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In vitro mutagenesis and characterization of mutants through morphological

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Indian Journal of Experimental Biology
Vol. 56, June 2018, pp. 385-394

In vitro mutagenesis and characterization of mutants through morphological and

genetic analysis in orchid Aerides crispa Lindl.
Deepti Srivastava1,2*, Gayatri MC1 & SK Sarangi2
1
Plant Biotechnology Unit, Department of Botany;
2
Department of Microbiology and Biotechnology, Bangalore University, Bangalore-560 056, Karnataka, India

Received 18 August 2015; revised 19 February 2018

Mutation breeding techniques combined with tissue culture and molecular marker methods provide a powerful tool for
improvement of slow growing plants such as orchids. In this study, we developed a protocol for in vitro mutagenic studies in
a medicinal orchid of Western Ghats, Aerides crispa Lindl., commonly called the Curled Aerides. In vitro grown 60 day old
protocorms treated with low concentrations (0.025–0.03%) of colchicine, ethyl methane sulphonate (EMS), and low doses
(1–4Gy) of gamma radiations, and 27 combinations resulted in healthy multiple shoot bud formation with 40–60% survival
frequency. Higher singly or combined dose treatments caused protocorm or shoot initials death, after 20–30 days. Healthy
2500 in vitro seedlings were screened for phenotypic changes in shoots and roots owing to a mutagenic effect. In total, 206
in vitro seedlings of 52 variant lines were identified on the basis of their unique leaf shape, colour, white stripes, thickness,
length, and width and their root length and thickness. These variant lines were multiplied, established in an orchidarium, and
compared with the control for genetic variability by using Random Amplified Polymorphic DNA method. Only 15
genetically distinct mutant lines were identified, which exhibited disparity in growth rate, leaf shape, leaf length, width,
chlorophyll variegation, stomatal density, and/or pigment contents.

Keywords: Colchicine, Ethyl methanesulfonate, Gamma radiation, In vitro mutation, Orchidarium, Protocorms, Western
Ghats

