Effect of some pre-sowing treatments on Sapindus laurifolius seed germination

Keywords: H2SO4, Sapindus laurifolius Vahl, seed germination, seed scarification.

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Journal of Research in

Plant Sciences An International Scientific

Research Journal

Authors:

Vishal R. Kamble1,

Bazegah K. Sayed2 and

Shrinath P. Kavade3.

Institution:

1-2. Department of Botany,

Bhavan‟s College Andheri

(West), Mumbai (MS)

400 058 India.

3. Department of Botany,

A.C.S. College, Lanja,

Ratnagiri, Maharashtra

416701, India

Corresponding author:

Vishal R. Kamble.

Email:

Web Address: http://plantsciences.info/ documents/PS0056.pdf.

Dates: Received: 02 Apr 2013 Accepted: 07 June 2013 Published: 04 July 2013

Article Citation: Vishal R. Kamble, Bazegah K. Sayed and Shrinath P. Kavade. Effect of some pre-sowing treatments on Sapindus laurifolius seed germination Journal of Research in Plant Sciences (2013) 2(2): 205-212

An International Scientific Research Journal

Original Research

Jou

rn

al of R

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in

Plan

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Journal of Research in Plant Sciences

205-212 | JRPS | 2013 | Vol 2 | No 2

www.plantsciences.info

ABSTRACT: Present paper deals with the effect of some pre-sowing treatments on the seed germination of Sapindus laurifolius Vahl. (Sapindaceae). The physical and chemical scarification treatments were given to S. laurifolius seeds in order to test, identify, and recommend suitable pre-sowing treatments. In-depth analysis of data obtained in the present work has proved that, sulphuric acid promotes seed germination in S. laurifolius comparatively within shorter time without affecting growth performance of saplings obtained.

INTRODUCTION

International Seed Testing Association (ISTA)

has long recognized the need to develop germination

testing methods for tropical species and thus assists in

the development of agriculture in low latitudes (Meadly,

1972 and Harty, 1983). Various workers have

contributed in research on tropical plant species

reproduced by seed, and suitable for wider exploitation

(National Research Council, 1975). Seed germination is

important as a method of increasing the food and

economic value of grains and there is a need for studies

of sprouting techniques. Sapindus laurifolius Vahl.

(Syn S. trifoliatus) [Family Sapindaceae], is a 5-10m

high, medium size to large deciduous tree. The berries of

tree are used as a substitute for soap; hence the name

„Soapnut tree‟ which is given to them by Anglo-Indians

(Cook, 1967). Soapnut tree is native to Indo-Gangetic

plains, Shivaliks and Sub-Himalayan tracts in India and

found at an altitudes ranges from 200m to 1500m

(Anonymous, 1988). Although, it is one of the most

important trees of tropical and subtropical regions of

Asia (Meena Devi, et al., 2012), it is commonly found in

the Western Ghats and plains of South India (Chopra

et al., 1956). It is also cultivated in Bengal, Southern and

Western part of India for its useful properties (Cook,

1967). It is most commonly distributed in southern

tropical dry deciduous forest of Maharashtra with annual

mean temperature 27.3°C, and annual rainfall 825 mm,

(Mahabale, 1987) particularly at Osmanabad, Vidharbh,

Konkan, Matheran and Kolhapur region.

According to Sharma et al., 2011, “fruits of

S. trifoliatus have been considered as a tonic, stomachic,

alexipharmic, astringent and sedative to the uterus and

also useful in chronic dysentery, diarrhea, cholera,

hemicranias, tubercular glands, paralysis and epileptic

fits of children; while roots are used as collyrium in sore

eyes and opthalmia”; whereas in Konkan region of

Maharashtra (India) the fruits are employed in native

medicine (Cook, 1967). The seeds of S. trifoliatus have

stimulatory effect on uterus and are used in childbirth as

well as to increase menstruation (Pelegrini et al., 2008).

