Micropropagation of Anubias barteri var. Nana

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Not Bot Horti Agrobo, 2012, 40(2): 148-151

Print ISSN 0255-965X; Electronic 1842-4309

Notulae Botanicae Horti AgrobotaniciCluj-Napoca

Micropropagation ofAnubias barterivar.Nanafrom Shoot

Tip Culture and the Analysis of Ploidy StabilityKantamaht KANCHANAPOOM1*, Panyaros CHUNUI2 , Kamnoon KANCHANAPOOM2

1Prince of Songkla University, Faculty of Science, Department of Molecular Biotechnology and Bioinformatics,Hat Yai, Songkhla, 90112 Tailand;[email protected](*corresponding author)

2Prince of Songkla University, Faculty of Science, Department of Biology, Plant BiotechnologyResearch Unit, Hat Yai, Songkhla, 90112 Tailand;[email protected]

Abstract

Plant regeneration of Anubias barteri var. Nanawas achieved through organogenesis in shoot tip cultures. Multiple shoots wereinduced from cultured shoot tips on a modified MS (Murashige and Skoog, 1962) medium supplemented with BA and kinetin. Temaximum green shoot numbers were best obtained on MS medium containing 3 mg/L BA with 5 shoots. Rooting in all regeneratedshoots was promoted on MS medium devoid of plant growth regulators or kinetin singly. Acclimatization and survival when transferredto field conditions were shown to be 100% in the regenerated plants. Cytological and flow cytometric analyses of the mother plants andin vitrogrown plants derived from 5 years old cultures showed no differences in ploidy level, they were all diploid (2n = 2x = 48) with a2C peak indicating that ploidy alteration did not occur.

Keywords:aquatic plant,Araceae, flow cytometry, nuclear DNA content

Introduction

Te genus Anubias o the amily Araceae is dividedinto many varieties such as Anubias barteri var. Barteri,A. barteri var.Angustifolia,A. barteri var. Caladiifolia,A.barterivar.glabra, andA. barterivar.Nana (Kasselmann,

2003). Te most cultivated and commercially importantspecies isA. barteri var.Nanawhich is commonly grownin aquaria.Anubiascan be propagated vegetatively usingstolon division; however, stolon division is an inefficient

propagation method or commercial purposes since theplanting material has a very low multiplication rate. Mi-cropropagation is currently applied to aquatic plants as atool or large scale multiplication o elite plants (Carteret al., 2011; Myung et al., 2010). However, inormationconcerning details o media and growth regulator amend-ments is still a undamental requirement o the intensecommercial production oA. barteri.

Te chromosome number o A. barteri var. Nana isvery difficult to assess since they are small and numerous.Flow cytometry is being used to analyze DNA contentin a number o plant species; which require only a smallamount o tissue and is thereore non-destructive. In thiscontext, this study investigated an efficient protocol orA.barteri var. Nana multiplication. Te study also investi-gated the effects o plant growth regulators on number ochromosomes using flow cytometry as rapid methods or

detecting ploidy levels in regenerated plants.

Materials and methodsPlant materials

Young plantlets oA. barteri var.Nanawere obtainedrom the Aquatic Plant Center Co., Ltd., Tailand. Teywere surace sterilized using 0.5% (w/v) mercuric chlo-ride solution containing 2 drops o ween-20 emulsifier

per 100 ml solution or 3 min. Te treated plantlets werewashed three times with sterile distilled water to removetraces o disinectant. Te explants were then surace ster-ilized again using a dilution o 10% (v/v) commercial Clo-rox which yields 5.25% NaOCl and 2 drops o ween20 per 100 mL solution or 15 min, ollowed by 5% (v/v)Clorox or 5 min. Afer the surace decontamination wascompleted the explants were rinsed 3 times with steriledistilled water. Following disinection 3-5 mm shoot tip

explants were excised prior to culture on MS (Murashigeand Skoog, 1962) basal medium containing 3% sucrose togrow the explants.

