In vitro mutation breeding of Anthurium by gamma radiation

Abstract

In vitro plantlet regeneration in a number of Anthurium (Anthurium andreanum) varieties was achieved from callus cultures derived from young, tender leaf explants on Nitsch's (1969) medium. Apart from the time period for callus induction, no differences in terms of regeneration were noted amongst the three varieties tested. Callus induction was found to be more rapid and prolific when Nitsch medium containing BA (1 mg L -1) and 2,4-D (0.1 mg L -1) with a reduced concentration of ammonium nitrate (200 mg L -1) was used. Shoot formation occurred when BA concentration was reduced to 0.5 mg L -1 and the ammonium nitrate level increased to 720 mg L -1 . Regenerated shoots rooted readily on Nitsch medium containing IBA (1.0 mg L -1). Rooting was improved significantly by the addition of activated charcoal (0.04%) to the medium. When explants (leaves, seeds, in vitro plantlets) were irradiated, best response was observed with the 5 Grays (Gy) treatment in terms of callus formation and regeneration while the 15 Gy dose was lethal to the Anthurium tissues. The phenotypic results indicated a boosting effect of the 5 Gy dose on the leaf tissues. The variability in the responses observed seemed to indicate some mutation, both positive and negative, at the cellular level of the tissues. However, no difference in RAPD profiles were noted between the DNA fingerprints of the mother plant and that of the irradiated tissues using a limited number of primers.

Full text

INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2005/07–1–11–20
http://www.ijab.org
In Vitro Mutation Breeding of Anthurium by Gamma Radiation
D. PUCHOOA
Faculty of Agriculture, University of Mauritius, Réduit, Mauritius
Author’s e-mail: sudeshp@uom.ac.mu
ABSTRACT
In vitro plantlet regeneration in a number of Anthurium (Anthurium andreanum) varieties was achieved from callus
cultures derived from young, tender leaf explants on Nitsch’s (1969) medium. Apart from the time period for callus
induction, no differences in terms of regeneration were noted amongst the three varieties tested. Callus induction was
found to be more rapid and prolific when Nitsch medium containing BA (1 mg L-1) and 2,4-D (0.1 mg L-1) with a reduced
concentration of ammonium nitrate (200 mg L-1) was used. Shoot formation occurred when BA concentration was
reduced to 0.5 mg L-1 and the ammonium nitrate level increased to 720 mg L-1. Regenerated shoots rooted readily on
Nitsch medium containing IBA (1.0 mg L-1). Rooting was improved significantly by the addition of activated charcoal
(0.04%) to the medium. When explants (leaves, seeds, in vitro plantlets) were irradiated, best response was observed with
the 5 Grays (Gy) treatment in terms of callus formation and regeneration while the 15 Gy dose was lethal to the
Anthurium tissues. The phenotypic results indicated a boosting effect of the 5 Gy dose on the leaf tissues. The variability
in the responses observed seemed to indicate some mutation, both positive and negative, at the cellular level of the
tissues. However, no difference in RAPD profiles were noted between the DNA fingerprints of the mother plant and that
of the irradiated tissues using a limited number of primers.
Key Words: Mutation; Breeding; Anthurium; Gamma; Radiation
INTRODUCTION
Species within the genus Anthurium, family Araceae,
are highly prized for their exotic flowers and foliage which
make demand for propagating material and new cultivars
high. In Mauritius commercial cultivation of anthurium
started about 30 years ago and over the years, anthurium has
undoubtedly become one of the most economically
important crops in Mauritius. In the year 2000, 95% of the
flowers exported consisted of anthurium, showing its
significance in our horticultural industry. The number of
blooms produced kept on increasing, with about 9.2 million
exported in the year 1990 and increasing to over 15 million
in 1995. The horticulture industry, in general, has flourished
within a few decades. Statistics show that in the year 1996,
income from flower export was only Rs 99,000 while by the
year 2000; it increased to Rs 127 million. At present,
however, Mauritius is facing severe competition from
countries such as the Netherlands, Hawaii and China which
is emerging as a potential producer of anthurium. Recently,
there has been a continued decline in production and the
price fetched on the world market. Our main anthurium
export markets are Japan, Italy, France and Taiwan and a
smaller amount is exported to Hong-Kong, South Africa,
the United States and Germany. The main constraints faced
by the anthurium sector presently are its high cost of
production, the high air-freight charges, lack of proper
marketing system, lack of adequate technical support,
scarcity of growing medium, no insurance cover to protect
against damage and no cold chain distribution facility. Also,
the traditional locally grown commercial varieties are
running out of fashion and have low market value. Earlier,
Mauritius was over-dependent on the Dutch varieties.
However, an imposition of ban on import of tissue-cultured
plantlets in the early 1990’s made access to new varieties
difficult and growers were unable to respond to the
changing market.
Anthurium is conventionally propagated by seeds and,
therefore, cultivation is hindered by problems due to the
inherent heterozygosity. Although traditional techniques of
vegetative propagation such as the use of stem cuttings and
suckers exist, they are tedious and not practical when carried
out on a large scale. Tissue culture greatly increases the
normal multiplication rate of plants and can provide a
source of clean material which has become increasingly
important due to outbreak of bacterial and other diseases
such as anthracnose, blight, leaf spot, root knot and bacterial
wilt caused by Xanthomonas campestris pv. diffenbachiae.
