Short communication

PQM-1: A newly developed superior clone of pineapple for northeastern Indiaas evident through phenotype, fruit quality and DNA polymorphism

Jai Prakash a, S. Bhattacharyya b, Krishnendu Chattopadhyay a,*, S. Roy b, S.P. Das a, N.P. Singh a

a ICAR Research Complex for NEH Region, Tripura centre, Lembucherra 799210, Tripura (West), Indiab Crop Research Unit, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252, India

Scientia Horticulturae 120 (2009) 288–291

A R T I C L E I N F O

Article history:

Received 3 February 2008

Received in revised form 22 September 2008

Accepted 7 November 2008

Keywords:

Pineapple

Clone

Fruit quality

Plant characters

ISSR

RAPD

A B S T R A C T

Queen is the most preferred table purpose variety in India. However, small fruit size, deep set eyes in

large numbers and weak fruit stalk make this less preferred in processing industries. A completely

different plant type with distinct fruit character was identified from a clonal population of Queen which

seems to be a natural mutant. The identified mutant was maintained and multiplied through vegetative

propagation at Lembucherra farm of ICAR Research Complex, Tripura centre. The present study clearly

established the fact that this new clone (PQM-1) is distinct from ‘queen’ and other cultivars of Tripura.

Distinctness of this new clone was confirmed on the basis of standard morphological and biochemical

descriptors as well as through DNA polymorphism. It was observed that PQM-1 diverged from Queen in

respect of pattern of vegetative growth, leaf shape, size and colour, flowering time, fruit shape, size and

colour, eye shape, fruit weight, juice content, total soluble solid (TSS) and acidity. PQM-1 was found

uniform at DNA level and complement the information of phenotypic and quality parameters by

showing stable differences with Queen in ISSR banding pattern. Natural mutation in DNA level might be

the probable cause of its origin. It could be empathetically stated that PQM-1 would have certain

competitive advantage over all existing cultivars in this region due to its late maturity, fruit quality and

usefulness in both canning and fresh consumption. This certainly would emerge as new alternative to

pineapple growers of this region.

� 2008 Elsevier B.V. All rights reserved.

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1. Introduction

Pineapple is one of the most important fruit crops of tropicaland humid subtropical regions. Productivity of pineapple innortheastern states of India, especially in Tripura is high. Queenis the most delicious table purpose variety among cultivarsgrown in India. The leaves of Queen are characterized as spinywith small closely spaced spines and serrated leaf margin.Cylindrical fruits of this variety are small with an averageweight of 1.2 kg and having deep set prominent and irregulareyes (Sen and Mitra, 2001). During regular observation ofsuckers grown plants of pineapple cultivar ‘queen’, a morpho-logically distinct plant type was observed, which on floweringproduced a clearly distinct fruit. Through selection and multi-plication over a period of five years, a unique clonal populationof pineapple, namely, PQM-1 was developed. Intra-specific andintra-varietal variability in pineapple has been reported earlierby many workers (Perez et al., 1998; Kato et al., 2005). Under

* Corresponding author.

E-mail address: chatterjee2@yahoo.com (K. Chattopadhyay).

0304-4238/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2008.11.016

this context, experiments were designed to validate PQM-1 as anew superior clone of pineapple, derived from cv. Queen, on thebasis of its distinctness from Queen as well as rest of thecultivars grown in this region, uniformity in clonal population,stability in expression of various important morphological andbiochemical characters as well as in genetic loci. Besides usingstandard morphological and quality parameters, clones weresubjected to molecular marker assay using Inter SimpleSequence Repeat (ISSR) and Random Amplified PolymorphicDNA (RAPD) primers.

2. Materials and methods

2.1. Plant materials and data recording

PQM-1 along with three established cultivars, viz. Queen,Mauritius and Kew were grown at experimental farm of ICARResearch Complex for NEH Region Tripura centre, Lembucherra forthe last five years (2002-2007). 100 suckers of each clone wereplanted in 30 cm � 60 cm � 75 cm spacing under double rowsystem (Chadha et al., 1975) in each plot. The clones were plantedin Randomized Block Design with five replications. Data were

Table 1List of ISSR and RAPD primers with their sequences, annealing temperature and reaction to pineapple clones used in the study.

