Studies on the flowering phenology and pollen biology of ......10. Pollen ovule ratio 3802 : 4 11. Pollen type Tricolporate 12. Pollen shape Ovoid 13. Stigma type Semi dry 14. Nectar

Current Biotica 5(4): 405-412, 2012 ISSN 0973-4031

_________________________________________________________________________ www.currentbiotica.com

405

Studies on the flowering phenology and pollen biology of

Indigofera barberi Gamble, an endemic plant of Tirumala hills

P.L. Padmavathi

1*, K. Michael David

1, M. Subba Rao

2 and G. Rama Gopal

1

1Department of Botany, S.V. University, Tirupati – 517 502, A.P., India

2Agricultural Research Station, Perumallapalle – 517 505, Tirupati, A.P., India

*E-mail: [email protected]

ABSTRACT

A study of floral phenology and pollen biology of Indigofera barberi Gamble, an

endemic and rare plant of Eastern Ghats (Tirumala hills) was carried out in wild conditions. The

flowers are small, purplish red and are arranged in axillary recemes. Flowering got initiated in

July and ended up in February with peak flowering in 3rd

week of September. Pollen

germination/viability was maximum between 6.00 to 8.30 AM and no germination was noticed

after 1.00 PM. Germination and pollen tube growth were maximum at 6.30 AM in 12.5%

sucrose with Brewbakers medium. Anthesis of flowers was observed between 6.30 and 8.30 AM.

Anthers however, dehisced 12 hours before anthesis. It reproduced by means of cross pollination

aided by insects. Its narrow habitat range, fragmentation of population, habitat destruction and

low germination percentage of seed could be the reasons for its limited distribution in the wild.

KEY WORDS: Flowering phenology, Indigofera barberi, pollen biology, Tirumala hills

INTRODUCTION

Most of India’s natural vegetation

has been greatly modified by agriculture,

forestry and urbanization. Hence, enormous

plant wealth present in our country is

eroding fast due to habitat loss,

fragmentation, over exploitation, invasion of

exotics, pollution and climate change.

Nearly 5725 angiosperm species belonging

to 140 genera are considered as endemic

and/or endangered (33.5%). Tirumala hills

are one of the most outstanding vegetation

zones in Eastern Ghats, with rich floristic

diversity, some of them having great

medicinal, botanical and economic

importance. Eleven different plant species

are considered to be endemic to this region

and their conservation is very much essential

to preserve their diversity. Both in situ and

ex situ conservation approaches of plant

resources have to be based on the in-depth

study of plant reproductive biology.

Understanding reproductive biology of

plants is of immense practical importance

for biotechnology, for the conservation of

biodiversity and the control of invasive

species (Spencer C.H. Barret, 2010). Studies

on reproductive biology will help not only in

developing genetic potential of rare species

but also help in the re-introduction of rare

species.

The genus Indigofera is represented

by about 700 species which are found in the

tropical and sub tropical regions of the

world (Wills, 1985). This genus is

comprised of many trees, shrubs, annuals,

and perennials. Indigoferas prefer full sun

and moist, well-drained soil. Indigofera

barberi Gamble is a rare, endemic plant

mainly found in Eastern Ghats and is

distributed in Tamilnadu and Andhra

Pradesh. In Andhra Pradesh it is found in



406

Kadapa, Kurnool and Chittoor districts at an

elevation of 300 to 700 meters. In Chittoor

district, it is found in Sheshachalam hills. It

is an erect, perennial under shrub, having

high medicinal value. Entire plants including

flowers of this plant are well known in

treating jaundice and renal diseases. It also

has nephroprotective action (Palani et al.,

2008). The population of this plant species

is rapidly declining due to various factors

such as habitat destruction, fragmentation of

population and lower percentage of seed

germination etc. A comprehensive study on

the reproductive biology of this plant has not

so far been attempted due to its habitat

specificity. Hence, the present study on

understanding of flowering phenology and

pollen biology was under taken to find out

possible reasons for its limited distribution

in Tirumala hills.