The fruits are often used for the preparation of hair

tonics, herbal shampoos, homemade herbal cosmetics

and skin creams (personal observations). Thus after

consulting with literature, soapnut tree apparently

concern with human health problems. It is a species that

presents a great diversity of chemical compounds like:

saponins, sapindosid, fatty acids such as ofarachidic,

behenic, linoleic, oleic, palmitic, stearic, oleanolic acid,

and sapindic acid etc (Prajapati et al., 2003); phenolic

acids such as proto catechuic acid, cis-pcoumaric acid,

p-hydroybenzoic acid and cinnamic acid (Naidu et al.,

2000).

During past few decades, various pre-sowing

treatments viz., cow dung slurry and acid (Brahmam

et al., 1996; Sheikh 1979); hot water treatment (Jackson,

1994; Campbell 1983) have been applied for the

improvement in soapnut seeds germination. According to

Thapa and Gautam 2006 “identification of suitable

pre-sowing treatment is necessary for quicker and higher

seed germination and thereby to reduce the shortage of

quality seedlings during plantation in many nurseries in

Nepal”. In India few workers have tried to understand

mechanism of seed germination in soapnut. Kumar and

Devar (2003), studied correlation of morphometric, fruit,

seed and germination traits in soapnut. Naidu et al.,

(1999 and 2000) showed effect of temperature and acid

scarification, nitrogenous salts and plant growth

regulators, on seed germination of soapnut. However, all

these reports show wide variations in germination results

and time required for establishment of saplings.

Therefore, in present work an attempt has been made to

study the effect of some pre-sowing treatments on seed

germination including physical and chemical

scarification in order to test, identify and recommend

suitable pre-sowing treatment for Sapindus laurifolius.

206 Journal of Research in Plant Sciences (2013) 2(2): 205-212

Kamble et al., 2013

MATERIALS AND METHODS

Seed index and Viability:

Seeds of S. laurifolius were collected from

Bhavan‟s Botanical Nursery, R. T. M. Nagpur University

campus Nagpur (Vidharbh) India, and also from local

market of Andheri particularly from Ayurvedic and

Herbal medicine suppliers. Seed index was analyised

with reference to colour, size, viability, weight, shape at

least for 100 seeds. Viability percentage of seed was

determined by soaking 100 seeds in water, floated seeds

were discarded and considered as non viable. Viable

seeds were used for further experiments.

Breaking of seed dormancy:

The pre-sowing treatments were given to

S. laurifolius seeds so that to break dormancy with the

help of physical and chemical scarification.

Physical scarification:

Seed coat was mechanically scarified by

methods viz. 1) Needle pin: the hilum region of seed was

punctured with the help of pointed metal needle pin and

the seeds were placed for germination in Petri plates.

2) Sand paper: the matured seeds in groups were rubbed

in the sand paper to break the hard testa. After that, the

seeds were kept for germination in petriplates.

3) Mortar and pestle: the testa of seeds was broken

gently and seeds were kept for germination in petriplates.

Chemical scarification:

The seeds were treated with different

concentrations of Sulphuric acid (H2SO4) viz., conc.,

1N and 2N H2SO4 by keeping seeds in for the time

interval of 0.15, 0.30, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0

minute. After that the seeds were kept for germination in

petriplates.

Determination of germination percentage:

Treated seeds were kept in germination chamber

for 25 days under observations. Emerging of radical and

plumule were considered as indication of seed

germination. Observation were recorded daily, days of

maximum germination percentage were recorded for

each treatment and germination percentage (g) was

determined by using the formula: g = Ng / Nt X 100

where (Ng) Number of seeds germinated, (Nt) Total

number of seeds.

Post germination study:

After 25 days of germination seedlings of each

treatment were planted separately in pots filled with

garden soil free from any fertilizer. All pots were kept in

nursery condition for the study of post germination effect

if any and watered regularly once in a week. For each

treatment study minimum thirty plants were maintained.

Leaf surface area (LSA) of post germinated saplings was

recorded by graphical method at regular time intervals.

The LSA (cm2) results obtained at the end of 8th week

were used for interpretation. Leaf protein content (LPC)

was estimated from the same leaves used for

determination of LSA for each treatment grade by

following Lowry‟s method (1951) and expressed as

mg/100g fresh leaves.

