IJABR, VOL.

9 (1) 2019: 68-74 ISSN 2250 – 3579

MICROPROPAGATION OF ANTHURIUM (Anthurium andreanum Lind.)

*
Rocky Thokchom, aSoumen Maitra, bSubhendu Shekhar Gantait, cArup Sarkar and dSanasam Sanjay Singh
*Department of Horticulture, Pandit Deen Dayal Upadhyay Institute of Agricultural Sciences, Utlou, Bishnupur, Manipur 795134
a
Department of Floriculture, Medicinal and Aromatic Plants, Faculty of Horticulture, Uttar Banga Krishi Viswavidyalaya,
Pundibari, Coochbehar, West Bengal-736165.
b
Department of Floriculture and Landscaping, Faculty of Horticulture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia,
West Bengal-741252
c
Department of Genetics and Plant Breeding, Faculty of Agriculture, Uttar Banga Krishi Viswavidyalaya, Pundibari, Coochbehar,
West Bengal-736165.
d
ICAR, NEH Region, Lamphelpat, Manipur- 795004
*Corresponding author’s email: rockythokchomaau@gmail.com

ABSTRACT
Anthurium (Anthurium andreanum Lind.), one of the most valued ornamentals is propagated conventionally through
vegetative means which is very slow and need attention to develop elite, genuine, true-to-type quality planting materials at
a faster rate. The present study attempts the micropropagation of Anthurium through callus culture followed by
organogenesis and rhizogenesis in vitro including subsequent acclimatization using leaf, petiole and inflorescence explants
at Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar, West Bengal, India during 2013-16. Results revealed that
immature coppery leaf lamina of Anthurium were highly responsive towards callus production than that of petiole and
inflorescence explants. Culture of leaf explants in MS medium supplemented with 2,4-D (2.0 mg/l) and TDZ (0.1 mg/l)
showed better response in respect of earliness in callusing (52.28 days), percentage of callusing explants [78.67%
(62.50%)] and weight of produced calli at 15 (0.404 g) and 30 days (2.944 g) after culture. Young growing calli when
cultured in MS medium containing Kinetin (3.0 mg/l) + BAP (1.0 mg/l) developed in vitro regeneration in greater
percentage of explants [98.07% (83.67%)] with higher number of micro shoots (17.27) per culture. Earlier regeneration
(26.93 days) was observed with MS culture medium having Kinetin (2.0 mg/l) and BAP (1.0 mg/l). Length of microshoot
(9.00 cm) and leaf production (20.67) were found higher with the culture medium MS + BAP (1.0 mg/l) whereas, higher
leaf length (2.07 cm) with greater width (1.52 cm) was obtained from the culture media MS + Kin (2.0 mg/l) + BAP (1.0
mg/l). In vitro regenerated microshoots when cultured in rooting medium containing MS + IBA (1.0 mg/l) + NAA (1.0
mg/l) initiated earlier (32.98 days) in vitro rooting in cent percent of microshoots with higher number of roots per shoot
(7.36) having greater length (5.29 cm) and diameter (1.225 mm). Coconut husk was found as the effective hardening media
showing earlier acclimatization (11.77 days) with greater survivability [92.75% (74.89%)].

KEY WORDS: Anthurium andreanum, micropropagation, explants.