Primer name Sequence (50–30) Annealing temperature (8C) Reaction to pineapple DNA

ISSR primers

IS-6 GAG AGA GAG AGA GAG AC 52 Positive, reproducible and polymorphic

IS-7 GTG TGT GTG TGT GTG TA 50 Negative

IS-8 AGA GAG AGA GAG AGA GC 52 Positive and reproducible, but did not show intra-clonal polymorphism

IS-9 TGT GTG TGT GTG TGT A 46 Positive, reproducible and polymorphic

IS-10 CGA GAG AGA GAG AGA GA 52 Positive but not reproducible

IS-11 CAC ACA CAC ACA CAC AG 52 Positive, reproducible and polymorphic

IS-12 GTG TGT GTG TGT GTG TC 52 Positive and reproducible but not polymorphic

IS-61 GAGAGAGAGAGAGAGAT 50 Positive, reproducible and polymorphic

IS-63 AGAGAGAGAGAGAGAGC 52 Positive, but not polymorphic

IS-65 AGAGAGAGAGAGAGAGT 50 Positive, but not polymorphic

RAPD primers

RA1 AGCGCCATTG 36 Positive, reproducible, but not polymorphic

RA2 CATCCGTGCT 36 Positive and reproducible

RA4 GTGTGCCCCA 36 Positive, reproducible and polymorphic

J. Prakash et al. / Scientia Horticulturae 120 (2009) 288–291 289

recorded on pattern of vegetative growth, plant characters, fruitcharacters, etc. following the standard procedures (Collins, 1960).

2.2. Biochemical analysis

Biochemical parameters like total juice content, total solublesolids (TSS), titrable acidity were measured from ten randomlyselected plants in each plot following Ranganna (1986).

2.3. DNA extraction and ISSR reaction

From 1 g tender leaves of pineapple plant, genomic DNA wasextracted by procedure described by Chattopadhyay et al. (2008).Bulked DNA of PQM-1 and three others cultivated varieties were

Fig. 1. Mature fruit of (a) P

subjected to PCR reaction using 10 ISSR primers and 3 RAPDprimers (Table 1). Each bulk DNA was constituted by genomic DNApooled from 10 plants. The 25 ml PCR reaction mixture contained4 ml 2.5 mM dNTPs, 20 ng DNA in 2.5 ml 10X Taq polymerase assaybuffer having 1.5 mM MgCl2 and 1.5U Taq polymerase enzyme and100ng of primer (all from Genei, Bangalore, India). The PCRproducts were analyzed on a 1.5% agarose gel (SRL) in 1X TBEbuffer. Bands were visualized by ethidium bromide staining(0.5 mg/ml) and photographed on UV-trans-illuminator.

3. Results and discussion

PQM-1 was found diverged from Queen in respect of pattern ofvegetative growth, leaf shape, size and colour, flowering time, fruit

QM-1 and (b) Queen.

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eta

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nd

fru

itch

ara

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rist

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of

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M-1

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ue

en

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au

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ew

va

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ura

.

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ty

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t(c

m)

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mb

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of

lea

ve

sa

t

red

he

art

sta

ge

Nu

mb

er

of

suck

ers

Nu

mb

er

of

slip

s

Flo

we

rin

g

(%)

Ma

turi

typ

eri

od

red

he

art

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arv

est

(da

ys)

Cro

wn

we

igh

t(g

)

Fru

it

we

igh

t(g

)