Orchids are one of the most beautiful creations on the
Earth, comprising a unique group of plants valued
mainly for the ornamental purpose, but some of them
are used from time immemorial in traditional
practices to treat various medical conditions. Orchids
are seen as the “pandas” of the plant kingdom because
of their rareness and charisma1. Western Ghats are
one of the world's ten "Hottest biodiversity hotspots"
loaded with more than 300 medicinal and ornamental
orchid species. Aerides crispa Lindl., commonly
called the Curled Aerides, is one of the epiphytic
orchids, endemic to tropical forest of the Western
Ghats and known for its medicinal and ornamental
properties. Many orchids including A. crispa are
vulnerable or endangered due to habitat loss by
frequent changes in climate or poaching of plants for
use in local traditional medicine, horticulture and
national/international floriculture trade2. A significant
literature exists on the causes of rarity in orchids,
drivers of rarity, in vitro mass propagation and their
——————
*Correspondence:
Phone: +91 80 22961312
E- mail: deeptibiotech@rediffmail.com; deeptibiotech@gmail.com
conservation. But deliberation on breeding strategies
for improvement of orchid’s economical and genetic
traits for their better conservation and
commercialization is scanty3,4.
Conventional breeding programs are difficult to
execute for orchids due to their poor seed germination
capacity and slow growth rate under natural
environmental condition5. Therefore, for mutation
induction in orchids, the combination of breeding
technology with tissue culture could be a successful
method because they have many evident advantages,
such as (a) effective use of chemical and physical
mutagens at lower doses; (b) high mutation rate; and
(c) simple, rapid, and cost effective in vitro clonal
propagation of individual mutant that provides
adequate materials for further screening as well as
reduction in chimera formation6.
It is well known that the use of ionizing radiations
like gamma rays and chemical mutagens such as ethyl
methanesulfonate (EMS) can generate unpredictable
mutations that do not exist in nature7. Colchicine, a
polyploidy agent can also initiate mutation by causing
mitotic irregularities, quantitative change in nuclear
volume, etc.8. Possibility of isolating beneficial
386 INDIAN J EXP BIOL, JUNE 2018
mutants increases when low doses and wider
spectrum of mutagens and their combinations are
used9. It is documented that treatment of seeds with
colchicine solution prior to physical mutagens
treatment caused the origin of rare mutation process
as well as large sized mutants10. Many studies showed
treatment of seeds with gamma radiations coupled
with EMS causes micro-mutations affecting polygenic
characters11. In addition, the use of molecular marker
technology provides a tool that can assist in
identifying mutants in mutation induction studies.
Random Amplified Polymorphic DNA (RAPD)
analysis is an efficient and economical method to
assess the mutation specific genetic variations.
However, so far, there is no report stating the use of
mutagens for Aerides breeding. Therefore in the
present study, we evaluated the effect of in vitro
mutation induction through colchicine, gamma
radiations, EMS and their combinations as a means to
improve the economic traits of A. crispa.
Materials and Methods
Plant material and establishment of in vitro culture
A. crispa pods were washed under running tap
water and 5% (v/v) laboline, a commercial liquid
detergent. These pods were disinfected under laminar
air flow by dipping in 0.5%w/v mercuric chloride
solution for 2 min followed by their washing
thoroughly in sterile distilled water. Subsequently,
these were sterilized by dipped in 70% alcohol for a
minute and flamed12. The sterilized pods were split
opened with sterile surgical blade and powdery seeds
of A. crispa were inoculated on Knudson C Basal
Medium (KCBM) fortified with 15% coconut water
(CW), 8.88 μM 6-benzyl amino purine (6-BAP) and
500 mg/L peptone for seed germination and
protocorm formation. All the chemicals (Tissue
culture grade) were purchased from Himedia, India.
The cultures were incubated at temperature 25±2C
under built-in white fluorescent light at a photon
density of 30-50 EM-2S-1 with 50-55% Relative
Humidity (RH) under a photoperiod regime of 16 h
light and 8 h dark cycles.
In vitro induction of mutation
Sixty day old 6000 protocorms obtained from seed
cultures were treated with colchicine, EMS, gamma
radiation, or their combinations to induce mutations.
For individual treatments, 15-20 protocorms were
treated in triplicate with different concentrations of
colchicine or EMS for five days or exposed to 1 Gy to
6 Gy gamma radiations. Sterile EMS (Himedia, India)
or colchicine (Himedia, India) solutions (0.025, 0.05,
0.1, 0.2, 0.3 and 0.4%) were added to autoclaved
nutrient media (KCBM supplemented with 8.88 μM
6-BAP) containing 8% agar, on which protocorms
were grown for five days. Protocorms were
subsequently rinsed thrice with sterile water, dried in
sterile filter paper, and transferred to multiplication
media (KCBM supplemented with 8.88 μM 6-BAP)
for 30 days. For irradiation, culture bottles containing
protocorms were exposed to different doses (1Gy-
6Gy) of 60Co γ-rays at Kidwai Memorial Institute of
Oncology, Bangalore, followed by their transfer to
multiplication media bottles. For combination
treatments, protocorms were grown on media
containing 100 different permutations of colchicine
(0.025, 0.050 0.100, 0.200 and 0.300%) and/or
exposure to gamma irradiation (1Gy-4Gy) and/or
EMS (0.025, 0.050, 0.100, 0.200 and 0.300%) as
described above. These protocorms were further
subcultured on multiplication media for 30 days.
Well surviving shoot buds were transferred to the
