Journal Identification = HORTI Article Identification = 3935 Date: May 20, 2011 Time: 2:34 pm

Sg

NS

a

ARRA

KBSGIRM

1

eBwow2

agwAbharroMaa

e

0d

Scientia Horticulturae 129 (2011) 390–395

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

tability of micropropagated Musa acuminata cv. Grand Naine over clonalenerations: A molecular assessment

ilesh Borse, Vivek P. Chimote ∗, Ashok S. Jadhavtate Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri, India

r t i c l e i n f o

rticle history:eceived 4 January 2011eceived in revised form 28 March 2011ccepted 4 April 2011

a b s t r a c t

The present work was undertaken to investigate clonal fidelity of banana (Musa acuminata cv. GrandNaine) regenerants from six different in vitro subculture generations and in the explant suckers by usingISSR and REMAP molecular markers. Both types of markers revealed high degree of monomorphism. Verylow variation was observed up to the eighth subculture generation with polymorphic bands being low inboth ISSR (0.96%) and REMAP (0.95%) markers. Epigenetic stability was studied by DNA methylation anal-

eywords:anana micropropagationubculture generationenetic stability

SSREMAP

ysis of the eighth subculture generation samples. Single 570 bp methylation sensitive band was absentin two of the fifteen MspI predigested samples, while it was present in HpaII predigested and undigestedsamples. The results of the investigation confirmed that the micropropagation of banana up to the eighthsubculture generation show low variation.

© 2011 Elsevier B.V. All rights reserved.

ethylation

. Introduction

Banana (Musa spp.) and plantain are important fruit and veg-table crop of India. It is also known as a universal fruit crop of India.anana is a crop grown in tropical and subtropical regions of theorld. Bananas provide a staple starch in some of the poorest parts

f the world. In India, the area under banana was 6.47 lakh hectaresith the production of 2.32 million tons in the year 2007–08. It is

9% of the world’s total production of this crop (Anonymous, 2008).In vitro propagation of banana has played a key role in obtaining

large number of homogeneous regenerated plants. Micropropa-ation has played a key role in Musa improvement programmesorldwide (Rowe and Rosales, 1996; Vuylsteke et al., 1997).s compared to the conventional propagules, micropropagatedanana plants establish faster and grow more vigorously. They yieldigher in shorter duration with more uniform crop cycle (Vuylstekend Ortiz, 1996). Maximum yield gains from in vitro derived plantsange from 20% in bananas to 70% in plantains. However, this supe-ior field performance does not appear to be consistent and requiresptimum field practices. The application of micropropagation ofusa spp. has been reported using different explant sources such

s shoot tips meristem (Ma and Shii, 1972; Banerjee et al., 1986),

nd suspension culture (Roux et al., 2001).

Somaclonal variation is usually observed when plants are regen-rated from cultured somatic cells, mostly during callus formation

∗ Corresponding author. Tel.: +91 9689901911; fax: +91 2426243578.E-mail address: vivekchimote@rediffmail.com (V.P. Chimote).

304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2011.04.001

and suspension culture. However, even in absence of de-/re-differentiation stress, as during the micropropagation, off-types areobserved that reduce commercial values of resultant plants. Fur-thermore, most of the variants are inferior to the original cultivarfrom which they are derived. For example, bunch and fruit of thesevariants are often smaller (Smith and Drew, 1990), which offsetsthe potential benefits of variants. Dwarfism in ‘Cavendish’ bananasand inflorescence variations in plantains are often observed aftermicropropagation of respective mother genotypes.

The appearance of somaclonal variants may not be a processlimited to in vitro propagation but it may occur naturally in plantsomatic and reproductive tissues (Cullis and Kunert, 2000). Ratesof somaclonal variations in plants derived from shoot-tip culturevary from 0 to 70% according to genotype (Israeli et al., 1995; Smith,1988; Vuylsteke et al., 1991). This genetic instability may be a riskassociated with the application of in vitro culture techniques forgermplasm handling and storage. Various factors have all beenshown to influence both the quantity and the type of somaclonalvariation in micropropagated banana (Smith and Hamill, 1993).These factors include genotype, origin of shoots in vitro (adven-titious or auxiliary buds), number of subcultures, the choice ofexplants and the degree of dedifferentiation of the tissues in cul-ture.