The method for in vitro production of plantlets of
Anthurium andreanum was first developed by Pierik et al.
(1974). The production of in vitro plants directly from
proliferating axillary buds (Kunisaki, 1980), adventitious
buds (Cen et al., 1993), leaf or petiole organogenic callus
culture (Pierik et al., 1974; Pierik, 1975, 1976; Finnie &
Van Staden, 1986; Kuehnle & Sugii, 1991) and from
somatic embryos derived from in vitro grown leaf blade
explants (Kuehnle et al., 1992) has been reported. Geier
(1982) working on Anthurium scherzeranium was also able
to develop plantlets from spadix explants and later from
leaves. All workers found that there was great variation in
the requirements of different genotypes. Methods for several
other varieties may have been worked out by commercial
establishments but these are not available for general use.
Hence, we have undertaken to develop suitable methods or

PUCHOOA / Int. J. Agri. Biol., Vol. 7, No. 1, 2005
12
modify existing ones for tissue culture of three varieties of
Anthurium. This will be of great help in micropropagation
and for future in vitro breeding work to develop resistant
varieties.
Mutagenic agents have been used to induce useful
phenotypic variations in plants for more than 70 years
(Foster & Twell, 1996). A large number of mutant lines
have been isolated from many plants and these have been
used for plant research and crop breeding purposes (Evans,
1962). For anthurium, new techniques are needed for further
improving crop cultivars apart from the traditional plant
breeding. Mutation breeding is therefore being proposed as
a means to create additional variation. The application of
ionizing radiation, chemical mutagens as well as somaclonal
variation from tissue culture is quite common in the creation
of genetic variation. Novel plant mutants like maize, alfalfa,
potato, banana, and barley among others and other cell lines
of agricultural and industrial interests generated from tissue
culture have also been quite popular (Collin & Dix, 1990).
Different sources of plant tissues such as seeds, pollen,
and other cell systems have been employed in studies
related with mutagenesis of a number of plant species
(Redei, 1970; Lindgren, 1975; Feldmann et al., 1994). For
seeds, both chemical and irradiation methods for inducing
mutations have proved to be highly efficient (Redei, 1970;
Koornneef et al., 1982; Haunghn & Somerville, 1987).
Physical mutagens like ionizing radiations (X-rays, gamma
rays and neutrons) and UV light, and also a series of
chemical agents are common examples of mutagenesis
techniques that have a high efficiency at generating
mutations in plants, animals as well as bacteria.
Furthermore, the outcomes of these treatments can at least
be predicted to a certain extent.
As yet, relatively few sequences of irradiation-
mutagenised genes in plants have been published. However,
recent work with irradiation mutants of Arabidopsis
supports the general assumption that irradiation causes
single- and double-strand breaks which result in
chromosomal rearrangements. These studies have also
indicated that deletions may be accompanied by other local
chromosomal alterations, such as insertions and inversions,
not detectable at the cytological level (Wilkinson &
Crawford, 1991). The commonly used mutagenic agents
cannot produce new genes but in fact they only alter those
present in the treated genotype. Ionizing radiation, for
instance, generates chromosomal breaks which, following
DNA repair, result in a variety of chromosomal aberrations.
Gene mutations are less frequent than chromosomal
mutations, which include translocations, inversions,
deletions and deficiencies. Mutations in the narrow sense
affect parts or sections of a gene, either single base pairs or
groups of them. Exchange of base pairs or alterations of
their sequence may change the primary gene product and by
way of a more or less complicated chain reaction of events
ultimately lead to a modified phenotypic expression of one
or several traits. Sometimes the gene is affected in such a
way that it cannot code for any product. The recovery of
mutants induced by high levels of mutagens is limited by
somatic effects, such as reduced viability, growth
abnormalities and reduced fertility. Therefore, every
mutagen has a most effective dose, which produces the
maximum level of mutagenesis with minimal somatic
effects. Mutated genes often appear to be somewhat weak in
their phenotypic expression and under heterozygous
conditions, can be classified as recessive.
The successful outcome of a mutation depends on the
efficient induction of mutations as well as the efficient
recognition and recovery of the desired mutant plants or
mutant genes. From the variety of mutagenic agents that are
available, each has their particular merits. For example,
some may induce predominantly point mutations and others
chromosomal rearrangements. Also, some can penetrate
multi-cellular plant structures, while others cannot and they
may be more easily available or safer than other mutagens.
Apart from the choice of the proper mutagenic agent, the
dosage and the treatment conditions are important.
Consideration must also be given to the plant materials
treated such as the stage in the life cycle of the plant or plant
organs, the sensitivity of the plant species to the effects of
the mutagenic agents and the possible genotypic differences
in sensitivity to the mutagenic treatments.
MATERIALS AND METHODS
Tissue Culture Studies
Plant materials. Anthurium andraeanum plants grown in
the University farm shadehouse were used for the
experiments. All plants were fertilized monthly with
13:13:20:2 soluble fertilizer containing micronutrients. The
plants were also treated with a fortnightly spray of a mixture
of 4 g L-1 Welgro, 3 g L-1 Microthiol, 2 g L-1 Peropal, 4 g L-1
Lannate and 4 mL L-1 Fenitrothion.
