Indian Journal of Exprimental Biology
Vol. 52, August 2014, pp. 825-834
In vitro flowering – A system for tracking floral organ development in
Dendrocalamus hamiltonii Nees et Arn. ex Munro†
Devinder Kaur, Pooja Thapa, Madhu Sharma, Amita Bhattacharya* & Anil Sood
Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology (IHBT)
Palampur 176 061, India
Received 3 February 2014; revised 28 April 2014
Dendrocalamus hamiltonii plants are slender and tall (15-25 m) thereby, rendering tagging, sampling and tracking the
development of flowers difficult. Therefore, a reproducible system of in vitro flowering was established for tracking the
stages of flower development. MS medium supplemented with 2.22 µM 6-benzylaminopurine, 1.23 µM indole-3-butyric
acid and 2% sucrose was optimized as the flower induction medium (FIM) wherein 28 and 42 days were required for the
development of gynoecium and androecium, respectively. Six distinct stages of in vitro flower development were identified,
and the flowers were comparable with that of in planta sporadic flowers. Pollen viability of the in vitro flowers was higher
than those of in planta ones. The in vitro system developed in the present study facilitates easy tracking of different stages of
flower development under controlled environmental conditions. It can also be used for medium- or long-term storage of
pollens and manipulation of in vitro fertilization.
Keywords: Bamboo, Flower induction medium, In vitro flowers, Somatic embryo derived plants
Dendrocalamus hamiltonii Nees et Arn. ex Munro is
a 15-25 m tall, commercially important bamboo with
a dominant central solid culm and several slender
and pendulous branches. The plant is popular for its
multiple utilities in rural livelihood, eco-restoration
and also in food, wood, fuel, paper and
textile industries1,2. D. hamiltonii propagates both
vegetatively as well as through sexual reproduction.
The sexual mode of reproduction is dependent upon
sporadic as well as gregarious flowering3. In case of
the former, one or two culms may flower sporadically
several times in its life time, and die after setting a
low amount of seeds. In contrast, all the culms/plant
from the same but physiologically mature stock
flower gregariously after 40 years and die after
seed set, irrespective of geographical locations4,5.
Sporadically as well as gregariously produced
flowers in D. hamiltonii have been reported to be
dichogamous and protogynous in nature5. However,
there are no detailed studies on their development as
it is extremely difficult to tag, sample and track
flower development on a regular basis in a 25 m tall
——————
*
Correspondent author
Telephone: 91-1894-23339-394
Fax: 91-1894-230433
E-mail: [email protected]
†
CSIR-IHBT publication number 3447
and slender plant like D. hamiltonii. In this regard, a
system of in vitro flowering was considered to be
particularly useful.
In vitro flowering has the potential to serve as
a convenient tool for time-effective studies on
various aspects of flowering from initiation to organ
development6. It has been reported to remarkably
compress the life cycle of different bamboos from
40 years to 3-6 months only7-11. It can facilitate easy
monitoring and sampling at each stage of floral
transition and flower development. Therefore, the
aim of the present study is to first develop a simple
and reproducible system of in vitro flowering in
D. hamiltonii and then use it for a detailed
understanding of the different stages of flowering
from initiation to floral organ development.
Materials and Methods
Induction of in vitro flowering in shoots derived
from somatic embryos—Somatic embryogenesis was
induced on leaf base of D. hamiltonii as per the
method of Godbole et al12. A bunch of 5-7 healthy
shoots (3.0–4.0 cm long) were excised from somatic
embryo derived plants and grown on SMM [MS
supplemented with 17.76 µM 6-benzylaminopurine
(BAP) and 9.84 µM indole-3-butyric acid (IBA)]
for shoot multiplication. The shoots were transferred
826
INDIAN J EXP BIOL, AUGUST 2014
to 0.8% (w/v) agar solidified basal MS13 medium
supplemented with 1.11-22.2 µM BAP either singly or
in combination with 1.23-4.92 µM IBA, 1.34-5.37 µM
1-naphthalene acetic acid (NAA) and 1.14-4.54 µM
phenyl-N'-(1,2,3-thiadiazol-5-yl) urea (TDZ). The
plant growth regulators were chosen on the basis of
earlier reports on different bamboo species, and a
total of 66 combinations were tested. These were
designated as induction media (IMs).