Somaclonal variation can be either genetic or epigenetic (i.e.non-heritable). There may be variability even among clonally prop-

agated plants of a single donor clone. Somaclonal variation includespoint mutation, gene duplication, changes in number and thestructure of chromosomes, transposable element movement andchanges in DNA methylation (Jain, 2001). Epigenetic variation is

Journal Identification = HORTI Article Identification = 3935 Date: May 20, 2011 Time: 2:34 pm

rticult

aEgs

vctlRrasmcetbtptoub(as

m2igaoDtDalpc

abcwompw

2

2

ecPfiaaafe

N. Borse et al. / Scientia Ho

n important mechanistic basis of somaclonal variation in plants.pigenetic aspects of somaclonal variation involve mechanisms ofene silencing or gene activation that are not due to changes inequence or chromosomal aberrations.

Several methods have been used to investigate the geneticariability present in Musa germplasm. Development and appli-ation of technologies based on molecular markers provideools that are able to reveal polymorphism at DNA sequenceevel which is adequate to detect genetic variability. ISSR andEMAP markers are preferred PCR-based markers as they areeproducible, highly polymorphic, applicable for any crop andmenable to large scale throughput demands necessary forcreening large plant populations. Inter-simple sequence repeatsarker involves PCR amplification of the region between two

losely placed simple repeat sequences that are inversely ori-nted. They are identified using primers designed from withinhe repeated region (Zietjiewicz et al., 1994). This technique isased on PCR amplification of intermicrosatellite sequences. Retro-ransposons are abundant and dispersed components of mostlant genomes and comprise over 50% of nuclear DNA con-ent in many species. Genome diversifies through the insertionf new copies, but old copy persists. Retrotransposons can besed as markers because their integration creates new jointsetween genomic DNA and their conserved ends. The REMAPRetrotransposons–Microsatellite amplified polymorphism) showmplification between proximate retrotransposons and simpleequence repeats (SSR) to produce the marker bands.

DNA methylation represents conversion of cytosine to 5-ethylcytosine in plant genomes (Grant-Downton and Dickinson,

005). DNA methylation does not change the DNA sequence orts function, but changes its expression level, referred as an epi-enetic change. HpaII and MspI isoschizomer restriction enzymesre frequently used to detect cytosine methylation. They both rec-gnize the same sequence 5′-CCGG. Such differentially digestedNA fragments can be detected by various techniques. However,

hese methods are appropriate for displaying a global picture ofNA methylation changes within a genome. But they are laboriousnd need locus specific primers and PAGE analysis. Use of methy-ation sensitive restriction enzymes in combination with arbitraryrimers based PCR is the simplest method of detecting methylationhanges in genomic DNA (Nakamura and Hosaka, 2009).

There is lot of data available on banana somaclonal variationt the phenotypic level in micropropagated bananas. However, theasis of this variation and its extent during multiple cycles of in vitroulture remains unknown. In the present investigation, attemptsere made to study the clonal fidelity of regenerants in banana

ver different clonal generations using ISSR and REMAP moleculararkers. Study on epigenetic stability of micropropagated banana

lants was undertaken by ISSR analysis of genomic DNA digestedith methylation sensitive isoschizomers i.e. HpaII and MspI.

. Material and methods

.1. Plant material

Fifteen banana leaf samples from each of the five subculture gen-ration (i.e. 2nd, 5th, 6th, 7th and 8th) were collected from tissueulture laboratory of State Level Biotechnology Centre, Mahatmahule Krishi Vidyapeeth, Rahuri, India. Shoot tip explants fromeld suckers were established in vitro, multiplied on Murashigend Skoog’s medium (Murashige and Skoog, 1962) + 5 mg/l BAP

nd rooted on MS media + 3 mg/l NAA, solidified with agar (8 g/l)t 24 ± 2 ◦C. Five clumps, each with 3 shoots, were transferred toresh medium in jamjars every 3 weeks for multiplication. Fromach sample 100 mg plant material was used for genomic DNA

urae 129 (2011) 390–395 391

isolation. Banana suckers (thirteen samples) were collected fromBanana Research Station, Jalgaon, India, for micropropagation pur-pose and they were also used for genomic DNA isolation.

2.2. Isolation of plant genomic DNA

The genomic DNA from tender leaves (100 mg) of differentclonal generations of micropropagated banana were isolated byHiPurA Plant Genomic DNA Miniprep Purification Spin kit (M/SHiMedia Ltd.) after crushing in liquid nitrogen. Concentration andpurity of isolated DNA was measured using UV visible spectropho-tometer (NanoDrop ND-1000, USA) at 260 and 280 nm wavelengthsand by visualization under 0.8% (w/v) agarose gel electrophoresis(BioRad Sub-Cell Model 96, USA).