Explant preparation and sterilization. Young unfolded
leaves were collected and briefly washed under running tap
water. Pre-sterilization was done on the whole leaf by
soaking in a solution of 0.6% Benlate (benomyl) for 30 min.
Sterilization consisted of washing the leaves with diluted
liquid soap and thorough rinsing with tap water followed by
a dip in 70% alcohol for 30 sec and soaking in 1.5% sodium
hypochlorite, containing two drops of Tween 20. After 20
min of gentle agitation, the leaves were rinsed three times in
sterile distilled water with 15 min in each rinse and a final
rinse for 30 min. The leaves were then cut into explants of 1
to 2 cm2 and inoculated abaxially onto callus induction
medium.
Culture medium and culture conditions. The following
culture media were used:
(1) Modified MS (Murashige & Skoog, 1962) medium with
macronutrients at half strength, full-strength MS
micronutrients, 100 mg L-1 myo-inositol and MS vitamins
and (2) Modified Nitsch (1969) medium and vitamins. For
callus culture, the ammonium nitrate concentration of this

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medium was reduced to 200 mg L-1 but for regeneration and
rooting, it was increased to 720 mg L-1. All media contained
8.0 g L-1 agar (Oxoid, Technical Grade No.3), 30 g L-1
sucrose and different concentrations and molar ratios of 2,4
- D and BA as shown in Table I. Medium pH was adjusted
to 5.8 with KOH before adding the agar. Media were
autoclaved for 15 min at 1210C and dispensed as 25 mL
aliquots into 125 mL glass jars. In an attempt to speed up
rooting, activated charcoal (0.04%) was added to the media.
For callus induction, explants were grown in a culture
environment at 25 ± 2
0C with continuous darkness. For
regeneration experiments, the calli were grown at 25 ± 20C
with a 16 h photoperiod and a light intensity of 5.0 Wm-2
provided by daylight-type fluorescent lamps. Plantlets were
hardened by potting in vermiculite and growing them in a
mist house with very low light intensity and keeping the
humidity as high as possible.
A completely randomized design was used for all
experiments. Each treatment consisted of six replications
(jars) with two explants in each jar. All the experiments
were run twice. The final data are reported as an average of
12 replications with 24 explants in each treatment. Data
were recorded as the number of explants per jar producing
callus after two months in culture, their fresh and dry
weights and the percentage of shoots regenerated from the
callus.
Irradiation Studies. The irradiation was carried out at the
‘Entomology Department’ of the Agricultural Research and
Extension Unit (AREU). For the irradiation studies, seeds,
callus and leaves of tissue-cultured plantlets of anthurium,
variety Nitta, were used throughout the experiment. The
apparatus used for irradiation is kept in a highly protected
area with restricted access as strong radiation may be
emitted. It consisted of a radiation cell and the dose rate had
to be calculated. The cell was rotated to enable the dose
rates to be uniform throughout. The gamma rays doses used
were 5, 10 and 15 Grays and had to be regulated according
to the time of exposure of the plantlets to the ionising
radiation emitted from 137Cs (Caesium) radioactive
compound. The equivalent time lapse were obtained
through a series of calculations as the irradiation fluctuates
with time due to the changing half - life of the compound
and with the capacity of the apparatus.
Following irradiation, the plantlets were kept in
culture for at least 2-4 weeks before any molecular work
was performed. DNA was extracted from each irradiated
explants and their profiles compared to that of the mother
plant.
Molecular Studies
DNA Extraction
Solutions. Extraction buffers consisting of 3% Sarkosyl
(v/v), 0.2 M Tris-HCl (pH 8.8), 50 mM EDTA (pH 8.8), 0.5
M NaCl, 0.1% and 1% β-mercaptoethanol, and 2.5%
polyvinylpyrrolidone (PVP - Mr 10,000), were prepared. In
addition, chloroform : Isoamyl alcohol (24:1), 70% and
80% ethanol, sodium acetate and a TE buffer consisting of
10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0) were
also needed.
DNA Isolation and Purification Procedures. Unbruised
tender pieces of leaves were ground in liquid nitrogen into a
fine powder. Two grams of leaf material was weighed and
placed on a pre-cooled mortar. Liquid nitrogen was poured
onto the sample and allowed to evaporate completely. The
leaf sample was macerated with the pestle to produce small
pieces. The latter were added to 15 mL of pre-heated (650C)
extraction buffer. The mixture was incubated for 4 h at 650C
with constant shaking at intervals followed by cooling to
room temperature (R.T) with gentle shaking on a shaker at
45 rpm. An equal volume of chloroform : isoamyl alcohol
was added to the mixture. The tubes were mixed gently for
5 min at R.T to produce a uniform emulsion. The latter was
centrifuged at 5000 g for 10 min at R.T. The supernatant
was transferred to a new Corning tube using a micropipette.
Second chloroform: isoamyl alcohol extraction was
performed. The supernatant was carefully decanted and
transferred to a new tube followed by precipitation with 2/3
volume of isopropanol. The precipitated nucleic acids were
collected and washed twice with the buffer (70% ethanol, 10
mM sodium acetate, TE (1X): 1 mM Tris, 0.1 mM EDTA,
pH 8.0). The pellets were air dried and re-suspended in TE.