INTRODUCTION shoot tips (Harb et al., 2010). Plantlet regeneration of

Anthurium, belonging to plant family Araceae and order Anthurium andreanum has been achieved through
Spathiflorae, is one of the most important ornamental adventitious shoot formation from callus (Jahan et al.,
plants consisting of 108 genera and approximately 3750 2009) and direct shoot regeneration from lamina explants
monocotyledonous species. Anthuriums are commonly (Martin et al., 2003).
cultivated for its long-lasting and unusually attractive heart Pierik et al. in 1974 first reported the tissue culture of
shaped spathe with finger like spadix borne on a long Anthurium where they used liquid culture to proliferate
stalk. Among the several commercially important callus (Martin et al., 2003) while Teng (1997) used the
Anthurium species, Anthurium andreanum is one of the liquid or raft culture instead of solid medium to regenerate
ten most cultivated ornamental plants for cut flowers in the into adventitious shoots from leaf explants. Vargas et al.
world (Jahan et al., 2009) and is valued next to next to (2004) obtained in vitro plants from germinated seed and
orchids among tropical flowers. Moreover, potted plants plantlets obtained from culture of micro-cuttings. These
are generally appreciated for export in world market plantlets showed callus at the stem base. Micropropagation
(Ullah, 1995). Anthurium is conventionally propagated by of Anthurium in vitro through indirect method is a difficult
seed and division of suckers (Dufour and Guerin, 2003; step and time consuming. However, for en masse
Maitra et al., 2012). Since the conventional method of multiplication of Anthurium, credible proliferation of
propagation is time consuming, micropropagation appears callus and subsequent plant regeneration is important. This
as an alternative to increase the production on a article describes the detailed protocol of Anthurium
sustainable basis (Hamidah et al., 1997; Martin et al., andreanum for establishment of rapid method for
2003). It has been achieved with various tissues including regeneration of Anthurium andreanum from callus tissue
leaf (Farsi et al., 2012), petiole (Raad et al., 2012), spadix, through organogenesis.
spathe, seed (Matsumoto et al., 1998), lateral bud and

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 Micropropagation of Anthurium

MATERIALS AND METHODS were given for plantlet acclimatization. Each treatment
The experiment was conducted at plant tissue culture was done in 3 replications for in vitro plant regeneration
laboratory, Department of Floriculture, Medicinal and and 5 replications were given in case of plantlets
Aromatic Plants, UBKV, Cooch Behar during the year acclimatization. All experiments were repeated three
2013-2016. Immature coppery-brown coloured leaf, times. The percentile data of the experiments were
immature petiole and young inflorescence explants of assumed and subjected to square root transformations.
Anthurium andreanum was collected from the net house of
the department. Explants were previously subjected to RESULTS AND DISCUSSION
running tap water for about half an hour using two to three A regeneration protocol for Anthurium andreanum was
drops of Tween-20 to remove excess dirt from the leaf established using three different explant sources viz., leaf,
surface. They were later treated with fungicides and petiole and inflorescence in thirteen aseptic MS basal
bactericides (1% each) consecutively for 10-20 minutes to media containing different concentrations of either 2,4-D
minimize fungal and bacterial contamination. Inside the alone or combination with different doses of TDZ for
laminar flow, the explants were sterilized with 0.1 per cent callus induction. Results pertaining to Table 1 revealed
(v/v) HgCl2 for 2-3 minutes followed by 70 per cent (v/v) that amongst all explant sources, the leaf explant
ethyl alcohol for 30 seconds. Explants were then rinsed performed the best by producing the maximum callusing