MATERIALS AND METHODS

Plant material for the present study

was collected form Tirumala hills from

elevations ranging from 1512 ft. to 2259 ft.

above MSL located between 13º 39’ 2.3” N

latitude and 79º 38’ 45.3” E longitude. Ten

plants were selected randomly in the natural

habitat for observations on phenology,

pollination and pollen biological studies

during the year 2010-11. Flowering

phenology was observed at weekly interval

with respect to flower initiation,

development, anthesis, pollen biological

studies etc. Laboratories studies on pollen

morphology and other characters were

carried out in the Department of Botany,

S.V. University, Tiruapti and SEM studies

were carried out at Ruska Laboratory, S.V.

Veterinary University, Hyderabad. Pollen

size and anther size were measured with an

ocular stage micrometer under light

microscope. Pollen viability was assessed

by Flouro Chromatic Reaction (FCR) and

2,3,5-Triphenyl Tetrazolium Chloride (TTC)

test. To study the pollen germination in

vitro, pollen grains collected from the fresh

flowers were incubated in sucrose medium

of different concentrations (2.5 to 30%) and

Brewbakers medium (BBM). Pollen

morphology was studied by acetolysis

(Erdtmen, 1963) and SEM (John J. Bozzala

and Lonnie D. Russel, 1998). Pollen histo-

chemical studies were carried out by using

Iodine potassium iodide (IKI) solution for

the starch contents (Jensen, 1962) and Sudan

IV solution for the lipid contents (Vaissiere,

1991) in the pollen sample.

RESULTS AND DISCUSSION

Indigofera barberi started flowering

from the 3rd

week of July and extended up to

the end of February with a maximum bloom

in September 3rd

week to end of October

(Table 2 and Fig. 1). In plants, the

phenomenon of flowering involves a

transition from vegetative phase to

reproductive phase. Initiation of flowering

and duration of flowering largely depends

on environmental conditions to which the

plants are exposed. The transition from the

vegetative to reproductive buds is usually

triggered by an environmental signal,

typically photoperiod or temperature. This

signal synchronizes flowering to

environmental events. Thus, this is a type of

timing mechanism that plants use to

coordinate actions with the season. If

flowers are produced at the wrong time of

the year, the pollinator may not be available,

or it may be too dry (or wet), or there may

not be enough time before winter to allow

time for successful seed set. The flower

buds of I. barberi took 20 – 24 days from

initiation to full bloom. Detailed floral

characters are presented in table 1.

FCR test and TTC test confirmed

that the viability of pollen is 80% and 92%

respectively. Assessment of pollen viability

is a pre-requisite and also important in

studies on pollen storage, reproductive



407

biology and hybridization (Heslop-Harrison

et al., 1984). TTC and FCR tests are most

reliable and proven satisfactory in assessing

pollen viability in a number of species

(Shivanna and Heslop-Harrison, 1981). In

the present study, pollen grains are viable at

the time of anthesis and gradually lost

viability as the time progressed.