The dissolved nucleic acids were brought to 1.4 M NaCl
and re-precipitated using 2 volumes of 70% ethanol (If the
pellet obtained was hard to re-suspend, this step was
repeated one more time). The pellets were washed twice
using 80% ethanol, dried and re-suspended in 100 μL of TE
buffer. The tube was incubated at 650C for 5 min to dissolve
genomic DNA followed by RNase treatment.
Measurement of Amount and Purity of DNA. The yield
of DNA per gram of leaf tissue extracted was measured
using a UV-VIS Spectronic Genesys 5 (Milton Roy)
spectrophotometer a 260 nm. The purity of DNA was
determined by calculating the ratio of absorbance at 260 nm
to that of 280 nm. Pure DNA has a ratio of 1.8 ± 0.1 (Clark,
1997). Polysaccharide contamination was assessed by
calculating the ratio of absorbance at 260 nm to that of 230
nm.
Method for PCR (RAPD analysis). Reagents used - Target
DNA (10-100ng), oligonucleotide primers (10-mers Primers
OPA 18, OPB 17, OPB 18, OPB 20, OPC 05, OPD 01 and
OPW 04), sterile de-ionised distilled water, Taq polymerase,
dNTP mix (dATP, dCTP, dGTP, dTTP), light mineral oil,
agarose (Sigma, Molecular biological grade), TBE buffer
(X0.5), Molecular marker VI, Gel-loading buffer (ULB –
0.25% bromophenol blue, 0.25% xylene cyanol FF, 30%
Table I. Plant Growth Regulators additives (in mg L-
1) for each of the stages of the culture
Additive Callus initiation Shoot development Rooting
BA 1.0 0.5 0.0
2,4 - D 0.1 0.0 0.0
IBA 0.0 0.0 0.1
Experimental design, data collection and analyses

PUCHOOA / Int. J. Agri. Biol., Vol. 7, No. 1, 2005
14
glycerol in water; Stored at 4 0C), Ethidium bromide (10 mg
mL–1).
The reaction mix was prepared on ice for the PCR
analysis. Table II gives the reagents used per PCR tube.
The reaction mix was dispensed into the reaction tubes. The
Taq Polymerase was added last. One drop of mineral oil
was added to each tube to prevent evaporation during
reaction. The tubes were placed in the thermal cycler and
the PCR program for RAPD was then run - reaction
initiation at 94oC for 2 min followed by cycles at: 94oC for 1
minute, 35oC for 1 min and 72oC for 1 min. Forty such
cycles were done.
RESULTS
Tissue Culture Studies
Pre-sterilization and sterilization. Contamination was a
major problem encountered during this study. Fungal
contamination appeared during the first week of inoculation
while contamination due to the presence of internal
contaminants, appeared after three weeks in culture. The
pre-sterilization and sterilization methods devised, reduced
the contamination level considerably (<10%), independent
of variety. However, the concentration of Benlate used
during pre-sterilization and the number and duration of
rinses in sterile distilled water following sterilization was
crucial. Benlate at concentrations higher than 0.6% caused
the leaves to become chlorotic while residual sodium
hypochlorite caused the explants to become necrotic.
Effect of media and plant growth regulators
concentrations. Various concentrations of cytokinin (BA)
and auxin (2,4 - D) added to either modified Murashige and
Skoog (1962) medium or modified Nitsch (1969) medium
were tested in a preliminary experiment. Of the two media
tested, Nitsch (1969) with reduced ammonium nitrate
concentration (200 mg L-1), proved to be the best for callus
induction (Table III). For regeneration, best results were
again obtained with Nitsch medium but with the ammonium
nitrate concentration increased to 720 mg L-1 (Table IV) The
same response was noted for all varieties under
investigation.
BA (1 mg L-1) and 2,4 - D (0.1 mg L-1) induced callus
in all three varieties. Callussing also occurred at lower and
higher concentrations but at much lower frequencies. Callus
induction began after two weeks in culture and was
produced along cut edges of the leaf explants. Incubation in
continuous darkness was found to enhance callussing. The
calli were firm and pale yellow in colour. Callus formation
was more prominent when veins, major or minor, were
present on the explants. This can be explained by the
presence of metabolically active phloem tissues which are
capable of growth in culture as well as retaining some of
their endogenous growth factors for additional stimulation
of explant growth (Finnie & Van Staden, 1986). Division
and subculture of the callus was done every 12 weeks. Fig. 1
shows the mean fresh and dry weights of callus of the three
varieties over a period of 70 days.
Transferring callus from the callus induction medium
to Nitsch basal medium (720 mg L-1 NH4NO3)
supplemented with BA (0.5 mg L-1) and culturing for 16h
per day to an illumination of 5.0 Wm-2, caused shoot
formation and a depression in callus growth. The shoots
were both adventitious and axillary in nature. Table VI
shows the effect of growth regulators on shoot formation.
The number and size of shoots produced per culture varied
considerably in all three varieties.
Allowing the regenerated shoots to stand for over two
months on Nitsch (1969) basal medium supplemented with
BA (0.5 mg L-1) caused spontaneous rooting to occur.
