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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.