INTRODUCTION

Most of the flowering plants are bisexualhaving flowers with both male and female re-productive organs and only less than 4% plantspecies are dioecious in nature (GUTTMAN andCHARLESWORTH 1998). Some dioecious plantspecies have distinct X and Y chromosomesand in some species no chromosomal distinc-tion has been recorded in relation to sex(GRANT et al. 1994; ROY et al. 1966; WESTER-GAARD 1958). Coccina indica Wight and Arn.and Trichosanthes dioica Roxb. are the twodioecious vegetatively propagated species ofcucurbitaceae which shows widespread distri-bution in the tropical and sub-tropical regionsof the world (CHAKRAVARTY 1959).Earlierworkers have found that a series of polyploidy(= 24, 36, 48) exists in C. indica with distinctheteromorphic pair of sex chromosomes(CHAKRABORTY 1948; D’CRUZ et al. 1972; ROY

and ROY 1971; SEN and DATTA 1977) in whichY chromosome plays a decisive role in the dif-ferentiation of the male sex (KUMAR andVISHVESWARAIAH 1952). Dioecism also existsin T. dioica and there is a record of polyploidseries from diploid to tetraploid with a basicnumber of n = 11 chromosomes (BHADURI andBOSE 1947). Inter-specific and intra-specificvariation and constancy in the amount of nu-clear DNA content of flowering plants havebeen reported by several authors (Bennett,1985; BENNETT and SMITH 1976; MUKHERJEEand SHARMA 1986; PRICE 1976; RIANA andREES 1983; SINHA et al. 1997). Data on nuclearDNA content was meagre in the sex forms ofC. indica and T. dioica (Chattopadhyay &Sharma, 1991) and so far, no comparativeanalysis of the karyotype in relation to DNAcontent was done in these plants. The overallcompaction of DNA molecules into the so-matic chromosome complements described asaverage packing ratio could be estimated (SIN-HA et al. 1996) to find out the measure of con-densation at mitotic metaphase of the sex

CARYOLOGIA Vol. 57, no. 1: 117-120, 2004

Average packing ratio as a parameter for analyzingthe karyotypes of dioecious cucurbitsANUPAM GUHA, RABINDRA KUMAR SINHA and SANGRAM SINHA*Department of Life Sciences, Tripura University, Agartala 799130, India.

Abstract - Chromosome studies and in situ estimation of 4C nuclear DNA con-tent were carried out on dioecious Coccinia indica and Trichosanthes dioica tounderstand the differential condensation of mitotic chromosome in relation tosex. The somatic chromosome number for each sex form of C. indica and T. dioicawas found to be 2n = 24 and 22 respectively. The karyotypes of the sex forms ofboth species show high homogeneity, though a distinct hetermorphic pair of sexchromosomes is found in male plants of C. indica. Interrelationship between the4C nuclear content and the chromosome length has been explained in terms ofaverage packing ratio.The data suggests that average packing ratio is a determi-nant of distinction between two sexes and therefore could be used as a parame-ter for analyzing the karyotypes of dioecious plants.

Key words: Average Paking Ratio, Karyotype, Dioecius, Coccinia indica, Tri-chosanthes dioica.

* Corresponding author: fax +91 0381-2374801; e-mail:sinhas_s@rediffmail.com.in

forms of each taxon. The present work dealswith chromosome studies and 4C nuclearDNA content of the sex forms of C. indica andT. dioica. The differential rate of average pack-ing ratio (APR) in relation to sex has been an-alyzed as well.

MATERIALS AND METHODS

Tuberous roots of the sex forms of C. indicaand T. dioica, collected from west Tripura wereplanted in the experimental garden of the Depart-ment of Life Sciences, Tripura University. Toanalyse the somatic chromosomes root tips werepre-treated in saturated solution of paradichlorobenzene for 4 hr at 10o-15oC followed by overnightfixation in 1:3 acetic acid-ethanol mixture. Theroot tips were stained overnight in 2% aceto-or-cein after hydrolysis in 5N HCl at 10oC for 15 min.Somatic chromosome number and total chromo-some length were determined from 5 well spreadmetaphase plates. For estimation of 4C nuclearDNA content young root tips were fixed in aceticacid-ethanol mixture 1:3, hydrolysed in 1N HClfor 15 min at 60oC, washed with distilled waterand stained with Feulgen solution for 1-2 hr fol-lowed by squashing in 45% acetic acid. Cytopho-tometric analysis was done using a Leitz WetzlarAristophot with microspectrometer and the singlewave length method was followed (SHARMA andSHARMA 1980). Estimations were done on10metaphase plates per root tip and 5 root tips foreach sample were used. The relative arbitrary unitsof absorbance were converted to picograms (pgs)using 4C nuclear value for Allium cepa L. as a stan-dard (VAN’T HOF 1965). Average packing ratio ofDNA was calculated by the following formula(SINHA et al. 1996).