However, transferring the shoots to Nitsch (1969) basal
medium supplemented with IBA (1.0 mg L-1), improved
Table II. Reagents for PCR reactions
Reagents Stock molarity Molarity required Volume used
(μL)
Water (Nanopure) _ _ 19.4
PCR Buffer 10 x 3.0
dNTP mix 100 mM 200 μM 2.4
Primer 50 μM 20 picomoles 4.0
Taq polymerase 250 U (5 U/μL) 1 μL 0.2
DNA Template Variable Variable (10-100 ng) 1.0
Total Volume/tube 30.0
Table III. Influence of ammonium nitrate
concentration on callus initiation from leaf segments
of different varieties of A. andraeanum after 4 weeks
in culture in complete darkness and supplemented
with BA (1 mg L-1) and 2,4 - D (0.1 mg L-1).
Variety NH4NO3 conc. (mgL-1) % Explants forming callus
Osaki 200* 100
720** 12.5
825*** 0
Nitta 200* 100
720** 12.5
825*** 0
Anouchka 200* 100
720** 8.3
825*** 0
* Modified Nitsch medium, ** original Nitsch medium, ***modified
MS medium
Table IV. Influence of ammonium nitrate
concentration on shoot development from callus,
obtained from leaf explants grown on modified
Nitsch medium, of different varieties of A.
andraeanum cultured under low light intensity and
supplemented with 0.5 mgL-1 BA
Variety NH4NO3conc.
(mgL-1) % callus forming
shoots No of shoots per
callus
Nitta 200* 16.7 1-5
720** 100 >10
825*** 8.3 1-5
Osaki 200* 20.8 1-5
720** 91.7 >10
825*** 8.3 1-5
Anouchka 200* 12.5 1-5
720** 87.5 >10
825*** 0 0
*modified Nitsch medium, **original Nitsch medium,*** modified
MS medium.

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15
rooting and this was further enhanced when activated
charcoal (0.04%) was added to this medium. The level of
ammonium nitrate used (720 mg L-1) was essential for
rooting as a reduced level (200 mg L-1) delayed rooting.
(Table VII)
Illumination was also found to be an important factor
in the rooting of shoots as callus of all three varieties, grown
in the dark on the above medium, did not produce any
shoots. As observed by Geier (1982) while working with
leaf explants of Anthurium scherzeranium, the time of
rooting was related to the extent of shoots and leaflet
development; the larger shoots with more prominent leaflets
forming roots quicker. Plantlets with well-developed roots
were hardened by transplanting in vermiculite and growing
in a mist house with very low light intensity and high
humidity. No losses were observed and the plantlets were
transferred to the shade house after two months. However,
considerable losses were observed in plantlets without roots
or poorly developed roots when transferred to vermiculite.
Irradiation studies. Apart from the control where the
explants were not irradiated, the other explants were
subjected to three doses of irradiation (5, 10 & 10 Gy). The
parameter to determine dose-dependent irradiation damage
was the survival of the explants after irradiation. The effects
of irradiation on seeds after eight weeks following the
treatments are shown in Fig. 2.
The higher the dose of radiation, the higher was the
mortality rate of seeds. Seeds irradiated at 5 Gy also showed
better growth response in the modified Nitsch’s (1969)
medium. They grew faster and more vigorously, producing
shoots within six weeks of culture.
The effects of increasing doses of gamma rays on
callus during eight weeks in culture are shown in Fig 3. By
the 4th week, the numbers of calli from the 5 Gy treatments
were highest as compared to the 10 Gy, 15 Gy and even the
control. By week 8, all the explants treated at 15 Gy died. In
Table V. Comparison of growth regulator
supplements to modified Nitsch medium in callus
induction from three A. andraeanum varieties
incubated in complete darkness.
Medium BA (mgL-1) 2,4 - D (mgL-1) % Callusa
1 0.5 0.0 0
2 1.0 0.0 0
3 1.5 0.0 16.7
4 0.0 0.1 25
5 0.5 0.1 33.3
6 1.0 0.1 100
7 1.5 0.1 75
8 0.0 0.5 75
9 0.5 0.5 83.3
10 1.0 0.5 75
11 1.5 0.5 75
a Expressed as a mean for the three varieties.
Table VI. Effect of growth regulators on shoot
formation from callus obtained from leaf explants
grown on modified Nitsch medium supplemented
with BA (1 mgL
-1) and 2,4 - D (0.1 mgL-1) and
transferred to original Nitsch medium.
BA (mgL-1) 2,4 - D (mgL-1) % Shootsa
0.5 0.0 100
1.0 0.0 66.7
0.5 0.1 50
1.0 0.1 50
0.5 0.5 33.3
1.0 0.5 33.3
a Expressed as a mean for the 3 varieties. Evaluation after 2 months
under low light.
Table VII. Effect of growth regulators on rooting (%
response) after two months in culture on original
Nitsch medium (720 mgL-1) at low light intensity
BA (mgL-1)
0 0.5 1.0
0 0 (0) 66.6 (16.6) 20.8 (4.1)
IBA (mgL-1) 0.5 58.3 (20.8) 50 (12.5) 25 (8.3)
1.0 83.3 (25) 50 (12.5) 41.6 (8.3)
Figures expessed as a mean for the three varieties. Figures in brackets
represent% response using 200 mgL-1 NH4NO3. All media contained
0.04% activated charcoal.
