EFFECT OF SOMB GROWTH REGULATORS ON CERTAIN BIOCHEMICAL PARAMETERS DURING SEED DEVELOPMENT IN CHICKPBA UNDER SALINITY

~;.

r'­

I

EFFECT OF SOMB GROWTH REGULATORS ON CERTAIN BIOCHEMICAL PARAMETERS DURING SEED DEVELOPMENT IN CHICKPBA UNDER

SALINITY

GURBAKSH SINGH AND SUMAN JAIN

Department of Botany. Punjab Agricultural University. Ludhiana

Revised May 1,1981

SUMMARY

Biochemical analysis of fruit wall and seed was done in order to see the effect of some growth regulators on chlorophyll, total free amino acids, proteins and total soluble sugars under different salinity levels in chickpea. Kinetin, GAa and 2,4,5-T

jmproved the chlorophyll contents. Salinity decreased the Chl'a'/Chl'b' ratio.

Total free amino acids were more in fruit wall during early stage of pod development

but later on decreased~ On the contrary. protein contents increased in fruit wall and

seed with the advancement of pod age. With an increasc in salinity, protein content decreased in both the cases. Among the growth regulators kinetin improved the protein content in both the components of pod at different stages of development. Chlorophyll and sugars were not detectable in the fruit wall of mature pod.

INTRODUCTION

Chickpea (Cicer arietinum L.) is an important pulse crop because of its high

nutritive value and its use in various forms. One of the main causes of low yield of

this crop is an increase in salinity levels of soil. It has been estimated that about

14 per cent soils in Punjab are salt affected (Abrol and Bhumbla. 1971) and contain higher amount of chlorides and sulphates. Theirelectrical conduc­ tance (EC) varies from 0.2-10 mmhos/cm on the basis of 1:2 ratio of soil: water (personal commullication from Extension wing of Punjab Agricultural

University, Ludhiana). This crop is considered to be most sensitive to salinity

(Bhumbla and Singh, 1965). Endogenous levels of hormones under stress conditions are disturbed and particularly ABA production is triggered (ltai and Vaadia. 1965;

Jones and Mansfield 1970; Bengston et. aI., 1978). It was, therefore, considered

worthwhile to study the effect of some growth regulators on certain biochemical paI'ameters viz., chorophyll, amino acids, proteins and sugars during various stages

168 GURBAKSH SINGH AND SUMAN JAIN

MATERIALS AND METHODS

Enamelled pots of uniform size (25 em

x

20 em) were selected for this experiment.

Each pot was filled with 8 kg of dry soil having pH 8.7 and electrical conductance (EC) 0.4 mmhos/cm. A combination of chloride and sulphate ions was used to

prepare different salinity levels. Sodium chloride (1.16 g), sodium sulphate (0.71 g)

and calcium chloride (0.73 g) were dissolved separately in the ratio of 2:1:1 meq. respectively per litre for preparing a solution of 4 m mhos/cm. Other salinity level (8 m mhos/cm) was the mUltiple of this quantity. Each pot was irrigated with 2 litres (calculated on the basis of saturation of the dry soil which was 25%) of required solution. The salts were added in this ratio to prepare different salinity levels because the sub soil water contains these salts in this proportion. When the soil in the pots was worth pulverizing, seeds of pure line 'G-130, were inoculated with

Rhizobium culture, dried under shade and sown 10 to a pot on October 22. The

plants were raised under optimum conditions of water and natural day-length. Thinning was done after one month of sowing and only 5 uniform plants were

maintained in each pot. A total of 90 pots were arranged on the ground in the

culture house in 3 rows each representing pots of one salinity level. The pots in each row were further divided into 5 lots each representing one growth regulator

treatment viz., Tl : control (water spray); T_{z :} GAs 25 ppm; Ta : kinetin (Kn) 10 ppm;

T" : TIBA 25 ppm; Ts : 2,4,5-T 5 ppm. The growth regulators were applied in the form offoliar spray with the help of a polyethylene hand sprayer. First spraying was done at 90 days from the date of sowing (just at the start of flower appearance) and the second spray after 10 days from the first spraying.

~

The pod samples were taken at three stages of its development

viz.,

at the start

of pod development (5 day old), full size of the pod (20 day old) and mature stage (45 day old). Pod was further divided into fruit wall and seed (except for the first stage when there was no seed). Chlorophyll contents were determined by the method of Anderson and Boardman (1964). The method of Lee and Takahashi (1966) was follo­ wed for the estimation of total free amino acids. Soluble proteins were determined according to the method of Lowry et al., (1951). Total soluble sugars were estimated by the method of Loewus (1952).

