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Research Article

Stigma receptivity of Corchorus aestuans L. with reference to in vivo

pollen germination and peroxidase activity

Pradyut Biswas

Department of Botany, Asutosh College, 92, S. P. Mukherjee Road, Kolkata 700026

pradyutbiswas79@yahoo.com

Article Info

Abstract

Received: 15-06-2017,

Revised: 16-07-2017,

Accepted: 17-07-2017

Stigma receptivity of Corchorus aestuans L., a wild edible and medicinally

important plant belonging to Tiliaceae is carried out with a view to find out the

optimum stigma receptive period in terms of in vivo pollen germination and

peroxidase activity in order to get information for successful fertilization after

completion of post-pollination events. Such feature of stigma is important in the

sexual life of a plant and it is also a basis for successful plant breeding

programme. The plant flowers during July to November. Flowers open at 08:45

h to 10:45 h. Anther dehisces by longitudinal slit prior flower opening. Stigma

showed maximum receptivity (60%) with mean 245±93.39 µm pollen tube

length after 3 h of flower anthesis. Intense amount of peroxidase are present all

over the stigmatic surface during high receptive period. The non-specific

peroxidase remains in scattered on the tri-lobed stigma. Such exudation on

stigma influences its receptivity towards the ability of stigma to support in vivo

pollen germination and tube growth for optimum success of fertilization.

Keywords:

Corchorus aestuans L., in

vivo pollen germination,

peroxidase activity, stigma

receptivity.

INTRODUCTION

The stigma is the localized part of the pistil and

pollen grains are trapped on the stigmatic surface

during pollination. Receptivity of the stigma is a

critical factor for successful completion after the

post-pollination events (Joshirao and Saoji, 1989).

Stigma receptivity confers the ability of the stigma

to support germination and tube growth of viable

pollen (Shivanna, 1998). Any successful breeding

event chiefly depends on the receptivity of stigma

(Joshirao and Saoji, 1989). Stigma receptivity

greatly influences the success of post-pollination

events at different stages in the life cycle of the

flower (Barrett, 2002). Mostly the stigma

receptivity is optimum soon after anthesis, but it

may vary from species to species and depends on

several environmental factors, such as temperature

and humidity (Shivanna and Johri, 1985). The

receptive surface of the stigma possesses some

proteins either as a pellicle in the dry stigma or as

an exudate in the wet stigma (Heslop-Harrison and

Shivanna, 1977; Heslop-Harrison, 1981; Shivanna

and Johri, 1985). Peroxidase is the important

component of the stigmatic exudation and its

copious presence is related to stigma receptivity.

Thus, receptivity of stigma in terms of in vivo

pollen germination with reference to peroxidase

activity of Corchorus aestuans L. belonging to the

family Tiliaceae has prime importance in the

biology of sexual reproduction. The plant is

medicinally important and used as leaf vegetables

(Biswas and Mondal, 2012). Objective of this study

is to determination of receptive period of the stigma

correlated with in vivo pollen germination and

peroxidase activity towards stigma receptivity

(Stone et. al., 1995; Lavithis and Bhalla, 1995;

Bhattacharya et. al., 2004, Choudhury et al., 2012,

Biswas and Mohammad, 2016).

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Pradyut Biswas

MATERIALS AND METHODS

Plants species of Corchorus aestuans L. growing at

Hooghly district (22°53´ N latitude and 87°50´ E

longitude) of West Bengal, India were selected.

Stigma receptivity was examined by the method of

Martin (1959) and Joshirao and Saoji (1989). For

this purpose 10 pollinated stigmas were fixing in

acetic alcohol (1:1) and softening with 4N NaOH.

Finally softening stigmas were stained with aniline

blue (water soluble aniline blue 0.05% in 0.05 M

Na2HPO4). The evolution of oxygen bubble per 2

minutes on the stigma as an indicator of peroxidase

activity was recorded by using 4% aqueous solution

of Hydrogen peroxide (Kearns and Inouye, 1993).

Microphotographs were taken by Olympus

Trinocular Microscope (Model: CH20i B1ME, No:

iIID 195) at low magnification (10X×10X) as well

as high magnification (10X×40X). For the study of

SEM, fresh stigmas were collected during their high

receptive period, washed with 0.015 M sodium

phosphate buffer (pH 7.2) and fixed in 2%

glutaraldehyde for 4 h. After that the stigmas were

dehydrated in an ethanol series of 50%, 70%, 80%,

90% v/v and absolute for 10 min in each grade. The

dehydrated stigmas were passed through a mixture

of ethanol and amylacetate at 1:1, 1:2 and 1:3 ratio

respectively for 5 min in each grade. The

dehydrated stigmas were preserved in pure

amylacetate solution. After critical point drying and

gold coating (IB2-ion Coater), photograph were

taken in Scanning Electron Microscope (Hitachi S-

530 SEM, Japan) to examine the surface pattern and

described following the terminology of Heslop-

Harrison (1992).

