v) will be added. The samples will be allowed to

stand for 20 mts. for maximum colour development. It will

be diluted with 5 ml of double distilled water and read at

540 nm using 'spectronic-20' colorimeter. A blank will be

run simultaneously consisting of 4.4 ml of double distilled

water plus 0.3 ml each of sulphanil amide and NED-HCl. A

standard curve will be plotted by taking known graded

dilutions of standard potassium nitrite solution. The

transmittance of test extract will be compared with those of

the calibration curve and NRA will be calculated in terms of

}x mole NO"^g~-'-(F.W. ) h"""".

3.11.6 ESTIMATION OF THIAMINE (Vitcimin-B )

Thiamine will be estimated in a given sample

according to the methodology of Horwitz (1972).

77

To 2 gm of leaf powder, 0.1 N HCL acid will be added

and agitated vigrously so that all particles will come in

contact with the liquid. Then the side walls of the flask

will be washed with 0.1 N Hydrochloric acid and the content

will be digested for 30 mts at 95-100 degree centigrade on a

water bath with frequent shaking. The liquid will be cooled

and filtered through an ashless filter paper (which will not

absorb vitamin B, ) . The filtrate will then be diluted to

100 ml with 0.1 N Hydrochloric acid and will proceed for

oxidation of thiamine to thiochrome. 1.5 gm potassium

chloride will be taken in a test tube and to it 5 ml

thiamine standard solution (Appendix vJO will be added. It

will be vigrously shaken and 3 ml oxidising reagent

(Appendix vi) will be added with a quick delivering pipette.

The tube will be swirled to ensure adequate mixing.

Immediately, 13 ml Iso-butanol will be added and tube will

be shaken vigrously for more than 50 seconds. Similar

treatment will be given to another tube by using the same

chemicals and solutions except the oxidising reagent which

will be replaced by 15% sodium hydroxide solution. This

tube will be treated as standard blank.

The same treatment will be given to two other tubes

for reading the oxidised and blank solution of the sample.

Isobutanol, will be added and all the tubes will be shaken

for 2 mts. Then all tubes will be centrifuged at low speed.

78

a clear supernatant will be obtained. 10 ml Isobutanol

extract (upper layer) which will be bluish in colour will be

pipetted into colorimetric tube from each tube. Thiochrome

flourescence will be observed at 365 nm using a Bausch and

Lamb 'spectronic-20' spectrophotometer.

The flourescence of the Isobutanol extract from

oxidised solution will be measured (A). Similarly, the

flourescence of the extract which will be treated with 3 ml

of 15% sodium hydroxide (b) and the flourescence of the

extract from oxidised standard thiamine solution will be

measured (S), finally, the flourescence of the extract from

standard vitamin-B, solution which will be treated with 3 ml

of 15% sodium hydroxide solution will be measured (d) and

the quantity of thiamine (vitamin-B,) in the sample will be

calculated by the following formula.

pg Vitamin-B, in 5 ml solution = (A-b) (S-d) from

this, the percentage of vitamin-B, in the leaf powder will

be calculated.

3.12 STATISTICAL ANALYSIS

The experimental data will be statistically analysed

according to the design of experiment chosen (Panse and

Sukhatme, 1985). Error due to replication will be determined

by applying 'F' test. CD will be calculated out if 'F'

vlaue will be found significant upto 5% probability level.

The results will be discussed in the light of other

researchers.

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APPENDIX

A P P E N D I X

PREPARATION OF REAGENTS

The reagents which will be used in the chemical

analysis will be prepared in the following ways:

1.0 REAGENTS FOR ESTIMATION OF REDUCING AND NON-REDUCING

SUGARS

1.1 Acetate buffer solution

3 ml HOAc, 4.1 gm anhydrous NaOAc and 4.5 ml H2S0^

will be diluted in distilled water and volume will be made

upto 1 liter.

1.2 Sodium Tungstate Solution (12%)

12.0 gm Na2W0. .2H2O will be diluted with water to

make up the volume 100 ml.

1.3 Alkaline ferricyanide solution (O.IN)

33.0gm pure dry K-Fe(CN)^ will be mixed with 44.0gm 3 0

Na_CO^ and volume will be made upto 1 liter with distilled

water.

1.4 Acetic acid salt solution

200 ml HOAc, 70gm KCl and 40gm ZnSO^. 7H2O will be

dissolved in water to made up the volume 1 liter.

1. 5 Soluble starch-potassium iodide solution

2gm solution starch will be added to small amount of

cold water and will be poured slowly into boiling water with

constant stirring. It will be then cooled thoroughly (or

(ii)

resulting mixture will be dark coloured), 50 gm KI will be

added and diluted to lOOrnl with distilled water, one drop

NaOH solution will also be added (1+1). 1 ml of this

solution will be used.

1.6 Thiosulphate standard solution (0.1 N)

24.82 gm Na-S-O^.SH' 0 will be mixed with 3.8 gm

Na-B.O^.10H_0 and the volume will be made upto 1 liter. 2 4 7 2 '^

2.0 REAGENTS FOR THE ESTIMATION OF CARBOHYDRATES

2.1 Sulphuric Acid (1.5 N)

10.20 ml pure sulphuric acid (BDH) will be diluted

to 250 ml in a volumetric flask with double distilled water.