2.3. DNA amplification by ISSR and REMAP markers

Polymerase chain reaction was carried out in 103 samples byusing 12 ISSR and 12 REMAP primer pairs (3 retrotransposons basedprimers and 5 ISSR primers) as per protocol (specified by Teo et al.,2005; Kalendar et al., 1999). The annealing temperature in PCRwas standardized for each ISSR primer and for each combinationof REMAP primer pairs. PCR regime involved initial denaturation(94 ◦C) for 5 min, followed by PCR amplification cycles [40 cycles(for ISSR) and 30 cycles (for REMAP), respectively] of denaturation(94 ◦C), annealing (as specified in Tables 1 and 2) and elongation(72 ◦C) and then final extension at 72 ◦C for 10 min. In ISSR analysiseach step of this step was carried out for 30 s. In REMAP amplifica-tion, denaturation and annealing steps were carried out for 1 mineach, while elongation was performed for 2 min.

2.4. Agarose gel electrophoresis and data analysis of PCR products

For ISSR and REMAP analysis 1.2% and 2% (w/v) agarose gel elec-trophoresis was used for PCR product profiling, respectively. Theamplified PCR products were observed under UV transilluminatorin gel documentation system (Flour ChemTM Alpha Innotech, USA)and image was captured. The clearly resolved PCR amplified ISSRand REMAP bands of banana samples with 12 primer/primer com-binations each, were scored for their binary data. The similarity andclustering analysis were carried out using the computer packageNTSYSpc 2.02i (Rohlf, 1998).

2.5. DNA methylation analysis

DNA methylation was detected by predigesting with methy-lation sensitive restriction enzymes MspI and HpaII; then PCRamplification (ISSR) and then again followed by post-digestionwith the same restriction enzymes. DNA methylation was detectedaccording to the procedure described by Nakamura and Hosaka(2009) with some modifications.

All 15 genomic DNA samples (8th clonal generation) weredivided into three different lots, MspI digested, HpaII digested (M/SBangalore GeNei Pvt. Ltd.) and undigested, with volume of restric-tion digestion setup to 40 �l. The two digestion reactions wereincubated at 37 ◦C for 6 h. After digestion, the DNA was ethanol pre-cipitated, dried and dissolved in 40 �l autoclaved distilled water.

All 45 samples (3 treatments × 15 original 8th clonal generationsamples) were PCR amplified with ISSR marker i.e. IS-13 primer.PCR amplification products (75 �l) were ethanol precipitated, andfinally dissolved in 50 �l autoclaved distilled water.

For the second restriction digestion, each of the above 45 PCR

amplified reactions was further subdivided into three lots. ISSRamplification product of MspI genomic DNA digestion was subdi-vided in three different lots again like MspI digested, HpaII digestedand undigested respectively. The same subdivision procedure was

Journal Identification = HORTI Article Identification = 3935 Date: May 20, 2011 Time: 2:34 pm

392 N. Borse et al. / Scientia Horticulturae 129 (2011) 390–395

Table 1ISSR primers used and their amplification at different generation of subcultured banana.

No. ISSR primer Sequence of primer Tann (◦C) Generation

P 2nd 5th 6th 7th 8th

1 ISSR 827 ACACACACACACACACG 55 5 6 6 5 7 72 ISSR 841 GAGAGAGAGAGAGAGAYG 46 6 6 7 7 7 93 IS 12 GTGTGTGTGTGTGTTG 47 9 12 10 6 11 124 IS 13 GTGTGTGTGTGTGTGTCA 52 10 6(1*) 8(1*) 8 9 95 ISSR 857 ACACACACACACACYC 46 11 7 7 6 6 66 ISSR 834 AGAGAGAGAGAGAGAGYT 45 6(1*) 8 8 7 8 9(1*)7 ISSR 820 GTGTGTGTGTGTGTGTC 54 2 2 2 2 2 28 ISSR 813 CTCTCTCTCTCTCTCTT 46 4 6 6 6 7 89 ISSR 811 GAGAGAGAGAGAGAGAC 50 6 8 7(1*) 7 9 9

10 ISSR 817 CACACACACACACACAA 54 2 2 2 2 2 211 ISSR 816 CACACACACACACACAT 54 4 3 4 4 3 412 ISSR 822 TCTCTCTCTCTCTCTCA 50 2 2 2 2 2 2

ii

fDu

wt

2

fsPddwstr

3

gpi6eb

TR

ii

: Total number of bands amplified.i*: Polymorphic bands.

ollowed for the lot originally derived from HpaII digested genomicNA and the third lot of PCR amplified derived from originallyndigested genomic DNA.

In 90 out of these 135 reactions, post-PCR restriction digestionas performed as per conditions specified. Termination of diges-

ion reaction was done by incubating at 65 ◦C for 20 min.