RESULTS AND DISCUSSIONS

Chlorophyll: It is evident from the data given in Table I that fruit wall of pod in

chickpea had sufficient chlorophyll (a&b) contents. Salinity had adverse effect on the chlorophyll content. Both chlorophyll a and b as well as chl'a'/chl'b' ratio decreased with lDcrease in salinity both in fruit wall and seed. The chlorophyll contents in fruit

wall decreased with increase in age. Growth regulators particularly Kn, GAs

,,'" Table 1. Chlorophyll contents (mg!c fresh wt.) o/fruit wall at two stages of pod development "{, Growth rc.ulators Salinity 5 days 20 days levels' ChI 'a' Chl'b' ChI 'a' Chl'a' Chl'b' ChI 'a' Q (m mhos/em)

---l1li 0

Chl'b'

ChI

'b'

~ oi

=

I. Control 0 0.54 0.27 2.00 0.28 0.18 LS6

~

(Water spray) 4 0.46 0;24 1,91 0.24 0.16 LSO

c::: I'"

8 0.44 0.25 1.76 0.22 0.15 1.47

> oi ~

""

2. GAa 25 ppm 0 0.56 0.26 2.16 0.31 0.19 1.63 0 :z: 4 0.48 0.27 1.78 0.27 0.18 LSO til 8 0.42 0.29 1.45 0.24 0.16 LSO

R

;;,

3. Kn 10 ppm 0 0.60 0.27 2.22 0.40 0.20 2.00

!

n

4 0.54 0.28 1'93 0.36 0.21 1.71 >' I'" 8 0.48 0.32 1.50 0.30 0.20 LSO

-a ~

4. TIBA 25 ppm 0 0.52 .. 0.24 2.17 0.27 0.16 1.69

>

s::

~

4 0.45 0.24 1.88 0.23 O.IS LS3 8 0.40 0.27 1.48 0.20 0.14 1.43

~

go c:::

S. 2,4,5-T 5 ppm 0 0.56 0.29 1.93 0.32 0.17 1.88

:z: t:::I

4 0.52 0.28 1.86 0.28 0.17 1.65

~ l1li

8 0.45 0.31 1.48 0.25 0.16 1.56

rn > !: :z:

Mean 0.50 0.27 1.83 0.28 017 1.61

.... oi 0< 0\

-

IQ

..

..."",

. . . • . . .

~

1·

_,

GURBAKSH SINGH AND SUMAN JAIN

170

,

in fruit wall ,during pod development. No tangible amount of cblorophyll in furit wall could be found in 45 day old pod (Table II). There are evidences to show that accumulation of salts in leaves, cause a decrease in the extractability of·

pigment from the chloroplasts especially when the chloride ions predominat~ in the

soil (Strogonov, 1964; Varshney and Baijal, 1977). It is also possible that chlorophylase

activity is increased under stress conditions. Sabatar and Rodrigueg (1978) have also suggested that kinetin lowers chlororophyllase level responsible for the chlprophyll degradation in detached leaves of barley and oats under stress conditions. Fruit wall besides lowering photosynthetic activity also effectively translocates the product to developing seeds. Similar observations were reported by Sinha (1971). Salinity ad­ versly affected the chlorophyll contents in developing seeds also. All the, growth

regulators improved the chlorophyll contents in seeds of 20 day old pod. It has been

shown by other workers that seed coat itself contribute 11 percent of its total re.serves (Verma et al., 1964).

Amino acids and Proteins:-Total free amino acids in fruit wall decreased with

age of the pod (Table Ill), while in seed (Table IV), there was some increase in total

free amino acids under saline conditions. It may be explained on the basis of their

higher synthesis in the fruit wall during early stages due to production of more photosynthates and later on amino acids were translocated from fruit wall to seed and simultaneously there might be incorporation of amino acids into proteins in the fruit wall itself and thus protein content increased in the fruit wall at later stages (Table V). Even the synthesis of amino acids can continue in the advanced stage of the pod but to a lesser extent. Seed itselC can synthesize the amino acids and also the acids from the fruit wall are transported to the seed as it acts as a sink. Salinity adversely affected the protein content particularly in seed (Table VI). Among the growth regulators, Kn significantly increased the protein content both in fruit wall as well as in seed. This is perhaps due to the fact that Kn may have alleviated a part of stress affected reduction in amino acid incorporation. These results conform with the

chickpea.