Percent induction of in vitro flowering was
recorded at 7 day interval for up to 60 days. The time
taken for the emergence of reproductive organs on
these media was also recorded. The average values
of all data were tabulated for inference(s). Finally, the
medium and the PGR combination yielding the
best response was selected as the ‘flower induction
medium’ (FIM) and used in all subsequent
experiments.
The pH of all media was adjusted to 5.8 prior to
autoclaving at 121 °C for 20 min. For each treatment,
five replicates of 4 flasks each (250 mL Erlenmeyer)
were taken. Each flask contained a clump of
5-7 shoots. All cultures were maintained under a
photoperiod of 16 h light (70±5 µmol m-2 s-1) and 8 h
dark at 25±2 °C, and the shoots were sub-cultured
regularly at 28 day interval.
Development of floral organs—The stages of
flower development on FIM and the time taken
thereof was recorded. For this, observations on
morphological features of individual spikelets and
florets were recorded both visually and under
stereozoom microscope (Nikon SMZ 15000, Japan).
These were also photographed using a digital camera
(Nikon Digital Sight DS-L1, Japan). A total of 6
heads-of-spikelets per treatment were used to study
the development of individual spikelets, florets and
dissected floral parts at 7 day interval for 60 days.
Histological studies—In order to understand the
organization of (i) florets in a spikelet and
(ii) individual organs of a floret, 5-6 in vitro spikelets
and carpels were fixed in FAA (formaldehyde: acetic
acid: 50% ethanol, 1:1:18) for 5 days. These were
then dehydrated in t-butyl alcohol series. After
dehydration, these were infiltrated and embedded
in paraffin wax (m.pt. 56-58 °C). Serial sections
(15 µm thick) were cut using a rotary microtome
(Shandon Finsse ME, Thermo Electron Corp., UK).
The sections were stretched on glass slides at 50 °C
using 4% formaldehyde as a stretching medium and
1% gelatin jelly as an adhesive. These were then
de-waxed in xylene series, stained with Safranin/Fast
green and mounted in DPX [Distrene, 8-10 g (British
resin product), 5 mL dibutylpthalate and 35 mL
xylene]. The sections were finally observed under a
microscope [Nikon (Biophot) No. 78508, Japan] and
photographed using a digital camera (Nikon DXM
1200). Based on the recorded observations and
reported literature on members of Poaceae, a floral
diagram and a floral formula for D. hamiltonii were
also constructed.
Comparative morphology of in vitro and in planta
sporadic flowers—The morphology of the in vitro and
in planta flowers was compared in order to ascertain
their similarity. For this, 15 year old plants showing
sporadic flowering were tagged in the field.
Preferentially, plants with an average of 8-10
flowering culms were selected and spikes were
collected from these. From each of these spikes,
around 100 spikelets were grouped into different
developmental stages. These were compared with 20
in vitro spikelets (five replicates of 4 spikelets each)
collected from shoots growing on FIM at 7 day
interval for 60 days.
In both the in vitro and in planta flowers,
parameters such as (i) number of spikelets per head,
(ii) number of florets per spikelet, (iii) morphological
development of individual floret, (iv) dissected floral
parts, and (v) their size were recorded both visually
and under stereozoom microscope (Nikon SMZ
15000, Japan). The developmental stages of in vitro
and in planta florets were compared on the basis of
their morphological features.
The morphology and the percent viability of
pollens from in vitro and in planta florets were also
compared at each stage of development. For this,
anthers of both types of florets (30 each) were teased
and fresh pollens were collected on a glass slide.
These were immediately stained with Alexander’s
stain14 for 30 min and observed under the microscope.
Photographs of each stage were taken using a digital
camera (Sony Cyber-shot T7, Japan).
Statistical
analysis—Data
were
analyzed
statistically by Advance Linear/Non-linear models
of General Linear Model (STATISTICA release 7,
statsoft Wipro, Bangalore, India). Analysis of
variance (ANOVA) followed by Duncan’s multiple
range tests were used to analyze the results of
each experiment which were repeated thrice.
