8/12/2019 Micropropagation of Anubias barteri var.

Nana

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Print ISSN 0255-965X; Electronic 1842-4309

Not Bot Horti Agrobo, 2012, 40(2): 148-151  Notulae Botanicae Horti Agrobotanici
Cluj-Napoca

Micropropagation of Anubias barteri  var. Nana from Shoot

Tip Culture and the Analysis of Ploidy Stability 
Kantamaht KANCHANAPOOM1*, Panyaros CHUNUI2 , Kamnoon KANCHANAPOOM2
1
 Prince of Songkla University, Faculty of Science, Department of Molecular Biotechnology and Bioinformatics,
 Hat Yai, Songkhla, 90112 Tailand; kantamaht@hotmail.com (*corresponding author)
2
 Prince of Songkla University, Faculty of Science, Department of Biology, Plant Biotechnology
 Research Unit, Hat Yai, Songkhla, 90112 Tailand; kamnoon_k@yahoo.co.th

 Abstract

Plant regeneration of  Anubias barteri   var.  Nana was achieved through organogenesis in shoot tip cultures. Multiple shoots were
induced from cultured shoot tips on a modified MS (Murashige and Skoog, 1962) medium supplemented with BA and kinetin. Te
maximum green shoot numbers were best obtained on MS medium containing 3 mg/L BA with 5 shoots. Rooting in all regenerated
shoots was promoted on MS medium devoid of plant growth regulators or kinetin singly. Acclimatization and survival when transferred
to field conditions were shown to be 100% in the regenerated plants. Cytological and flow cytometric analyses of the mother plants and
in vitro grown plants derived from 5 years old cultures showed no differences in ploidy level, they were all diploid (2n = 2x = 48) with a
2C peak indicating that ploidy alteration did not occur.
 Keywords: aquatic plant, Araceae , flow cytometry, nuclear DNA content

Introduction Materials and methods 

 Plant materials

intoTe manygenus  Anubias

varieties   oasthe
such amily barteri
 Anubias Araceae is  Barteri
 var. divided, romYoung plantletsPlant
the Aquatic o  A.Center  var. Nana
barteriCo.,  were obtained
Ltd., Tailand. Tey
 A. barteri  var. Angustifolia, A. barteri  var. Caladiifolia,  A.  were surace sterilized using 0.5% (w/v) mercuric chlo-
barteri var. glabra, and  A. barteri  var. Nana ( Kasselmann , ride solution containing 2 drops o ween-20 emulsifier
 2003). Te most cultivated and commercially important  per 100 ml solution or 3 min. Te treated plantlets were
species is  A. barteri  var. Nana which is commonly grown  washed three times with sterile distilled water to remove
in aquaria. Anubias can be propagated vegetatively using traces o disinectant. Te explants were then surace ster-
stolon division; however, stolon division is an inefficient ilized again using a dilution o 10% (v/v) commercial Clo-
 propagation method or commercial purposes since the rox™ which yields 5.25% NaOCl and 2 drops o ween
 planting material has a very low multiplication rate. Mi- 20 per 100 mL solution or 15 min, ollowed by 5% (v/v)
cropropagation is currently applied to aquatic plants as a Clorox™ or 5 min. Afer the surace decontamination was
tool or large scale multiplication o elite plants (Carter completed the explants were rinsed 3 times with sterile
et al ., 2011; Myung et al ., 2010).  However, inormation distilled water. Following disinection 3-5 mm shoot tip
concerning details o media and growth regulator amend- explants were excised prior to culture on MS (Murashige
ments is still a undamental requirement o the intense and Skoog, 1962) basal medium containing 3% sucrose to
commercial production o A. barteri. grow the explants.
Te chromosome number o  A. barteri  var.  Nana  is
 very difficult to assess since they are small and numerous.  Medium preparation and culture conditions
Flow cytometry is being used to analyze DNA content Afer 6 weeks o culture, well developed shoots were
in a number o plant species; which require only a small obtained. Te small shoots with a pair o leaves were trans-
amount o tissue and is thereore non-destructive. In this erred to MS medium supplemented either with 0, 1, 3, or
context, this study investigated an efficient protocol or A. 5 mg/L BA or 0, 1, 3, or 5 mg/L kinetin. All culture media
barteri  var.  Nana multiplication. Te study also investi- consisted o MS salts and vitamins supplemented with 3%
gated the effects o plant growth regulators on number o sucrose and 0.82% MermaidM agar. Te pH o media was
chromosomes using flow cytometry as rapid methods or adjusted to 5.8 with 1 N NaOH or 1 N HCl prior to auto-
2
detecting ploidy levels in regenerated plants. claving at 1.05 kg/cm , 121˚C or 20 min. Cultures were