finding of n:any workers (Mizrahi and Richmond, 1971; Eder and Huber, 1977). Maxi­

mum protein accumulation in seed was over by 20 days in Kn and GAll as compared

.MUii,. I.

qg

244iliM,X

##.=#11.

ua::S;;;UAA444 4 p:;:;;. Ii ~-"'=-~--'>, ~, ~-=:"-','.-. Table II. Chlorophyll contents (mg/g frest wt.) in seeds at two,stages of pod development Growth regulators

Salinity levels

Chi 'a' 20 days Chi 'b' Chi 'a' Chi 'a' 45 days Chl'b' Chi 'a'

G'l ~

m mhos/em

--­

Chi 'b'

---­

_Chi 'b'

~ '"'! ::: lilt !'I:I

1. Control (Water 0 0.24 0.14 1.71 0.12 0.08 1.50 G'l

c:

spray) 4 0.21 0.14 LSO 0.09 0.06 1.48

I:'" >

8

0.18

0.12

1.58

C1 lilt {Il

2. GA. 25 ppm 0 0.30 0.20 1.50 0.13 0.09 1.44

~

4 0.29 0.20 1.45 0 10 0.07 1.43 =' 8 0.25 0.18 140

~

3. ' Kn 10 ppm

0 4 8

0.29 0.26 0.23

9.18 0.18 0.17

1.61 1.44 1.3S

0.13 0.10

0.09 0.08

-1.44 1.2S

II: () > I:'" '"c:I ~

4.

TIBA

25

ppm

0 4 8

0.25 0.23 0.20

0.14 0.16 0.14

1.79 1.44 1.43

0.12 0.09 0.08 0.07

1.S0 1.29

j

f'l'

S. 2,4, SoT S ppm 0 0.27 0.16

.

1.70 0.13 0.08 1.63

c:

0.23 0.20

0.17 0.16

1.35 1.2S

0.12

0.09

1.33

~

1iI

{Il > I:'"

-Mean 0.24 0.16 LSO 0.11 0.08 1.43

~

...

-

-

l

\

~

"

Table

111.

Totalfree

amino

acids

(mg!g

dry

wt.)

in/ruit

wall

at

two

stages

0/

pod

development

.­

t::J

Growth

regulators

5

days

20

days

Salinity

levels

(m

mhos/em)

Salinity

levels

(m

mhos/em)

0

4

8

Mean

0

4

8

Mean

1.

Control

43.75

31.87

35.62

37.08

25.62

31.67

25.00

27.50

(Water

spray)

CI

2.

GA,

25

ppm

33.25

35.62

30.00

32.96

21.25

30.00

21.00

24.08

~

3.

Kn

10

ppm

30.62

43.40

33.43

35.82

24.87

30.62

28.12

27.87

>­

r!

4.

TIBA

2S

ppm

26.87

27.20

40.00

31.36

24.87

25.00

22.50

24.12

l:I:I

5.

2,4,

5·T

5

ppm

41.25

44.00

31.81

39.12

24.37

23.75

18.75

22.39

i

~

)0; Z

Mean

35.14

36.29

34.18

24.19

28.24

23.07

1;1 til

i

C.

D.

at

5%

for

>­ Z

(i)

Salinity

levels

N.S.

2.44

...

>­

-

Z

(ii)

Growth

regulators

2.91

1.90

(iii)

Interaction

5.07

3.20

(Salinity

X

Growth

re.ulators)

~

,

---Table

IV.

Total/ree

amino

acids

(mg/g

dry

WI.)

in

seeds

at

two

stages

o/pod

development

Growth

regulators

0

20

days

Salinity

levels

(m

mhos/em)

4

8

Mean

45

days

Salinity

levels

(m

mhos/em)

0

4

8

Mean

i

~

~

1.

Control (Water

spray)

51.25

40.80

37.50

43.18

38.12

49.37

42.50

43.33

==

•

'"

~

2. 3, 4.

GA. Kn TIBA

25

ppm

10

ppm

25

ppm

32.50 45.00 40.00

35.00 40.80 35.80

29.37 34.37 31.25

32.29 40.06 35.68

28.75 48.22 40.31

38.00 32.50 37.50

33.06 45.00 41.25

33.27 41.87 39.69

>­

S

i:

0 :z till

5.

2,4,5-T

5

ppm

45.62

32.50

26.25

34.79

35.62

40.31

42.00

39.31

~

Mean C.

