IN SITU 4C DNA CONTENT STUDY OF TWENTY-NINE HYBRID VARIETIES OF SOME SELECTED TAXA OF TRIBE PHASEOLAE (FABACEAE)

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1 Legume Res., 30 (4) : , 2007 IN SITU 4C DNA CONTENT STUDY OF TWENTY-NINE HYBRID VARIETIES OF SOME SELECTED TAXA OF TRIBE PHASEOLAE (FABACEAE) B. Bandyopadhyay and S. C. Santra Department of Environmental Science, University of Kalyani, Nadia, West Bengal , India ABSTRACT A quantitative study of in situ estimation of 4C nuclear DNA amount and chromosome counts were carried out on different genera, species and varieties of economically important legumes ( twenty nine hybrid varieties of Glycine max, Erythrina variegata, Lablab purpureus, Vigna unguiculata, Canavalia ensiformis, Canavalia gladiata and Cajanus cajan) of the tribe Phaseolae (family Fabaceae). The 4C nuclear DNA amount was estimated through in situ Feulgen microspectrophotometry expressed in arbitrary units of relative absorbance. Somatic chromosome number is studied which is constant at intra-specific level, but not at interspecific level. There were significant variations in 4C DNA amount at the inter-specific but not at the intra-specific level. To considering nuclear DNA amount in relation to total chromosome length, the diversity in DNA amount was also associated with structural differences of chromosomes among the different species. The variation in 4C DNA content was non significant at the varietal level, though in most of the cases the variations were significant at species and generic level. INTRODUCTION The legumes have economic importance as food, fodder, medicinal and ornamental plants. Aside from the review of morphology many recent contributions from the chromosome counts have led to a much fuller understanding of the tribe (Phillips and Strickland 1966; Rees and Hazarika, 1967; Lackey, 1977; Freckman and Wemple, 1963; Cubas and Pardo, 1997, 1998; Naranjo et al. 1998)..During the last few years, extensive work has been done to determine the variation in nuclear DNA amount in intergeneric, inter-specific and even at intra-specific levels. (Rees and Jones,1967; Price and Bachman,1975; Narayan and Rees,1976; Narayan, 1982, 1985; Watson 1987; Ignacimuthu and Babu, 1988; Bandyopadhyay, 1992; Bennett and Leitch, 1997; Laneray Burn, 1997, Jones et al 1998, Naranjo et al., 1998; Palomino and Sousa, 2000). The evolution and divergence of species of higher plants are often accompanied by large variations in nuclear DNA amount (Parida et al., 1990 and Cavallini et al., 1993). The estimation of DNA content has proved to be a useful tool in the study of evolution and affinity. Surveys have shown that even closely related species within the same genus vary from three to six fold in amount of nuclear DNA (Sharma and Mukhopadhyay 1984; Banerjee and Sharma 1985; Altamura 1991; Le-Coq et al 1992; Naranjo1998).In the family Fabaceae, extensive studies have been carried out on in situ DNA estimation of different genera at intergeneric and interspecific level (Mukhejee and Sharma, 1990; Galasso et. al 1997; Lanerayburn et. al. 1997; Naranjo et al. 1998; Palomino and Sousa 2000). The correlation between Feulgen absorbance and amount of nuclear DNA has been justified by several workers. The present study aims to estimate the in situ 4C DNA content in varieties of the species of tribe Phaseolae to explore the extent of its correlation at inter-specific and intraspecific levels.