Fig. 1. Mean fresh weight and dry weight of callus of
the three varieties
0
0.2
0.4
0.6
0.8
1
1.2
0 1428425670
Time ( Days)
Weight (g)
Fr. w t. (N)
Fr. w t. (O)
Fr. w t( A)
Dr y w t .( N)
Dr y w t .( O)
Dr y w t .( A)
Fr wt.: Fresh weight. N: Nitta. O: Osaki. A: Anouchka.
Fig. 2. Number of seeds surviving after irradiation
Response of seeds after irradiation
0
2
4
6
8
10
12
0 week
2 weeks
4 weeks
6 weeks
8 weeks
Time (weeks)
Number of seeds
0 Gy
5 Gy
10 Gy
15 Gy

PUCHOOA / Int. J. Agri. Biol., Vol. 7, No. 1, 2005
16
vitro grown leaf explants also showed similar response.
Fig. 4 shows some of the response observed when the
explants were treated at different gamma rays doses. Several
morphological changes were observed in plantlets
regenerated following irradiation. The variability in the
responses indicates that the radiation doses may have caused
mutation. Explants irradiated at 5 Gy gave very good
response indicating the boosting effect of the 5 Grays doses.
The 15 Gy dosages were lethal to most of the explants.
Molecular studies. The results of the different
spectrophotometer readings and the amount of DNA
obtained following extraction from leaves of non-irradiated
and irradiated plantlets are shown in Table VIII
A series of PCR reactions were carried out to
determine the optimum MgCl2, DNA template, primers and
dNTP concentrations to be used for the analysis. From the
results obtained, it was found that using 2.0 mM MgCl2, 200
µM dNTP and 20 picomoles of primer gave satisfactory
banding patterns. Using either 10 or 20 ng of genomic DNA
did not reveal any difference. Out of the seven primers of
arbitrary nucleotide sequences chosen to amplify genomic
DNA, all were able to amplify PCR products. Results of a
preliminary study to compare the banding patterns obtained
when using primer OPB 17 and OPC 05 are shown in fig.
5a and 5b respectively. Both monomorphic (Fig. 5b) and
polymorphic bands were observed (Fig. 5a).
The sharp bands given for the marker VI correspond to
base pairs of 2176, 1766, 1260, 1033, 653, 517, 453, 394
and 293, respectively).
The bands given in the DNA sample were compared
with that of the given marker through visual estimations.
The results obtained using other primers following
the optimization are shown in Fig 6a and 6b. Some of the
results obtained from the DNA extracted from irradiated
explants and using the limited number of available
primers, are shown in Fig 7 and 8.
After DNA extraction from the irradiated calli and
from leaves of anthurium, variety Nitta under the same
conditions, the above results were obtained. All four calli
DNA gave similar patterns, and these corresponded to that
from the mother plant. Hence, at this level no difference in
the banding patterns was observed.
Here again, the similar patterns that were obtained
showed that the polymorphisms at the genome level of the
Anthurium callus and leaf tissues were not demonstrable
from the RAPD analysis.
DISCUSSION
Several workers have reported the variation in the
requirements of different genotypes of Anthurium in tissue
culture (Fersing & Lutz, 1977; Kunisaki, 1980; Geier,
1982). Although methods for several commercial varieties
have been worked out, they are not available for general
use. The results reported in this paper demonstrate that a
single medium can be used for the in-vitro culture of three
different varieties of Anthurium andraeanum. Apart from
the time taken for callus induction, there were no genotypic
differences in the ability of the callus to regenerate shoots
and eventually plantlets.
The influence of the ammonium level on shoot
initiation from Anthurium andraeanum callus was first
reported by Pierik and Steegmans (1976) and Pierik et al.,
(1979). In this study, the level of ammonium nitrate used
had a significant effect on callus formation and
regeneration. Callus initiation was quicker on modified
Nitsch’s medium (200 mg L-1 NH4NO3) supplemented with
2,4 - D (0.1 mg L-1) and BA (1 mg L-1) than original
Nitsch’s medium supplemented with the same growth
regulators. The cultivar Nitta was the first to respond
followed by Osaki while callus initiation took longer in the
case of Anouchka. This beneficial effect of the low NH4NO3
level was also observed by Geier (1982) while working with
Anthurium scherzerianum. The inability of the leaf explants
to initiate callus on modified Murashige and Skoog’s (1962)
medium with half-strength macronutrients (825 mg L-1
NH4NO3) further confirms the efficiency of using a low
level of ammonium nitrate during callus induction in the
cultivars Nitta, Osaki and Anouchka. This is contrary to the
findings of Finnie and Van Staden (1986). Geier (1982)
obtained shoot regeneration from callus of A.scherzeranium
on modified Nitsch’s medium (200 mg L-1) supplemented
with BA (1 mg L-1) and 2,4-D (0.1 mg L-1). However, we
observed no regeneration in all the three varieties, using this
Table VIII. Spectrophotometric readings and DNA
concentration from irradiated explants
Gamma ray
dose (Gy) A 230 A 260 A 280 A 260/A 230 A 260/A 280 DNA conc
(μg/mL)
0 0.063 0.128 0.068 2.032 1.882 0.640
5 0.057 0.108 0.057 1.895 1.895 0.540
10 0.045 0.095 0.054 2.102 1.759 0.475
15 0.019 0.034 0.021 1.789 1.619 0.170
Fig. 3. Survival rate of calli
SURVIVAL OF CALLI AFTER
IRRADIATION
0
5
10
15
20
25
30
35
0 week 2
weeks 4
weeks 6
weeks 8
weeks
TIME (WEEKS)
NUMBER OF CALLI
0 Gy
5 Gy
10 Gy
15 Gy

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17
level of ammonium nitrate. Instead, shoot regeneration from
callus occurred when original Nitsch’s medium (720 mg L–1
NH4NO3) supplemented with BA (0.5 mg L-1) was used.