D.

at

5%

for

42.87

36.98

31.75

36.18

39.54

40.76

m I:

r;

>­ t""

(i) (ii) (iii)

Salinty

levels

Growth

regulators

Interaction (Salinity

X

Growth

regulators)

2.08 2.46 4.25

3.16 3.16 5.49

~

>­ I:

!

_c::

:z

i

!ill >­

t:

!

~ Nl

-

_c:;1

~

'·--1:,

<'"

Table

V.

Protein

content

(mg!g

dry

wt.)

infruit

wall

at

different

stages

of

pod

development

<t'Ji .;lo

Growth

regulators

5

days

20

days

45

days

Salinity

levels

(m

mhos/em)

Salinity

levels

(m

mhos/em)

Salinity

I~vels

(m

mhos/em

0

4

8

Mean

0

4

8

Mean

0

4

8

1.

Control (Water

spary)

34.0

35.0

26.0

31.7

32.5

26.3

27.0

28.6

2.

GAs

25

ppm

27.0

29.0

22.0

26.0

35.0

32.0

34.0

33.7

3.

Kn

10ppm

45.0

29.0

31.5

35.2

39.0

42.0

41.0

40.7

C'l ~

4. 5.

TIBA

•

25

ppm

2,4,5-T

5

ppm

Mean

C.

D.

at

5%

for

31.5 22.0 31.9

22.0 24.0 27.8

20.0 27.0 25.3

24.5 24.3

25.0 36.0 33.5

29.9 30.0 31.9

23.0 34.0 31.8

25.5 33.3

tI.1

e

_II

i

0

=

> %

'='

(i) (ii)

Salinity

levels

Growth

resulators

1.16 2.25

3.19 3.40

!

~

(iii)

Interaction

3.90

(Salinity

X

Growth

resulators)

5.78

~

.','

....

'..:..

_..,.. __________________ ~-,-~~~'~ ... ~!""' .... ". '--"-__ ...,.~":""'" ... ~~n:>~~ ... """'1' •

-"'

--..

.... ~ -Table YI. Protein content (mg/g dry wt.) in seeds at two stages of pod development Growth reaulators 20 days 45 days Salinity Ievels(m mhos/em) Salinity levels (m mhos/em Q

-!

0 4 8 Mean 0 4 8 Mean

~

1. Control 167.0 163.0 155.0 161.7 182.5 173.' 167.0 174.2

~

(Water spray)

c::

!"'

2. GAs 25 ppm 182.0 166.0 145.0 164.3 169.0 161.0 156.0 162.0

a

~

3. Kn 10 ppm 189.0 178.0 160.0 172.3 187.5 178.0 173.0 179.5

!

~

4. TIBA 25 ppm 102.0 126.0 118.0 115.3 178.0 173.0 172.0 174.3 5. 2.4,5T 5 ppm 147.0 124.0 124.0 131.7 172.0 172.0 174.0 172.7

5

Mean 157.4 151.4 140.4 177.8 171.4 168.4

('l >­!"'

:

C. D. at 5% for

E

(i) Salinity level 14.77 7.24

i

(ii) Growth regulators 10.52 N.S. (iii) Interaction 18.20 N.S.

~

(Salinity X Growth realators)

;f"""~""" Table VII Sugar content (mg/g dry wt.) in/mit wall at different stages

0/

pod development

:!:i

Q\

Growth regulaton 5 days 20 days 45 days ~ -Salinity levels (m mhos/em) Salinity levels (m mhos/em) Salinity levels (m mhos/em

--'---­

0 4 8 Mean 0 2 8 Mean 0 4 8 ]. Control 33.0 33.0 28.7 31.5 27.5 27.0 23.0 25.8 (Water spray) 2. GAs 25 ppm 27'5 30.0 31.0 29.5 23.7 31.0 25.0 26.6 3. Kn 10 ppm 31.0 28.5 26.5 28.7 26.5 31.5 31.5 29.lJ

~

>­

4. TJBA 25 ppm 30.0 23.7 23.0 256 32.S 24.0 23.0 26.5

tl

==

5. 2,4,5T 5 ppm 24.5 21.0 19:0 21.5 30.0 25.5 21.2 25.6

tI>

2!

~

Mean 29.2 27.2 25.6 28.2 27.8 24.7

>

~

C,

D.

at

5%

(or

i

~

(i)

Salinity

levels

1.99

2.69

...

>­

(ii)

Growth

regulators

2.06

2.75

...