Table 1: Detailed floral characters of Indigofera barberi

Floral Characters Observations

1. Inflorescence Axillary receme

2. Flowering period July - February

3. Flower Bisexual, Zygomorphic with Papilionacious

corolla

4. Flower colour Purplish Red

5. Flower opening Between 6.30 AM to 8.30 AM

6. Number of anthers/flower 10 (9+1)

7. Anther dehiscence 12 hrs before flower opening

8. Average number of pollen grains/Anther 380.2 ± 3.56

9. Average number of ovules per flower 4

10. Pollen ovule ratio 3802 : 4

11. Pollen type Tricolporate

12. Pollen shape Ovoid

13. Stigma type Semi dry

14. Nectar glands A pair of nectar glands at the base of the

ovary and few pairs of extra nectarines on the

upper part of the stylar region

Table 2: Number of flowers per plant in I. barberi during the flowering period

Month I Week II Week III Week IV Week

Total No. of

flowers/month

July -- 8.5 ± 1.28 21.9 ± 3.85 63.3 ± 8.63 93.7

August 148.5 ± 7.81 241.4 ± 9.27 376.7 ± 16.24 552.9 ± 28.28 1319.5

September 800.8 ± 34.28 1079.7 ± 55.36 1703.2 ± 98.89 1579.3 ± 93.42 5163

October 1474.2 ± 98.24 1354.3 ± 95.75 1238.9 ± 92.77 1093.3 ± 85.35 5160.7

November 933.1 ± 81.5 768.3 ± 71.07 635.5 ± 64.36 534.1 ± 68.32 2871

December 430.5 ± 62.61 340.7 ± 57.51 256.9 ± 40.74 200.8 ± 33.26 1228.9

January 155.9 ± 27.39 113.4 ± 18.46 89 ± 13.88 56 ± 8.84 414.3

February 39.7 ± 6.38 26.3 ± 4.01 16 ± 2.23 9 ± 1.15 91



408

Fig. 1: Flowering phenology of I. barberi during 2010-11

Both maximum germination (88%)

and pollen tube growth (2618 ± 3.74 µm)

were noticed with BBM + 12.5% sucrose

after 30 minutes after initiation of

germination (Figures. 2 & 3). Germination

of pollen is the first morpho-genetic event

for the transport and discharge of sperm

cells into the embryo sac. To study the

structural and physiological details of

germination and tube growth in vivo studies

are difficult to perform due to the

involvement of pistillate tissue. In vitro

germination has been extensively used since

pollen grains of a large number of speicies

readily germinate in vitro on a simple

medium with a carbohydrate source, boron

and calcium (Shivanna and Rangaswamy,

1992).

Pollen were lipid rich and starch

poor (Fig. 4). Pollen morphological studies

through acetolysis and SEM revealed that

pollen grains were medium in size, oval

shaped, triangular in shape in polar axis,

isopolar, bilaterally symmetric, prolate,

tricolporate with thick nexine relative to

sexine. Exine surface was foveolate (Figure

5). The maximum average size of anther

(512 ± 5.60 X 500 ± 4.27 µm) and pollen

(40.5 ± 0.62 X 38.75 ± 0.67 µm) was

observed in the month of September.

Maximum pollen count i.e. 380.2 ± 3.56 was

also observed during September. Medium

size, triangular, tricolporate with psilate

exine was reported in different Indigofera

species by Marina Fernanda et al., 2008.

They have also reported that pollen of

different Indigofera species contained oil,

starch grains and protein bodies through

pollen histo- chemical studies.

Stigma receptivity test revealed that

the stigma was receptive 24 hours before

and after anthesis. In vivo pollen

germination was observed after six hours of



409

anthesis. Any success in plant breeding

experiments or artificial pollination

procedures should be accompanied by tests

during the period and duration of the

stigma’s receptivity. Stigma receptivity is a

crucial stage in the maturation of the flower

which might greatly influence the rate of

pollination, pollination success, the relative

importance of various pollinators, the

interference between male and female

functions, the rate of competition via

improper pollen transfer and the chances of

gametophytic selection (Evans, 1895).

Fig. 2: In vitro pollen germination of I. barberi pollen

in different sucrose concentrations in BBM

A. Bursting of pollen observed in BBM + 2.5% sucrose.

B. Pollen germination in BBM + 5% sucrose.

C. Pollen germination in BBM + 7.5% sucrose.

D. Pollen germination in BBM + 10% sucrose.

E. Pollen germination in BBM + 12.5% sucrose showing maximum pollen

germination (%) and pollen tube growth (without bursting).

F. Pollen germination in BBM + 15% sucrose.

G. Pollen germination in BBM + 17.5% sucrose.

H. Pollen germination in BBM + 20% sucrose.

I. Shrinked pollen in BBM + 25% sucrose.

A B

D F E

G H I

C



410

Fig. 3: Percent pollen germination of I. barberi at different timings of the day

Fig. 4: Pollen histo-chemical studies

A. Sudan III and Sudan IV test for lipids – Reddish Brown colour of pollen indicating that

the pollen are lipid rich

B. IKI test for carbohydrates – Absence of blackish blue colour indicating that the pollen

are poor in starch.