PQ

M-1

10

8.0

52

.32

1.7

82

.28

83

.36

12

4.2

32

0.0

13

32

.0

Qu

ee

n9

3.0

24

5.4

81

.38

4.5

80

.91

01

.42

94

.01

11

4.0

Ma

uri

tiu

s9

1.8

84

7.9

81

.22

.56

81

.42

11

0.6

28

1.0

10

64

.0

Ke

w9

8.8

45

6.4

61

.48

4.7

72

.44

13

0.8

41

5.4

16

06

.0

Me

an

97

.94�

0.7

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0.6

21

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0.0

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.64�

0.0

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9.5

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0.7

81

16

.75�

0.6

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7.6�

4.1

71

27

9�

20

.1

CV

(%)

2.0

53

.15

6.5

95

.67

2.5

1.3

33

.29

4.0

5

CD

(5%

)2

.82

.19

0.1

30

.28

2.7

2.1

41

4.8

37

1.3

3

Pin

ea

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Fru

it

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(cm

)

Fru

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(cm

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S

(Bri

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r(%

)A

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(%)

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(%)

Ca

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ry(%

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PQ

M-1

14

.68

17

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17

.51

2.9

60

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40

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.0

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ee

n1

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81

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.21

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3.6

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8.7

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4.0

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07

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.12

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.20

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0.2

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2

Ke

w1

5.5

81

4.3

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1.7

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4.8�

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41

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0.1

51

2.8

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0.1

30

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00

.21�

0.0

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0.3

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0.4

5

CV

(%)

4.0

62

.55

.86

2.3

2.6

1.7

71

0.7

23

.83

CD

(5%

)0

.86

0.5

18

.16

0.5

50

.46

0.0

20

.03

1.6

J. Prakash et al. / Scientia Horticulturae 120 (2009) 288–291290

shape, size and colour (Fig. 1), eye shape, fruit weight, juicecontent, TSS and acidity. Leaf margin of PQM-1 was spiny likeQueen and leaf colour was reddish and no way similar to Queen,Mauritius and Kew. From the Table 2 it was revealed that theaverage ‘D leaf’ height of PQM-1 was significantly higher ascompared to other three cultivars indicating its vigorous growthwhich could be considered as a desirable breeding trait. Thenumber of leaves at red heart stage (flowering stage) was littlehigher as compared to Queen and Mauritius while slightly lessthan Kew, which further proved the vigorousness of the PQM-1over Queen. The production of suckers was as usual in PQM-1 likeother cultivars and number of slips was less than Queen and Kew.The flowering time (April-May) and subsequently the fruitmaturity (July-August) was late in PQM-1 as compared to queen(February and May-June, respectively) and other varieties. The lateflowering might be due to vigorous nature of the plant. Latematurity and ripening of the fruits could offer an additional optionto fruit growers, consumers and canning industry to get fresh fruitover a longer period. The flowering percentage (Table 2) was alsohigher in comparison to other cultivars, this was highly desirableas pineapple plant had tendency not to produce uniform flowering(Perez et al., 1998).

The PQM-1 was also found distinct from Queen and othercultivars in respect to fruit character as presented in Table 2. Fruitcolour was deep orange in PQM-1, more attractive than Queen(light orange), Mauritius (goldern) and Kew (orange yellow). Theattractive colour is very much linked with the consumers’ choice infresh market for direct consumption. The cylindrical shape of thePQM-1 was also found suitable for table purpose and in canningindustry. Yellowish golden flesh colour of the fruits of PQM-1might be preferred over ‘queen’ by the canning industry to prepareslices. From the Table 2 it was also revealed that the higher crownweight indicated higher fruit weight. PQM-1 had greater valuethan ‘queen’ in respect of both the traits, but lower than the Kew.The average fruit length of PQM-1 was minimum (14.68 cm) butfruit diameter was maximum (17.66 cm) among the clones. Thehigher fruit diameter probably leads to higher fruit weight in PQM-1 than Queen (Table 2). Higher diameter of fruit is a majorconsideration while developing a new clone in pineapple. Thenumber of eyes is also very important quality parameter inpineapple. Minimum average number of eyes was observed inPQM-1 (84.6) among the four clones. Lower number of eyes leadsto boldness of eyes which was described earlier as desirable qualityof this fruit (Ray, 1999).

