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IV.3 Picrorhiza kurrooa Royle ex Benth.
(Scrophulariaceae)
IV.3a Study area:
The present study on Picrorhiza kurrooa Royle ex Benth. has been conducted in
Garhwal region of North - Western Himalayas. Several natural popualtions were
located around Kedarnath and Tungnath in Rudraparyag district of Uttrakhand,
India (Table IV. 32).
Table IV. 32 Different study sites of P . kurrooa in Garhwal Himalayas
S. No.
Name of the site
Altitude (m) (amsl)
1.
2.
3.
4.
5.
6.
7.
8.
9.
Vasuki taal -1 (VT-1)
Vasuki taal -2 (VT-2)
Vasuki taal- 3 (VT-3)
Bhairo Mandir 1 (BM-1
Bhairo Mandir 2 (BM-2)
Bhairo Mandir 3 (BM-3)
Sar parvat (SP)
Gandhisarovar (GS)
Tungnath (TN)
3758.8
3917
4446
3688.8
3739.5
3800
3540
3750
3680
IV.3b Habitat and Ecology:
At all the study sites, P. kurrooa was found growing on mountain slopes in the
alpine region which were dominated by herbaceous vegetation (Figs. 80a to f).
Most of these areas remain under snow between November to June and vegetation
appears after the snow melts. Soil of these habitats was found to be black, gravelly
and humus rich. Plants of this species grow in patches along the sides of glacier
fed springs or even inside these springs in loose gravelly soil (Figs. 81a to f).
During the study, three different morphoforms of P. kurrooa were observed.
These morphoforms had specific habitat preferences and were different from each
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other in several vegetative and floral features. The change in morphology was
observed along the altitudinal gradient as the habitat conditions change from open
sunny slopes at lower altitudes (Figs. 80 b to c) to shrub covered shady mid
altitudes (Fig. 80 d) and to open sunny slopes partially covered with snow on the
top most portions of slope (Figs. 80e to f).
IV.3c Population Structure:
This species propagates by vegetative means through stolons (Figs. 82a to b).
Stolons profusely branch and produce closely spaced offshoots which consist of
tuft of leaves above and adventitious roots at the lower side. Due to this mode of
propagation, its populations in nature occur in patches (Fig. 83). Each offshoot in
a patch is capable of establishing itself independently and was therefore,
considered as a single individual while making assessments of population size.
During the flowering season, inflorescences are produced by some of these
offshoots. However, the number of vegetative offshoots bearing the inflorescences
was found to be very low (Fig. 83). Ratio of the flowering to vegetative shoots in
a patch in different populations is given in table IV. 33.
IV.3d Associated plant species:
Habitat range of P. kurrooa was found to be very rich in diversity of annual as
well as perennial angiosperms. P. kurrooa grows in association with species of
Polygonum, Oxalis, Euphorbia, Potentilla, Rubia and many others. A species of
Rhododendron typically formed a canopy at mid altitudes over ground vegetation
in all habitats surveyed for the species (Fig. 84a). One morphoform of P. kurrooa
also grows under the canopy of this species (Fig. 84b).
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Table IV. 33. Ratio of flowering to vegetative shoots in different
populations of P. kurrooa
S. No. Population code
1.
BM-1
Ratio of flowering
to the vegetative shoots
3 : 113 (n = 14)
2.
BM-2
20 : 405 (n = 2)
3.
BM-3
57 : 194 (n = 4)
4.
VT-1
44 : 326 (n = 12)
5.
VT-3
67 : 490 ( n = 8)
6.
TN
0 : 700 ( n = 7)
7.
SP
1 : 444 (n = 9)
n - number of patches examined.
IV.3e Habit:
Picrorhiza kurrooa is a perennial stoloniferous herb (Fig. 82a). Stolons appear to
be the chief mode of propagation as well as perennation in this species. The
plants remain under the snow for a considerable period of time. Stoloniferous
mode of vegetative propagation leads to the formation of patches with densely
spaced offshoots in the species (Fig. 83).