The means of each treatment and their interactions
were compared at probability level (P) of ≤0.05.
KAUR et al.: IN VITRO FLORAL ORGAN DEVELOPMENT IN DENDROCALAMUS HAMILTONII
Results
Induction of in vitro flowering in shoots derived
from somatic embryos—Flowering was recorded in
12 out of 66 plant growth regulator combinations
(Table 1). When BAP alone was used, 60% shoots on
IM12 and 40% on IM8, IM16 and IM20 showed
flowering within 28 days. Induction also occurred
when BAP was combined with IBA (60% on IM6
and IM14, 40% on IM5, IM10, IM15 and IM23 but
only 20% on IM13). At 2:1 ratio of BAP and IBA,
flowering occurred invariably within 14 days on IM9
(20%) and IM14 (10%). After 28 days, flowering
increased to 80 and 60% on IM9 and IM14,
respectively. Further increase to 80% was recorded
after 56 days on IM14. Flowering did not increase
beyond 80%, irrespective of PGRs (Table 1).
Gynoecium emerged on 12 IMs but only 4 IMs
favoured the emergence of androecium (Table 2).
Earliest emergence of gynoecium was recorded on
827
IM9 and IM14 (21 days) followed by IM8 and IM12
(28 days), IM10 (35 days) and IM5-IM6, IM13,
IM15-IM16, IM20 and IM23 (42 days). Androecium
emerged on IM9 (35 days), IM8 and IM14 (42 days)
and also IM12 (49 days). The difference between the
time of gynoecium and androecium emergence was
14 days on IM8 and IM9 but 21 days on IM12 and
IM14. BAP and IBA at 2:1 ratio favoured the
emergence of both gynoecium and androecium.
Development of floral organs—Basal nodes of
in vitro shoots showed profuse flowering in the form
of heads-of-spikelets (Fig. 1a) and each spikelet had
3-4 florets. Emergence of purplish-pink stigma was
initiated in each floret after 21 days and completed
after 28 days (Fig. 1 b-d). Distinct yellow anthers
emerged only after 35 days (Fig. 1c and e) and were
found dangling after 56 days (Fig. 1f). This indicated
the protogynous nature of the flowers. The flowered
shoots senesced completely, whereas, the shoots that
Table 1—Induction of in vitro flowers in shoots growing on medium containing different concentrations of BAP and IBA
[Values are mean ± SE from 5 replicates of each combination]
Percent induction of in vitro flowers after
Medium
IM0
IM1
IM2
IM3
IM4
IM5
IM6
IM7
IM8
IM9
IM10
IM11
IM12
IM13
IM14
IM15
1M16
IM17
IM18
IM19
IM20
IM21
IM22
IM23
IM24
IM25
IM26
IM26
BAP (µM)
0.00
0.00
0.00
0.00
1.11
1.11
1.11
1.11
2.22
2.22
2.22
2.22
4.44
4.44
4.44
4.44
11.1
11.1
11.1
11.1
17.76
17.76
17.76
17.76
22.2
22.2
22.2
22.2
IBA (µM)
0.00
1.23
2.46
4.92
0.00
1.23
2.46
4.92
0.00
1.23
2.46
4.92
0.00
1.23
2.46
4.92
0.00
1.23
2.46
4.92
0.00
1.23
2.46
4.92
0.00
1.23
2.46
4.92
14 days
f
0
0f
0f
0f
0f
0f
0f
0f
0f
20d
0f
0f
0f
0f
10e
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
0f
28 days
f
0
0f
0f
0f
0f
40c
60b
0f
40c
80a
40c
0f
60b
20d
60b
40c
40c
0f
0f
0f
40c
0f
0f
40c
0f
0f
0f
0f
56 days
0f
0f
0f
0f
0f
40c
60b
0f
40c
80a
40c
0f
60b
20d
80a
40c
40c
0f
0f
0f
40c
0f
0f
40c
0f
0f
0f
0f
Mean values having different superscript are significantly different according to Duncan’s multiple range test at P ≤ 0.05; SEM 3.01
INDIAN J EXP BIOL, AUGUST 2014
828
Table 2 —Time taken for the emergence of gynoecium and androecium on different IMs favouring flower induction
Days taken for
Medium
IM5
IM6
IM8
IM9
IM10
IM12
IM13
IM14
IM15
IM16
IM20
IM23
BAP (µM )
IBA (µM )
1.11
1.11
2.22
2.22
2.22
4.44
4.44
4.44
4.44
11.1
17.76
17.76
1.23
2.46
0.00
1.23
2.46
0.00
1.23
2.46
4.92
0.00
0.00
4.92
Start of
flowering
28
28
21
14
28
21
28
14
28
28
28
28
Gynoecium
emergence
42
42
28
21
35
28
42
21
42
42
42
42
Androecium
emergence
42
35
49
42
-
Fig. 1—In vitro flowering. (a) head-of-spikelets after 14 days, (b-d) gynoecium with purplish coloured plumose bifid stigma, (b) after 21
days; (c-d) after 28 days, (e) emergence of yellow coloured anthers after 35 days, (f) an in vitro flower showing androecium and
gynoecium. The developing floral organs are indicated by arrows and circles. Bars a-c=1 cm, d-f=1 mm.