TSS is the most important quality trait of pineapple. The datapresented in Table 2 have revealed that total soluble solids of PQM-1 were comparatively higher (17.5) than Mauritius and Kew butless than Queen. However, PQM-1 might have been preferred thanQueen for its late maturity and its usefulness in both canning andfresh consumption. Almost, similar trend was noticed in averagesugar percentage where PQM-1 (12.96) exceeded Kew but couldnot supersede Queen. The average acidity was higher than Kew andslightly lesser than Queen and Mauritius (Table 2). In spite ofeverything, PQM-1 has great advantage over all existing cultivarsin this region due to its late maturity, its fruit quality which is farbetter than Kew and its usefulness in both canning and freshconsumption unlike Queen. The canning recovery was foundhigher compared to Queen and Mauritius and little lesser thanKew. PQM-1 was also free from major diseases (e.g., Heart rot) ofthis region; whereas, a fair percent of the fruit plant of Kew andMauritius was infected by heart rot (Table 2). The tolerance todiseases and higher canning recovery percentage indicatedsuperiority of PQM-1 over ‘queen’ and other cultivars grown inthis region.

Among the primers, except one (Table 1) all gave reaction to thepineapple DNA. Four ISSR and one RAPD primers produced stable

Fig. 2. ISSR profile using (a) IS-61 and (b) IS-6 primer of bulk DNA of pineapple

clones, viz., Mr1-3: Mauritius, Mq1-3: PQM-1, K1-3: Kew, Q1-3: Queen and M

denotes 50 bp DNA ladder. Arrows indicate the major banding difference of PQM-1

and Queen.

J. Prakash et al. / Scientia Horticulturae 120 (2009) 288–291 291

polymorphism among the clones, without any intra-clonaldifferences. Three ISSR primers (Table 1) were found capable todifferentiate particularly cv. Queen and PQM-1 (Fig. 2). The arrowsin the Fig. 2 indicated the stable differences in banding patternamong them. No RAPD primers revealed polymorphism amongthese two clones. There was no significant difference among thebulks of PQM-1 in DNA fingerprinting profiles. Therefore, the clonalpopulation of PQM-1 was found to be uniform and stable in itsgenetic makeup as revealed through markers.

ISSR was found to be of great use while identifying geneticdifferences among closely related individuals in pineapple as wellas in various fruit crops (Fang and Roose, 1997; Monte-Corvo et al.,2001; Carlier et al., 2004; Tapia Campos et al., 2005). In the presentstudy also, the applicability of ISSR markers in differentiation ofclones of pineapple could be accepted. ISSR analysis complemen-ted the morphological and biochemical results by establishingstable genetic differences of PQM-1 with cv. Queen.

4. Conclusion

From the Queen plant population, a plant was found havingsimilar plant morphology but with distinct fruit. Later plantgenerated from this fruit was found morphologically different fromQueen plant. The clonal population (PQM-1) derived from thatplant was found completely different from cv. Queen in respect ofplant architecture, fruit colour, shape, quality and maturity period

in the present observation. This phenotypic deviation might beeither due to somatic mutation which was reported to be veryfrequent in pineapple and useful for developing new clone. In thisstudy, highly reproducible banding pattern in ISSR analysis wasfound to be very effective in discriminating clones. Three ISSRprimers indicated the genetic differences of newly developedclone, PQM-1 with cv. Queen from which it was derived. Therefore,this clone might be the outcome of accumulation of many naturalmutations in cv. Queen. Those ISSR primers also could be used forassessing diversity among and within clones. The sequence dataof unique bands generated through ISSR-PCR reaction could beused in generating STMS and SNP primers for detecting minordifferences among and within clones of pineapple. Late maturity,superior fruit quality, suitability of both for direct consumptionand canning would definitely make this clone an alternative choicefor the pineapple growers of northeastern region of India.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.scienta.2008.11.016.

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