Reproductive Biology and Breeding System of Aconitum balfourii (Benth) Muk: A High Altitude Endangered Medicinal Plant of Garhwal Himalaya, India
Aconitum balfourii (Benth) Muk an endangered medicinal herb of high altitude region was studied for reproduction biology. Controlled pollination studies were also conducted on plants grown under hothouse. Observation reveals that ravine and scree wild habitats of alpine region had better flowers and seed production. Furthermore, hot house grown plants had far more superiority over wild populations for flowers and seed production. Protandry type of dichogamy was observed and is viewed as an anti-selfing mechanism. In general, higher pollen germination was achieved comparatively at low concentrations of GA3, IAA and IBA (1 ppm). Tube elongation was maximum upto 65 μm in IAA 1 ppm and 63 μm in IAA (5 ppm) and sucrose 5%. Dark condition along with violet color inhabits pollen germination whereas it enhances pollen tube elongation. Apomixis as well as autogamous self pollination was not observed in the species. However, fruit set differed significantly between the hand-selfed and hand-crossed treatments. Seed characteristics of open pollinated plants viz., number of seeds and seed yield per pod and plant were significantly at par than hand self pollinated flowers. Self-compatibility in the species may be a derived condition, considering that flowers are insect pollinated. The abundance and efficiency of pollinators may also affect mating patterns. The results of this study on the floral biology and breeding system of A. balfourii indicate reproductive potential of the species for cross-pollination, which would limit the production of selfed seeds and as such is likely to maintain sustainable levels of heterozygosity among the various populations.
to cite this article:
B.P. Nautiyal, M.C. Nautiyal, N. Rawat and A.R. Nautiyal, 2009. Reproductive Biology and Breeding System of Aconitum balfourii (Benth) Muk: A High Altitude Endangered Medicinal Plant of Garhwal Himalaya, India. Research Journal of Medicinal Plants, 3: 61-68.
Aconitum balfourii (Benth) Muk locally known as Meetha vish of
family Ranunculaceae is very important medicinal plant in Indian Traditional
System of Medicines (TSM) including Ayurveda as Vatsnabha. In fact, it
is cited by Susrutas (in Ayurveda) as one of the 13 bulb poisons and
4 varieties (species) of Vatasnabha among the 55 stable poisons. Information
on taxonomy (Stapf, 1905; Naitnani,
1984), chemical composition and active ingredients (Anonymous,
1985; Bahuguna et al., 2000) and uses (Thakur
et al., 1989; Uniyal, 1998) of the species
is available. As per the National Medicinal Plant Board (NMPB), Government of
India website, annual demand of the species during 2001-02 was 322.3 tons which
went up to 3426.8 tons during 2004-05 with an annual growth rate of 30%. Due
to great market demand, over and illegal exploitation from wild is going on
and the species is identified as an endangered (Nautiyal
et al., 2002) in Garhwal Himalaya and vulnerable (Vu) in entire Uttarakhand
by CAMP (2003). Therefore, to ensure sustainable utilization
as well as conservation of the species, cultivation is recommended (Nautiyal
and Nautiyal, 2004; Nautiyal et al., 2005).
Information on pollination biology not only required for comprehensive understanding
of the efficiency of breeding system of a species and its evolutionary success
but also for effective optimization of yield, conservation and rational genetic
improvement (Shivanna and Mohan Ram, 1993). Pollination
success in plants is determined by the timing of flower opening, anther dehiscence
and stigma receptivity. These depend on the environment/species interaction,
pollen vector accessibility and recognition events in the stigma and style.
