Karyotype Diversity in Three Cultivars of

Momordica charantia L.

Md. Yahia Zaman and Sheikh Shamimul Alam*

Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Received October 22, 2009; accepted December 20, 2009

Summary Two F1 hybrids namely TIA and PARROT and an open pollinated cultivar viz. TAJ-88of Momordica charantia L. (bitter gourd) were cytogenetically investigated. The 3 cultivars werefound to possess 2n�22 metacentric chromosomes. However, a plant specimen of cultivar TIA had2n�44 chromosomes. The extreme symmetric karyotypes revealed these cultivars as primitive.Three CMA positive bands were found in TIA and PARROT whereas 4 were found in TAJ-88. Onthe other hand, a plant specimen of TIA had 6 CMA positive bands. The distribution, location andintensity of CMA positive bands were different among the 3 cultivars. A number of DAPI positivebands were found in TIA and PARROT. It was possible to identify some marker chromosomes withCMA and DAPI staining specific to each cultivar. Fluorescent banding indicated the occurrence ofminute deletion and paracentric inversion in the genomes of these cultivars. The overall karyotypicfeatures revealed that a plant specimen of cultivar TIA was actually an auto-tetraploid. With the helpof fluorescent banding, the karyotype diversity among these 3 cultivars was determined.

Key words Fluorescent banding, Karyotype, Momordica, Cucurbitaceae.

The genus Momordica L. belongs to family Cucurbitaceae, which consists of more than 100species (Jackson 1946). Most of the species of this genus are economically important. Momordicacharantia L. is one of the most important species in this genus. This species is commonly known asBitter gourd, Bitter melon, Karela etc. This species is widely grown in tropical and sub-tropicalcountries of the world such as South-East Asia, China, Japan, etc. In Bangladesh, 3 species ofMomordica viz. M. charantia L., M. cochinchinensis (Lour.) Spreng. and M. dioica Roxb. ex Willdare available (Rahman 2008).

Bitter gourd grows into different shapes such as long spindle, short spindle, long rugby ball,oblong and olive. The bitter gourd fruits are usually used as vegetables and sometimes as roasted.The most important compound which is present in bitter gourd is known as Charantin and lowersblood sugar level. Due to its high medicinal and nutritive value this plant has become popular and apart of the everyday diet in Bangladesh. However, it is susceptible to many diseases and insectpests. For example, water melon mosaic which reduces the yield remarkably.

Different public and private sectors have been trying to overcome the disease susceptibility andto develop improved cultivar. Lal Teer Seed Company is one such private company in Bangladesh.They have collected different cultivars/germplasms from all over the country. They have been tryingto develop improved variety by classical breeding and selection. The different cultivars and releasedvariety of bitter gourd (Momordica charantia L.) are characterized solely on the basis of theirmorphological features. This kind of identification is not reliable and is confusing since phenotypicplasticity may change the morphology in different environments. This may create problem for thefarmers. Moreover, sometimes they are sprayed with hormones (planofix-female flower inducinghormone) to induce early flowering in order to save time (Dr. Shyamali Saha, Senior Plant Breeder,Lal Teer Seed Company Ltd.–personal communication). It is well established that hormones create

* Corresponding author, e-mail: ssalam81@yahoo.com

© 2009 The Japan Mendel Society Cytologia 74(4): 473–478, 2009

chromosomal aberration. Thus there must be authentic identification of each cultivars/germplasmsto screen before releasing to the farmers.

For authentic identification one must choose reliable and stable parameters. Karyotype analysiswould be an important parameter in this regard. Because it is stable and specific for each specimen.Varieties of a species have the same chromosome numbers. They may have even the samekaryotypes. In this case, classical karyotype analysis is not enough to characterize each variety.When varieties possess all small metacentric chromosomes, consideration of chromosomal length,arm ratio, position of centromere etc. are not sufficient to differentiate individual chromosomes.Moreover, minute changes in the heterochromatic regions, AT- and GC-rich repeats due to smalldeletion and paracentric inversion would not alter the gross karyotypes (Sumner 1990). In such asituation, different procedures for karyotype analysis should be undertaken. Fluorescentchromosome banding is a relatively modern and effective technique for critical karyotype analysis.Schweizer (1976) was the pioneer for this technique. There are 2 common fluorochromes viz. i)Chromomycin A3 (CMA) and ii) 4, 6� Diamidino 2-Phenyl Indole (DAPI). CMA binds with GC-rich repetitive sequences of the genome and gives characteristics yellow color bands. On the otherhand, DAPI binds with AT-rich repetitive sequences fluorescing characteristic blue band.Fluorescent chromosome banding was able to solve the taxonomic problems in different cases(Alam and Kondo 1995, Kondo and Hizume 1982). Even different categories such as form, variety,and sub-species could also be characterized with fluorescent banding technique (Huq et al. 2007,Khandaker et al. 2007, Sultana et al. 2006, Akter and Alam 2005, Alam and Zerin 1998).