same media for 70 days for multiple shoot formation.
Healthy shoots were subcultured on half-strength
KCBM supplemented with 2.45 μM indole-3-butyric
acid (IBA), (Himedia, India) for 35 days for root
formation. Seedlings showing distinct shoot/root
phenotypes were propagated as variant lines to
provide sufficient materials for secondary screening.
Mutation frequency was calculated as ratio of variant
per treatment/total number of protocorm treated
X100. Variant seedling leaves were excised and
inoculated on KCBM supplemented with 6-BAP
(8.88 μM) for 60 days to obtain protocorm-like bodies
(PLBs). These PLBs were further subcultured on
KCBM supplemented with 6-BAP (8.88 μM) and IBA
(2.45 μM) for 120 days to get healthy shoots and
roots. Variant lines were hardened for 90 days on
small plastic pots containing peat: perlite: vermiculite
(1:1:1 v/v) under controlled environment (225C,
twice watered, 60% Relative Humidity (RH) and
photoperiod regime of 14 h light and 10 h dark
cycles) for acclimatization. Survived plantlets were
maintained on potting mixture brick pieces: perlite:
charcoal (1:1:1w/v) in Orchidarium, Bangalore
University.
Primary screening of mutants under in vitro condition
The effects of colchicine, gamma radiations, EMS,
and their combinations were primarily evaluated
under in vitro condition. The survival of mutagen-
 SRIVASTAVA et al.: IN VITRO MUTAGENESIS IN AERIDES CRISPA
treated protocorms was confirmed at 30 days of final
treatment based on healthy shoot bud formation,
shrinkage or death of protocorms. The survival
frequency was calculated using formula: survival
frequency= number of plants survived/total number of
PLBsX 100. The surviving shoot buds were grown
in vitro as seedling and evaluated for phenotypic
changes in the shoots and roots after 105 days. Size of
shoots and roots were measured just before hardening
by millimeter scale and/or vernier scale13.
Secondary screening of mutants under ex vitro condition
Hardened and acclimatized 300 day old plants,
categorized into 52 variant lines, were screened for
genetic and morphological variations to identify
mutant lines.
Molecular characterization of mutants
Cetyl trimethyl ammonium bromide (CTAB)
method of plant genomic DNA extraction was
followed to isolate genomic DNA from juvenile
leaves of A. crispa control and mutants. A total of 28
arbitrary operon RAPD primers were used for initial
screening in control plants. The PCR reaction volume
was 25 µL containing 200 ng genomic DNA, 1 unit
Taq polymerase, 4 µL dNTP’s, 2.5 µL of 10X
reaction buffer with 3.5 µL random primers. All the
chemicals required for PCR were purchased from
Chromous Biotech, Bangalore, India. Amplification
was carried out for 40 cycles with initial heat
denaturation of the DNA at 94C for 3 min. The
thermal cycling was performed with the following
temperature regimes: 94C for 90 s, 30-40C for
2min-30sec (depends upon primer) and 72C for
3 min. The final extension step was performed at
72C for 5 min followed by cooling to 4C for
completion of the programme. RAPD amplification of
control and variants by using 11 operon RAPD
primers was successfully studied based on band
intensity and size. RAPD profiling was repeated
thrice to confirm its reproducibility.
Each informative RAPD band was scored
independently as 1 for presence and 0 for absence.
Percentage of polymorphism was calculated as the
proportion of polymorphic bands over the total
number of bands. Bands present in only one sample
were counted as unique band. Bands common in all
samples were considered as monomorphic bands.
Cluster analysis of polymorphic and unique bands
was conducted on the Euclidean distance matrix with
387
the Unweighted Pair-Group Method Arithmetic
average (UPGMA) using Soft Stat Mode, 1993
software to analyze genetic distance among variant
lines having morphological dissimilarity.
Morphological characterization of mutants
Comparative distinction in the morphological
parameters, such as plant height and leaf length and
width, were measured using a millimeter scale. The
number of leaves per plant, leaflet shape, and
chlorophyll spectrum of the control and mutant plants
were assessed by naked eye observation. Stomatal
density is the total number of stomata present per
millimetre square of tissue and was calculated using
an optical microscope. This morphological study was
conducted in triplicate for 8–10 plant replicas.
Estimation of Chlorophyll and Carotenoid Content
Total chlorophyll and carotenoid contents were
extracted using standard methods14. Total chlorophyll
and carotenoid concentration were calculated using
Porra equation (2002) and Lichtenthaler & welburn
equation (1983), respectively and were computed in
terms of fresh weight (FW)14,15: total chlorophyll =
17.76 (A646) + 7.34 (A663); carotenoid = {[1000 A470 –
3.27 Chl a] – [104 Chl b]}/ 227; where A=
absorbance
Statistical analysis
The experiments were set in completely
randomized design and repeated thrice in identical
physical conditions. The data obtained from the
results of all experiments were subjected to one-way
ANalysis Of VAriance (ANOVA) to determine the
variation between the treatments and Standard
Deviation (SD) to determine variations within the
treatments. The mean values were compared using
Duncan’s multiple range test16. Statistics Software
SPSS 15.0 (2006) was used for ANOVA and SD
determination.
Results
In vitro induction of mutation
Protocorm treatment (Fig. 1a) with various
concentrations (0.025–0.03%) of colchicine and EMS;
different doses (1–4 Gy) of gamma radiations; and 27
combinations of colchicine (0.025, 0.05 and 0.1%),
gamma radiations (1, 2 and 3 Gy) with EMS (0.025,
0.05 and 0.1%) caused protocorm swelling and
initiated shoot buds, after 10–20 days (Fig. 1 b and c).
388 INDIAN J EXP BIOL, JUNE 2018