.6. Gel electrophoresis conditions for methylation analysis

After termination of reaction, 1.4% (w/v) agarose gel was usedor resolution of the fragments. The nine treatments from eachample were loaded in order in the gel i.e. MspI predigested (PostCR digested: MspI, HpaII, undigested), HpaII predigested (Post PCRigested: MspI, HpaII, undigested) and pre-undigested (Post PCRigested: MspI, HpaII, undigested). The above digested samplesere observed under UV transilluminator in gel documentation

ystem (Flour ChemTM Alpha Innotech, USA) and image was cap-ured. The same procedure was followed for the next fourteenemaining samples and results were recorded.

. Results

In the current study molecular characterization of seven clonalenerations/subcultures of banana was carried out using 12 ISSRrimers and 12 REMAP markers for 88 DNA samples. This study

ncluded fifteen samples from each clonal generation (i.e. 2nd, 5th,th, 7th and 8th) (MS + 5 mg/l BAP) and thirteen samples from suck-rs. For each marker the annealing temperature was standardizedy gradient temperature PCR amplification (Tables 1 and 2). ISSR

able 2EMAP markers used and their amplification at different generation of subcultured banan

No. REMAP primer pair Tann (◦C)

LTR primer 1 retrotransposon ISSR primer 2

1 LTR 6149 IS 8081 542 LTR 6150 IS 8386 563 3′LTR IS 8386 564 Nikita IS 8386 565 Sukula IS 8386 566 LTR 6149 IS 8386 607 LTR 6150 IS 8081 568 3′LTR IS 8081 589 Sukula IS 8081 58

10 Nikita IS 8081 5611 Sukula ISSR 827 5412 Nikita ISSR 827 54

: Total number of bands amplified.i*: Polymorphic bands.

and REMAP analysis of amplified PCR products was done using 1.2%and 2.0% agarose gel for electrophoresis, respectively. The numberof bands amplified with each marker with their detail pattern isgiven in Table 3 (ISSR markers) and Table 4 (REMAP markers).

In the present study, ISSR amplification by using 12 primer paircombinations generated profile ranging between 62 and 79 ampli-cons over various subculture generations. IS 12 marker yielded thehighest mean of 10 bands per sample per generation; while theminimum of 2 bands per sample per generation were observed withISSR 817 and ISSR 822 markers. Only ISSR 13 (in 2nd and 5th gen-eration) ISSR 834 (in Parental Sucker and 8th generation) and ISSR811 (in 5th generation) each yielded a single polymorphic band inthe generation mentioned.

In the present study, REMAP amplification by using 12 primerpair combinations generated profile ranging between 113 and 135amplicons over various subculture generations. The average ampli-fication was 122.7 amplicons per generation. REMAP analysis didnot show much variation in banding pattern of the 1st to 7th gen-erations (113–125 amplicons). However, highest number of bandsi.e. 135 were observed in the 8th subculture generation plantlets.REMAP with LTR 6150 + IS 8081 primer combination yielded thehighest mean of 13.2 bands per sample per generation; while thelowest of 5.2 bands per sample per generation were observedwith REMAP (LTR 6149 + IS 8386) primer combination. REMAP (LTR6150 + IS 8386) primer combination yielded three, two and a single

polymorphic band in the 2nd, 5th and 7th generation, respectively.In addition, 3′LTR + IS 8386 REMAP yielded 3 polymorphic bands in8th generation samples. Rest of all REMAP markers were monomor-phic in their amplification profile.

a.

Generation

P 2nd 5th 6th 7th 8th

11 14 10 11 15 1610 11(3*) 12(2*) 7 11(1*) 11

7 6 6 6 7 10(3*)10 10 12 12 10 1010 10 9 9 9 10

2 2 8 8 2 913 14 13 13 13 1311 10 9 11 11 11

9 8 13 13 11 119 13 12 12 12 12

12 13 13 9 13 1310 9 8 14 8 9

Journal Identification = HORTI Article Identification = 3935 Date: May 20, 2011 Time: 2:34 pm

N. Borse et al. / Scientia Horticulturae 129 (2011) 390–395 393

Table 3ISSR analysis of different generations of micropropagated banana.