2.2 Phenol (5%)

5 ml of the distilled phenol will be pipetted into a

100 ml volumetric flask and final volume made upto the mark

with distilled water.

3.0 REAGENTS FOR THE ESTIMATION OF PROTEIN

3.1 Reagent A

2% sodium carbonate will be added to 0.1 N sodium

hydroxide in the ratio of 1:1.

3.2 Reagent B

0.5% copper sulphate will be added to 1% sodium

tartrate (in the ratio of 1:1).

(iii)

3.3 Reagent C (Alkaline copper sulphate solution)

It will be prepared by mixing 50 ml of reagent 'A'

and 1 ml of reagent 'B'.

3.4 Reagent D (Carbonate Copper Sulphate Solution)

It will be prepared in the same way as Reagent 'C

with the ommission of Sodium Hydroxide.

3.4 Reagent E (Folin's phenol reagent)

100 gm sodium tungstate and 25 gm sodium molibdate

will be dissolved in 700 ml distilled water to which 50 ml

of 85% phosphoric acid and 100 ml concentrated hydrochloric

acid will be mixed. The flask will be connected with a

reflux condenser and will boiled gently •&n a heating mentle

for 10 hrs. At the end of boiling period 150 gm Lithium

Sulphate, 50 ml distilled water and 3-4 drops of liquid

bromine will be added in the flask. The reflux condenser

will be removed and the solution in the flask will be boiled

for 15 mts, in order to remove excess bromine, cooled and

diluted the solution.

4.0 REAGENTS FOR THE ESTIMATION OF NITRATE

4.1 Phenol di-sulphonic acid reagent

25 gm pure white phenol (AR) will dissolved in 150

ml of pure concentrated sulphuric acid to which 75 ml

fuming sulphuric acid will be added. The solution will be

heated for about 2 hrs at 100 degree centigrade in water-

(iv)

bath and cooled. This solution will be kept in dark

coloured, bottle in a refrigerator.

4•2 Standard Nitrate solution

7.22 gm potassium nitrate will be dissolved in

sufficient distilled water and final volume will be

adjusted. 5 ml of this standard solution will further be

diluted to 1 litre with distilled water. 1 ml of this

solution will give 5 gm of nitrate nitrogen.

5.0 REAGENTS FOR THE ESTIMATION OF NITRATE REDUCTASE

ACTIVITY

5.1 Phosphate Buffer (pH - 7.5)

(A) 13.6 gm potassium hydrogen orthophosphate (KH2P0^)

will be dissolved in water and the final volume made upto 1

litre.

(B) 17.42 gm di-potassium monohydrogen orthophosphate

(K-HPO.) will be dissolved in distilled water and the final

volume will be made upto 1000 ml. This will give 0.2 M

solution.

(C) 160 ml of solution 'A' will be mixed with 840 ml of

solution 'B' in order to get phosphate buffer of pH 7.5.

5.2 Potassium Nitrate (0.2 M)

2.02 gm of potassium nitrate will be dissolved in

double distilled water and final volume made upto 100 ml.

(v)

5.3 Sulphanil amide solution (1%)

1 gm sulphanil amide will be dissolved in 3N HCl, to

complete the volume 100 ml.

5.4 Nephthyl ethylene diamine di-hydrochloric acid

(NED-HCl 0.02%)

20 mg N-1-Nephthyl ethylene diamine di-hydrochloric

acid will be dissolved in enough distilled water and the

final volume made upto 100 ml.

6.0 REAGENTS FOR THE ESTIMATION OF THIAMINE

6.1 Hydrochloric acid (O.IN)

2.2 ml pure HCl will be added to distilled water and

the final volume will be made upto 250 ml.

6.2 Aqueous Ethanol solution (20%)

20 ml ethanol will be mixed with 80 ml distilled

water.

6.3 Thiamine standard solution (Vitarain-B )

10 mg Vitamin-B, v/ill be dissolved in 1 liter 20%

aqueous ethanol solution to make 10 ug ml stock solution. pH

will be adjusted to 3.5-4.3 with hydrochloric acid and

stored at 10 degree centigrade in light resistant bottle.

10 ml of the stock solution will be mixed with 50 ml of

O.lN HCl, and will be digested at 100 degree centigrade on

steam bath for 30 mts. Cooled and will be diluted to 100 ml

with O.lN HCl to make 1 ug ml vitamin-B, standard solution.

(vi)

6.4 Potassium ferricyanide solution (1%)

1 gm of potassium ferricyanide will be dissolved in

100 ml distilled water.

6.5 Sodium hydroxide solution (15%)

15 gm of sodium hydroxide will be dissolved in IGOml

distilled water.

6.6 Oxidising reagent for thiamine (Vitamin-B,)

4 ml of 1% potassium ferricyanide will be mixed with

15% sodium hydroxide solution to made the volume upto 100ml.

The solution will be used within 4 hrs.