2 236 LEGUME RESEARCH TABLE 1. Representation of a comparative account of chromosomes and mean dna amount Name of Varieties Somatic Mean 4C DNA Range Total AVR the species Chromosome Amount in Chromosome Chromosome Length Number (2n) Picogram (μm) Length (μ) Length (μm) Glycine UPSM ± max (L) SOYA ± Merr. Erythrina ± variegata L. Lablab Sem IC ± purpureus Sem ± L. IC29253A ± Sem Black ± Vigna Hyd white ± unguiculata ± (L) walp ST-6B (brown) ± ST 1B (black) ± ST-6B (white) ± ST-6A (brown) ± ± A ST Canavalia IC ± ensiformis IC ± Canavalia T29/ ± gladiata IC ± Cajanus Rabi ± cajan B ± ± MATERIAL AND METHODS The material under this investigation are described in the Table I. For estimation of in situ DNA content and chromosome number study, fresh healthy young root tips of taken plant species were pretreated in PDB and aesculine in 2-3 hours fixed in Newcommer s fluid, hydrolyzed in (N) HCl for minutes at 58 C, fluid, stained in Schiff s reagent for 1 hour at 20 c and finally squashed in 45% acetic acid. The transmittance values of 100 different metaphase plates of each variety were recorded in a Leitz Wetzlar Aristophot micro-spectrophotometer through the single wave length method (500 μm) after adjusting the blank at 100. The radius of the aperture for the transmittance of light was kept constant at 20 for all the species studied 4C nuclear DNA amounts were calculated on the basis of optical density in terms of relative arbitrary units of absorbance, which were then converted to absolute units (picograms, that is, grams ) by using Van t Hof s (1965) 4C nuclear values for Allium cepa as a standard, i.e., grams. For each variety about 100 observations were taken of metaphase plates. The chromosome number was counted from the metaphase plates. (Table 1) RESULTS AND DISCUSSION Chromosome number : All the species of Lablab purpureus (L) Sweet studied showed 22 chromosomes in their diploid cells and

3 11 chromosomes in haploid cells. The number of chromosomes in the four varieties of Lablab purpereus (L) and six varieties of Vigna unguiculata (L) Walp was 2n=22 in their root system. In four varieties of Vigna unguiculat (L) Walp the chromosome number was 2n=20.In all the varieties of Glycine max (L) Merr studied, the chromosome number was 2n=40. In Erythrina variegat L the chromosome number was 2n=42. In Cajanus cajan (L) Mill. sp the chromosome number was 2n=22. in all the three varieties. The total chromosome length differed within the species having the same chromosome number. In the case of Lablab purpureus, it was found to range intra-specifically from 52 to 72.5 µm. In Vigna unguiculata the intra-specific variation was from to 52.5 µm whereas in the two varieties of Canavalia ensiformis, the chromosome size was from 67.5 to 70 µm and in varieties of Canavalia gladiata, the chromosome size varied from 58.5 to 78 µm. In Cajanus cajan, among the three varieties, the total length was found to range between and µm. A comparative account of chromosome number and size of all the studied species of the Vol. 30, No. 4, TABLE 2. ANOVA table showing analysis of variation of 4C DNA mean amounts per cell Source of Variation Degree of Freedom Sum of Squares (S.S) Mean (S.S) F Glycine max Between the groups Error or within the groups Lablab purpureus Between the groups Error or within the groups Vigna unguiculata Between the groups Error or within the groups Canavalia ensiformis Between the groups Error or within the groups Canavalia gladiata Between the groups Error or within the groups Cajanus cajan Between the groups Error or within the groups tribe Phaseolae are presented in the Table 1. The total and average length of the chromosomes in the studied species have been represented in the histogram (Fig. 1) for comparison. In situ 4C DNA content (unit) : The relative values of DNA were recorded from metaphase plates of root tips of the material studied (Table 1). These data were analyzed in detailed by variance test. For two varieties of Glycine max, the 4C DNA values were found to be 7.56 ± 0.84 p.g., 7.62 ± 0.84 p.g respectively in UPSM 19, Soya,. For Erythrina variegata the 4C DNA values were found to be 7.80 ± 0.74 p.g. For four varieties of Lablab purpurius,the 4C DNA values were found to be 7.60± 0.14 p.g; 7.20 ± 0.12p.g; 5.84 ± 0.14 p.g.; 7.02 ± 0.04 p.g., respectively in Sem IC 16843, Sem IC 22006, Sem IC 29253, Sem Black. For nine varieties of Vigna unguiculata, the 4C DNA values were found to be 4.26 ± 0.07 p.g., 4.6 ± 0.11 p.g., 4.90 ± 0.17 p.g., 4.91 ± 0.01 p.g., 4.06± 0.85 p.g., 4.98 ± 0.14 p.g., 4.37 ± 0.11 p.g. respectively in Hyd-White 1, ST 6B (Brown), ST-1B (Black), ST-6B (White), ST- 6A (Brown), 528, 526, 526A, ST-13. For two