Sr. no. Generation No. of bands Monomorphic bands Highly informativebands (20–80%)

Less informativebands (<20 or >80%)

% polymorphic bands

1 Parent 67 66 0 1 1.492 Second 68 67 1 0 1.473 Fifth 69 68 0 2 2.94

bNwPrrw3bIH

4

niwo

igpm

fvn(nrsoaaeNi1tdr

TR

4 Sixth 62 625 Seventh 73 736 Eighth 79 78

In order to initiate epigenetic studies in micropropagatedanana, the DNA methylation was studied as per protocol ofakamura and Hosaka (2009). DNA methylation study was doneith the fifteen 8th subculture generation DNA samples. It involved

CR analysis of genomic DNA digested using methylation-sensitiveestriction enzymes (MspI and HpaII). Overall 135 PCR (ISSR 13)eactions were performed comprising of 15 genomic DNA samples,ith 3 digestion treatments of each sample and followed by furtherpost-PCR digestion treatments. Single 570 bp methylation specificand was found polymorphic in two samples of the 8th generation.

t was absent in MspI predigested samples, while it was present inpaII predigested samples and undigested one as well.

. Discussion

The basic aim of banana micropropagation is to create largeumber of uniform and healthy planting material through rapid

n vitro culture, without any genetic variation. The present studyas conducted to study the occurrence of somaclonal variations

ver various clonal generations during banana micropropagation.Two types of molecular markers i.e. ISSR and REMAP were used

n the present study for analysis of all six clonal generations. Allenerations showed high monomorphism with a few exceptionalolymorphic bands. Even these polymorphic bands were less infor-ative ones (present or absent in <20% or >80% samples).There have been reports of morphological and vigour variation

rom field populations. In micropropagated bananas somaclonalariation is detectable at the level of phenotype. The range of phe-otypic variation has been reported to vary between 1 and 50%Israeli et al., 1995). Jambhale et al. (2000) had suggested that theumber of subcultures in micropropagation of banana should beestricted to eight. They observed that some plantlets were con-picuously distinct from the parental clones in the populationsf hardened plants after the 8th subculture. Morphological vari-tions were observed in the 10th, 12th and 14th subculture ofll the clones except Safed Velachi. Three other cultivars studiedxhibited moderate frequency of variation i.e. Basrai (1.0–5.5%),endran (15.87–36.49%) and Lal Kela (3.0–7.2%). Nendran exhib-

ted maximum variants in the 10th (15.87%), 12th (26.585%) and

4th (36.49%) subcultures. Gomez and Garcia (1997) also observedhat the percentage of phenotypic variants differed in betweenifferent Cavendish banana genotypes. Daniells and Smith (1993)eported as high as 91% variants in tissue cultured plants in banana.

able 4EMAP analysis of different generations of micropropagated banana.

Sr. no. Generation No. of bands Monomorphic bands

1 Parent 113 1132 Second 116 1163 Fifth 125 1234 Sixth 125 1245 Seventh 122 1216 Eighth 135 132

0 0 0.00 0 0.01 0 1.27

Earlier there have been reports of genetic stability analysis of micro-propagated banana cultivars using molecular markers (Martin et al.,1998; Ray et al., 2006; Lakshmanan et al., 2007; Venkatachalamet al., 2007). However, there is lack of molecular data regarding theeffect of the number of subcultures on stability of micropropagatedpopulation.

The twelve ISSR markers generated an average of 69.7 ampli-cons per generation. The early generations i.e. from 1st to 6thdid not show much variation in the banding pattern (no. ofbands/assay); the range of amplicons was 62–69 per generation.An increase was observed in the number of amplicons in the 7th(73 amplicons) and 8th (79 amplicons) generation. The mean ofmonomorphism observed till eighth generation was 98.81%. AllISSR markers showed monomorphic banding pattern in the 6thand 7th generations. Only one polymorphic band was observedin the explant suckers, 2nd and 8th generations. Two polymericbands were observed in the 5th generation (one each with ISSR811 and ISSR 13). Polymorphic amplicon were obtained with ISSR13 marker in 2nd and 5th generation, while ISSR 834 yielded apolymorphic amplicon in Parental (absent in 3 samples) and 8thgeneration (absent in 5 samples).

On ISSR analysis, Ray et al. (2006) reported 5.08% polymorphismbetween the mother and few micropropagated plants of Robustacultivar. Rout et al. (2009) concluded in vitro multiplication as thesafest mode for multiplying true-to-type plants. On ISSR analysis ofmicropropagated plantlets they observed that most of ISSR markersshowed monomorphic banding pattern. Very few plants showedvariation at the DNA level, but morphologically they were similar.Venkatachalam et al. (2007) observed homogeneous RAPD and ISSRpatterns on genetic analysis of micropropagated plants of banana.