thoroughly with sterile double distilled water to remove percentage [43.40% (41.21%)] in shortest time duration
the toxic residues from the surface. Well sterilized (52.28 days) and yielded the heaviest calli (2.944g). The
explants were reduced to the size of about 0.5 to 1.0 cm2 reason might be due to the choice explants that have
pieces for inoculation. adequate morpho-genetical plasticity leading to higher
Media division of cells and rapid callus induction. Morphogenetic
For indirect organogenesis, MS basal medium (Murashige plasticity dependent response to callus initiation was also
and Skoog, 1962) fortified with 30 g/L sucrose was used observed by Bejoy et al. (2008) in Anthurium; Trejgell et
adjusting the pH to 5.7 before autoclaving. al. (2009) in Carlina acaulis; Taha et al. (2011) in
Callus initiation ornamental fern. Among the different plant hormones
Explants were transferred into the culture tubes containing used, the best callusing ability was observed in the MS
30 g/L sucrose, with or without or either 2,4-D alone or in basal medium containing 2.0 mg/l 2,4-D + 0.1 mg/l TDZ
combination with TDZ. The pH was adjusted to 5.7 before [61.78% (52.05%)] producing callus at the earliest (42.38
autoclaving. The media differed in concentrations of plant days) with maximum weight (3.653g) at 30 days after
growth regulators and light conditions of culture. explant inoculation. The observations recorded from
Shoot regeneration interaction effect on the callusing ability showed that leaf
For in vitro shoot regeneration, calli of leaf explants were explants cultured in MS+1.0 mg/l 2,4-D + 0.1 mg/l TDZ
transferred to the basal medium fortified with or without yielded calli in maximum percentage of explants [77.78%
or different concentrations of Kinetin and BAP either (62.00%)] taking the minimum duration (43.78 days) to
alone or in combination which was kept at 16/8 light and callus initiation. However, MS basal medium containing
darkness photoperiod. 1.0 mg/l 2,4-D +0.1 mg/l TDZ generated heaviest calli
Rooting (5.004 g). It is obvious that the hormonal combination in
The regenerated shoots longer than 3 cm with a pair of callusing medium determines the callus formation which
leaves were transferred to the MS medium containing with varies from species to species and explant to explant (Tsay
or without plant growth regulators or different et al., 2006; Gitonga et al., 2010; Atak and Celik, 2015).
concentrations of IBA either alone or in combination with Hence, optimization of phytohormonal combination in
NAA. callusing media is an important aspect for any conclusive
Hardening opinion for understanding the mode of action at the
The well rooted anthurium plantlets were later transferred molecular level (Grozeva et al., 2006; Arab et al., 2014).
to various hardening medium for acclimatization and were For regeneration of Anthurium, both Kinetin and BAP was
maintained at 90 per cent relative humidity in the found suitable. Data depicted in Table 2 revealed that
hardening chamber for proper establishment of the culture of calli in MS basal medium fortified with 2.0 mg/l
plantlets. Kin +1.0 mg/l BAP recorded the earliest regeneration
Statistical analysis (26.93 days), producing the longest (2.07 cm) and broadest
The experiment was laid out under factorial completely leaf (1.52 cm) while MS basal medium containing only 1.0
randomized design for various explants callusing and mg/l BAP regenerated the tallest microshoots (9.00 cm)
completely randomized design for the rest of the and highest number of leaves per microshoot (20.67).
experiments. The data generated were analyzed by However, MS medium supplemented with 3.0 mg/l Kin +
Fisher’s analysis of variance (ANOVA) technique at 5 per 1.0 mg/l BAP recorded the maximum percentage of
cent level of significance. Ten explants were used in each regeneration[98.07% (83.67%)].
treatment for plant organogenesis while five treatments