These treated protocorms developed into healthy
multiple shoot buds, with 40–60% survival frequency.
Among the all treatments, Gamma rays (2Gy) and
EMS (0.05%) were found as LD50% doses for
individual treatments, whereas combination of 0.05%
Colchicine with 0.025% EMS and 0.025% colchicine
with 2Gy gamma radiation were found LD50 doses
for their combination treatments. Colchicine and EMS
(0.025% each) with 1Gy gamma rays were noted
LD50 dose for combination of three mutants.
Treatment with higher singly or in combination doses
of colchicines (0.4%), gamma radiations (5-6Gy), and
EMS (0.4%) resulted in the browning of protocorms
or shoot initials from the apical end (Fig. 1d) followed
by their shrinkage and death after 20–30 days (Fig. 1e).
The survival frequency of protocorms treated
with higher doses varied from 0–10%. In total, 2500
in vitro seedlings were obtained from mutagenic
treatment. Untreated protocorm were developed with
92±8% survival rate and 0.32±0.03% mutation
frequency. This minimal mutation frequency might be
found due to epigenetic variation. The mutation
frequency of healthy in vitro seedling developed by
mutagen treatment did not vary in a dose dependent
manner. Maximum mutation frequency (86.63±4.51%)
with 52±2% survival rate was observed on combination
treatment of 0.025% colchicine, 1-Gy gamma
radiation, and 0.025% EMS (Table. 1). In total, 206
healthy in vitro seedlings with diverse phenotypes
were obtained from 35 mutagenic treatments.
Primary screening of variants
Healthy seedlings were considered for variant
identification based on variation in shoot and root
phenotype. Screening of seedlings with naked eyes
discriminated 52 lines of mutant phenotypes under
culture conditions. The results revealed major
variations in the shape, colour, white stripes,
thickness, length, and width of leaves as well as the