IV.3f Plant Morphology:
As elaborated before, plant is a stoloniferous herb. Stolons grow horizontally
above the soil in morphoforms - 1 and just beneath the soil in morphoforms - 2
and - 3. Stem is herbaceous and non woody during young stages but becomes
woody later on. Dried leaf traces are present on stolons. Main root is taproot
arising from a swollen base whereas fibrous roots arise at the point of emergence
of leafy offshoots. Leaves cauline, sessile, spathulate with slightly sheathing base,
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margins dentate and apex acute. Venation is unicostate reticulate, leaves arranged
in rossettes with overlapping leaf bases. Leaf surface glabrous but sparse hairs or
trichomes (2-3 celled) are present along midrib on abaxial side.
Erect flowering axis or peduncles arise from the axils of the youngest pair of
leaves in some of the offshoots during flowering season. Peduncles bear nearly
sessile flowers arranged spirally around the axis thus forming dense spikes
(pedicel 0.5 - 1mm) above half of their length. A pair of sterile bracts is usually
present at the base of inflorescence. Fruit is a capsule (Fig. 85a), divided into two
locules by a thick septum. It is conical in shape with a broad base and narrow
tapering apex, which splits up vertically at maturity to form a short suture (Fig.
85b). Flowers are nearly sessile (Fig. 85c) but fruits are stalked as the pedicel
elongates upto 7mm. in fruit. Seeds are minute and membranous (Fig. 85d).
IV.3g Morphological Variants or morphoforms of Picrorhiza
kurrooa:
Three morphoforms of the species differing from each other in certain vegetative
and floral features were found in different habitats. Each of these morphoforms
conform to the same basic taxonomic description for the species, but vary in some
qualitative as well as quantitative traits (Tables IV. 34, 35 & 36). These
morphoforms remain associated with specific habitat conditions which seem to be
preferred by each of them. Each of them therefore, restricts itself to a portion of
the mountain slope along the altitudinal gradient. Middle parts of mountain slope
are dominated by a species of Rhododenron which grows as a trailing shrub and
forms a dense canopy all over the area. Due to the presence of this thick canopy
cover ground surface do not receive direct sunlight. Over such shady ground
surface morphoform - 1 was found (Figs. 86a to b). In the transitional zone
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between open slopes exposed to direct sunlight and parts where the shrub begins
to appear, morphoform - 3 was found (Figs. 86a to c). Lower as well as the top
most parts of the slope are exposed to sunlight and are not covered by shrub
canopy. In these parts morphoform - 2 was found (Figs. 86d to e). Morphological
differences in floral and vegetative characters were recorded and are tabulated in
tables IV. 34 and 35. Besides these, some basic qualitative traits were also
compared (Table IV. 36).
Table IV. 34 Comparative account of some morphological characters in three
morphoforms of Picrorhiza kurrooa
S. No Character
1.
2.
3.
4.
Morphoform - 1 Morphoform - 2
17.8 ± 3.2*
(11 - 23.5)**
n = 41
No. of leaves per 9 ± 2
offshoot.
(6 - 17)
n = 58
Leaf length (cm) 12.9 ± 2
(5.7 - 18)
n = 133
Leaf width (cm) 2.8 ± 0.4
(2 - 4.8)
n = 133
Height of spike
(cm)
*- Mean ± SD; ** - Range; n - sample size
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7 ± 2.7
(1.9 - 13.2)
n = 68
9 ± 1.8
(4 - 13)
n = 59
3.8 ± 1.5
(1.4 - 9.1)
n = 189
1.3 ± 0.3
(0.7 - 2.5)
n = 189
Morphoform - 3
12.9 ± 2.6
(6.6 - 20.1)
n = 51
10 ± 1.7
(7 - 14)
n = 58
7.1 ± 1.8
(4 - 11.5)
n = 133
2.1 ± 0.3
(1.5 - 3.1)
n = 133
4.3h Description of floral structure in different morphoforms:
3h (i) Morphoform - 1:
This type grows under canopy of a Rhododendron shrub (Figs. 86a to b). Average
leaf number and size observed in this type was 9 ± 2 (6 - 17) and 12.9 x 2.8 cm
respectively. Average inflorescence length in this from was 17.8 ± 3.2 cm (Fig.