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IJABR, VOL.9 (1) 2019: 68-74 ISSN 2250 – 3579

TABLE 1. Effect of culture media on callusing characteristics of Anthurium andreanum Lind. through various explant
sources
Percentage of Days to callus Weight of callus after 30
Explants
callusing (%) initiation (days) days of initiation (g)
Leaf 43.40 (41.21) 58.28 2.944
Petiole 37.16 (37.56) 64.82 2.242
Inflorescence 21.80 (27.83) 73.66 1.409
S. Em ± 0.15 0.42 0.018
CD at 5% 0.43 1.18 0.053
Culture Media
MS 10.17 (18.46) 142.42 0.239
MS+1.0 mg/l 2,4-D 35.41 (36.17) 54.06 2.505
MS+2.0 mg/l 2,4-D 42.02 (40.13) 52.72 2.684
MS+3.0 mg/l 2,4-D 26.22 (30.38) 67.74 1.619
MS+4.0 mg/l 2,4-D 15.60 (23.22) 84.26 0.887
MS+1.0 mg/l 2,4-D + 0.1 mg/l TDZ 51.26 (45.84) 47.18 3.303
MS+1.0 mg/l 2,4-D + 0.2 mg/l TDZ 55.46 (47.89) 46.22 3.514
MS+2.0 mg/l 2,4-D + 0.1 mg/l TDZ 61.78 (52.05) 42.38 3.653
MS+2.0 mg/l 2,4-D + 0.2 mg/l TDZ 53.04 (46.78) 46.46 3.255
MS+3.0 mg/l 2,4-D + 0.1 mg/l TDZ 36.44 (37.42) 53.16 2.789
MS+3.0 mg/l 2,4-D + 0.2 mg/l TDZ 27.31 (31.46) 63.31 1.907
MS+4.0 mg/l 2,4-D + 0.1 mg/l TDZ 21.33 (27.50) 73.04 1.285
MS+4.0 mg/l 2,4-D + 0.2 mg/l TDZ 17.53 (24.72) 79.67 0.937
S. Em ± 0.32 0.87 0.039
CD at 5% 0.90 2.45 0.110
Explant x Culture Media
Leaf x MS 12.89 (21.00) 122.91 0.381
Leaf x MS+1.0 mg/l 2,4-D 55.11 (47.94) 43.07 3.915
Leaf x MS+2.0 mg/l 2,4-D 47.11 (43.34) 42.71 3.014
Leaf x MS+3.0 mg/l 2,4-D 40.44 (39.49) 48.89 2.773
Leaf x MS+4.0 mg/l 2,4-D 16.59 (24.02) 81.00 0.669
Leaf x MS+1.0 mg/l 2,4-D + 0.1 mg/l TDZ 77.78 (62.00) 32.00 5.004
Leaf x MS+1.0 mg/l 2,4-D + 0.2 mg/l TDZ 70.16 (56.52) 38.13 4.692
Leaf x MS+2.0 mg/l 2,4-D + 0.1 mg/l TDZ 69.48 (56.50) 40.22 4.797
Leaf x MS+2.0 mg/l 2,4-D + 0.2 mg/l TDZ 61.19 (51.59) 41.24 4.571
Leaf x MS+3.0 mg/l 2,4-D + 0.1 mg/l TDZ 45.04 (42.15) 44.98 3.647
Leaf x MS+3.0 mg/l 2,4-D + 0.2 mg/l TDZ 38.67 (38.00) 58.24 2.930
Leaf x MS+4.0 mg/l 2,4-D + 0.1 mg/l TDZ 21.19 (27.39) 80.73 1.025
Leaf x MS+4.0 mg/l 2,4-D + 0.2 mg/l TDZ 18.96 (25.81) 83.49 0.854
Petiole x MS 8.89 (17.22) 138.51 0.153
Petiole x MS+1.0 mg/l 2,4-D 29.63 (32.970 59.69 2.002
Petiole x MS+2.0 mg/l 2,4-D 56.15 (48.54) 50.44 3.074
Petiole x MS+3.0 mg/l 2,4-D 22.37 (28.20) 69.04 1.441
Petiole x MS+4.0 mg/l 2,4-D 16.00 (23.55) 70.84 1.224
Petiole x MS+1.0 mg/l 2,4-D + 0.1 mg/l TDZ 51.56 (45.89) 53.40 3.016
Petiole x MS+1.0 mg/l 2,4-D + 0.2 mg/l TDZ 66.37 (54.55) 46.73 3.797
Petiole x MS+2.0 mg/l 2,4-D + 0.1 mg/l TDZ 74.07 (59.40) 44.78 3.992
Petiole x MS+2.0 mg/l 2,4-D + 0.2 mg/l TDZ 63.70 (52.96) 49.18 3.297
Petiole x MS+3.0 mg/l 2,4-D + 0.1 mg/l TDZ 46.80 (43. 29) 57.38 2.858
Petiole x MS+3.0 mg/l 2,4-D + 0.2 mg/l TDZ 24.74 (29.82) 62.18 1.694
Petiole x MS+4.0 mg/l 2,4-D + 0.1 mg/l TDZ 21.33 (27.50) 68.82 1.525
Petiole x MS+4.0 mg/l 2,4-D + 0.2 mg/l TDZ 17.33 (24.58) 71.67 1.067
Inflorescence x MS 8.74 (17.15) 165.84 0.180
Inflorescence x MS+1.0 mg/l 2,4-D 21.48 (27.60) 59.42 1.598
Inflorescence x MS+2.0 mg/l 2,4-D 22.81 (28.50) 65.01 1.965
Inflorescence x MS+3.0 mg/l 2,4-D 15.85 (23.44) 85.29 0.643
Inflorescence x MS+4.0 mg/l 2,4-D 14.22 (22.09) 100.93 0.770
Inflorescence x MS+1.0 mg/l 2,4-D + 0.1 mg/l TDZ 24.44 (29.62) 56.13 1.884
Inflorescence x MS+1.0 mg/l 2,4-D + 0.2 mg/l TDZ 29.04 (32.59) 53.80 2.054
Inflorescence x MS+2.0 mg/l 2,4-D + 0.1 mg/l TDZ 41.78 (40.26) 42.13 2.170
Inflorescence x MS+2.0 mg/l 2,4-D + 0.2 mg/l TDZ 34.22 (35.79) 48.96 1.898
Inflorescence x MS+3.0 mg/l 2,4-D + 0.1 mg/l TDZ 20.00 (26.81) 57.11 1.861
Inflorescence x MS+3.0 mg/l 2,4-D + 0.2 mg/l TDZ 19.50 (26.58) 69.51 1.098
Inflorescence x MS+4.0 mg/l 2,4-D + 0.1 mg/l TDZ 21.48 (27.59) 69.56 1.305
Inflorescence x MS+4.0 mg/l 2,4-D + 0.2 mg/l TDZ 16.30 (23.77) 83.87 0.889
S. Em ± 0.55 1.51 0.068
CD at 5% 1.56 4.25 0.191