RESULTS AND DISCUSSION

Seed index study reviled that seed weight is

0.7689 g (average), seed size is measured and was

ranging from 0.872±0.02 mm and seed colour exhibit

reddish brown to black, and shape was spherical. The

known empirical methods for breaking dormancy were

received by Maguire (1975) but it is evident that more

study is needed to develop effective germination

promotion techniques (Maguire, 1976). Recent studies

(Suresha et al., 2007) have proved that the seed size may

also responsible for variation in percentage of

germination in Sapindus emerginatus, where large size

seeds possessed higher germination percentage (98%)

than the medium (80%) and small (70%) and the biomass

production was found to be higher in seedlings produced

from larger seeds. However, seeds used in the present

investigation were not having any significant size

variation. Thus it is really seems to be very difficult to

make any firm conclusion that seed size may affect on

Journal of Research in Plant Sciences (2013) 2(2): 205-212 207

Kamble et al., 2013

S. laurifolius seed germination.

Pre-sowing treatments were given to seeds as

described earlier and seeds were considered germinated

when the coleoptile and radical extended the length of

the seed or more. The seed germination results after

pre-sowing treatments viz., physical seed scarification

and chemical seed scarification are presented in Table 1.

The analysis revealed that germination behavior differed

in all the treatments. Highest germination percentage was

observed in seeds scarified with needle pin technique

(76%), followed by sand paper treatment (56%). It is

very much vivid that needle pin technique has showed

best germination, but unfortunately applying this

technique for S. laurifolius seeds is very laborious as

well as time consuming. However, in the present work

physical scarification showed increase in germination

rate over controlled seeds (12%) (Figure 1). Although

physical scarification was enhancing the germination

percentage positively over control, however average time

duration required for germination was up to 25 days,

which was comparatively longer than the chemical

scarification method (14 days). Less germination

percentage was reported in mortar and pestle method

(31%) (Table 1), the most probable justification for it

might be that, most of the seed were prone to

mechanical injury caused during physical scarification.

The analysis for pre-sowing treatments of H2SO4

also revealed variation in germination behavior. In the

present work it was observed that, the rate of

germination percentage was declined with an increase in

the treatment time exposure beyond two minutes

duration. All the concentration grade of H2SO4 treatment

almost followed reciprocal increase in germination

percentage up to 2 min time exposure and then declined.

In general amongst the three grades of sulphuric acid

treatments germination was found very poor in

concentrated H2SO4 (34% /2.0 min-1), whereas highest

percentage germination (86% /2.0 min-1) was recorded in

2N H2SO4 followed by (80% /2.0 min-1) in 1N H2SO4

(Figure 2).

Laboratory methods for overcoming hard seeds

have been described for a range of species. Previous

studies suggested that, concentrated Sulphuric acid

scarification is more universally found to be effective in

removing seed coat impermeability ( Pe et al., 1975;

Febles and Padilla, 1971). In comparison with control,

our results also agree with these studies on breaking seed

dormancy with the help of concentrated sulphuric acid

scarification, however 2N H2SO4 and 1N H2SO4

treatments respectively were comparatively producing

best results. Recently, Fang et al., (2006) reported

a high seed germination percentage (98%) for

Cyclocarya paliurus (Batal) Iljinskaja seeds by applying

concentrated sulphuric acid for ten hours. In earlier

studies from tropical regions (Naidu et al., 1999) on

seeds of Acacia nilotica, Prosopis juliflora, and

Annona senegalensis including S. trifoliatus suggested

that some seeds have responded positively to Sulphuric

acid scarification. However, it is also suggested that,

such seeds should be collected directly from trees to

ensure the absence of holes prior to acid pre-treatment

(Mng‟omba, et al., 2007). Whereas, in present work

208 Journal of Research in Plant Sciences (2013) 2(2): 205-212

Kamble et al., 2013

Table 1: Effect of pre-sowing treatments on seed germination percentage of S. laurifolius.

Physical seed scarification Chemical seed scarification

Treatments Germination*

(%)

Treatments

(min-1)

Germination# (%)

0.15 0.30 1.0 2.0 3.0 4.0 5.0 6.0

Control 12 Control 12 12 12 12 12 12 12 12

Needle pin 76 Conc. H2SO4 18 26 28 34 30 28 12 12

Sand paper 56 1N H2SO4 34 48 57 80 16 12 12 10

Mortar & Pestle 31 2N H2SO4 20 28 44 86 24 20 24 16

(* observed at 25th day ; # observed at 14th day)

seeds were collected direct from trees at different places

as well as from local suppliers and it helped to evaluate

germination from randomly bulked seed sample.