In the present study, on REMAP analysis the average amplifi-cation was 122.7 amplicons per generation. REMAP analysis didnot show much variation in banding pattern from the 1st to7th generations (113–125 amplicons). However, higher numberof 135 bands were observed in the 8th generation plantlets. Themean of monomorphic bands observed till eighth generation was98.66%. The REMAP markers showed monomorphic banding inthe explant suckers, while in others until 8th generation only1–3 bands were polymorphic with 97.8–99.2% monomorphism.REMAP primer pairs amplified a total of 4 polymorphic bands,

of which 1 was highly polymorphic (in 7th generation) and 9were less polymorphic (LTR 6150 + IS 8386 yielded 3 and 2 poly-morphic bands, in 2nd and 5th generation respectively; similarly3′LTR + IS 8386 yielded 3 polymorphic markers in 8th generation).

Highly informativebands (20–80%)

Less informativebands (<20 or >80%)

% polymorphic bands

Nil Nil 0.0Nil 3 2.56Nil 2 1.62Nil Nil 0.801 Nil 0.82Nil 3 2.27

Journal Identification = HORTI Article Identification = 3935 Date: May 20, 2011 Time: 2:34 pm

3 rticult

A6Rpm

cvKmpttrRsppa

R5pmRppp1fiw

rliNlm

t1tpRDed

d(ueatlmaShs

gVog

94 N. Borse et al. / Scientia Ho

total of six polymorphic bands were observed with REMAP (LTR150 + IS 8386) primer combination till 7th generation samples.EMAP (3′LTR + IS 8386) primer combination yielded all 3 polymor-hic bands in the 8th generation. Rests of the REMAP markers wereonomorphic in their amplification profile.Ubiquitous distribution, high copy number and widespread

hromosomal dispersion of retrotransposons mobile elements pro-ide excellent potential for developing DNA-based marker systems.alendar et al. (1999) developed intertransposons amplified poly-orphism (IRAP) and retrotransposons microsatellite amplified

olymorphism (REMAP) fingerprinting techniques based on retro-ransposon to detect high level of polymorphisms. Using IRAPechnique, Teo et al. (2005) detected the integration of newetrotransposon into the banana genome during tissue culture.etrotransposons are generally thought to be activated by tis-ue culture. Using retrotransposon based IRAP markers (using LTRrimers derived from barley sequences) they found that theserimers generated specific amplification pattern showing universalcceptability.

In terms of comparative informativeness of polymorphic bands,EMAP primers amplified a total of 9 polymorphic bands as againstpolymorphic bands by ISSR primers. Two ISSR bands were highlyolymorphic (with IS 13 and IS 834 primers) and 3 were less poly-orphic bands (with IS 834, IS 13 and ISSR 811 primers). Single

EMAP band was highly polymorphic while eight of them were lessolymorphic. Only REMAP, LTR 6150 + IS 8386 and 3′LTR + IS 8386rimer combinations showed polymorphic bands. These REMAPrimers in addition to informative ISSR markers (IS 834 and IS3), indicate a shared region in genome which is likely more liableor stress induced variations during tissue culture. Oh et al. (2007)dentified tissue culture stress labile portion of the banana genome,

hich can be for identifying banana somaclonal variants.Tissue culture generates a variety of epigenetic changes in

egenerants. DNA methylation has been considered as an under-ying mechanism of micropropagation induced mutagenesis. Thiss due to high frequency of quantitative phenotypic variation.akamura and Hosaka (2009) developed a simpler method of ana-

yzing DNA methylation using RAPD of genomic DNA digested withethylation sensitive enzymes.In the current study, only a single 570 bp methylation sensi-

ive band was found in two samples of 8th generation, out of the5 bands amplified with ISSR 13 primer. This methylation sensi-ive band was absent in MspI predigested samples, while it wasresent in HpaII predigested samples and undigested one as well.eproducibility was confirmed by repeated PCR using the sameNA samples. PCR amplification products were cut again by thesenzymes. As expected such methylation sensitive band got cut andisappeared because PCR products were no longer methylated.

Tissue culture-induced methylation has previously beenetected in regenerants from various crops including bananaPeraza-Echeverria et al., 2001). Peraza-Echeverria et al. (2001)sed MSAP technique to detect the polymorphic methylationvents in micropropagated bananas derived from the vegetativepex of the sucker (polymorphic methylation events 1.7%) andhe floral apex of the male inflorescence (polymorphic methy-ation events 13%). Gimenez et al. (2006) identified four banana

ethylation sensitive amplification polymorphism markers associ-ted for resistance to toxin of Mycosphaerella fijiensis causing Blackhigotika disease. These MSAP markers showed high sequenceomology with resistance genes analog and retrotransposonsequences.