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 TABLE 2. Effect of culture media on the shooting parameters of Anthurium andreanum Lind. in vitro.
Days to Regeneration Number of Length of Number of Leaf length Leaf diameter
Treatments regeneration from Percent from microshoots/ microshoot leaves/microsh (cm) (cm)
callus (days) callus (%) culture (cm) oot
MS0 80.51 31.85 (33.96) 2.56 2.92 3.33 0.45 0.28
MS+0.5 mg/l Kin 37.73 81.33 (64.43) 9.98 5.64 6.47 0.82 0.56
MS+1.0 mg/l Kin 30.38 87.26 (69.12) 14.29 6.48 8.27 1.38 1.02
MS+2.0 mg/l Kin 33.36 96.89 (80.19) 9.91 5.63 7.18 1.10 0.78
MS+0.5 mg/l BAP 32.27 77.19 (61.52) 12.42 6.29 9.09 0.90 0.60
MS+1.0 mg/l BAP 28.73 82.52 (65.35) 17.07 9.00 20.67 1.65 1.27
MS+2.0 mg/l BAP 33.22 75.85 (60.57) 13.80 7.68 16.38 1.46 1.10
MS+2.0 mg/l Kin + 1.0 mg/l BAP 26.93 98.22 (83.37) 8.20 7.91 18.18 2.07 1.52
MS+2.0 mg/l Kin + 2.0 mg/l BAP 29.56 97.33 (82.62) 12.53 7.32 14.53 1.82 1.40
Micropropagation of Anthurium

MS+2.0 mg/l kin + 3.0 mg/l BAP 34.27 87.41 (69.27) 11.20 7.07 13.80 0.60 0.34
MS+3.0 mg/l Kin + 1.0 mg/l BAP 32.80 98.07 (83.67) 8.73 6.42 11.02 0.75 0.45
MS+3.0 mg/l Kin + 2.0 mg/l BAP 41.24 94.22 (76.57) 7.27 5.98 7.60 0.69 0.41
MS+3.0 mg/l Kin + 3.0 mg/l BAP 44.89 78.22 (62.23) 5.51 5.61 5.44 0.55 0.32
S.E(m)± 0.43 1.79 0.24 0.06 0.21 0.02 0.02
C.D. at 5% 2.10 8.67 1.15 0.30 1.04 0.09 0.09

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TABLE 3. Effect of culture media on the rooting parameters of Anthurium andreanum Lind. in vitro.
Days to root Percentage of rooted Number of roots Length of root Root diameter
Treatments
initiation (days) microshoots (%) per microshoot (cm) (mm)
MS0 82.78 27.70 (31.74) 2.00 1.88 0.684
MS+1.0 mg/l IBA 33.27 100.00 (90.00) 5.60 4.22 1.090
MS+1.5 mg/l IBA 36.71 100.00 (90.00) 4.24 3.79 1.070
MS+2.0 mg/l IBA 42.09 100.00 (90.00) 4.16 3.35 1.025
MS+2.5 mg/l IBA 45.91 98.67 (84.66) 3.73 2.92 0.965
MS+3.0 mg/l IBA 52.49 98.37 (83.183) 2.80 2.47 0.910
MS+3.5 mg/l IBA 56.16 97.04 (80.23) 2.44 1.88 0.856
MS+1.0 mg/l IBA + 1.0 mg/l NAA 32.98 100.00 (90.00) 7.36 5.29 1.225
MS+1.5 mg/l IBA + 1.0 mg/l NAA 34.16 100.00 (90.00) 6.09 5.03 1.144
MS+2.0 mg/l IBA + 1.0 mg/l NAA 37.24 100.00 (90.00) 5.22 4.79 1.052
MS+2.5 mg/l IBA + 1.0 mg/l NAA 42.33 99.85 (89.26) 4.49 4.58 0.969
MS+3.0 mg/l IBA + 1.0 mg/l NAA 45.89 98.96 (85.27) 3.69 4.06 0.933
MS+3.5 mg/l IBA + 1.0 mg/l NAA 52.87 96.74 (80.03) 2.73 2.81 0.856
S.E(m)± 0.79 0.72 0.14 0.08 0.01
C.D. at 5% 3.83 3.48 0.68 0.40 0.05
IJABR, VOL.9 (1) 2019: 68-74 ISSN 2250 – 3579