had not flowered continued to produce new shoots.
Six distinct stages of floret development were
recorded (Table 3). The florets were at immature bud
stage at S-I. A white ovary with a thin, light-yellow
coloured style and light pink, highly curled stigma
were observed upon opening each immature floret.
The androecium comprised of very small stamens
with short white filaments and small light green
anthers. By S-II, the ovary became pale yellow in
colour and had a slightly thicker style with purplishpink stigma that started unfolding. There was also a
slight emergence of the stigma. Although the anthers
KAUR et al.: IN VITRO FLORAL ORGAN DEVELOPMENT IN DENDROCALAMUS HAMILTONII
became yellowish-green, they were small and did not
emerge out of the floret. At S-III, the ovary increased
in size and became yellow in colour. It had a yellow,
turgid style with fully elongated, unfolded, plumose
(hairy), bifid and purple stigma. The filaments were
also elongated with long, yellowish green anthers and
purple tips. By S-IV, the ovary was dark yellow with
thick, turgid and pale-yellow style and dark brown
stigma that started withering. The filaments became
further elongated and the anthers started protruding.
As a result, each floret with six yellow anthers having
purple apical portions became visible. At S-V, the
ovary became yellowish-red in colour and had a
degenerated style with completely withered stigma.
At this stage, the filaments of the stamens were
completely elongated with prominently visible
distinct yellow anthers. At S-VI, the yellowish red
ovary had a totally degenerated brown coloured style
and stigma. The androecium was highly prominent
with elongated filaments and yellowish-brown,
versatile anthers dangling from the florets.
Inflorescence development and organization of
floral organs—Visual (Fig. 2a) and histological
(Fig. 2b-e) studies revealed that four florets were
oppositely and alternately arranged one above
the other. Histology confirmed the racemose
829
inflorescence of the spikelet and also showed the
presence of lemma and palea on either side of
each floret. Two glumes enclosed each spikelet.
The floret was hermaphrodite with 4 bracts (2 glumes,
1 lemma and 1 palea), 2-3 lodicules, six stamens
and a monocarpellary, superior ovary with basal
placentation (Fig. 2f-g).
Based on the above observations, the floral
diagram of a spikelet was drawn (Fig. 2h) and the
floral formula for each floret was written as: Br
P(lodicules) 2-3 A6 G1.