Fig. 1 — Effect of mutagenic treatment on in vitro protocorms of A. crispa. (a) In vitro grown 60 days old protocorms; (b) Swelling of
protocorms after 10 days of 0.05% colchicine treatment; (c) Initiation of healthy shoot buds after 20 days of 0.05% colchicine treatment;
(d) Browning of protocorm from apical end after 25 days of 0.05% colchicine with 0.1%EMS treatment; and (e) Shrinkage and death of
leaf primordium after 30 days of 0.3% EMS treatment

Table 1 — Effect of different doses of mutagens on in vitro induction of mutation

Variant Colchicine Gamma rays Percent Percent Root length
EMS (%) Shoot length (cm)
line (%) (Gy) survival Mutation (cm)
Control 92±8k 0.32±0.03a 1.29±0.03c 0.50±0.05a
ACM2 0.025 58±3i 81.50±3.10l 1.56±0.23e 1.14±0.19f,g
d k d
ACM4 0.05 47±2 75.75 ±2.54 1.40±0.03 0.79±0.09c,d
ACM7 1 60 ±11j 45.02±3.31f 1.64±0.21e 0.65±0.13b
f i b
ACM8 2 50 ±6 62.20±4.70 1.14±0.20 2.15±0.22j
d e a
ACM10 3 47 ±3 38.95±3.41 0.72±0.03 1.20±0.17g,,h
i c h
ACM11 0.05 58±7 26.18 ±2.72 2.10±0.27 1.16±0.19f,g
d b c
ACM13 0.1 46±8 19.13 ±3.52 1.29±0.15 2.30±0.21k
e d i
ACM17 0.05 0.025 49±5 32.67±2.10 2.36±0.12 0.94±0.07e
ACM20 0.025 0.05 42±7b 39.84 ±2.51e 1.79±0.03f 1.12±0.20f,g
ACM25 0.025 2 49±3e 44.70±3.20f 2.00±0.20g 0.70±0.08b,c
a g i
ACM29 0.025 3 41±2 50.95 ±2.80 2.30±0.14 0.88±0.16d,e
h h j
ACM39 1 0.025 54±10 57.22±3.32 2.56±0.17 1.25±0.09h,i
a k k
ACM41 1 0.05 40±7 75.21±2.51 2.77±0.15 1.31±0.15i
g m m
ACM49 0.025 1 0.025 52±2 86.63 ±4.51 3.40±0.09 1.19±0.16g,h
ACM52 0.025 2 0.05 44±7c 68.10±5.42j 2.98±0.19l 1.07±0.18f
[Values are means ± SD. Values followed by different letters are significantly different at P≤0.05 according to Duncan’s multiple range test]
 SRIVASTAVA et al.: IN VITRO MUTAGENESIS IN AERIDES CRISPA 389