The timing of these events generally differs between species and cultivars,
but their synchronization governs fruit set, yield and quality (Nautiyal
et al., 2009). A detailed understanding of the relationship between
these flowering events in A. balfourii is needed given that breeding
program efficiencies and production of new varieties/hybrids relies on manipulation
of processes such as pollination and the pollen-pistil interaction. Pollen germination
studies are essential for the estimation of quality of the pollen required for
controlled pollination. Artificial germination of the pollen grains is a surest
test of pollen fertility, which is important for the understanding of any breeding
programme. These aspects of reproductive biology in A. balfourii remain
unclear so far, only the pollen structure is studied by Sharma
(1985). Keeping in view all the above facts in mind, this study was aimed
at understanding the phenology and breeding system of A. balfourii so
that further genetic improvement can be done to meet the demand of TSM and pharameutical
MATERIALS AND METHODS
Four wild populations of A. balfourii i.e., Tungnath, Dayara, Kilpur and Panwali-Kantha, in alpine region of Garhwal Himalaya, India were selected and the reproductive phenology viz., time of anthesis, anther dehiscence, stigma receptivity, pollination and fruit and seed setting were recorded daily on ten randomly selected plants from August to October 2005. In order to estimate flower production, the total number of flowers per plant were counted manually in all the selected plants (N = 10) of each populations. The same procedure was carried out to quantify production of pods. Seeds per pod were estimated on ten selected pods per plant (N = 10 plants). These observations were aimed to identify the populations as best seed source for future conservation and crop improvement study.
Reproductive biology of species was studied at Alpine Field Station of High
Altitude Plant Physiology Research Centre (HAPPRC), located at Tungnath (3550
m.a.s.l.), Uttarakhand, India. The area lies between 30°14 N Latitudes
and 79°13 E Longitudes of Western Himalaya. Observations on flowering
time and duration, time of anthesis, anther dehiscence, stigma receptivity,
pollination, fruit and seed setting, were recorded daily from August to October
2005. Receptivity of stigma was analyzed with H2O2 method
(Dafni, 1992) However, controlled pollination studies
were conducted on plants those had been brought into cultivation in the hothouse
as a protective measure since hail storm and frosting is common feature of alpine
environment during this period.
Pollen counts were made on 5 anthers from different flowers. The anthers were
obtained from closed flower just prior to anthesis, placed in the small vial
containing 1 mL of glycerin 1%, smacked and pollen grains were suspended. From
this concentrate, five 10 μL droplets were removed and pollen grains were
counted under the microscope. Production of pollen grains per flower was estimated
by multiplying number of pollen grains per anther by the number of anthers per
flower. A factorial experimental design (Tuinstra and Wedel,
2000), was used to evaluate the effects of sucrose, IAA, IBA and GA3
(1, 5 and 10 ppm), thiourea and KNO3 on pollen germination. Sucrose,
boric acid and calcium nitrate have been shown to be key substrates for pollen
germination in other alpine species (Raina et al.,
2003; Nautiyal et al., 2009). Sucrose was
initially tested at 1, 5 and 20%; IAA, IBA and GA3, thiourea and
KNO3 were tested at 1, 5 and 10 ppm. The experiment was blocked in
time with five replications in a randomized complete block design. Pollen was
collected from undehisced anthers. Bulk of the pollen was distributed onto germination
media in cavity slides and placed at room temperature (15°C) for 52 h. Germination
was quantified as the percentage of germinated pollen grains per 100 evaluated.
Pollen grains were considered germinated when the pollen tube length was greater
than the diameter of the pollen grain (Tuinstra and Wedel,
2000). Pollen germination at different light conditions, viz., dark, blue,
green, violet and red, was also evaluated on optimal germination medium (5%
sucrose) to test the effect of light on pollen germination.
The assessment of breeding system involved randomly selected five plants in
each treatment. Following treatments were performed: (1) natural pollination-flowers
were not manipulated, (2) autogamous self pollination-buds were bagged throughout
their flowering period, (3) hand self pollination - bagged flowers were hand
pollinated with their own pollen, (4) open cross pollination- anthers were emasculated
and stigmas left for open pollination, (5) hand cross pollination-emasculated
bagged flowers were pollinated with pollen from another plant. Emasculation
were done with methods of Verma et al. (1979) approximately
16 h before anthesis and (6) apomixes-anthers and stigma of buds were clipped.
Fruit setting among hand-selfed and hand cross treatments and among open pollinated
and hand-cross treatments were also compared. An indirect measure of self-incompatibility
was obtained by dividing the average fruit set after self pollination by the
average fruit set after cross-pollination (Lloyd and Schoen,
1992). The value of one indicates complete self-compatibility.