In the present study, two hybrids namely, TIA and PARROT and an open pollinated cultivarviz. TAJ-88 were investigated. The aim was to:

(i) characterize the karyotype of each cultivar after staining with orcein, CMA and DAPI;(ii) screen out chromosomal aberrated plants; and(iii) develop a karyotype patent for each cultivar.

Materials and methods

Three cultivars of Momordica charantia L. were used in this study. The–Hybrids (F1):(i) Momordica charantia, TIA;(ii) M. charantia, PARROT;and the Open pollinated cultivar:(iii) M. charantia, TAJ-88.Healthy roots were collected and pretreated with cold water overnight at 4°C followed by

15 min fixation in 45% acetic acid at 4°C. These were then hydrolyzed in a mixture of 1 N HCl and45% acetic acid (2 : 1) at 60°C for 7 s. The root tips were stained and squashed in 2% aceto-orcein.For fluorescent banding, Alam and Kondo’s (1995) method was followed with slight modification.After hydrolyzing and dissecting, the materials were squashed with 45% acetic acid. The coverglasses were removed quickly on dry ice and allowed to air dry for at least 48 h before study. Theair-dried slides were first pre-incubated in McIlvaine’s buffer (pH 7.0) for 30 min followed byDistamycin A (0.1 mg/ml) treatment for 10 min in a humid chamber. The slides were rinsed mildlyin McIlvaine’s buffer supplemented with MgSO4 (5 mM) for 15 min. One drop of CMA (0.1 mg/ml)was added to the materials for 15 min in a humid chamber and rinsed with McIlvaine’s buffer withMg2� for 10 min. Slides were mounted in 50% glycerol and kept at 4°C for overnight beforeobservation. These were observed under Nikon (UFX-IIA) fluorescent microscope with Blue Violet(BV) filter cassette. For DAPI-staining, after 48 h of air drying, the slide was first pre-ineubated inMcIlvaine’s buffer (pH 7.0) for 20 min in a humid chamber. The slides were treated in actinomycinD (0.25 mg/ml) for 10 min. The slides were immersed in DAPI solution (0.01 mg/ml) for 20 minand mounted with 50% glycerol. These were observed under Nikon (UFX-IIA) fluorescent

474 Cytologia 74(4)M. Y. Zaman and S. S. Alam

2010 475Karyotype diversity in Momordica charantia L.

Figs. 1–16. Mitotic chromosomes of 3 cultivars of Momordica charantia L. 1. Orcein-stained mitoticmetaphase of TIA, 2. Orcein-stained mitotic metaphase of a plant specimen of TIA, 3. Orcein-stained mitotic metaphase of TAJ-88, 4. Orcein-stained mitotic metaphase of PARROT, 5.CMA-stained mitotic metaphase of TIA, 6. CMA-stained mitotic metaphase of a plantspecimen of TIA, 7. CMA-stained mitotic metaphase of TAJ-88, 8. CMA-stained mitoticmetaphase of PARROT, 9. CMA-stained karyotype of TIA, 10. CMA-stained karyotype of aplant specimen of TIA, 11. CMA-stained karyotype of TAJ-88, 12. CMA-stained karyotype ofPARROT, 13. DAPI-stained mitotic metaphase of TIA, 14. DAPI-stained mitotic metaphase ofa plant specimen of TIA, 15. DAPI-stained mitotic metaphase of TAJ-88 and 16. DAPI-stainedmitotic metaphase of PARROT. Bar�10 mm.

microscope with Ultra Violet (UV) filter cassette.

Results and discussion

Orcein-stained karyotypesExcept a plant specimen of TIA, the 3 cultivars possessed 2n�22 metacentric chromosomes

(Figs. 1, 3, 4). However, 2n�44 chromosomes were found in a plant specimen of variety TIA (Fig.2). The chromosomes of this specimen were too small to determine the position of the centromere.Since this is a plant specimen of variety TIA and TIA has all metacentric chromosomes, it may beconsidered that this specimen had all metacentric chromosomes. The range of chromosomal lengthand total length of 2n chromosome complements were more or less similar in these 3 cultivars(Table 1). The orcein stained karyotypic features revealed that these cultivars possessed symmetrickaryotypes. Stebbins (1971) stated that symmetric karyotypes were primitive in nature. Therefore,these cultivars conserved primitive karyotypes even after long domestication and cultivation.

CMA-bandingThe number, location, distribution and intensities of CMA-bands varied in different cultivars

(Figs. 5–12). CMA-bands correspond to GC-rich (Guanine-Cytosine) regions of chromosomes(Schweizer 1976). Most of the CMA-bands were present at the terminal regions of respectivechromosomes (Figs. 9–12). It indicated a tendency of accumulating GC-rich repetitive sequences atthe chromosomal ends. The percentage of GC-rich regions was also different (Table 1). The presentstudy therefore, indicated that each cultivar has its own characteristic CMA-banded karyotype.