Z

,,~

Table

VIII.

Sugar

content

(mgJg

dry

wt.)

in

seeds

at

two

stages

of

pod

development

Growth

regulators

---­

20

days

4S

days

---­

Salinity

levels

(m

mhos/em)

Salinity

levels

(m

mhos/em)

0 ~

0

4

8

Mean

0

4

8

Mean

~

5!

1. 2, 3.

Control (Water

spray)

GAa

2Sppm

Kn

10

ppm

14.0. 9.0 9.7

12.0 9.7 _11.2

8.7 8.S 8.7

11.6 9.1 9.9

6.7 _{6.S 8.0}

9.S 6.S _10.0

6.0 6.0 8.0

7.4 6.3 8.7

l1:li

g

i

0 Z

4. S.

TIBA 2,4,

S-T

25

pym

S

ppm

13.0 14.0

10.0 7.0

7.0 8.S

10.0 9.8

6.0 8.0

6.0 8.0

4.7 6.S

S.6 7.S

CII

§

111

Mean

11.9

10.1

8.3

7.0

8.0

6.2

§

1"'

::

C.

D.

at

S%

for

~

(i) _(ii)

Salinity

levels

Gtowth

regulators

1.17 2.23

1.72 0.72

Ie

a

i:

(iii)

Interation

3.98

1.24

~

(Salinity

x

Growth

regulators)

111 l1:li tn >

t:: z

...

o.,j 0<

•

.­ ~ ~

;i!1

178 GURBAKSH SINGH AND SUMAN JAIN

ins by en hen cement of amino acids incorporation. This view is also supported from

the work of Benzioni et al. (1967).

Total soluble sugars: During pod and seed development the metabolic activity

is high and sugars are used as a source of energy thereby decreasing their level with the advancement of age. Salinity lowered the sugar content in fruit wa)) as we)) as selfls (Table VII and VIII) of <IitTerent pod age. Growth regulators etTect on

this parameter was not conspicuous. It is just possible that under salt stress conditions

th" translocation of sugars from other parts of the plant to the poor sink (pod) may be less. Ramana and Rama (1978) also reported a delayed translocation of carbohydrates from cotyledons to embryonic axis in pea under salt treatments.

?

ACKNOWLEDGEMENTS

Authors are thankful to Dr. C.P. Malik, Professor and Head, Department, of Botany, Punjab Agriculrural University, Ludhiana, for his encouragement and for providing the necessary facilities in carrying out these studies. We are also thankful tQ the Pulse Breeder of this University for supplying the pure line seed of 'G-130'.

REFERENCES

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Anderson, J. M. and Boardman, N. K. (1964). Studies on greening of dark brown bean plants. VI.

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Bengston, C., Klokare. B., Klokare, R., Larsoon, S. and Sundquist, C. (1978). The after effect of

water stress on chlorophyll formation during greening and the levels of ABA and proline

in dark grown wheat seedlings. Phpsiol. Plant., 43: 205-12.

Benzioni, A., Jtai, C. and Vaadia, Y. (1967), Kinetin and protein synthesis in tobacco leaves under

ter and salt stress. Plants. Physiol., 42 : 361-65.

Bhumbla, D. R. and Singh, N. T. (1965). Effect of salt on seed germination. Curro Sci., 31: 96-97.

Eder, A. and Huber, W. (1977). Effect of ABA and kinetin on biochemical changes in Pennist!lum

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Itai, C. and Vaadia, Y. (1965). Kinetic like activity in root exudate of water stressed sunflower

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Jones, R. J. and Mansfield, T. A. (1970). Suppression of stomatal opening in leaves treated with

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GROWTH REGULATORS ON BIOCHEMICAL PARAMETERS UNDER SALINITY 179

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Sabater. B. and Rodrigues, M. T. (1978). Control of chlorophyll degradation in detached leaves of barley and oat through effect of kinetin on chlorophyllase levels. Physlol. Plant., 43: 274-76.

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Strogonov. B. P. (1964). Physiological basis of salt tolerance of plants. Translated from Russian. Jerusalem Israel programme for scientific translations.

Varshney. K. A. and Baijal, B. D. (1917). Effect of salt stress on chlorophyll content of some

grasses. Indian J. Plant Physiol., 20 : 161.63.

Verma. S. C. and Lal. B. N. (1966). Physiology of Bengal gram seed II. Changes in phosphorus

compound during ripening of seeds. J. Sci. Fd. Agric., 17 : 4346.

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