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maintained at 25±1˚C in a culture room with a 16-h light top o ice in a bucket and nuclei were mechanically iso-
 photoperiod. All explants were subcultured at 8-week in- lated by chopping lea materials using a sharp razor blade.
tervals. Cultures were maintained at 25±1˚C in a culture Afer chopping, the suspension was filtered through a 42
room with a 16/8 h light/dark photoperiod under an il- µm nylon mesh and CyStain UV Ploidy (DAPI staining
lumination o 20 µmolm-2 s -1 photosynthetic photon flux solution) was added. Te fluorescence o a minimum o
intensity provided by cool white fluorescent light. Plant 5000 DAPI-stained nuclei per sample was estimated using
materials were stored in glass-capped culture jars (115 ml a PA-II flow cytometer (Partec, Germany). Te reerence
capacity) each containing 20 ml o medium. standard plant ( Zea mays cv. CE-777; 2C = 5.43 pg) was
kindly provided by Dr. Jaroslav Dolezel, Laboratory o
Chromosome counting  Molecular Cytogenetics and Cytometry, Institute o Ex-
o determine an accurate ploidy level, chromosome  perimental Botany, Czech Republic. Te reerence stan-
counting was carried out on young root tips o in vitro  dard peak was adjusted to show at channel 100 o relative
grown plants. Actively growing root tips ca. 5-10 mm in fluorescence intensity or instrument calibration. Te 2C
length were excised and pretreated with saturation solu- DNA content was calculated according to the ormula
tion o Para dichlorobenzene or 24 h at 4°C. Tey were Sample G1 peak mean × Standard 2 C DNA content
fixed in resh solution o Carnoy’s fluid (3 parts 95% etha- 2 CDNA =
nol and 1 part glacial acetic acid) or 24 hours and stored Standard G1 peak mean
 

in 70% ethanol at 4°C. Tis treatment was ollowed by hy-

drolysis in 1N HCl at 60°C or 5-6 min. Finally, they were Statistical analysis
rinsed with tap water and stained in carbol uchsin. Te All experiments were carried out at least 3 times with
stained regions o root tips (0.5-1 mm long) were cut and 5-10 replicates per treatment. Te fluorescence histograms
squashed on a slide and cover with a cover slip. Te chro-  were resolved into G0/G1 (2C), S and G2/M (4C) cell-
mosomes counts were carried out at 1000x magnification cycle compartments with a peak-reflect algorithm using
under light microscope (Olympus model CH 30, Japan) two Gaussian curves (WinMDI version 2.8). Data were
and the chromosomes o 7-8 cells were counted in three analyzed by ANOVA and the differences among the means
replications.  were compared using Scheffe’s test at p<0.05.

 Flow cytometry analysis Results and discussion

Approximately 20-30 mg o ully expanded young
leaves o the
harvested mother
and plants to
transerred andglass
in vitro grown
Petri dish plants were  Nana
containing Teareresults
shown oininab.  organogenesis
vitro1. Both BA andinkinetin
 A. barteri  var
supple-
nuclei extraction buffer. Te glass Petri dish was placed on ments resulted in different morphogenetic responses in
ab. 1 Effect o different concentrations o BA and kinetin combinations on shoots leaves and root regeneration o Anubias barteri 
 var. nana 
Root
BA Kinetin Number o shoots per Number o leaves Number o leaves Number o roots per
ormation
(mg/L) (mg/L) explant (Mean±SD) (Mean±SD)  per shoot shoots (Mean±SD)
(%)
0 1.4±0.89c 3.8±2.16 c 3.0±1.87abcd 2.0±2.00abc 60
1 1.2±0.45c 4.4±1.34c 4.0±1.41ab 2.4±1.95ab 80
0
3 1.2±0.45c 4.2±2.38c 3.7±2.44abcd 3.8±4.27a  60
c c cd abc

50 1.4±0.89
1.6±0.89c 3.8±3.19
5.4±2.79c 2.1±1.52abcd
3.47±0.51 2.2±3.03
0.0±0.00c 40
0
1 1.2±0.45c 4.4±1.14c 4.4±1.14a  0.0±0.00c 0
1
3 1.4±0.55c 5.8±1.78bc 4.4±1.08a  0.0±0.00c 0
5 1.0±1.58bc 7.8±4.43bc 2.9±0.96 abcd 0.0±0.00bc 0
0 5.0±2.12a  13.4±5.52a  2.9±0.42abcd 0.0±0.00c 0
1 2.4±1.14bc 5.0±2.12c 2.0±0.71d 0.0±0.00c 0
3
3 1.8±1.06bc   5.2±2.80c 3.87±1.04abc 0.0±0.00c 0
5 1.8±1.10bc 7.2±4.65bc 4.1±1.02ab 0.0±0.00c 0
0 3.4±1.34b 10.4±3.04ab 3.3±0.97abcd 0.0±0.00c 0
1 3.0±2.00bc 8.20±3.76 bc 3.1±0.82abcd 0.0±0.00c 0
5
3 2.0±1.41bc 6.6±4.27bc 3.5±0.50abcd 0.0±0.00c 0
5 2.4±0.89bc 6.0±3.46 bc 2.4±0.55bcd 0.0±0.00c 0
Te different letters within column show significant difference (Mean ± SE.) analyzed by Scheffe’s test at  p<0.05.