RESULTS

In the present investigation the somatic chro-mosome number for each sex form of C. indicaand T. dioica was found to be 2n = 24 and 22 re-spectively (Figs.1a,b; 2a,b). 4C nuclear DNAcontent of the sex forms of C. indica showedmarked variation and the amount was 10.17 +0.24 pgs and 8.37 + 0.14 pgs in male and femaleplant respectively. However, no significant vari-ation was recorded in T. dioica as the amount of4C nuclear DNA of male and female plant is11.05 + 0.14pgs and 10.87 + 0.11pgs respec-tively. The average packing ratio of the sex formsof two dioecious species was calculated and itwas noted that the ratio was comparatively highin the male plant of each species (Table 1).

DISCUSSION

Somatic chromosome number 2n = 24 wasfound to be constant in the male and femaleplants of C. indica as was reported by earlier re-searchers (BHADURI and BOSE 1947; CHA-KRABORTY 1948; KUMAR and VISHVESWARAIAH1952; ROY and ROY 1971). In both sexes therewere 11 pairs of metacentric and one pair ofsub-metacentric chromosome having secondaryconstriction. The present cytological investiga-tion also revealed the presence of a distinct het-eromorphic pair of sex chromosome in maleplants. Diploid males are, therefore, heteroga-metic with 22 + XY and the females are ho-mogametic with 22 + XX.

The male determining Y chromosome isconspicuously large, nearly metacentric anddoes not bear secondary constriction as re-ported by SINGH (1988). Detailed karyologicalstudies suggest that apart from the sex chro-

4C DNA content/cell (pgs) x 0.965 x 109 x 3.4 (µm)APR =

2 x Total chromosome length at metaphase/cell x 104 (µm)

118 GUHA, SINHA and SINHA

Table 1 – Chromosome complements, 4C DNA content and average packing ratio (APR) in dioecious C. indica and T. dioica.

Somatic Karyotype Total Chromosome Amount of APRTaxon Sex Chromosome TF% formule Chromosome arm symmetry 4C DNA (pgs) (Mean ± SE)

number lenght (Mean ± SE) index (SI) (Mean ± SE)

Male 24 44.81 A2B20CX1DY1 40.96±0.394 82.15 10.17±0.245 40786±411C. indica Female 24 45.14 A2B20CX2 38.46a±0.211 71.68 8.37b±0.140 35703c±195

Male 22 44.64 A2B20 30.13±0.295 79.62 11.05±0.145 60388±586T. dioica Female 22 43.60 A2B20 31.34d±0.225 76.77 10.87e±0.113 56902f±403

Significant at ap, bp, cp<0.05 (t=5.60; t=5.68; t=11.15 respectively), dp, fp<0.005 (t=3.25, 4.897 respectively) ep>0.005 (t=1.064)

mosomes, the karyotypes of male and femaleplants exhibit similarity in the types of chro-mosome present, TF% and the number ofchromosomes with secondary constriction. Thesomatic chromosome number of the sex formsof T. dioica was found to be 2n = 22 with onepair of chromosomes bearing secondary con-striction. Diploid males are homogameticwhich negates the possibility of XY mechanismin relation to sex. There were ten pairs of meta-centric and one pair of sub-metacentric chro-mosomes in each of them. The distinction be-tween sexes of T. dioica cannot be resolved atchromosome level since the karyotype of maleand female plants shows gross similarity in thetypes of chromosome with secondary constric-tion, chromosome arm symmetry index, TF %and total chromosome length.

The 4C nuclear DNA content of male and fe-male plants of C. indica shows a marked varia-tion and the increase in amount of DNA is as-sociated with the corresponding increase inchromosome length. On the other hand, the 4C

DNA content in relation to sex does not differmuch in T. dioica and the apparent increase inchromosome length is not associated with thecorresponding increase in amount of DNA.Therefore, in one dioecious taxon C. indicawhere XY mechanism is involved with sex ex-pression there is a positive linear relationship be-tween the amount of DNA and the total chro-mosome length. But, in T. dioica where the ex-pression is thought to be under genic control nosuch positive linear relationship could be estab-lished between the amount of the DNA and thetotal chromosome length. This paradoxical sit-uation can be explained by the fact that in C. in-dica the maleness determining Y chromosome isconspicuously large and is nearly 3-4 times larg-er than majority of the chromosomes present insomatic chromosome complement of the femaleplant. Though, in most of the works (CHAT-TOPADHYAY and SHARMA 1990; RAMCHANDRANand NARAYAN 1985) attempts have been madeto find out the co-relation between the 4C DNAcontent and the total chromosome volume, inthe present analysis the dimension of chromo-

AVERAGE PACKING RATIO FOR ANALYZING THE KARYOTYPE 119

Figs. 1 – Metaphase plates of C. indica showing chromosomes 2n = 24; a. Male (Arrowindicates Y chromosome), b. Female.Fig. 2 – Metaphase plates of T. dioica showing chromosomes 2n = 22; a. Male, b. Female.

some is taken as unit “length” instead of chro-mosome volume because it signifies the rate ofoverall compaction of DNA molecules (Lewin,1994). Utilising this principle it has been notedthat the APR of mitotic chromosome of maleand female plants significantly (P< 0.005) differsand the ratio is comparatively high in male sexforms. This suggests that a differential conden-sation is involved with the organisation of themitotic chromosome in relation to sex. Thus therelationship between the amount of DNA andthe total chromosome length in presence or ab-sence of any direct association can be explainedin terms of average packing ratio. The data alsosuggests that in dioecious species like T. dioica,even in the absence of chromosome mediated(XY) sex expression, the karyotypes of male andfemale plants can easily be distinguished usingaverage packing ratio as a parameter.

