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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
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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
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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
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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
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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
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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
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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
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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.
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[MS received 18 January 2012;
MS accepted 21 March 2012]
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