Kaeppler and Phillips (1993) and Kaeppler et al. (1998) sug-

ested that tissue culture represents a unique form of stress.uylsteke et al. (1991) had suggested that in genus Musa, the extentf somaclonal instability was a result of the interaction between theenotype and the tissue culture conditions. Fukui (1983) observed

urae 129 (2011) 390–395

that if rice regenerants remain in the in vitro culture condition forlong periods of time, mutations are likely to occur more frequently.Li et al. (2008) suggested that the culture period itself is a likelymutagenic factor in methylation, and that these mutations are notsimply pre-existing in the explant source.

The current study concludes that during banana micropropa-gation there is little variation till the 8th subculture generation,as reported previously by a report from our university (Jambhaleet al., 2000). Usually plantlets from commercial firms show highmorphological variation in field. They go for more subcultures inexcess to reduce their economic cost as with fresh explant usagethere are more chances of contamination associated losses. Recy-cling of sucker explants from previously micropropagated bananatransplanted to field back to lab, further accumulates variation.Number of subculture, explant source and hormonal concentra-tion should be strictly followed to improve the genetic stability ofmicropropagated banana.

Acknowledgements

The authors are grateful to authorities of Mahatma Phule KrishiVidyapeeth, Rahuri for providing necessary facilities to undertakethis study. Help rendered by Dr. S.V. Pawar and Mr. G. Newaskarduring this study is also gratefully acknowledged.

References

Anonymous, 2008. In: Kumar, B., Mistry, N.C., Singh, B., Gandhi, C.P. (Eds.), IndianHorticulture Database. National Horticulture Board, Ministry of Agriculture,Government of India, 298 pp.

Banerjee, N., Vuylsteke, D., Langhe, E., 1986. Meristem tip culture of Musa: histomor-phological studies of shoot bud proliferation. In: Plant Tissue and its AgriculturalApplication. Butterworths, London, pp. 139–147.

Cullis, C.A., Kunert, K.J., 2000. Isolation of tissue culture induced polymorphisms inbananas by representational difference analysis. Acta Hortic. 530, 421–428.

Daniells, J.W., Smith, M.K., 1993. Somatic mutations of bananas—their stability andpotential. In: Valmayor, R.V., et al. (Eds.), International Symposium on RecentDevelopments in Banana Cultivation (INIBAP/ASPNET). Los Banos, Philippines,pp. 162–171.

Fukui, K., 1983. Sequential occurrence of mutations in a growing rice callus. Theor.Appl. Genet. 65, 225–230.

Gimenez, C., Palacios, G., Colmenares, M., 2006. Musa methylated DNA sequenceassociated with tolerance to Mycosphaerella fijiensis toxins. Plant Mol. Biol. Rep.24, 33–43.

Gomez, I.H., Garcia, E.G., 1997. Agronomic studies of Cavendish bananas grown byin vitro culture. InfoMusa 6, 23–26.

Grant-Downton, R.T., Dickinson, H.G., 2005. Epigenetics and its implications for plantbiology. 1. The epigenetic network in plants. Ann. Bot. 96, 1143–1164.

Israeli, Y., Lahav, E., Reuveni, O., 1995. In vitro culture of bananas. In: Gowen, S. (Ed.),Bananas and Plantains. Chapman & Hall, London, pp. 147–178.

Jain, S.M., 2001. Tissue culture derived variation in crop improvement. Euphytica118, 153–166.

Jambhale, N.D., Patil, S.C., Jadhav, A.S., Pawar, S.V., Waghmode, B.D., 2000. Effect ofnumber of subcultures on in vitro multiplication of four banana clones. InfoMusa10, 38–39.

Kaeppler, S.M., Phillips, R.L., 1993. DNA methylation and tissue culture inducedvariation in plants. In vitro Cell. Dev. Biol. Plant 29, 125–130.

Kaeppler, S.M., Phillips, R.L., Olhoft, P., 1998. Molecular basis of heritable tissueculture-induced variation in plants. In: Jain, S.M., et al. (Eds.), SomaclonalVariation and Induced Mutations in Crop Improvement. Current Plant Sci-ence and Biotechnology in Agriculture, vol. 32. Kluwer Acad. Publ., Dordrecht,Netherlands, pp. 465–484.

Kalendar, R., Grab, T., Regina, M., Suoniemi, A., Schulman, A.H., 1999. IRAP andREMAP: two new retrotransposon based DNA fingerprinting techniques. Theor.Appl. Genet. 98, 704–711.

Lakshmanan, V., Venkataramareddy, S.R., Neelwarne, B., 2007. Molecular analysisof genetic stability in long-term micropropagated shoots of banana using RAPDand ISSR markers. Electron. J. Biotechnol. 10, 106–113.