RESULTS AND DISCUSSION

Maximum 60% stigma showed in vivo pollen

germination along with mean 245±93.39 µm pollen

tube length over the stigmatic surface after 3 h of

flower anthesis towards its receptivity (Table 1 and

Figure 1). Prominent presence of peroxidase

enzyme (13.70±1.94 oxygen bubbles/2 minute) was

observed within 3 h of flower opening (Table1 and

Figure 1). Presence of copious peroxidase coincided

with its receptivity. Prominent presence of

peroxidase enzymes was observed during higher

receptive period of stigmas (Table 1).

Stigma is the ideal place for post pollination event

after successful placement of pollen. It is known

that stigmas pass through different functional

phases during their maturation- immature, receptive

and degenerated (Heslop-Harrison, 2000).

Receptive phase is more crucial in the sexuality.

Stigma stimulates most of the viable pollen to

germinate over the receptive surface and guide the

pollen tube for effective fertilization during pick

receptive period. Stigma receptivity is varied from

species to specie. Moreover, duration of stigma

receptivity is variable; sometimes it may last for a

day or may remain receptive for many days

(Montoya-Pfeiffer et al., 2016). In a condition of

protogyny and protandry, stigma remains receptive

for one or a few days before or after anthesis (Lloyd

and Webb, 1986; Williams et al., 1991). The wet-

type stigma secrets some exudates during their

receptive period, which contain lipids, phenolic

compounds, proteins, carbohydrates, lectins, amino

acids, phosphatase in addition to esterase and

peroxidase (Mattsson et al., 1974; Shivanna and

Rangaswamy, 1993; Lavithis and Bhalla, 1995;

Bhattacharya et. al., 2004; Bhattachary and Mandal,

2004; Chuodhury et. al., 2008; Kulloli and

Sreekala, 2009). Peroxidase activity has become

widely used as an indicator to assess the stigma

receptivity due to high enzymatic expression (Dafni

and Motte Maues, 1998). But active release of

stigmatic fluids including peroxidase depends on

morphology of stigma, vigor of the stigma and its

receptivity (Ghosh and Shivanna, 1984). In the

present investigation, maximum stigma receptivity

in terms of in vivo pollen germination and

peroxidase activity were obtained on the day after

anthesis. Similar findings on stigma receptivity was

investigated by Bhattacharya and Mandal (2004),

Choudhury et al. (2012), Dey et al. (2016), Biswas

and Mohammad (2016) and Choudhury et al.

(2017) in Moringa oleifera Lamk., Carissa

carandas L., Grewia asiatica L., Clerodendrum

inerme and Abrus precatorius L. respectively. Thus,

during high receptive period, peroxidase expression

becomes significant in particular time of anthesis.

Copious presence of peroxidase at the time of pick

receptive period of the stigma suggests contribution

of peroxidase towards stigma receptivity. So,

stigma receptivity in terms of in vivo pollen

germination with reference to peroxidase activity at

different time after flower anthesis is important for

successful breeding (Stone et al., 1995). A

successful breeding programme perhaps depends on

the timing and duration of the stigma receptivity.

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Bioscience Discovery, 8(3): 628-632, July - 2017

Table 1: Stigma receptivity of Corchorus aestuans L.

Period after flower

opening

Time of flower anthesis

09.00 h

(Bud stage)

10.00 h 11.00 h 12.00 h 13.00 h 14.00 h

(Closing

stage)

Total number of stigmas

observed

10 10 10 10 10 10

Total number of pollen

retained on the stigma

- 50 205 323 300 290

Average number of

germinated pollen

- 10 82 194 156 132

Percentage of germinated

pollen

- 42 40 60 52 45

Mean pollen tube

length(µm)

- 130±19.72

(R 100-

150,

N=10)

140±39.44

(R 75-200,

N=10)

245±93.39

(R 100-

375, N=10)

212±98.07

(R 100-400,

N=10)

180±76.19

(R 100-375,

N=10)

Number of evolved

bubbles/ 2 min in H2O2

solution

4.10±0.87

(R 3-5,

N=10)

5±0.81

(R 4-6,

N=10)

7.50±1.08

(R 6-9,

N=10)

13.70±1.94

(R 10-16,

N=10)

12.30±1.42,

(R 10-14,

N=10)

5.40±0.96

(R 4-7,

N=10)

Intensity of stigma

receptivity (Peroxidase

activity)

+ + ++ +++ ++ +

± Standard Deviation, N= Number of observation, R= Range. + = Low, ++= Moderate, +++=High and -

=Absent

ACKNOWLEDGEMET

The author is thankful to the Principal of Netaji

Mahavidyalay, Arambagh, Hooghly for providing

laboratory facilities. Technical help received from

Dr. Srikanta Chakraborty of University Science

Instrumentation Centre (USIC), The University of

Burdwan for Scanning Electron Microscopy is duly

acknowledged.

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Pradyut Biswas

Figure 1: Corchorus aestuans A. Plant habit, B. Single flower, C. SEM of stigma showing in vivo pollen

germination (×200), D. In vivo pollen germination (LM 10X), E. In vivo pollen germination (LM 40X) and

F. Peroxidase activity of stigma during pick receptive period. PG= Pollen Grain and PT= Pollen Tube

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How to Cite this Article:

Pradyut Biswas, 2017. Stigma receptivity of Corchorus aestuans L. with reference to in vivo pollen

germination and peroxidase activity. Bioscience Discovery, 8(3):628-632.