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terms o shoot, lea and root ormation, number o leaves taining MS medium (control) or kinetin singly (ab. 1).
 per shoot, and percentage o root ormation. At low con- Te roots arising rom the basal end o shoots were large
centration o BA (0, 1 mg/L) regenerated single shoot and vigorous (Fig. 1a). Te explants were subcultured or
 while an increase in BA concentration rom 3 to 5 mg/L 5 years successully and did not show any morphological
resulted in increased number o shoots per explant. Te abnormality when compared with the non tissue cultured
data in ab. 1 revealed that the maximum number o  plants.
5±2.12 shoots per explant was obtained on MS medium Te cytological study o root tips at metaphase o long
supplemented with 3 mg/L BA term cultures revealed complete accurate counts o 48
Te results show that BA used singly was important chromosomes (Fig. 1b). An occurrence o chromosome
or induction o axillary bud outgrowth in  A. barteri var. changes has been ofen observed during application o
 Nana. By successive subculture on MS medium contain- tissue culture, especially in plants, callus or cells that were
ing 3 mg/L BA, masses o prolierating shoot cultures were maintained or long-term cultures in vitro (Hao and Deng,
established (Fig. 1a). 2002). Te distribution o the nuclei extracted rom both
A significant difference in the number o leaves was de- the mother plants and in vitro grown plants displayed a
tected among the treatments containing BA and kinetin.  prominent peak at 2C indicating that they consisted o
Regenerated shoots had higher number o leaves on MS cells with G0/G1 phase o cell cycle thus no ploidy varia-
medium supplemented with kinetin alone. Leaves were tion occurred (Fig. 2). Te mean 2C DNA content o the
ormed at a high requency o 13.4 ( p≤0.05, ab. 1) on mother-1 plants and in vitro grown plants are 5.43 and 5.45
MS medium supplemented with 3 mg/L BA. No lea was  pg 2C , respectively. Tis finding confirmed that the al-
observed on the control explants. Te eature o leaves teration o DNA content was not observed among in vitro 
developed on MS medium containing 1-5 mg/L BA and grown plants compared to the mother plants probably due
1-5 mg/L kinetin showed broad and dark green leaves to the reason that plants regenerated rom well-developed
(Fig. 1a). BA seemed to inhibit root ormation since the meristematic tissues that had minimum tendency o ge-
root ormation was recorded only on culture media con- netic variation (Rout et al ., 1998).

Fig. 1.  In vitro propagation o  Anubias barteri var.  Nana (a) Multiple shoots ormation rom a
single shoot explant afer eight weeks cultured on MS medium supplemented with 3 mg/L BA
(Scale bar =10 mm) (b) Mitotic metaphase o root tips showing diploid 2n = 2x =48 chromo-
somes (Scale bar = 50 µm)

Fig. 2.
loid Flow cytometric
profiles o standardhistograms o theprofile
plant (b)diploid relativeonuclear
5 yearsDNA
old incontent (in channel
vitro grown plant numbers) o A. barteri var. Nana with (a) dip-

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In conclusion, we demonstrated that the establishment Kane ME, Greg L, Davis1 GL, McConnell DB, Gargiulo JA
o rapid in vitro plant propagation o A. barteri var. Nana  (1999). In vitro propagation o Cryptocoryne wendtii Aquat
can be achieved. Te nuclear DNA content value or  A. Bot 63:197-202.
barteri var. Nana was provided. Ploidy variations were not Kasselmann C (2002). Aquarium Plants. Malabar, FL: Krieger
observed during subculture in 5 years as detected by cyto- Publishing Co., 104 p.
logical study and flow cytometry. A combination o cyto- Murashige , Skoog F (1962). A revised medium or rapid
logical study and flow cytometry achieved better results in growth and bioassays with tobacco tissue culture. Physiol
terms o both accuracy and rapidity. Planta 15:473-479.
Myung JO, Hye RN, Hong-Keun C, Jang RL, Suk WK (2010).
 Acknowledgements High requency plant regeneration system or  Nymphoides
Tis research was financially supported by the Faculty coreana  via somatic embryogenesis rom zygotic embryo-
o Science, Prince o Songkla University, Tailand. Te au- derived embryogenic cell suspension cultures. Plant
thors are grateul to Dr. Brian Hodgson or assistance with Biotechnol Rep 4:125-128.
English and useul comments on this manuscript. Predieri S (2001). Mutation induction and tissue culture in
improving ruits. Plant Cell iss Org Cult 64:185-210.
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