87a) (Table IV. 34). Flowers bracteate, shortly stalked, pentamerous, complete,
zygomorphic, hermaphrodite and hypogynous (Fig. 87b). Flower has one linear
bract with entire margins and tapering, acute apex (Fig.87c). Calyx lobes 5,
polysepalous, 4 lobes equal, 1 on the anterior side of flower narrower and shorter
(almost half) than the rest. Corolla green, gamopetalous, 5 linear, acute lobes
radiate from a very short rim like corolla tube (Fig. 88b). The two anterior petals
showed a tendency to fuse along their length, which is demarcated by a deep
notch between these petals. Corolla lobes valvate at base but margins slightly
overlap. Corolla pubescent on interior surface and gynoecium consists of a
bicarpellary syncarpous superior ovary placed on a small cushion like disc. It has
axile placentation with multiple ovules in each locule. Style single, upright
terminating to a simple and wet stigma. Androecium consists of 4 stamens of
same length, obdiplostemonous, anthers bilobed, basifixed, extrorse, and
dehiscing by longitudinal slits. Calyx and style are persistent in fruit. Usually
smaller lobe is ephemeral and fruit has 4 calyx lobes only.
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Table IV. 35 Floral morphometry of three morphoforms of Picrorhiza kurrooa
S. No
1.
Character
No. of flowers
per inflorescence
2.
Length of Bract
(mm)
3.
Width of bract
(mm)
4.
Length of corolla
(mm)
5.
Width of corolla
(mm)
6.
Length of pistil
(mm)
7.
Length of stamen
(mm)
8.
Length of calyx
(mm)
9.
Width of calyx
(mm)
10.
Stigma size (μ)
Morphoform - 1
32 ± 12.4*
(7 - 55)**
n = 42
10 ± 1.3
(8 - 12.5)
n = 30
2.3 ± 0.5
(2 - 3.5)
n = 30
5.6 ± 0.8
(4 - 7)
n = 30
4.6 ± 1
(3 - 7)
n = 30
19.8 ± 2.3
(13 - 24)
n = 40
16.6 ± 2.4
(6 - 20)
n = 30
9.5 ± 1
(8 - 120)
n = 30
2 ± 0.1
(1.5 - 2.5)
N = 30
56.75 ± 5.8
(35 - 87.5)
n = 37
*- Mean ± SD; **- Range; n - Sample size
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Morphoform - 2
11 ± 2.3*
(6 - 18)**
n = 80
6.5 ± 2
(4 - 11)
n = 30
3.5 ± 1
(0.5 - 6)
n = 30
10.6 ± 0.9
( 9 - 12)
n = 30
5.5 ± 0.8
(4 - 7)
n = 30
10.8 ± 2.3
(8 - 17)
n = 30
Upper pair= 8.2 ± 2.2
(5 - 13)
n = 30
Lower pair= 9.2 ± 1.7
(5 - 13)
n = 30
6.12 ± 0.9
(5 - 8)
n = 33
2.8 ± 0.4
(2.5 - 4)
n = 33
170.35 ± 3.7
(150 - 187.5)
n = 14
Morphoform - 3
17 ± 5.3*
(10 - 37)**
n = 73
8.25 ± 0.79
(6 - 10)
n = 33
3.2 ± 0.5
(2.5 - 4.5)
n = 33
7.26 ± 0.9
(5 - 9)
n = 30
4 ± 0.6
(3 - 5)
n = 30
15.5 ± 2.5
(11 - 20.5)
n = 40
15.45 ± 3.7
(13 - 18)
n = 20
9.1 ± 0.9
(6 - 10)
n = 20
2.2 ± 0.2
(1.5 - 2.6)
n = 20
77.37 ± 7
(25 - 110)
n = 40
Table IV. 36 Comparison of some distinct characteristics of 3 morphoforms
of P. kurrooa.
Character
Pigmentation
Bract
Morphoform - 2
Inflorescence axis,
Morphoform – 3
Inflorescence axis,
staminal filaments,
staminal filaments,
style and corolla
style and corolla
deep purple.
light purple.
Linear, straight,
Ovate, twisted along
Linear, straight with
margins entire.
length and
entire margins in
margins dentate.
most flowers ,
Morphoform - 1
Green on all parts.
in a few basal
flowers, ovate
with dentate margins.