4 238 LEGUME RESEARCH TABLE 3. Manova table (Multivatiate analysis of variance) for comparing population mean vectors Source Matrix of sum Degrees of squares and of freedom crossproducts (ssp) (d.f.) Treatment [ ] Residual [ (Error) ] Total [ (Corrected for the mean) ] Δ* = W = B+W (Σhi g-1) (Σni L*) F obs = = g-1 L* F = > F 0 now obs 0.05,46,524 therefore, the test is significant; MANOVA results indicate that the species and varieties are significant by different from each other. varieties of Canavalia ensiformis, the 4C DNA values were found to be ± pg., 8.70 ± pg. respectively in IC and IC For two varieties of Canavalia gladiata, the 4C DNA values were found to be 9.70 ± pg. and 9.58 ± pg. respectively in T 29/1 and IC For three varieties of Cajanus cajan, the 4C DNA values were found to be 6.8 ± pg., 6.8 ± pg., 0.79 ± pg. respectively in rabi., B 7 and 517.The Table 1 shows the values of 4C DNA in all the materials included in the study. Histogram (Fig. 2) shows different amount of 4C nuclear DNA in different species and varieties of material studied. The estimation of DNA content has proved to an useful tool in the study of evolution and estimation of 4C nuclear DNA contents of root tip of strains of Glycine max,, Lablab purpureus, Canavalia ensiformis, Canavalia gladiata and Erythrina variegata to explore the extent of its correlation at intra-specific level. In situ estimation of 4C nuclear value varies from ± 0.89 p.g. to 8.70 ± 0.09.g., the highest in Cannavalia ensiformis var. IC and the lowest in Vigna unguiculata ST-6B (White)(Table1). On the basis of nuclear 4C DNA amounts estimated, we can differentiate only the species from one another and suggest a close association at the varietal level. Variation in the total amount of 4C DNA content was nonsignificant at varietal level. The range of chromosome counts of 24 species and varieties of the tribe Phaseolae resolved in the present study was between 2n=20 and 2n=42 chromosomes. The present reports of chromosome number confirmed the previous ones which were discussed in the results of cytological analysis. Homogeneity in chromosome number was observed in different species studied, excepting Vigna unguiculata where both 2n=20 as well as 22 chromosomes were observed. The chromosomal system of legumes that have survived to the present are suggestive of three major periods of evolutionary radiation within the family (Goldblatt, 1981). All the materials included under the study are under the same tribe Phaseolae but under different sub-tribes of Phaseolae. The previous study showed that with the exception of Erythrinae and some genera, the Phaseolae as constituted by Lackey (1977) comprised of a family of uniform assemblage cytologically with n=11 predominant in all sub-tribes of Phaseolae. The base number of Phaseolae was represented to be n=11, which was also probably basic to all sub-tribes; some exceptions were found in Glycine max (sub-tribe Glycinae), Erythrina variegata (sub-tribe Erythrinae) and some varieties of Vigna unguiculata (sub-tribe Phaseolinae). In the present investigation, the range of chromosome counts of 24 varieties in the tribe Phaseolae was between 2n=20 and 2n=42 chromosomes. Previous workers reported 2n=20, 22 and 24 in V. unguiculata. The present study of different varieties of Vigna unguiculata confirmed this, though in this investigation, 2n=22 chromosomes were dominant. The haploid chromosome number in Lablab purpureus was n=11 and in Vigna unguiculata,

5 Vol. 30, No. 4, LENGTH IN NM TOTAL CHROMOSOME LENGTH AVERAGE CHROMOSOME LENGTH 0 LABLAB PUR. SEM IC22006 LABLAB PUR. SEM IC29253A LABLAB PUR. SEM BLACK LABAL PUR SEM PLS29 VIGNA UNG. HYD-WHITE 1 VIGNA UNG. 514 VIGNA UNG. ST-6B (BROWN) VIGNA UNG. ST-11B (BLACK) VIGNA UNG. ST-6B (WHITE) GLY. MAX UPSM 19 GLY. MAX SOYA ERYTHR. VARIEGATA VIGNA UNG. ST-6A (BROWN) VIGNA UNG. 526A VIGNA UNG.ST-13 CANAVALIA ENS. IC34212 VIGNA UNG. 528 CANAVALIA ENS. IC21134 CANAVALIA GLAD. T29/1 NAMES OF MATERIALS UNDER STUDY Fig. 1. Comparative Account of Total Chromosome Length and Average Chromosome Length in The Tribe Phaseolae CAJANUS CAJAN RABI CAJANUS CAJAN B7 CAJANUS CAJAN 517 CANAVALIA GLAD. IC45270 n=10, which were also the basic numbers of the genus (Polhill and Raven, 1981). From the observed results it was clear that there was a distinct similarity among the studied