DAPI-bandingA number of different DAPI bands was observed in the cultivar TIA and PARROT (Figs. 13,

16). Few chromosomes of both the cultivars were fluoresced entirely (Figs. 13, 16). However, noDAPI band was observed in a plant specimen of cultivars TIA and TAJ-88 (Figs. 14, 15). DAPIbands correlated with AT-rich repetitive sequences of chromosomes (Schweizer 1976). The presentfindings revealed that the former two cultivars were rich in AT-rich repetitive sequences. In respectof DAPI banding, each cultivar could also be distinguished.

Marker chromosomesSince few chromosomes of each cultivar possessed characteristic CMA-positive bands (Figs.

9–12), these chromosomes could easily be considered as marker chromosomes of the respectivecultivar. In TIA and PARROT, 3 and 2 entirely DAPI fluoresced chromosomes were observed,

476 Cytologia 74(4)M. Y. Zaman and S. S. Alam

Table 1. Comparative orcein, CMA- and DAPI-stained karyotype analysis in 3 cultivars of Momordicacharantia L.

CMA-positive DAPI-positive

Range of Total

No. of No. ofBanded Banded

Name of 2n

chromosomallength of 2n

CMA- DAPI-region region

chromo- chromosomecultivars

some no.length

comple-positive positive

Total Total(mm)

mentsbands bands

Length % Length %(mm) (mm)

TIA (Common) 22 1.00–1.58 27.85 3 13 1.24 4.45 15.5 55.56TIA (a plant specimen) 44 0.73–1.27 41.77 6 — 2.15 5.14 — —TAJ-88 22 0.92–1.67 25.53 4 — 1.67 6.5 — —PARROT 22 0.98–1.58 28.73 3 10 0.87 3.02 13.33 46.40

respectively (Figs. 13, 16). These chromosomes may be considered as marker chromosomes ofthese cultivars.

SatelliteA pair of satellites was observed in only PARROT when stained with orcein (Fig. 4, arrow).

However, no satellite was found after CMA and DAPI-staining in this cultivar. Several well spreadmitotic metaphase cells were observed after staining with CMA and DAPI. No satellite could bedetected. Therefore, absence of satellite in CMA and DAPI was not a technical error. Alam andKondo (1995) reported stain specific satellite and chromosomes in Drosera spp. The satellites ofPARROT may have some stain-specificity, and therefore could not be detected with CMA andDAPI.

A plant specimen of TIAAmong the germinated plants from seeds of TIA, a plant showed completely different

characteristic features. It was not alike other TIA plants for a number of reasons:(i) this plant was found to possess 2n�44 metacentric chromosomes whereas other plants of

this cultivar had 2n�22 metacentric chromosomes (Figs. 1–4, Table 1);(ii) the total length of 2n chromosome complements was almost double to that of other plants

in this cultivar (Table 1);(iii) this plant had 6 CMA-positive bands, whereas 3 CMA-positive bands were observed in

case of other plant of this cultivar (Figs. 1, 2);(iv) total length and percentage of CMA-banded regions were more than the other plants of

this cultivar (Table 1).The above results clearly indicate the polyploidy nature of this plant. Since 2n�44

chromosomes were present in this plant, it was a tetraploid. Now the question is whether it was anauto-tetraploid. The number and position of CMA bands clearly revealed the exact duplication ofgenome. Therefore, this plant was an auto-tetraploid.

Structural aberrationThe comparative CMA-banded karyotypes revealed the occurrence of structural chromosomal

aberration among these 3 cultivars. Both the members in chromosome pair I of TAJ-88 hadterminal CMA-positive bands. Only 1 member of this pair in cultivar TIA and PARROT showedterminal CMA-bands (Figs. 9–12). Lacking of a CMA-band in a chromosome of pair I wasprobably due to deletion of banded regions.

Two terminal CMA-positive bands were found in both members of pair V in a plant specimenof TIA (Fig. 10). In PARROT, the same pair showed centromeric bands (Fig. 12). These findingsindicated the occurrence of homozygous inversion either from centromeric region to terminal orvice versa.

In a plant specimen of TIA, a pair of terminal CMA-bands was found on pair VII. On the otherhand, 1 member of the same pair had a terminal and another centromeric CMA band in TAJ-88(Figs. 10–11). The latter banding suggested a heterozygous inversion. Structural aberration played arole in the diversification of karyotype. However, minute deletion and paracentric inversion did notcreate a gross change in the karyotype.

Diversification in cultivarsAlthough the 3 cultivars possessed almost similar orcein stained karyotypes (except a plant

specimen of TIA), they differed sharply in respect of CMA and DAPI banding patterns. Thesecultivars had undergone minute changes in CMA and DAPI banding followed by deletion andinversion. Moreover, polyploidization was another cause for karyotype diversification.

2010 477Karyotype diversity in Momordica charantia L.

Acknowledgements

We are grateful to Dr. M. Akhtaruzzaman, Professor of Botany, University of Dhaka,Bangladesh for constructive criticism and going through the manuscript.

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