Recently, Kumar and Devar, (2003) conducted study to

improve the germination percentage of soapnut by

treating the seeds with nitrogenous salts and recorded

improvement in germination percentage.

In the present work, post-germination effects on

saplings of S. laurifolius was also checked with reference

to leaf surface area (LSA) and leaf protein content. The

leaf surface area observed for saplings obtained from

physical scarification as pre-sowing treatments showed

non-significant variations but LSA values recorded

[Needle pin (3.10 cm2); Sand paper (2.55 cm2) and

3.20 cm2 for Mortar and Pestle] were comparatively less

significant over the control 3.55cm2 (Figure 3).

However, LSA recorded in pre-sowing treatments by

sulphuric acid scarification was showing, best results at

2 min time exposure [6.23 cm2: Conc. H2SO4, 4.12 cm2:

2N H2SO4 and 3.59cm2: 1N H2SO4] over the control.

Further than these values were declined with an increase

in treatment time exposure (Figure 4). In general

amongst the three grades of sulphuric acid treatments

best value of LSA was recorded in concentrated H2SO4

(Table 2).

The results obtained from pre-sowing treatments

on Leaf protein content (LPC) in post germinated

S. laurifolius saplings are presented in (Table 3). Leaf

protein content plays an important role in various

physiological reactions in plants and is also

corresponding to LSA values. In present work LPC

values observed for saplings obtained from

physical scarification as pre-sowing treatments were

non-significant over control (33.0mg/100g) such as

31.1 mg/100g (Needle pin); 30.6 mg/100g (Sand paper)

and 32.4 mg/100g (Mortar and Pestle) respectively

(Figure 5). However, LPC values observed for saplings

obtained from chemical scarification as pre-sowing

treatments were showing comparatively significant

results over control at 2 min time exposure

[41.5 mg/100g: Conc. H2SO4, 36.2 mg/100g: 2N H2SO4

and 38.5 mg/100g: 1N H2SO4]. Further these values were

declined with an increase in treatment time exposure

(Figure 6). In general amongst the three grades of

sulphuric acid treatments best value of LPC was

recorded in concentrated H2SO4.

Journal of Research in Plant Sciences (2013) 2(2) : 205-212 209

Kamble et al., 2013

Table 2: Effect of pre-sowing treatments on Leaf surface area (LSA) in post germinated S. laurifolius saplings.

Physical seed scarification Chemical seed scarification

Treatments LSA (cm2) Treatments-time

(min-1)

LSA (cm2)

0.15 0.30 1.0 2.0 3.0 4.0 5.0 6.0

Control 3.55 Control 3.55 3.55 3.55 3.55 3.55 3.55 3.55 3.55

Needle pin 3.10 Conc. H2SO4 3.15 3.52 3.35 6.23 5.24 4.62 4.42 3.76

Sand paper 2.55 1N H2SO4 2.22 2.25 2.37 3.59 2.96 2.46 2.21 2.15

Mortar & Pestle 3.20 2N H2SO4 2.65 2.80 3.10 4.12 3.83 3.40 3.23 2.83

Table 3: Effect of pre-sowing treatments on Leaf protein content (LPC) (mg/100g) in

post germinated S. laurifolius saplings.

Physical seed scarification Chemical seed scarification

Treatments LPC Treatments-time

(min-1)

LPC

0.15 0.30 1.0 2.0 3.0 4.0 5.0 6.0

Control 33.0 Control 33.0 33.0 33.0 33.0 33.0 33.0 33.0 33.0

Needle pin 31.1 Conc. H2SO4 31.3 33.7 34.5 41.5 39.0 35.0 37.5 36.0

Sand paper 30.6 1N H2SO4 28.5 29.0 31.5 36.2 30.8 30.3 27.5 26.4

Mortar & Pestle 32.4 2N H2SO4 30.6 30.5 31.2 38.5 36.8 35.1 32.8 30.2

Thus post germination data in the present work

comprising leaf surface area and leaf protein content

strongly supports that both the pre-sowing seed

treatments viz., physical and chemical scarification were

not causing any kind of adverse effect on growth

performance of saplings obtained. An earlier study on

Panicum maximum Jacq suggested that, seed coat act as

a competitor with the embryo for oxygen and probably

serves to explain the promoting effect of sulphuric acid

on germination of a number of species, (Smith, 1971).