genera, species and varieties. In the subtribe Erythrinae, Erythrina is perhaps the most isolated genus with the very distinctive base number n=21 now recorded for more than half the species (Lewis, 1974). The suggestion of Raven (1977), Lackey (1977), that Erythrina be segregated in a mono-generic tribe may be valid in view of its increasingly apparent isolation in Phaseolae. The chromosome number was constant in the genera studied. Goldblatt (1981) emphasized that in Leguminosae, the chromosome number alone was seldom of use and cytology must always be used judiciously in conjunction with other data in order to arrive at the best classification possible. The bulk of the Phaseolae including the sub-tribe Diocleinae (genus Canavalia), Phaseolinae (Dolichos biflorus, which is now known as Vigna unguiculata and D. lablab which is known as Lablab purpureus) and more remotely the Glycinae formed one natural group. The classification presented in this treatment was a modification of a traditional one given by Bentham (1865), Taubert (1894) and Hutchinson (1964). As revealed in the 24 species of the tribe Phaseolae studied here, the absolute chromosome length showed a wide range of variation from mm in Vigna unguiculata var. ST-6B (brown) to 104 mm in Glycine max (in UPSM 16). Inter-generically, inter- and intraspecifically, there were also marked differences in total chromosome length in the tribe Phaseolae (Table No. 2). There was a close relationship between chromosome number and size. But as shown by the present investigation the chromosome number was not necessarily related

6 240 LEGUME RESEARCH NUCLEAR DNA AMOUNT 4C MATERIALS Fig. 2. Different Amounts of 4C Nuclear DNA in Different Species of Family Fabaceae with the chromosome size and this observation matched with that of others (Mukherjee and Sharma, 1986). Numerical changes are not always responsible for variation. The variation of chomosome length is greatly dependent on differential spiralization and difference of protein together with DNA (Mukherjee and Sharma, 1986, 1990; Mukhopadhyay and Sharma, 1987). Minute structural alteration might have an important role in evolution (Nandini et al 1998; Lanerayburn et al, 1997; Palomino and Sousa, 2000). On the basis of chromosome length, we can differentiate the variation as well as species from one another in the analysis (ANOVA Table.2 and 3), this indicates that species and variation are significantly different from each other. As based on the absorption data, minute differences were seen in 4C nuclear DNA amount at an intra-specific level such differences were found to be non-significant (Table 2). The variation in DNA amount was statistically significant at an inter-specific level in case of Lablab purpureus, Vigna unguiculata (formerly known as Dolichos lablab and Dolichos biflorus respectively and placed under the same genus Dolichos) but not in case of Canavalia ensiformis and C. gladiata. The 4C nuclear DNA amount was found to be the highest in Canavalia ensiformis (variety IC 21134: ± pg.) and the lowest in Vigna unguiculata ST-6B (White) : 4.07 ± pg. On the basis of chromosome length we can diffentiate the variation as well as species from one another in the analysis. (Manova Table 3), this indicates that species and variation are significantly different from each other. The increase or decrease in DNA amount may be a basic feature of evolution. Inter-generic and inter-specific variations of the DNA content of legumes have been found to be significant by several observers (Mukherjee and Sharma, 1986; Narayan 1987,1988; Mukherjee and Sharma, 1990; Bennett and Leich, 1997; Lanerayburn et al 1997; Nandini et.al 1997; Naranjo et al 1998; Palomino and Sousa, 2000), but the fact that quantitative estimation of DNA at intra-specific level in case of Lablab purpureus and Vigna unguiculata varieties showed no statistical significance indicated a varietal constancy in the amount of DNA present in the roots. Hence, the total form percentage differences at intra-specific level can be interpreted on the basis of structural changes only, without any significant change in DNA content. This result was the same also in cases of Canavalia ensiformis, Canavalia gladiata, Glycine max and Cajanus cajan. The present study of different genera, species and varieties revealed that the genus Canavalia, whose somatic chromosome number 2n=22, had the highest amount of in situ DNA content; the amount of DNA content was lower in Lablab purpureus, though its somatic chromosome number was also 2n=22. The ANOVA studies (Table 2) clearly showed that a highly significant