Acknowledgements – The authors are gratefulto Prof A. K. Sharma, CAS in Botany, Calcutta Uni-versity, for providing laboratory facilities.

REFERENCES

BENNETT M.D., 1985 – Intra-specific variation inDNA amount and the nucleotypic dimension inplant genetics. In: M. Freeling (ed.), “Plant Ge-netics”, pp. 325-341. Acad. Press.

BENNETT M.D. and SMITH J.B., 1976 – NuclearDNA amount in angiosperms. Phil. Trans. R. Soc.London B, 274: 227-274.

BHADURI P.N. and BOSE P.C., 1947 – Cytogeneticalinvestigation in some common cucurbits with spe-cial reference to fragmentation of chromosomes asphysical basis of speciation. J. Genet., 48: 237-256.

CHAKRABORTY A.K., 1948 – Cytology of Coccinia in-dica W and A. with reference to the behaviour ofits sex-chromosomes. Proc. Indian. Acad. Sci.,B27: 74-86.

CHAKRABARTY H.L., 1959 – Monograph on Indiancucurbitaceae. Rec. Bot. Survey. India. 17: 1-234.

CHATTOPADHYAY D. and SHARMA A.K., 1990 –Chromosome studies and microspectro-photo-metric estimation of nuclear DNA in differentstrains of Coriandrum sativum L. Cytobios, 64:43-51.

–, 1991 – Sex determination in dioecious species ofplants. Feedes Repertorium (Berlin), 102: 29-55.

D’CRUZ R., VYAHALKAR G.R. and UGALE S.D., 1972– Cytogenetic studies in tetraploid Coccinia indi-ca W. and A. Caryologia, 25: 505-512.

GRANT S., HOUBER A., VYSKOT B., SIROKY J., PANW.H. and MACAS J., 1994 – Genetics of sex de-termination in flowering plants. Devel. Genet.,15: 214-230.

GUTTMAN D.S. and CHARLESWORTH D., 1998 – AnX- linked gene with a degenerate Y - linked homo-logue in a dioecious plant. Nature, 396: 263-266.

KUMAR L.S.S. and VISHVESWARAIAH S., 1952 – Sexmechanism in Coccinia indica Wight and Arn.Nature, 170: 330-331.

LEWIN B., 1994 – Genes. Oxford Univ. Press. Inc.New York.

MUKHERJEE S. and SHARMA A.K., 1986 – Estimationof in situ DNA content in organs of differentstrains of Pisum sativa L. Nucleus, 28: 236-239.

PRICE H.J., 1976 – Evolution of DNA content inhigher plants. Bot. Rev., 43: 27-52.

RAMCHANDRAN C. and NARAYAN R.K.J., 1985 –Chromosomal DNA variation in Cucumis. The-or. appl. Genet., 69: 497-502.

RIANA S.N. and REES H., 1983 – DNA variation be-tween and within chromosome complements ofVicia sp. Heridity, 5 : 335-346.

ROY R.P. and ROY P.M., 1971 – Mechanism of sexdetermination in Coccinia indica. J. Indian Bot.Soc., 50 A: 391-400.

ROY R.P., THAKUR V. and TRIVEDI R.N., 1966 – Cy-togenetical studies of Momordica L. J. Cytol.Genet., 1 : 33-40.

SEN R. and DATTA B.K., 1977 – Chromosomal basisof sex expression in some dioecious cucurbits. Nu-cleus, 22: 241.

SHARMA A.K. and SHARMA A., 1980 – Chromosometechniques: Theory and practice. Third Edition.,Butterworths Ltd. London.

SINGH A.K., 1988 – Cytogenetics and evolution in thecucurbitaceae. In: Bates, Robinson and Jeffery(Eds.), “Systematics and evolution of cucur-bitaceae”, pp. 10-28. Academic press, New York.

SINHA S., DEBNATH B. and SINHA R.K., 1996 –Karyological studies in dioecious Momordicacochinchinenis (Lour.) with reference to averagepacking ratio. Cytologia, 61: 297-300.

SINHA S., DEBNATH B. and SINHA R.K., 1997 – Differ-ential condensation of chromosome complements ofdioecious Momordica dioica Roxb. in relation toDNA content. Indian J. Exp. Biol., 35: 1246-1248.

VAN’T HOF J., 1965 – Relationship between mitoticcycle duration, S period duration and average rateof DNA synthesis in root meristem cells of sever-al plants. Exp. cell Res., 39: 48-58.

WESTERGAARD M., 1958 – The mechanism of sex de-termination in dioecious flowering plants. Adv.Genet., 9: 217-281.

Received November 27, 2003; accepted December 12, 2003

120 GUHA, SINHA and SINHA