This level of ammonium nitrate was also found to be
necessary for rooting, which was more prominent when
original Nitsch’s medium supplemented with 1.0 mg L-1
IBA was used. This was true for all the three varieties
tested.
In this experiment, rooting was enhanced by the
addition of activated charcoal (0.04%) to the medium.
Although the precise role of activated charcoal in tissue
cultures is unknown, it seems to be involved in the removal
of substances from media that promotes unorganised growth
(Friedborg & Eriksson, 1975). Growing cells excrete large
amounts of phenyl acetic acid and derivatives of benzoic
acid (Friedborg et al., 1978), which accumulate in the
medium and possibly have negative effects on
differentiation. Phenolic compounds have also been
demonstrated in plant tissue cultures (Butcher, 1977) and
have been shown to affect differentiation in tobacco callus
(Lee & Skoog, 1965). It is possible that phenyl acetic acid
and benzoic acids are not in themselves responsible for the
inhibition of root formation in tissue cultures, but their
presence indicates a block of a biosynthetic pathway, which
is necessary for normal organ development. Other
compounds, alone or in combination, may also be active as
inhibitors in plant tissue cultures and activated charcoal
adsorbs these as well.
Regulation of auxin and cytokinin balance has long
been recognized as a key factor in the control of cell
division and organogenesis in tissue culture (Murashige,
1977). Our research demonstrated that exogenously applied
BA (1.0 mg L-1) and 2,4 - D (0.1 mg L-1) was essential for
callus induction from leaf explants of all the three varieties
Fig 4 (a). Irradiated seeds
(0 Gy, 5 Gy, 10 Gy & 15
Gy) after 8 weeks in
culture
Fig. 4 (b). Plantlet
regeneration on the 5 Gy
treated explants
Fig. 4 (c). Plantlet
showing sign of necrosis
following 10 Gy treatment
Fig. 4 (d). Plantlet
following 15 Gy dose
treatment
Fig. 5a. Results using Primer OPB 17
LANES 1 2 3
(Lane 1: DNA template (10 μg); Lane 2: Negative control; Lane 3:
Marker VI)
Fig. 5b. Results using Primer OPC 05
LANES 1 2 3
(Lane 1: DNA template (20 μg); Lane 2: Negative control; Lane 3:
Marker VI).

PUCHOOA / Int. J. Agri. Biol., Vol. 7, No. 1, 2005
18
of Anthurium. Shoot regeneration from the callus occurred
when BA (0.5 mg L-1) alone was used while rooting
required removal of the BA from the medium and addition
of IBA (1 mg L-1). The difference in regeneration capacity
and mode of regeneration at concentrations higher and
lower than optimum may be explained on the basis of
variation in the endogenous levels of these growth
hormones in leaf tissues. Similar observations regarding the
role of endogenous hormone levels in determining the shoot
forming-capacity of tomato leaf disks have been reported
(Kartha et al., 1976; Frankenberger et al., 1981). Another
Fig. 6(a) Amplification product using primer
OPB 18
Lanes 1 2 3 4 5 6
Lane 1: Molecular marker VI; Lanes 2 – 5: DNA samples from
the two replicates; Lane 6: Negative control.
Fig 6 (b) Amplification product using primer
OPW 04 (Lanes 2 – 5) and OPD 01 (Lanes 6 – 9)
Lanes 1 2 3 4 5 6 7 8 9 10
Lane 1 & 10: Molecular marker VI; Lanes 2 – 9: DNA samples
from the different replicates.
Fig. 7. Using Primer OPB 20
Lanes 1 2 3 4 5 6 7
Lane 1: Marker VI, Lane 2: DNA from leaf explants, Lane 3: DNA
5 Gy (irradiated), Lane 4: DNA 10 Gy (irradiated), Lane 5: DNA 15
Gy (irradiated), Lane 6: DNA 0 Gy (not irradiated)/ control, Lane 7:
Negative control
Fig. 8.Using Primer OPA 18
Lanes 1 2 3 4 5 6
Lane 1: Marker VI, Lane 2: DNA from leaf sample, Lane 3: DNA 5
Gy, Lane 4: DNA 10 Gy, Lane 5: DNA 15 Gy, Lane 6: DNA 0 Gy
(control)

MUTATION BREEDING OF ANTHURIUM BY GAMMA RADIATION / Int. J. Agri. Biol., Vol. 7, No. 1, 2005
19
study (Elliot et al., 1987) has also demonstrated that a
critical endogenous level of growth regulators has to be
attained before cell division and organogenesis could occur.