B A



411

Fig. 5: Scanning Electron Microscopic (SEM) images of pollen and image showing

pollen viability (FCR test) in Indigofera barberi

A. Release of pollen grains from dehiscing anther

B. View of pollen grains in different planes

C. Pollen grain in Equatorial view

D. Polar view of pollen grain

E. Surface view of pollen grain (pitted surface)

F. FCR test - Fluorescent images of viable (V) and Non viable (NV) pollen

A B

C D

E F

V

NV



412

CONCLUSION

Knowledge of phenology and floral

morphology are essential for conducting studies

on breeding systems, particularly on pollination

syndrome, if any. Therefore, reproductive

biology helps in developing strategies to

preserve genetic potential of rare species which

are crucial for restoration programmes. In the

present study extent of occurrence of I. barberi

is restricted to a very limited area; hence, the

availability of disjunct viable population is a

limiting factor for gene flow which greatly

affects the sexual reproduction. Further, the

seed germination is also poor in natural

conditions which may be one of the reasons for

poor seedling recruitment. Indigo barberi is

limited in distribution in the wild because of

narrow environmental niche, fragmentation of

populations, low percentage of seed germination

and heavy anthropogenic pressures. All these

causal factors either alone or in combination

with others are responsible for the endemism of

I. barberi.

REFERENCES

Erdtman, 1963. Pollen morphology and plant

taxonomy. The Cronica Botanica Co:

Waltham, Mass., U.S.A

Evans, M.S. 1895. The fertilization of the

Loranthus kraussianus and L. drgeri.

Nature. Lond. 51; 235-236.

Heslop-Harrison, J.,Heslop – Harrison, Y. and

Shivanna, K.R. 1984. Theor. Appl.

Gen., 67, 367.

Jensen, W.A. 1962. Botanical Histochemistry,

Freeman, San Francisco. Pp. 201.

John J. Bozzala. and Lonnie D. Russel. 1998. In:

Elelctron Microscopy - Principles and

Techniques for Biologists 2nd ed. Jones

and Bartlett Publishers, Sudbury,

Massachusetts. Pp. 19-24,54 -55 and 63

– 67.

Marina Fernanda Bortolin Costa, Juliana Villela

Paulino and Simone de Padua Teixeira.

2008. XX International Congress on

Sexual Plant Reproduction. Brasilla,

Brazil. P. 137 & 138.

Palani,S., Senthil Kumar,B., Praveen Kumar, R.,

Devi, K., Venkatesam, D and Raja

Sathendra. 2008 Effect of the ethanolic

extract of Indigofera barberi (L) in

acute Acetaminophen – Induced

Nephrotoxic Rats. Advanced Biotech.

7(3):28-31

Shivanna, K.R. and Heslop – Harrison, J. 1981.

Membrane state and pollen viability.

Ann. Bot. 47: 759-770.

Shivanna, K.R. and Rangaswamy, N.S. 1992.

Pollen Biology: A Laboratory Manural,

Narosa Publ. House, New Delhi, India.

Spencer C. H. Barrett. 2010.

Understanding

plant reproductive diversity. Philos.

Trans. R Soc. Lond B Biol. Sci.;

365(1537): 99–109.

Vaissiere, B.E. 1991. Honey bees, Apis mellifera

L. (Hymenoptera : Apidae), as

pollinators of upland cotton, Gossypium

hirsutum, L (Malvaceae), for hybrid

seed production, Ph.D. thesis, Texas A

and M. University, College Station.

Wills, J.C. 1985. A Dictionary of the Flowering

Plants and Ferns. Cambridge Univ.

Press, Cambridge

[MS received 18 January 2012;

MS accepted 21 March 2012]

.

Disclaimer: Statements, information, scientific names, spellings, inferences, products, style, etc. mentioned in

Current Biotica are attributed to the authors and do in no way imply endorsement/concurrence by Current Biotica.

Documents

Studies on the flowering phenology and pollen biology of ......10. Pollen ovule ratio 3802 : 4 11. Pollen type Tricolporate 12. Pollen shape Ovoid 13. Stigma type Semi dry 14. Nectar