Other species showed little response to sulphuric acid

(Cresswell and Nelson, 1972), where sulphuric acid is

promotive and there were complex interaction with

temperature, light and KNO3 (Smith, 1979), the possible

involvement of auxins was indicated by responses to

boron (Cresswell and Nelson,1972). Previously Thapa

and Gautam (2006), studied seed germination in

Sapindus mukorossi Gaertn, and suggested that, acid

scarification is the most effective pre-treatment for

quicker and higher germination of this species. They also

suggested that germination was increased with the

increase in the duration of soaking in acid, as there was

90% germination in 90min soaking in acid for

3 weeks. Our results also strongly agree with these

210 Journal of Research in Plant Sciences (2013) 2(2) : 205-212

Kamble et al., 2013

12

76

56

31

0

20

40

60

80

Control Needle pin Sand paper Mortar & Pestle

Figure 1

Physical seed scarification Germination (%) 0

20406080

100

0.15 0.30 1.00 2.00 3.00 4.00 5.00 6.00

See

d g

erm

inat

ion

(%

)

Seed treatment time (min)

Figure 2 Chemical scarification Germination (%)

Control

Conc. H2SO4

1N H2SO4

2N H2SO4

3.553.1

2.55

3.2

0

1

2

3

4

Control Needle pin

Sand paper

Mortar & Pestle

Figure 3

Physical seed scarificationLeaf surface area (cm2) 0

2

4

6

8

0.15 0.3 1 2 3 4 5 6

Leaf

su

rfac

e a

rea

(cm

2)

Seed treatment time (min)

Figure 4Chemical scarification & Leaf surface area

Control

Conc. H2SO4

1N H2SO4

2N H2SO4

33

31.130.6

32.4

29

30

31

32

33

34

Control Needle pin

Sand paper

Mortar & Pestle

Figure 5

Protein content (mg/100g fresh leaves)

01020304050

0.15 0.3 1 2 3 4 5 6

Leaf

Pro

tein

co

nte

nt

Seed treatment time (min)

Figure 6Chemical scarification & protein content (mg/fresh leaves)

Control

Conc. H2SO4

1N H2SO4

2N H2SO4

Figure 1-6 showing effect of pre-sowing treatments in S. laurifolius: Figure 1. Physical seed scarification and

seed germination percentage; Figure 2. Chemical seed scarification and seed germination percentage;

Figure 3. Physical seed scarification and Leaf surface area (LAC); Figure 4. Chemical seed scarification and

Leaf surface area (LAC); Figure 5. Physical seed scarification and Leaf protein content (LPC) in post

germinated saplings and Figure 6. Chemical seed scarification and Leaf protein content (LPC) in post

germinated saplings.

H2SO4

H2SO4

H2SO4

H2SO4

H2SO4

H2SO4

H2SO4

H2SO4

H2SO4

findings (i.e. acid scarification is the most effective

pre-treatment) with reference to S. laurifolius. According

to Thapa and Gautam (2006) the acid treatments up to

75-90min in concentrated hydrochloric acid can be used

to have higher and quicker germination in

S. mukorossi. However, in the present work we have

strongly proved that sulphuric acid treatment time

required for seed germination in S. laurifolius was

significantly very less (2min) and acid scarification

technique was not causing any kind of adverse effect on

saplings obtained.

CONCLUSION

A general problem of germination testing was

observed throughout the world in the variation between

testes performed in different laboratories. In the tropics

the problem is particularly acute because of variation in

available controlled environment facilities, in adequate

sampling procedure, substrata phytotoxicity or lack of

uniform judgment (Verma et al., 1976) which might be

responsible to create variations in germination results.

However, based on in-depth analysis of data obtained in

the present work, it is proved that, sulphuric acid is

promoting seed germination in S. laurifolius

comparatively in shorter time.

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