Flowering and seed production potential of wild populations, cultivated in
an alpine garden and hothouse is presented in Table 1. It
appears that populations dominated by tree canopy had minimum flowers as well
as seeds per fruits, followed by scrub dominated populations. However, ravine
and scree habitats of alpine comparatively had better flowers and seed production.
Cultivation further improved flowers as well as seed production while hothouse
grown plants showed far more superiority over wild populations. Flowering is
asynchronous and lasts for 43 days (7 August-20 September). Flowers arranged
in straight racemose and the flowers at base bloom first which remain up to
10-15 days and gradually decreased up to 5 days in terminal flowers (Table
2). Floral display has been summarized in Table 3. Flowers
are many, tomentellous and bluish in color. Pedicels erect or lower ascending,
bracteoles if any, dentate and small. Sepals blue, pubescent, uppermost helmet
shaped, semi orbicular in profile, slightly convex in front and shortly beaked.
||Flowering potential of A. balfourii in different wild
locations, alpine garden and hothouse condition at Tungnath during 2006
|| Flowering phenology of A. balfourii (out of 50 plants
|| Floral biology of A. balfourii
The anthesis was highly temperature dependable and observed between 7:00 to10:00
am. The temperature in hot house conditions in between 08:00-10:00, 10:00-12:00
and 12:00-14:00 h of the day averaged at 10.7±0.5, 22.2±0.9 and
18.1±0.9°C, respectively. Whereas, the mean daily minimum and maximum
temperature in alpine condition was recorded as 6.5±0.3 and 20.7±0.7°C,
respectively. At low temperature and during night, corolla remains closed, therefore,
are temperature sensitive. This process continues till fertilization when stigma
lobes become dry and shedding of corolla start. Nectar is glabrous and odorless.
The number of flowers per plant in natural populations varied from 12-35, whereas
the plants grown under hot house conditions produce massive flowers and the
number goes up to 65 flowers plant-1.
Anthers dehisced longitudinally between 7.30 to 11.00 am; strongly depend on
higher level of temperature. Anthers numbered 20 per flower and the pollen grains
per anther varied between 43500-46500, which means an average of 90000 pollen
grains per flower (Table 3). Pollen remains viable only up
to 3 days after dehiscence. The anthers densely surround stigma up to 3 days
before anthesis and moves towards corolla and attain maturity thereafter dehisce
to discharge of pollen. Small gap thus separate stigma and anther which provide
passage of insect (mainly bumble bee) for pollination. Colour pattern of corolla
attract insect vectors to effect pollination. Anther dehiscence was not synchronous
rather they dehisced at different time for 5-8 days. The stigmatic lobes at
the time of anther dehiscence remain in adpressed conditions. After the completion
of anther dehiscence, stigmatic lobes start opening till 3-6 days, which was
the stage of stigma receptivity for pollen germination (Table
4). Table 5 and 6 summarize the data
on effect of different growth hormones, nitrogenous compounds (thiourea, KNO3)
and different light colors on pollen germination and tube elongation.
|| Different sequences in development of pollen and stigma receptivity
in A. balfourii
|| Effect of growth hormones and other medium on pollen germination
and tube elongation
|| Effect of light on pollen germination and tube elongation
The results revealed that pollen germination was higher in 5 and 20% sucrose
with significant variation. Among growth hormones treatments, the maximum germination
was observed in GA3, 1 ppm. In general, higher germination was achieved
comparatively at low concentrations of growth hormones. The low concentrations
of the nitrogenous medium (KNO3 and thiourea) also proved as better
germination medium but success rate of germination was lower than the growth
hormones and sucrose. Tube elongation was maximum upto 65 μm
in IAA 1 ppm. Analysis of data (single factor ANOVA) on pollen germination and
tube elongation revealed significant improvement due to different concentrations
|| Effect of different pollination methods on fruit/seed set
in A. balfourii species
These concentrations represent the requirements for optimal pollen germination
and pollen tube elongation. Even at optimal media composition, only 55% pollen
germination was observed.
The effect of different light colors on pollen germination and tube elongation
was also observed (Table 6) as the alpine region experiences
high intensity of solar radiation (Korner, 1999). Observations
revealed that dark condition and violet light inhibited pollen germination whereas,
dark condition enhanced tube elongation. The maximum pollen germination and
tube elongation was observed in red light. Furthermore, variation in pollen
germination (F = 14.37; p = 0.0) and pollen tube elongation (F = 51.30; p =
0.0) was found highly significant using single factor ANOVA.