Li, X., Wang, X., He, K., Ma, Y., Su, N., He, H., Stolc, V., Tongprasit, W., Jin, W., Jiang,J., Terzaghi, W., Li, S., Deng, X.W., 2008. High-resolution mapping of epigeneticmodifications of the rice genome uncovers interplay between DNA methylation,histone methylation, and gene expression. Plant Cell 20, 259–276.

Ma, S.S., Shii, C.T., 1972. In vitro formation of adventitious buds in banana shoot apexfollowing decapitation. J. Chin. Soc. Hortic. Sci. 18, 135–142.

Martin, M.J.G., Grillo, G.S., Dominguez, A.M., 1998. The use of randomly amplifiedpolymorphic DNA (RAPD) for the study of genetic diversity and somaclonalvariation in Musa. Int. Soc. Hortic. Sci. 490, 445–454.

Journal Identification = HORTI Article Identification = 3935 Date: May 20, 2011 Time: 2:34 pm

rticult

M

N

O

P

R

R

R

R

N. Borse et al. / Scientia Ho

urashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassayswith tobacco tissue cultures. Physiol. Plant. 15, 473–497.

akamura, S., Hosaka, K., 2009. DNA methylation in diploid inbred lines of potatoesand its possible role in the regulation of heterosis. Theor. Appl. Genet. 120 (2),205–214.

h, T.J., Cullis, M.A.K., Engelborghs, I., Swennen, R., Cullis, C.A., 2007. Genomicchanges associated with somaclonal variation in banana (Musa spp.). Physiol.Plant. 129, 766–774.

eraza-Echeverria, S., Herrera-Valencia, V., Andrew James, K., 2001. Detec-tion of DNA methylation changes in micropropagated banana plants usingmethylation-sensitive amplification polymorphism (MSAP). Plant Sci. 161,359–367.

ay, T., Dutta, I., Saha, P., Das, S., Roy, S.C., 2006. Genetic stability of three econom-ically important micropropagated banana (Musa spp.) cultivars of lower IndoGangetic plantains, as assessed by RAPD and ISSR markers. Plant Cell TissueOrgan Cult. 85, 11–21.

ohlf, F.J., 1998. Numerical Taxonomy and Multivariate Analysis System, Version 2.0(Exeter Software, New York).

out, G.R., Senapati, S.K., Aparajita, S., Palai, S.K., 2009. Studies on genetic identifi-

cation and genetic fidelity of cultivated banana using ISSR marker. Plant OmicsJ. 2, 250–258.

oux, N., Dolezel, J., Swennen, R., Zapata-Arias, F.J., 2001. Effectiveness of threemicropropagation techniques to dissociate cytochimeras in Musa spp. Plant CellTissue Organ Cult. 66, 189–197.

urae 129 (2011) 390–395 395

Rowe, P., Rosales, F.E., 1996. Bananas and plantains. In: Janick, J., Moore, J. (Eds.), FruitBreeding, vol. 1: Tree and Tropical Fruits. John Wiley, New York, pp. 167–211.

Smith, M.K., 1988. A review of factors influencing the genetic stability of microprop-agated bananas. Fruits 43, 219–223.

Smith, M.K., Drew, R.A., 1990. Current applications of tissue culture in plant propa-gation and improvement. Aust. J. Plant Physiol. 17, 267–289.

Smith, M.K., Hamill, S.D., 1993. Early detection of dwarf off-types from microprop-agated bananas. Aust. J. Exp. Agric. 33, 639–644.

Teo, C.H., Tan, S.H., Ho, C.L., Kalender, R., 2005. Genome constitution and classifica-tion using retrotransposon-based markers in orphan crop banana. J. Plant Biol.48, 96–105.

Venkatachalam, L., Sreedhar, R.V., Bhagyalakshmi, N., 2007. Genetic analyses ofmicropropagated and regenerated plantlets of banana as assessed by RAPD andISSR markers. In vitro Cell. Dev. Biol. Plant 43, 267–274.

Vuylsteke, D., Ortiz, R., 1996. Field performance of conventional vs. in vitro propag-ules of plantain (Musa spp. AAB group). Hortscience 31, 862–865.

Vuylsteke, D., Ortiz, R., Ferris, R.S.B., Crouch, J.H., 1997. Plantain improvement. PlantBreed. Rev. 14, 267–332.

Vuylsteke, D., Swennen, R., De Langhe, E., 1991. Somaclonal variation in plan-

tains (Musa spp. AAB group) derived from shoot tip culture. Fruits 46,429–439.

Zietjiewicz, E., Rafalski, A., Labuda, D., 1994. Genome fingerprinting by sim-ple sequence repeat (SSR)-anchored polymerase chain reaction amplification.Genomics 20, 176–183.