Corolla
Stamens
Green, pentamerous,
Deep purple, distinctly
Same as in M - 1, except
gamopetalous, corolla
bilipped, 2 anterior lobes
for light purple colour
tube short, sparsely
form hood, Densely
and larger size, apical
pubescent, 2 anterior
pubescent. Apical
notch marking fusion
lobes fused having a
notch inconspicuous.
of 2 anterior petals is
deep notch.
.
shallower than M - 1.
All the 4 of same length, Slightly didynamous,
All the 4 of same length,
extended out of corolla,
confined to the corolla
extended out of
about 3 times the length
tube.
corolla, about 3 times
of corolla tube.
the length of corolla
tube.
Pistil
Stigma simple,
Stigma capitate, style
Stigma simple,
style remains
straight initially but
style remains
straight throughout,
becomes deflected
straight throughout
flower development.
downwards after anther
flower development.
dehiscence.
3h (ii) Morphoform - 2:
This type grows on exposed sunny slopes both at the bottom and top of the mountain
(Figs. 86d to e). Average leaf number and size observed in this type was 9 ± 1.8 and 3.8 x
1.3 cm respectively. Average inflorescence length in this morphoform was 7 ± 2.7 (Table
84
IV. 34). Flowers bracteate, with a small pedicel, complete, zygomorphic, pentamerous,
hermaphrodite and hypogynous (Figs. 87a to c). Bract 1, ovate with dentate margins and
acute apex, twisted along the length. Calyx lobes 5, unequal, smaller lobe anterior. Sepals
ovate, have dentitions in some of the inflorescences and also slightly twisted like the
bract. Mostly the anterior sepal sheds at early stages of flower maturation and mature
flowers are observed with only 4 sepals.
Corolla deep purple and gamopetalous (Fig. 87a). Anterior pair of petals united to form
hood where as posterior most petal forms the lower lip, 2 petals are lateral in position. Tip
of the hood is marked with 2 - 3 dentitions; however, the notch is inconspicuous.
Gynoecium consists of a superior, bicarpellary, syncarpous ovary placed on a cushion like
disc. Style single, upright, terminating to capitate and wet stigma. Placentation axile,
ovules many in each locule. Androecium consists of 4 slightly didynamous stamens, 2
upper stamens are at each margin of the hood petal and lower 2 at each margin of the lip.
Anthers bilobed, basifixed, dehiscence extrorse by longitudinal slits. Calyx and style
persistent in fruit.
3h (iii) Morphoform - 3:
In the transitional zone between open slopes exposed to direct sunlight and parts where
the Rhododendron shrub begins to appear, morphoforms - 3 was observed (Figs. 86a to
c). Average leaf number and size observed in this type was 10 ± 1.7 and 7.1 x 2.1 cm
respectively. Average inflorescence length in this from was 12.9 ± 2.6 (Fig. 87a) (Table
34). Floral structure of morphoforms - 3 is similar to that of morphoforms - 1 except for
the light purple tinge and somewhat larger corolla (Fig. 87b). The notch representing the
fusion of 2 anterior petals is also comparatively shallower in morphoform - 3 than that in
morphoform - 1.
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IV.3i Flowering:
Flowering initiates in the 2nd week of June at lower altitudes and 2nd to 3rd week of July at
higher altitude regions of the slope. Thus there is a temporal separation in the flowering
time in morphoforms growing at these parts of slope. By the time flowering begins in
populations on top most altitudes beyond the shrub cover, fruit set gets completed in
populations located at the lower most altitudes of a slope. However, morphoforms – 1 and
- 3 which are located in the middle part of slope in transition zone or under the shrub
show partial overlapping in their flowering time both with lower and top most
populations.
IV.3j Pattern of flowering within an inflorescence:
Within an inflorescence flowering proceeds from base to the top i.e. in acropetal
succession. This pattern is same for all the 3 morphoforms. In morphoforms - 2 after the
basal most flowers open, the entire inflorescence blooms within 2 - 3 days and during this
period all the flowers of inflorescence remain open. In morphoforms - 1 and - 3, owing to
the larger size of inflorescence, by the time flowers of the upper half of the inflorescence
begin to open, corolla of the flowers in the lower half of inflorescence is shed and fruit set
begins.