The results from controlled pollination are summarized in Table
7. Flowers used to test for apomixes, did not set fruit. Fruit set differed
significantly between the hand-selfed and hand-crossed treatments with an ISI
value of 0.37 (Lloyd and Schoen, 1992). Seed characteristics
viz., number of seeds and seed yield per pod and plant were significantly at
par than hand self pollinated flowers.
Observations reveal that shade is detrimental factor for flowering and seed
production in wild habitats. Furthermore, domestication of the species in the
hot house showed optimal flowering and seed production. These results support
earlier observations (Nautiyal and Purohit, 2000) on
the species. Likewise, variation in the time of anther dehiscence and stigma
receptivity indicate protandry forms of dichogamy in individual flower to present
the potential for cross-pollination. Protandry in particular, is viewed as an
anti-selfing mechanism because it provides opportunities for the receipt of
outcross pollen before self-pollen is shed and is more common in self-compatible
than self-incompatible taxa (Lloyd and Webb, 1986; Bertin,
It appears that different growth hormones and nitrogenous compounds influenced
pollen germination and tube elongation differently (Setia
et al., 1985). Chhabra and Malik (1978) interpreted
that quiescent pollen contains RNAs and proteins needed for the tube emergence.
IAA pretreatments stimulate the synthesis of new RNAs and thereby increase proteins
needed for tube growth. Mascarenhas and Mermelstein (1981)
also emphasized the need of newly synthesized protein for tube growth. Over
the years, an array of plant growth hormones and other chemicals have been empirically
added to the culture medium to promote pollen germination and tube growth and
the positive effect of some of these substances have led to speculation about
their biochemical functions. Further, pollen germination and tube elongation
are two independent processes governed by separate set of conditions (Malik,
1985). Maximum germination and tube elongation in red colour suggest the
involvement of phytochromes, as red colour synthesize phytochrome protein and
its biological manifestation (Sharma and Malik, 1978;
The results on controlled pollination suggest predominantly self-incompatibility
in A. balfourii, as no fruit setting was observed from autogamous self
pollination which may be due to the existence of protandry as also observed
earlier in two high altitude herbs, Gentiana kurroo (Raina
et al., 2003) and Aconitum heterophyllum (Nautiyal
et al., 2009) although, few fruits developed from selfing. Such fruits
were smaller than the fruits produced by open pollinated and from hand-crossed
flowers and most aborted early in development. In general, flowering plants
possess a wide array of morphological and physiological mechanisms that influence
mating patterns, particularly the degree of self fertilization (Eckert
and Barrett, 1994). Temporal separation of male and female function (protandry
in this case) is one of the most widespread morphological mechanisms as found
in >75% co-sexual angiosperm species (Griffin et al.,
2000). Further, self-compatibility in the species may be a derived condition,
considering that its flowers are insect pollinated (Gituru
et al., 2002). In addition, the abundance and efficiency of pollinators
may affect mating patterns. However, observations on pollinators were not undertaken
during the course of this study. Nevertheless, aim of this study was to observe
floral and reproductive biology and breeding behaviour in A. balfourii
so that crop improvement programmes could be undertaken in future for better
in situ and ex situ conservation of genetic variability and cultivation
for commercial purposes, as the species, already has already been specified
vulnerable to endangered (CR) in the wild (Nautiyal et
al., 2002; CAMP, 2003).
The results of this study on the floral biology and breeding system of A. balfourii indicate the species reproductive potential for cross-pollination, which would limit the production of selfed seeds and as such is likely to maintain sustainable levels of heterozygosity among the wild populations. This fact furthermore could be useful in future crop improvement study considering the fact that there already exist a great heterozygosity among the wild populations of this very important medicinal herb of high altitude Himalaya.
Author (BPN) is thankful to researchers Dr. Rajeev K. Vashitha, Gunjan, Poonum, Anurag Dhyani and Karan Singh for their help during observations. The author BPN gratefully acknowledges the SERC division, DST, GOI, New Delhi for partial financial support.
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