Medium preparation and culture conditionsAfer 6 weeks o culture, well developed shoots were

obtained. Te small shoots with a pair o leaves were trans-erred to MS medium supplemented either with 0, 1, 3, or5 mg/L BA or 0, 1, 3, or 5 mg/L kinetin. All culture mediaconsisted o MS salts and vitamins supplemented with 3%sucrose and 0.82% MermaidMagar. Te pH o media wasadjusted to 5.8 with 1 N NaOH or 1 N HCl prior to auto-

claving at 1.05 kg/cm2

, 121C or 20 min. Cultures were


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top o ice in a bucket and nuclei were mechanically iso-lated by chopping lea materials using a sharp razor blade.Afer chopping, the suspension was filtered through a 42m nylon mesh and CyStain UV Ploidy (DAPI stainingsolution) was added. Te fluorescence o a minimum o5000 DAPI-stained nuclei per sample was estimated usinga PA-II flow cytometer (Partec, Germany). Te reerencestandard plant (Zea mayscv. CE-777; 2C = 5.43 pg) waskindly provided by Dr. Jaroslav Dolezel, Laboratory oMolecular Cytogenetics and Cytometry, Institute o Ex-

perimental Botany, Czech Republic. Te reerence stan-dard peak was adjusted to show at channel 100 o relativefluorescence intensity or instrument calibration. Te 2CDNA content was calculated according to the ormula

Statistical analysisAll experiments were carried out at least 3 times with

5-10 replicates per treatment. Te fluorescence histogramswere resolved into G0/G1 (2C), S and G2/M (4C) cell-cycle compartments with a peak-reflect algorithm usingtwo Gaussian curves (WinMDI version 2.8). Data wereanalyzed by ANOVA and the differences among the means

were compared using Scheffes test atp


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terms o shoot, lea and root ormation, number o leavesper shoot, and percentage o root ormation. At low con-centration o BA (0, 1 mg/L) regenerated single shoot

while an increase in BA concentration rom 3 to 5 mg/Lresulted in increased number o shoots per explant. Tedata in ab. 1 revealed that the maximum number o52.12 shoots per explant was obtained on MS mediumsupplemented with 3 mg/L BA

Te results show that BA used singly was importantor induction o axillary bud outgrowth in A. barterivar.

Nana. By successive subculture on MS medium contain-ing 3 mg/L BA, masses o prolierating shoot cultures wereestablished (Fig. 1a).

A significant difference in the number o leaves was de-tected among the treatments containing BA and kinetin.Regenerated shoots had higher number o leaves on MSmedium supplemented with kinetin alone. Leaves were

ormed at a high requency o 13.4 (p0.05, ab. 1) onMS medium supplemented with 3 mg/L BA. No lea wasobserved on the control explants. Te eature o leavesdeveloped on MS medium containing 1-5 mg/L BA and1-5 mg/L kinetin showed broad and dark green leaves(Fig. 1a). BA seemed to inhibit root ormation since theroot ormation was recorded only on culture media con-

taining MS medium (control) or kinetin singly (ab. 1).Te roots arising rom the basal end o shoots were largeand vigorous (Fig. 1a). Te explants were subcultured or5 years successully and did not show any morphologicalabnormality when compared with the non tissue cultured

plants.Te cytological study o root tips at metaphase o long

term cultures revealed complete accurate counts o 48chromosomes (Fig. 1b). An occurrence o chromosomechanges has been ofen observed during application otissue culture, especially in plants, callus or cells that weremaintained or long-term cultures in vitro(Hao and Deng,2002). Te distribution o the nuclei extracted rom boththe mother plants and in vitro grown plants displayed a

prominent peak at 2C indicating that they consisted ocells with G0/G1 phase o cell cycle thus no ploidy varia-tion occurred (Fig. 2). Te mean 2C DNA content o the

mother plants and in vitrogrown plants are 5.43 and 5.45pg 2C-1, respectively. Tis finding confirmed that the al-teration o DNA content was not observed among in vitrogrown plants compared to the mother plants probably dueto the reason that plants regenerated rom well-developedmeristematic tissues that had minimum tendency o ge-netic variation (Rout et al., 1998).

Fig. 1.In vitro propagationo Anubias barterivar.Nana(a) Multiple shoots ormation rom asingle 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 m)

Fig. 2. Flow cytometric histograms o the relative nuclear DNA content (in channel numbers) oA. barterivar.Nanawith (a) dip-loid profiles o standard plant (b)diploid profile o 5 years old in vitrogrown plant


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In conclusion, we demonstrated that the establishmento rapid in vitroplant propagation oA. barterivar.Nanacan be achieved. Te nuclear DNA content value or A.barterivar.Nanawas provided. Ploidy variations were notobserved during subculture in 5 yearsas detected by cyto-logical study and flow cytometry. A combination o cyto-logical study and flow cytometry achieved better results interms o both accuracy and rapidity.

AcknowledgementsTis research was financially supported by the Faculty

o Science, Prince o Songkla University, Tailand. Te au-thors are grateul to Dr. Brian Hodgson or assistance withEnglish and useul comments on this manuscript.

References

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Micropropagation of Anubias barteri var. Nana

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