IV.3k Floral biology:
3k (i) Time and pattern of anthesis:
Flowers begin to open early in the morning at about 6 am. Generally anthesis begins
simultaneously in 3 - 5 flowers in sequence of maturity i.e. from bottom to the top of the
inflorescence. Anthesis continues to occur up to 2 - 3 pm in flowers from base to apex of
the inflorescence. All the 3 morphoforms of P. kurrooa exhibit herkogamy (Fig. 87).
Protogyny is predominant in all of them (Figs. 87b to d). Occasionally in some of the
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inflorescences of morphoform - 2, a few flowers were found to be protandrous (Fig. 88a).
In typically protogynous flowers of all morphoforms, anthesis is marked with the
emergence of stigma out of the folded corolla in the bud (Fig. 88b). In morphoforms - 1
and - 3, stigma is simple and inconspicuous (Figs. 89a and c), but in morphoform - 2,
stigma is very prominent, capitate (Fig. 89b) and pale yellow in colour at anthesis. At this
stage, length of staminal filaments is negligible and all the 4 anthers lie at the base of
corolla tube surrounding the ovary. After 6 -7 h of beginning of anthesis, one pair of
stamens (anterior one) starts elongating and emerges out of corolla tube. By this time
style also attains its maximum length i.e. about 3 times the length of corolla tube in case
of morphoforms - 1 and - 3 and just under the tip of hood petal in morphoform - 2. In
later, colour of stigma also changes from pale yellow to purple and sticky exudates also
appear. The two elongating stamens reach the level of stigma in another 6 - 7 h. Anther
dehiscence also begins in this pair. Meanwhile the 2nd pair of stamens also emerges out of
corolla tube and at this stage stamens appear didynamous in morphoforms - 1 and - 3.
Corolla opens due to the force exerted by the elongating stamens. It takes about 30 - 31
hours for all the stamens to reach the same level i.e. at the level of stigma. Anther
dehiscence is complete in all the 4 anthers in about 33 hours. In morphoforms - 1 and - 3,
style remains positioned along the central axis of flower whereas 4 stamens are directed
outwards. However, in morphoform - 2, style takes a sharp bend (Figs. 90a to c) thus
deflected away from the closely placed anthers. Style remains healthy and intact for about
45 hours after which it withers and droops. Stigma also turns brown and shrivels. Anthers
are shed and corolla tube is detached in 50 - 55 hours. A few flowers in some
inflorescences of morphoform - 2 were observed in which stamens have elongated up to
87
maximum, anthers dehisced, but style is still in the corolla tube (Fig. 91a). The style
emerges late and takes a position just under the hood or even below that point (Fig. 91b).
3k (ii) Stigma receptivity:
Stigma receptivity was checked by manual pollinations of stigmata after different time
intervals right from bud condition to senescence of the flower revealed that in all the
morphoforms stigma turns receptive with the onset of anthesis i.e. when they are just 1 - 2
mm outside the corolla tube. However, stigma is partially receptive even in the buds
ready to open. Peak receptivity was found to be between anthesis and 52 hours (even after
the anthers are shed and corolla tube has detached) after which the receptivity declines
and after 60 hours of anthesis, styles whither completely. Randomly collected pistils from
different populations were checked for pollen load on stigma under open pollination
conditions. Average pollen load per stigma was maximum for morphoform - 2 (49) and
minimum for morphoform - 3 (9). For morphoform - 1, it was 20 on an average.
3k (iv) Pollen ovule ratio:
Pollen - ovule ratio in this species was observed to be very high. Average number of
pollen grains produced per flower is 36,420, 36,336 and 33,680 respectively for
morphoforms - 1,
- 2 and - 3. Average number of ovules per ovary is respectively 50, 54
and 50 for these morphoforms (Table IV. 37). Thus, number of pollen grains available for
siring one ovule is respectively 728, 673 and 674 for the three morphoforms.
3k (v) Pollen shape and size:
Pollen grains appear as deep yellow powdery mass sticking to the dehisced anthers.
Pollen grains were observed to be spherical, with a smooth exine and 3 germ pores when
examined under a compound microscope. Pollen grains measured 23 μ, 25 μ and 22.5μ
respectively in morphoforms - 1, - 2 and - 3 respectively (Figs. 92a to c).