Comparative morphology of in vitro and in planta
flowers—Both in vitro and in planta flowers were
organized in heads-of-spikelets (Fig. 3a, b and c) and
were largely similar (Table 4). The in planta headsof-spikelets were 1.7 to 3.2 cm in diameter and
contained an average of 46.28 straw coloured
spikelets arranged in racemose pattern. The heads-ofspikelets were in turn organised as racemose
inflorescence on an average of 100-120 cm long
spike(s). In contrast, a single in vitro head-of-spikelet
was produced at the base of a shoot and consisted of
an average of 9.26 spikelets. About 2-3 green headsof-spikelets (average of 1.0 to 2.5 cm diameter) were
produced per flask. The in vitro spikelets were turgid,
green, hairy and significantly large with loosely
Table 3—Developmental stages of in vitro flowers of D. hamiltonii
Stage
S-I (14 days)
S-II (21 days)
Ovary
*Small, white in
colour
*Pale yellow in
colour
S-III (28 days)
*Slightly larger in
size than S-II and
yellow in colour
Style
*Thin and light
*Slightly thicker Thick, turgid and
yellow in colour than S-I and light yellow in colour
Gynoecium
yellow in colour
Emergence and
Fully elongated,
Stigma *Light pink in
colour and highly unfolding; purplish dark purple in
curled
pink in colour
colour, plumose,
unfolded and bifid
Filament *Small and white *Small and white *Elongated than
in colour
in colour
S-II
Anthers *Immature, small *Immature, small *Elongated than
Androecium
light green in
yellowish-green S-II. Yellowishcolour
in colour
green with purple
apical portion
[*Visible only after dissection]
S-IV (35 days)
S-V (42 days)
S-VI (56 days)
*Dark yellow in
colour
*Yellowish-red
in colour
*Yellowish-red
in colour
Thick, turgid
and pale yellow
in colour
Withering of dark
brown stigma
initiated
Degenerated and
pale yellow in
colour
Withered
completely
Totally
degenerated and
brown in colour
Withered
(not visible
in floret)
*Elongated than Fully elongated
S-III
filaments
Initiation of anther Yellow in colour
protrusion. Yellow and prominently
anthers with light visible
purple apical
portions
Highly elongated
filament
Yellowish-brown
coloured versatile
anthers
830
INDIAN J EXP BIOL, AUGUST 2014
Fig. 2—Arrangement of florets and floral organs in a spikelet. (a) dissected spikelet showing alternate and opposite arrangement of florets
(F1=floret 1, F2=floret 2, F3=floret 3, F4=floret 4, Gl=glume, L=lemma, P=palea, A=anther(s), G=gynoecium), (b-f) serial transverse
sections of a spikelet (b) F4, (c) F1, (d) F3, (e) F2, (f) close-up of F2, (g) TS of ovary showing basal placentation (h) floral diagram of
a spikelet. Bars a-c=1 cm, d-f=200 µm.
Fig. 4—Stages of floral development. (a) in vitro (b) in planta.
Fig...3—Heads-of-spikelets showing racemose inflorescence.
(a) spike on a flowering culm of 15 year old plant (b) an excised
spike (c) in vitro head of spikelet.
arranged florets (Fig. 4a). The in planta spikelets
were comparatively compact, small, dry and straw
coloured with scanty short hairs (Fig. 4b).
Dissected in planta and in vitro florets had a
similar arrangement (Fig. 5a-c), and were bracteate
with lemma and palea covering the lodicules. Each
floret was encased by flowering-glumes with lemma
on one side and a smaller, transparent, thin palea on
the other (Fig. 5b). These enclosed the gynoecium and
the androecium in their axils. The androecium and
gynoecium were structurally similar with a
monocarpellary, superior ovary and androecium with
six stamens and versatile anthers (Fig. 5b and c). The
florets differed slightly in their size, texture
and colour (Table 4). The lodicules though not visible
to the naked eye or easily under the stereozoom
microscope, were nevertheless present at the base
of the ovary.