length and thickness of roots among 52 in vitro variant
lines. Prominent white stripes were observed on the
leaves of the ACM2 variant (Fig. 2b), whereas light
green stripes were observed on those of the ACM4,
ACM7, ACM8, and ACM10 variants (Fig. 2 c-f).
Variations in the leaflet shape, leaf length, root length,
and root thickness were noticed among many of the
variants (Fig. 2 a-p).
The longest shoot (3.40±0.09 cm) was observed in
ACM49, a variant developed using a combination
treatment of 0.025% colchicine, 1 Gy gamma
radiation, and 0.025% EMS. In contrast, the shortest
shoot (0.72±0.03 cm) was observed in the ACM10
variant, which was evolved using gamma irradiation
(3 Gy) (Table 1). Mutagenic treatments showed a
complex effect on the root phenotype. The root length
of in vitro seedlings varied between 0.65±0.13 cm and
2.30±0.21 cm among the ACM7 and ACM13
variants, which were developed by treating
protocorms with 1 Gy gamma radiation and 0.1%
colchicine, respectively (Table 1; Fig. 2). The shoot
and root lengths of the variants were not correlated.
Secondary screening of mutants under ex vitro condition
The 52 in vitro variant lines, which had
morphological anomaly relative to the control plants,
were hardened, acclimatized, and maintained in an
Orchidarium, with 70–80% survival frequency. These
variant lines and control plants were grown under
identical physical conditions. Some desirable
attributes, such as an increase in the height of the
plant and number of leaves per plant were observed in
all the variant lines compared with the control plants.
One or few remarkable variations, such as leaf sizes,
leaflet shapes, chlorophyll spectrum of leaves
[maculate (spots where chlorophyll and/or carotene
has been destroyed), striata (white longitudinal bands
alternating with green bands), and viridis (light green
in colour)], and stomatal density were noticed among
variant lines and control plants. Root length and
thickness did not vary prominently among most of the
variants and control plants. These variant lines were
compared for their genetic diversity before identifying
and confirming morphological mutant lines.
Molecular characterization of variants
DNA was extracted from juvenile leaves of control
and variants. The DNA yielded 0.2–0.5 mg/g fresh
weight. All DNA samples showed A260/A280 ratio
averaged between 1.46 and 1.76, indicating that the
DNA samples were suitable for RAPD analysis and
long-term storage. Of 28 Operon RAPD primers used
for the initial screening of control, 11 were chosen for
genetic diversity analysis based on their
reproducibility, robustness of amplification, and
scorability of bands among control and mutants. The
DNA amplification of variant lines and control
revealed that five primers produced monomorphic
bands for all variant lines, whereas three primers
OPA-12, OPD-07, and OPG-09 showed 63, 96, and
162 polymorphic bands and 7, 4, and 2 unique bands,
respectively (Fig. 3 a–c; Table 2) among control and
15 variant lines. Thus, among 52 variant lines
studied, RAPD profiling confirmed genetic variability
in 15 morphological variant lines of A. crispa: ACM2,
ACM4, ACM7, ACM8, ACM10, ACM11, ACM13,
ACM17, ACM20, ACM25, ACM29, ACM39,
ACM41, ACM49, and ACM52 (Fig. 3).
The results are clearly depicted in the dendrogram,
which was constructed with the combined data of

Fig. 2 — In vitro mutagenesis of A. crispa: identification of variant lines through distinct morphology. (a) Control; (b) ACM2: white
strips; (c) ACM4: lack of chlorophyll; (d) ACM7: thicker root; (e) ACM8: thicker and longer roots; (f) ACM10: lessen growth; (g, h, i, j)
ACM11, ACM13, ACM17, ACM20:abnormal shoot and root length and shapes; (k, l) ACM25, ACM29: abnormal shoots; (m) ACM39:
broader and thicken leaves; (n) ACM41: Broader waxy and thicken leaves; and (o, p) ACM49, ACM52: faster growth— longer and
broader leaves [Scale Bar = 1cm is used for fig. 2A to 2P]
390 INDIAN J EXP BIOL, JUNE 2018

primers OPA-12, OPD-07, and OPG-09 (Fig. 4). The

dendrogram analysis, performed using the UPGMA
method, displayed the clustering of mutant populations
with a moderate range of genetic polymorphism, where
genetic distance varied from 2.4 to 5.1.
Characterization of mutants based on morphology, stomata and
pigments
The clustering pattern corresponded well with the
variation in morphology, stomatal density, and
pigment contents among mutants and control (Table 3;
Fig. 5).The morphology of the control leaf and plant
is illustrated in Fig. 5 a and b. Cluster analysis
separated ACM25 and ACM29 mutant lines away
Table 2 — Polymorphism information of 11 operon primers
responded during RAPD analysis of colchicine, EMS, gamma
radiations and their combinations induced mutants of A. crispa
Primer Sequence 5’-3’ Amplified Number of Per cent
Bands polymorphic Polymorphism
bands
OPA-04 GGGTAACGCC 36 0 0
OPA-08 AGGTGACCGT 48 0 0
OPA-09 GTGTGCCCCA 88 4 4.5
OPA-12 GGGTAACGCC 70 63 90
OPA-13 CTGACGTCAC 29 0 0
OPA-18 AGGTGACCGT 82 8 10
OPD-03 AGTCAGCCAC 35 0 0
Fig. 3 — RAPD profile of morphological variants in A. crispa OPD-07 AGTCAGCCAC 100 96 96
using operon primers: (a) OPA-12; (b) OPD-07; and (c) OPG-09.[
OPG-09 GACTAAGCCC 164 162 98.8
Lanes L. Ladder; C- control; 1-15 mutants: ACM2, ACM4,
ACM7, ACM8, ACM10, ACM11, ACM13, ACM17, ACM20, OPZ-13 CTACGGAGGA 34 0 0
ACM25, ACM29, ACM39, ACM41, ACM49, ACM52] OPZ-14 GACTAAGCCC 52 7 13.5