88
3k (vi) Pollen stainability and viability:
Pollen viability of the species was found to be very high when tested using 1%
acetocarmine and FCR test (Figs. 92a to d). Average stainability of pollen was found to
be 95.6% for morphoform-1, 90.15% for morphoform - 2 and 91.75% for morphoforms 3 (Table IV. 37).
Table IV. 37 Observations on pollination biology in three morphoforms of
Picrorhiza kurrooa
S. No. Parameter
1.
Pollen per flower
2.
Ovules per flower
3.
4.
5.
6.
Pollen load on open
Pollinated stigma
Pollen size
Pollen stainability in
1 % acetocarmine
Morphoform - 1
36, 420
49.5 ± 9.8*
(33 - 68)**
n = 50
19.8 ± 19.5
(0 - 48)
23 ± 0.3
95.6 % ± 6.1
(76.1 - 100)
Pollen viability in FDA 91.3%
*- Mean ± SD; **- Range; n - Sample size
Morphoform - 2
36, 336
53.9 ± 10.9*
(30 - 71)**
n = 50
49.45 ± 28.3
(5 - 107)
24.95 ± 0.3
90.15 ± 14.5
(56.3 - 98.6)
Morphoform - 3
33, 680
49.6 ± 16.6*
(31 - 122)**
n = 50
9.4 ± 9.9
( 0 - 43)
21.9 ± 0.9
91.75 ± 9.2
(67.3 - 98)
89.2%
90.7%
IV.3l Pollination:
Mechanism of pollination in this species is not clear. Even after repeated field visits
during different times of day no insect activity was noticed in any of the habitats. A
species of Bombus was the only insect that could be occasionally viewed on clear, sunny
days with calm atmosphere in the whole habitat range of this species. But this insect did
not visit the flowers of P. kurrooa as it was attracted by other bright, scented and nectar
rich flowers of many other herbaceous species that grow in close association to P. kurroa
in all the habitats (Figs. 93a to c). Aphids were observed in association with the flowers
of this species during later periods of the season. Fluorescence microscopic studies of
open pollinated and manually pollinated pistils to study the pollen-pistil interaction,
89
revealed that pollen tubes originating at stigmatic surface traverse through the style
without any hindrance and enter both the locules of ovary and enter the ovules
successfully.
IV.3m Compatibility status of different morphoforms of P. kurrooa:
Analysis of pistils of different morphoforms that were subjected to manual selfing and
inter morphoform pollinations under Fluorescence microscope showed that species is self
as well as cross compatible and no pre-fertilization compatibility barrier exists among the
morphoforms (Figs. 94a to f).
IV.3n Reproductive output:
Average fruit set per inflorescence under open pollination conditions was calculated for
each morphoform. In morphoforms - 1 it was 70%, in morphoform - 2 it was 47.2% and
in morphoform - 3 it was 57%. Average seed set per capsule was 38%, 64.8% and 74%
respectively for the three morphoforms.
IV.3o Fruit and seed dispersal in the species:
During surveys of the natural habitats, it was observed that autonomous fruit as well as
seed dispersal mechanisms are lacking in this species. This was evident from the fact that
in most of the populations surveyed, dried inflorescences with fruit formed persisted up to
next flowering season with most of the fruits intact and filled with seeds (Figs. 95a to b).
Although an apical suture for seed dispersal is present in all these fruits, due to upright
position and absence of biotic agents in these regions the seeds are retained within
capsules for as long as one year. This is inspite of the heavy snowfall occurring in the
region. Only a few fruiting spikes that come to lie within the swiftly flowing glacier fed
springs are washed away and taken downstream by water. Some of the fruiting
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inflorescences were found to be grazed upon by livestock in lower parts of the slope
accessible to them.
IV.3p Seed germination and seedling survival in the species:
Seeds of Picrorhiza kurrooa showed very poor germination under ex situ conditions.
Germination experiments conducted using different substrates like sand, moist filter paper
and garden soil mixed with manure yielded poor results as out of about 700 seeds tried for
germination, only 19 germinated between March - May at room temperature in garden
soil mixed with manure. None of these seedlings survived beyond 10 days. In nature also
no juvenile patch or seedling was observed. A few small patches were however found
growing at sites mostly along the stream, far away from the main populations. But
whether these patches are remnants of a previously large population or have appeared as a
result of the germination of seeds brought downstream by water was unclear.