Apparently, callus induction from leaf was not
dependent on light, which is contrary to the findings of
Finnie and Van Staden (1986), where light was found to be
essential. However, we found that low light intensity levels
considerably enhanced shoot regeneration. These results are
consistent with the studies of Pierik et al., 1974, 1979 and
Geier (1982) on plant growth regulator and light action in
organ differentiation in a number of Anthurium varieties.
Increased shoot formation with a slight increase in light
intensity levels has also been reported in other plant species
(Hughes, 1981).
In this study, it is also demonstrated that no genotypic
variation exists in terms of regeneration from callus for the
varieties Nitta, Osaki and Anouchka. Regeneration occurred
in all genotypes via an intermediary callus phase; no direct
shoot regeneration from leaf explants was observed. The
phenomenon of genotypic differences in callus formation
and regeneration capacity in other varieties of Anthurium
has been reported earlier (Kunisaki, 1980; Geier, 1982;
Kuehnle & Sugii, 1991).
A matter of interest for plant breeders is the use of
mutagens, in combination with in-vitro cultures, to create
genetic variation. The Anthurium variety “Nitta” was
maintained for the irradiation studies since it had responded
earlier than the other tested varieties under in vitro
conditions. Here, explants like seeds, leaf pieces and
plantlets of the same variety were subjected to the three
doses of gamma-rays (5, 10 & 15 Gy) from the 137 Cs
radioactive compound. Similar results obtained for the
different explants showed the reproducibility of the
radiation effects of the radiation on Anthurium tissues.
From the experiments on seed culture for example, the
5 Gy treatment showed a higher survival rate (70% at week
8) as compared to the other. The results were even better
than those obtained from the “control” experiments, both in
terms of survival rate (60%) and in the morphological
variation of the seeds in culture. The calli and plantlets also
expressed better responses at the 5 Gy but lethality at the 15
Gy doses, whereas the 10 Gy treated explants were
moderately lethal to the radiation. These observations
demonstrated the probable mutations that have taken place
in the anthurium tissues due to the gamma radiation. Apart
from the dose effects, the responses were controlled by a
number of parameters, including the genotype, the type of
explant, the orientation of the explant on the culture
medium, and the origin of the explant from the mother plant
(Douglas, 1985).
Tissue-cultured based mutagenesis has been employed
for many years to generate novel plant mutants and cell
lines of agricultural and industrial interest (Collin & Dix,
1990). Somaclonal variation, which can induce a range of
gross chromosomal alternations, (as well as more limited
gene mutations) has been studied as possible route for
generation of novel genetic variation (Evans & Sharp,
1986). The factors responsible for the in vitro origin of
chromosomal structural changes (or chromosome
mutations) and gene mutations are not known. In several
cases, hormones, especially 2,4-D or other hormone
combinations are suspected of causing such changes. In fact,
it is more likely that these hormones may act as mutagens
and favour mutation by influencing metabolism (D’Amato,
1985). Moreover, the physical mapping and DNA
sequencing of loci mutagenised by irradiation and chemicals
in plants has provided more precise information about these
different types of DNA alterations. For irradiation by
gamma rays, the occurrence of insertion or inversion
changes could explain the occurrence of mutation.
However, the mutation frequency may be influenced
by a number of factors such as the mechanism of mutagen
action (Sparrow, 1961; Griffiths et al., 1993), target gene
size and nucleotide composition (Haughn & Somerville,
1987; Bichara et al., 1995), genomic location (Swoboda et
al., 1993; Brown & Sundaresan, 1991), chromatin structure
(Jackson, 1991; Shaffer et al., 1993), replication timing
(Salganik, 1983), efficiency of DNA repair (Britt et al.,
1993; Veleminsky & Gichner, 1978) and transcriptional
activation (Schlissel & Baltimore, 1989; Zehfus et al., 1990;
Lindahl, 1991). The frequency of a particular mutation can
be underestimated if a degree of elimination occurs (Butler,
1977; Dellaert, 1980; Vizir et al., 1994). The recovery of
mutants induced by high levels of mutagens is limited by
somatic effects, such as reduced viability, growth
abnormalities and reduced fertility. Therefore, every
mutagen has a most effective dose, which produces the
maximum level of mutagenesis with minimal somatic
effects.
This could be the case for the 5 Gy treatment that
produced minimal damages to the Anthurium tissues. In
fact, the better responses observed could suggest that the
type of chromosomal alterations that took place eventually
produced a change in the morphology. This was expressed
under in vitro conditions. The higher gamma-ray doses may
have produced other modifications that caused necrosis of
the tissues and calli. However, from the RAPD-profiles,
these genomic changes could not be detected. The base-pair
sequences for the DNA extracted from tissues irradiated at
the three doses gave similar banding patterns. From these
results, it showed that the RAPD was inefficient in detecting
the more precise genomic alterations that have occurred due
to the gamma rays. In fact, from the use of RAPD, it was
expected that polymorphisms resulting from mutations or
rearrangements either at or between the primer binding sites
could be detected as the presence or absence of
amplification products.
Acknowledgements. The author wishes to thank the
Mauritius Research Council for funding this project, Mrs
S Malhotra who initiated this project, the technicians and
technical assistants at the Faculty of Agriculture and the
University of Mauritius for support of this work.

PUCHOOA / Int. J. Agri. Biol., Vol. 7, No. 1, 2005
20
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(Received 01 September 2004; Accepted 10 November 2004)