Table 3 — Characterization of 15 mutant lines of A. crispa for distinct morphological and stomatal traits
Mutant lines Plant height Number of Leaf length (cm) Leaf width (cm) Leaflet Chlorophyll Stomatal
(cm) leaves shape spectrum density
Control 2.00±0.15a 2.03±0.08a 2.57±0.06b 0.69±0.11b Emarginate - 95.70±2.57a
ACM2 2.31±0.05c 2.53±0.08c 3.96±0.05f,g 1.15±0.12i Lanceolate Straita 121.72±4.55f
ACM4 3.17±0.07f 2.95±0.06e 4.15±0.08h 1.10±0.2h,i Emarginate Maculate 99.81.±1.52b
ACM7 2.57±0.01d 3.78±0.02i 3.55±0.11d 0.89±0.08d,e Emarginate Maculate 102.82±3.99 b,c
ACM8 2.90±0.01e 3.09±0.39f 3.28±0.14c 1.06±0.12g,h Oblong Viridis 105.09±5.34c
ACM10 2.20±0.08b 2.56±0.37c 2.20±0.25a 0.81±0.12c,d Oblong Viridis 110.92±6.32d
ACM11 3.69±0.13g 3.90±0.09j 4.24±0.41i 0.56±0.12a Lanceolate - 99.21±3.66 b
ACM13 3.20±0.02f 4.02±0.02k 4.02±0.09g 0.70±0.12b Lanceolate Higher 93.35±6.04a
Chlorophyll
ACM17 4.14±0.12i 3.66±0.33h 4.03±0.04g 0.91±0.09d,e Emarginate - 115.91±6.32e
h m f k
ACM20 3.90±0.02 4.70±0.1 3.88±0.14 1.36±0.11 Emarginate Higher 120.27±7.88f
carotenoid
ACM25 2.59±0.09d 2.76±0.41d 3.31±0.41c 0.97±0.09e,f Lanceolate - 212.35±8.43h
e b b d,e
ACM29 2.97±0.06 2.44±0.41 2.91±0.05 0.89±0.11 Lanceolate - 189.94±5.40g
g h g i
ACM39 3.75±0.09 3.60±0.51 4.00±0.59 1.16±0.18 Oblong - 113.63±8.30d,e
g g e j,k
ACM41 3.71±0.14 3.49±0.31 3.79±0.09 1.30±0.18 Oblong - 94.06±5.00a
j l k j
ACM49 4.79±0.18 4.42±0.36 5.39±0.54 1.27±0.14 Emarginate - 111.90±6.36d,e
ACM52 5.29±0.18k 5.08±0.2n 4.99±0.25j 1.17±0.14i Emarginate - 121.00±7.82f
[Values are means ± SD. Values followed by different letters are significantly different at P≤0.05 according to Duncan’s multiple range test]
 SRIVASTAVA et al.: IN VITRO MUTAGENESIS IN AERIDES CRISPA 391

from control and other mutants. These mutant lines
showed higher stomatal density (122 and 98%,
respectively) than control in addition to morphological
variations (Table 3; Fig. 5 c and d). Mutant lines
ACM11 and ACM13, evolved by treatment of
protocorms with colchicine: 0.05 and 0.1%,
respectively were noted least genetically distant to each
other (2.4) among all mutant lines (Fig. 4). They had
longer (4.24±0.41 cm and 4.02±0.09 cm, respectively)
lanceolate leaves (Fig. 5 e and f) compared with the
emarginated small leaves of the control. These leaves
can be differentiated to each other based on variations
in the total chlorophyll contents (Fig. 6). The

Fig. 4 — Dendrogram illustrating genetic relationship among

control and 15 morphological variants of A. crispa using UPGMA Fig. 6 — Total chlorophyll and carotenoid contents (mg/gFw) in
method of clustering mutants and control of A. crispa