IV.3q Ex situ conservation:
Attempts to raise the plants under ex situ conditions both through seed as well as
vegetative means did not succeed because of stringent and very specific habitat
preference of the species. Plants deposited in the Lead Botanic Garden from wild
survived for two seasons but did’t bear flowers.
IV.3r Analysis of genetic diversity among three morphoforms of the
species using ISSR markers:
Total genomic DNA was extracted from representative samples of three morphoforms
following the CTAB method (Doyle and Doyle, 1990) with minor modifications and
checked for its quality and quantity on 0.8% agarose gel (Fig. 96). Accordingly
dilutions were made and subjected to primer screening. Total 20 random ISSR
primers were tested, of which 12 selected on the basis of robustness of amplification,
clarity and scorability of banding patterns, were employed for diversity analysis.
91
Among 41 genotypes from 6 populations, 80 bands were generated of which 7 to 38
(8.75% to 47.5%) were polymorphic within populations. The range of bands
generated per primer was 4 - 9, in the size range of 250 – 2000 bp, with the average of
6.6 bands per primer.
Analysis of ISSR data revealed lowest frequency of polymorphic bands (8.7%) in
Long Vasukitaal (LV) morphoform while Long Bhairo Mandir (LB) morphoform had
the highest polymorphism (47.5%) (Fig. 97; Table IV. 38). Shannon information
index and Nei’s gene diversity were lowest for LV population (H = 0.0437; I = 0.060)
and highest for LB population (H = 0.1583; I = 0.240) (Table IV. 38).
Shannon index based analysis calculated the total species diversity (HT) 0.1908,
average diversity within morphoforms (populations) (HS) 0.0943 and among
morphoforms (populations) (GST) 0.5057 (Table 39). The value of gene flow (Nm)
was 0.4888. Intermediate Bhairomandir (IB) morphoform and Long Bhairomandir
(LB) morphoform showed highest (0.9588) while Intermediate Vasukitaal (IV) and
Small Vasukitaal (SV) morphoforms showed least genetic identity (0.7570) (Table
IV. 40). Neighbour joining tree showed two major clusters, one comprising Small
Vasukitaal (SV) and Small Bhairomandir (SB) morphoforms and the other comprising
the rest (Figs.98).
.
92
Table IV. 38 Polymorphic percent, Nei’s genetic diversity (H) and Shannon’s
information index (I) for ISSR in P. kurrooa
Population
H (mean)
I (mean)
H(SD)
I (SD)
IV morphoform
Polymorphic
%
21.2
0.0720
0.1089
0.1521
0.2223
IB morphoform
23.7
0.0940
0.1383
0.1740
0.2529
LB morphoform
47.5
0.1583
0.2403
0.1899
0.2744
LV morphoform
8.7
0.0437
0.0607
0.1422
0.1971
SB morphoform
36.2
0.1194
0.1840
0.1675
0.2521
SV morphoform
25
0.0786
0.1222
0.1455
0.2200
Table IV. 39 Shannon’s estimates of genetic diversity within and among
populations of 3 morphoforms of P. kurrooa
HT
HS
Proportion of diversity Proportion
of
diversity
within morphoforms
among morphoforms (GST)
0.1908
0.0943
0.4943
0.5057
Table IV. 40 Nei's Unbiased Measures of Genetic Identity and Genetic distance
in 6 populations of 3 morphoforms P. kurrooa
Pop ID
IV
IB
0.9447
LB
0.9608
LV
0.8977
SB
IV
0.0000
IB
0.0568
0.0000
0.9687
LB
0.0400
0.0318
0.0000
LV
0.1079
0.0847
0.0724
0.0000
0.8909
0.8395
SB
0.1636
0.1649
0.1207
0.1155
0.0000
0.9160
SV
0.2731
0.2267
0.2041
0.0878
0.0000
0.9188
0.9302
0.1749
0.8491
SV
0.8479
0.8863
0.7610
0.7972
0.8154
Nei’s genetic identity (above diagonal) and genetic distance (below
diagonal).
93