The development of in vitro and in planta florets
was also similar. However, the gynoecium of in vitro
florets was more turgid and smaller at S-III, and it
emerged slightly later (Fig. 6a, b; Table 4). Both
in vitro and in planta anthers had no pollens at S-I
KAUR et al.: IN VITRO FLORAL ORGAN DEVELOPMENT IN DENDROCALAMUS HAMILTONII
831
Table 4—Comparison of in vitro and in planta inflorescence
[Values are mean ± SE from 20 replicates of each stage]
in vitro
in planta
Colour
Diameter (cm)
Number of spikelets/head
Colour
Length (cm)
Number of florets/spikelet
Number of glumes
Length of glume (cm)
Racemose
Green
1.0-2.5
9.26 ± 1.00b
Green
0.94 ± 0.02a
3.60 ± 0.07a
Two
1.2 ± 0.02a
Racemose
Straw
1.7-3.2
46.28 ± 2.78a
Straw-green
0.87 ± 0.01b
3.22 ± 0.06b
Two
0.6 ± 0.02b
Floret
Colour
Turgidity
Length of lemma (cm)
Length of palea (cm)
Number of stamens
Number of carpels
Green
High
0.79 ± 0.02a
0.54 ± 0.02a
Six
One
Straw-green
Low
0.68 ± 0.02b
0.58 ± 0.02a
Six
One
Gynoecium
Colour
Average length (cm)
Period between gynoecium
and androecium emergence
Whitish to yellow
0.67 ± 0.01b
14 days
Pale yellow to yellow
0.79 ± 0.02a
8-12 days
Androecium
Colour
Average length (cm)
S-I
S-II
S-III
S-IV
S-V
Green to yellow with purple tips
0.48 ± 0.01a
No pollens
Few monoporate pollens
Increased number of pollens
Large number of pollens
Anthers filled with pollens having
68% viability
Brown coloured anthers filled with pollens
Yes
Green to yellow with purple tips
0.49 ± 0.01a
No pollens
Few monoporate pollens
Increased number of pollens
Large number of pollens
Anthers filled with pollens
having 47% viability
Brown coloured empty anthers
Yes
Type of Inflorescence
Head-of-spikelets
Spikelet
S-VI
Desiccation of flowered
shoots
Mean values having different superscript are significantly different according to Duncan’s multiple range test at P ≤ 0.05
(Fig. 6c) but monoporate pollens developed from
S-II onwards to completely fill the anthers at
S-V. Both viable (pink stained) and non-viable
(green stained) pollens were observed (Fig. 6e and f)
and their percent viability was >68% (in vitro) and
47% (in planta). At S-VI, innumerable pollens were
aligned along the sutured anther lobes of in vitro
florets, whereas the in planta anthers (Fig. 6d) were
completely empty.
Discussion
Routine tagging, sampling and tracking of flower
development from initiation to organogenesis is
difficult in D. hamiltonii plants growing in situ.
This is largely due to their long vegetative phase of
40 years, slender form and height of about 15-25 m.
In contrast, in vitro flowering can serve as an ideal
and alternative system for such studies6,15-16.
Therefore, a simple, time effective and reproducible
system of in vitro flowering was developed in the
present study.
The presence of BAP (up to 17.76 µM) or its
combination with IBA (1.23-4.92 µM) supported the
induction of in vitro flowering. Lower concentrations
of BAP and IBA at 2:1 ratio invariably induced early
in vitro flowering. Any change in this ratio increased
the duration of flower induction (Table 1). The best
response (20 and 80% flowering after 14 and 28 days,
respectively) was obtained when the basal MS
medium was supplemented with 2.22 µM BAP, 1.23
µM IBA and 2% sucrose (IM9). This medium also
supported the earliest emergence of gynoecium and
androecium, and was designated as the ‘flower
induction medium’ (FIM). Earlier, Chambers et al.8
832
INDIAN J EXP BIOL, AUGUST 2014
Fig. 6—Gynoecium and androecium of in vitro and in planta
florets. (a-b) gynoecium at S-IIIg (a) in vitro (b) in planta
(c) anthers at S-I, in vitro (left) and in planta (right) (double head
arrow showing anther tips, and single head arrows showing suture
of bilobed anthers), (d) anthers at S-VI, in vitro (left) and
in planta (right) (black arrow showing open lobe and blue arrow
showing pollens), (e-f) pollens stained with Alexander’s stain at
S-V (e) in vitro (f) in planta. Bars a-d=1 mm
Fig. 5—Comparison between in vitro and in planta inflorescence.
(a) heads-of-spikelets, (b) dissected florets, (c) dissected spikelets.
had demonstrated the requirement of 91 days for the
induction of in vitro flowering in 47% D. hamiltonii
shoots. This duration was 6.5 times higher than that
recorded in the present study (Table 1). The percent
shoots showing flowering was also 1.7 times higher
than that reported earlier. All other plant growth
regulators (IBA, NAA and TDZ) failed to induce
flowering when used singly (Table 1). High
auxin/cytokinin ratio is known to reduce in vitro
reproductive growth in B. edulis17.