Fig. 5 — Morphological characterization of in vitro induced mutants of A. crispa. (a) Control leaf; (b) Control plant; (c) mutant line
ACM25; (d) ACM29; (e) ACM11: lanceolate leaflet, thin and long leaf with less chlorophyll; (f) ACM13: lanceolate leaflet, thin, long
leaves with high chlorophyll; (g) ACM17: long emarginated leaf with high chlorophyll; (h, i) ACM20: long, emarginated leaf with high
carotenoid; (j) ACM2: long, lanceolate, straita leaves; (k) ACM4: long emarginated, thicker maculate leaves; (l) ACM7: long,
emarginated, maculate leaf; (m) ACM8: long, oblong, waxy, Viridis leaf; (n, o) ACM10: short, oblong, Viridis leaf; (p) ACM39: long,
oblong and broad leaves; (q) ACM41: long, oblong, thicker and broader leaves; (r) ACM49: longer plant with more number of longer
leaves; and (s) ACM52: longest plant with more number of broader leaves
392 INDIAN J EXP BIOL, JUNE 2018
dendrogram (Fig. 4) showed maximal dissimilarity
(4.65) in the ACM17 and ACM20 mutant lines, which
were morphologically bigger in size than control but
had same leaflet shape. They were dissimilar because
of extremely high carotenoid content in ACM20
(Fig. 5 g–i; Fig. 6).
Morphological characterization revealed that the
five mutant lines (ACM2, ACM4, ACM7, ACM8,
and ACM10), which had evolved from the individual
dose treatments of gamma radiations and EMS,
showed moreover altered leaflet shapes, leaves size,
number or variegation than those of the control. These
mutant lines had lesser chlorophyll and carotenoid
than the control. Leaves of ACM2 exhibited
lanceolated leaflet shape with straita (white stripes)
chlorophyll spectrum (Fig. 5j). ACM4 and ACM7
leaves had maculata spectrum (spots of missing
chlorophyll and/or carotenoid) but were emarginated
similar to the control (Fig. 5 k and l). ACM8 and
ACM10 leaves were notably oblong in shape with
viridis (light green in colour) chlorophyll spectrum,
and visually distinguishable leaf size (Fig. 5 m–o).
ACM39 and ACM41 lines had oblong, long, broad,
and markedly thicker leaves, whereas ACM41 lines
had distinct uneven pigmented waxy leaves (Fig. 5 p
and q). Abnormal plant growth (a significant increase
in plant size) occurred among the ACM49 and
ACM52 lines, unlike the control. Among mutants,
ACM52 displayed maximum plant height and number
of leaves, whereas ACM49 showed maximum leaf
length (Fig. 5 r and s). The study results confirmed
that plant growth, leaf structure, and stomata- and
pigment-producing or regulating genes are majorly
affected by in vitro induced mutations.
Discussion
Mutation frequency depends on the gene position
in the genome and the treatment conditions during
mutagenesis17; therefore, the selection of a mutagen
and its optimum dose is a crucial step in artificially
induced mutation breeding programmes. In this study,
extremely low concentrations of colchicine and EMS
(0.025–0.3%) as well as gamma radiation doses (1–4 Gy)
induced variations in the treated protocorms, whereas
higher doses (0.4% of colchicine and EMS; 5 and 6 Gy
of gamma radiation) caused destruction or death of
the treated protocorms in A. crispa. Similar doses
were effective for in vitro mutation induction in
Lilium longiflorum and Asteracantha longifolia18,19. In
this study, combinations of mutagenic treatments for
in vitro mutagenesis were used in the sequence:
colchicine, gamma radiation, and EMS. Treatment
started with colchicine because other than improving
the growth rate, low colchicine concentration
enhances plant growth hormone synthesis and
changes the specific activity of enzymes, which
reduces lethality10. Furthermore, gamma irradiation
treatment stimulate hormonal signalling network
which control EMS uptake ensuing fewer alkylations
in the macromolecular fraction and reduced frequency
of mitotic and meiotic chromosomal aberrations20. In
this study, permutations of low doses of mutagens
were found more effective than individual doses for
inducing random favourable mutations at a higher
frequency. Dose-independent random mutation
frequency were possibly noted because of irregular
chromosomal, chromatid, or subchromatid
aberrations; changes in chromosome number;
inhibition of cell division; and induction of mitotic
activity8,21.
In this study, protocorm treatment with low doses of
colchicine, EMS, and gamma radiation revealed the
anomalous development of shoots and/or roots, leaflet
shape, and chlorophyll spectrum among the mutated
in vitro plantlets. Similar stimulatory effect at low
gamma radiation doses was noted in lettuce21. The
synergistic effects of physical mutagen coupled with
chemical mutagens are comprehensively studied in
various crop plants9,11. Mutant phenotypes of the control,
possibly accounted for somaclonal variations was
caused by in vitro stress22,23. Mengli et al.17 observed
similar distribution of mutation frequency in control.
Morphological traits are the product of gene and
environmental interactions. So, they do not determine
the actual level of genetic variation22-24. Genetic
variations among in vitro morphological variants were
confirmed using a molecular marker technique
(RAPD), in this study. Among 52 in vitro variant lines
based on variation in morphology, only 15 mutant
lines could be established on the basis of genetic
diversity. This is possibly because most
morphological variations were due to somatic or
epigenetic mutation and might be distributed in the
noncoding region of the genome and less but
permanent variations occurred in the coding regions25.
Clustering pattern of mutant lines inter se were well
correspond to morphological, stomatal and pigment
variations in this study. Similar dendrogram analysis
using UPGMA method exhibited clustering of control
and EMS mutants in Asteracantha26, radiomutants of
 SRIVASTAVA et al.: IN VITRO MUTAGENESIS IN AERIDES CRISPA
Chrysanthemum inter se27 and morphologically,
ecologically and geographically closer populations of
Rhynchostylis retusa species28.
Borzouei et al.29 hypothesised that low irradiation
induces growth stimulation by changing the hormonal
signalling network in plant cells or by increasing the
antioxidative capacity of the cells to overcome stress
factors easily. These reasons may be responsible for
faster growth of plants in mutant lines than in the
control. Leaf phenotype is the most prominent and
easily detectable characteristic of induced mutations.
Low doses of EMS and gamma radiations,
individually and in combinations, induced leaflet
shape mutations and/or variations in the leaf length
and width. Emarginated, oblong, and lanceolate leaf
shapes were noted in different dose treatments of
EMS and gamma radiations. Mutated leaves were
longer than control leaves, except at comparatively
high gamma radiation dose (3 Gy). In addition,
conflicting leaf width was noticed after different
treatments. Leaf structure and shape mutations
through gamma radiations have also been reported in
many crops like wheat and mungbean29,30. The present
findings showed differential effects of physical and
chemical mutagens in inducing chlorophyll
variegations, such as maculate and straita, after EMS
treatment and maculata and viridis spectrum after
gamma radiations treatment. However, no effect was
observed after the combination treatment. The
predominance of various mutants in the present study
may be attributed to involvement of polygenes in
chlorophyll formation31.
Colchicine effectively induced growth rate at
cytological, genetical, biochemical, physiological, and
morphogenetic levels and the amount of pigments in
different genotypes32,33. In this study, treatments with
colchicine and a combination of colchicine with gamma
radiations and/or EMS did not show any chlorophyll
variegation; however, increased pigment concentration
and growth parameters, such as plant height, number of
leaves, and leaf length, were noted. Similar beneficial
variability on the growth behavior, pigments, and
volatile oil products of Melalenea amillaris smith
explants was noted due to various gamma irradiation
and colchicine combination treatments34. Photosynthetic
pigments are known as appropriate indicators of
mutations, stress and nutritional state35. The irregular
dose-independent distribution of chlorophyll and
carotenoid contents, in the mutants of present study, are
observations analogous with those reported in Triticum
393
aestivum L. and Capsicum annuum L.29,36. Few reports
revealed that point mutation in a single gene of a
signaling pathway can cause specific alterations in the
stomatal density and size37,38. This may explain the
unusual increase in stomatal density and the apparent
variation in the leaf morphology of ACM25 and
ACM29 mutants.
Conclusion
In the present study, we successfully evaluated the
effect of in vitro mutation induction through
colchicine, gamma radiations, EMS and their
combinations as a means to improve the economic
traits of 15 mutant lines of A. crispa. Combination of
mutation technology under in vitro conditions and
RAPD method proved successful with A. crispa,
exhibiting variations in terms of growth rate, leaf
shape, leaf length, width, chlorophyll variegation,
stomatal density, and/or pigment contents.
Acknowledgement
Junior research fellowship provided by Council of
Scientific and Industrial Research (CSIR), New Delhi,
India to one of the authors, Dr. Deepti Srivastava is
gratefully acknowledged. Co-author Dr. MC Gayatri
thankfully acknowledges the University Grants
Commission (UGC) for providing UGC-BSR Faculty
Fellowship. The authors report no conflicts of interest.
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