a) Callus from leaf explants b) Callus from petiole explants c) Callus from inflorescence explants

d) Caulogenesis e) Organogenesis f) Shoot induction

g) Shoot multiplication h) Rooted plantlets i) Rooted microshoots ready for

hardening

j) Dividing shootlets from clump k) Plantlets ready to transfer l) Hardened plantlets

FIGURE 1. Different phases of micropropagation of Flowering Anthurium (Anthurium andreanum Lind.)

The pivotal factors that governing the regeneration of Rhizogenesis occurred satisfactorily when the cultures
callus like auxin: cytokinin ratio, presence of meristematic were allowed to stand for long period under artificial
tissue, absence of apical dominance, nature of totipotency illumination. Isolation of microshoots from a mass
were also observed by Su et al., (2011). Higher cytokinin resulted rapid and consistent rooting when cultured in
requirement during regeneration from callus was observed specified rooting medium in vitro. For this purpose a
by Maitra et al. (2012) while enhancement of shoot supplementation with IBA and NAA in MS medium was
growth and development in vitro of anthurium using found essential. Data presented in Table 3 revealed that
kinetin and BAP was also examined by Bejoy et al. MS + 1.0 mg/l IBA + 1.0 mg/l NAA took the minimum
(2008); Atak and Celik (2009); Murillo-Gómez et al. days (32.98 days) to initiate roots in vitro, maximum
(2014). number of roots per microshoot (7.36), longest root (5.29

72
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 Micropropagation of Anthurium

cm) and maximum root diameter (1.225 mm). Cent simply (Raad et al., 2012). This may be due to the
percent rooting were observed in almost all the basal enhancement of auxin environment inside the regenerated
medium fortified with either IBA alone or the combination microshoot and auxin transport in the basipetal manner
of both IBA and NAA. Hormone free MS basal medium leading to higher auxin accumulation at the lower end of
developed least percentage to root initiation [27.70% microshoots results higher, rapid and consistent rooting in
(31.74%)] of microshoots. It has been observed in several vitro. Use of a combination of IBA and NAA fortified in
crops that NAA when used in the rooting medium along basal MS medium for in vitro rooting was also observed
with IAA or IBA induced in vitro rooting with higher by Zhang et al. (2001); Joseph et al. (2003).
magnitude as compared to application of IAA or IBA

TABLE 4. Effect of hardening media on plantlets acclimatization of Anthurium andreanum Lind. ex-vitro.
Days required for Survivability
Treatments
acclimatization (days) percentage (%)
Vermiculite + Sand (1:1) 14.20 63.08 (52.63)
Sand + Sawdust (1:1) 18.47 55.42 (48.12)
Coconut husk 11.77 92.75 (74.89)
Vermiculite + coconut husk (1:1) 18.98 73.67 (59.23)
Vermiculite 21.32 68.92 (56.21)
S.E(m)± 0.62 1.05
C.D. at 5% 3.05 3.05

The success of micropropagation lies in acclimatization of REFERENCES

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ACKNOWLEDGEMENT Regeneration in Tomato (Lycopersicon esculentum Mill.)
The authors gratefully acknowledge Mr. Kamal Das, in vitro. Bulgarian Journal of Agricultural Science, 12,
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