Nadgauda et al.18 had reported the morphology
of in vitro flowers and the time taken for anthesis in
Bambuse arundinacea but the actual stages of flower
development were not studied in any bamboo species.
In this regard, the present study revealed that the
process of in vitro flowering comprised of six distinct
stages, wherein stages of maturity were identifiable
and invariably completed within 56 days (Table 3).
The morphological features observed at S-I indicated
immature reproductive organs. The gynoecium
emerged after 21 days at S-II but attained maturity
after 28 days at S-III when its dark-purple, hairy and
bifid stigma became fully elongated. The stigma
underwent withering and drying at S-IV. Progressive
development of androecium was also evident from
(i) the absence of pollens in the immature anthers at
S-I, (ii) the presence of few monoporate pollens at
S-II, (iii) elongation of stamens and an increased
number of pollens at S-III, (iv) emergence of anthers
at S-IV, (v) versatile, pollen-filled, yellow coloured
anthers at S-V indicating androecium maturation and
finally (vi) browning of anthers at S-VI. The in planta
anthers at this stage were completely devoid of
pollens, indicating thereby, dehiscence. Thus, the
period between S-IV and S-V was identified as the
stage of androecium maturity. This information can
prove useful for easy manipulation of in vitro
fertilization where two separate batches of in vitro
flowers (i.e., one at S-III and the other at S-V)
can be simultaneously readied for the concomitant
maturation of gynoecium and androecium.
The development of the in vitro as well as the
in planta flowers (collected from field grown plants)
was also similar (Figs 4 and 5; Table 4). Comparable
development of in vitro and in planta flowers were
earlier reported in B. arundinacea and B. oldhamii18,19.
In the present study, extrusion of the stigma much
before the anthers confirmed the protogynous nature of
KAUR et al.: IN VITRO FLORAL ORGAN DEVELOPMENT IN DENDROCALAMUS HAMILTONII
both in vitro and in planta flowers of D. hamiltonii.
The protogynous nature of in vitro flowers of
D. hamiltonii was also reported by Chambers et al8.
The inflorescence of in vitro and in planta
flowers were largely indistinguishable except for
the (i) absence of a long spike, (ii) presence of only
heads of spikelets and the (iii) size of the spikelets
being larger under in vitro conditions. Further, the
same number of glumes (2), lemma (1), palea (1),
stamens (6) and gynoecium (1) were present in
both in vitro and in planta florets. The size of the
different parts of in vitro and in planta florets was
also comparable. However, the gynoecium of
in planta florets was larger and their lemma was
smaller than those of the in vitro florets (Table 4).
Histological studies revealed that the arrangements
of floral organs were characteristically similar to that
of different members of the family Poaceae20.
Although in vitro and in planta pollens were similar,
their viability in the former was 68% as compared to
47% in the latter. As opposed to the report on
B. arundinacea18, the in vitro florets in the present
study showed higher pollen viability than the
in planta ones. The reason for this difference could
be the collection of pollens at the right stage of
development in this study. This can facilitate
medium- and long-term storage of pollens.
The transition of plants from vegetative to
reproductive phase is known to involve a series
of morphological, physiological, biochemical and
molecular changes21,22. These changes are actually
the manifestations of complex biological events
that unfold sequentially in response to certain
environmental and biological stimuli23,24. In this
regard, the system of in vitro flowering developed
in the present study offers the advantages of
constant environmental conditions while facilitating
easy tracking of changes associated with
floral-transition and flower development in a time
effective manner.
Acknowledgement
The authors acknowledge the Director, CSIR-IHBT
for providing the necessary infra-structure for
carrying out this work and the Council of Scientific
and Industrial Research (CSIR), New Delhi for
financial assistance. The authors thank Mr. Sanjoy
Chanda for assistance in histological studies and
Mr. Pabitra Gain for his photographic inputs.
DK gratefully acknowledges the CSIR for Senior
Research Fellowship.
833
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