7 difference existed among all the species of Lablab purpureus, Vigna unguiculata, and the two species of Canavalia. The significant difference in DNA content between Lablab purpureus and Vigna unguiculata indicated that the amount of DNA can be utilized as a measure of the diversity at the inter-specific level. But again, the intra-specific constancy showed a close association at the varietal level in case of Vigna unguiculata, Glycine max and Cajanus cajan. The diversity in DNA amount was also associated with structural differences of chromosomes among the different species. On the basis of nuclear DNA amounts estimated, one can differentiate only the species from one another and suggest a close association at the varietal level. Inter-specific variation and similarities in amount of DNA content per genome have been noted between Lablab purpureus and Vigna unguiculata. Knowledge of such variation is useful to cyto-taxonomical studies. The diploid and haploid chromosome numbers and other chromosome details of the materials under study were determined. Diploid chromosome numbers studied here complied with the previous claim that the base number for all the species of Lablab, Vigna, Canavalia and Cajanus was 11 except for some varieties of Vigna unguiculata where n=10. In Glycine and Erythrina, the base numbers were 20 and 21 respectively. The range of chromosome length showed marked variation also among species and varieties in some cases. The 4C nuclear DNA amount was found to be the highest in Canavalia ensiformis (var. IC 2113): ± pg. and the lowest in Vigna unguiuculata (ST-6B, White): 4.07 ± pg. The increase or decrease in DNA amount may be a basic feature of evolution. As based on absorption data, minute differences were seen in 4C nuclear DNA amount at an intra-specific level such differences were found to be nonsignificant. The variations in DNA amount were Vol. 30, No. 4, found to be highly significant at an interspecific level in case of Lablab purpureus and Vigna unguiculata but not in case of Canvalia ensiformis and C. gladiata.intergeneric and interspecific variation in the DNA content of legumes have been found to be significant by several observers (Mukherjee and Sharma, 1986; Mukherjee and Sharma, 1990), but quantitative estimation of DNA at intra-specific level in case of Lablab purpureus and Vigna unguiculata showed no statistical significance, indicating varietal constancy in the amount of DNA present in the roots. Nuclear DNA amount in relation to total chromosome number and length: The present study of different genera, species and varieties of the tribe Phaseolae revealed that the genus Canavalia had the highest amount of in situ DNA content; its somatic chromosome number was 2n=22, the same as Lablab purpureus which had a much lower DNA content. So, the diversity in DNA amount was also associated with structural differences of chromosomes among the different species. Taxonomical implications of the present study are that the different genera, species and varieties for the cytological and biochemical analysis included Glycine max. of the subtribe Glycinae, Erythina variegata of the subtribe Erythrinae, Lablab purpureus and Vigna unguiculata of the sub-tribe Phaseolinae, Canavalia ensiformis and C. gladiata of the sub-tribe Diocleinae and Cajanus cajan of the sub-tribe Cajanae. The parameters utilized in the present investigation bring out clear similarities in certain aspects in all the genera under the tribe Phaseolae, though certain dissimilarities too were recorded in certain parameters. The data obtained do not permit an assessment of placing the above-mentioned genera under the sub-tribe Glycinae, Erythrinae, Dioclenae, Phaseolinae and Cajanae respectively (Lackey, 1977). However, it indicates their affinity in certain respects, validating their position under the tribe Phaseolae.

8 242 LEGUME RESEARCH REFERENCES Altamura, M.M,. (1991). Cytobios (Cambridge) 67 (269) : Bandyopadhyay, B. (1992). In : Perspectives in Cytology and Genetics (Manna, G.K. and Roy, S.C. eds.) 7: Banerjee, N. and Sharma, A.K. (1985).. Proc. Indian natn. Sci. Acad. 51(4) : Bennett, M.D. and Leitch, I.J. (1997 )Ann Bot. 80: Bentham, G. (1865)., Genera Plantarum. In Bentham and J.D. Hooker. Vol. 1. Reeve, London. Cavallini, A et al (1993). Heredity 70 : Cubas, P. et al(1998). Bot. J. Linn. Soc. 128: Cubas, P. and Pardo, C. (1997). Bot. J. Linn. Soc. 125: Freckman, R.W. and Wemple, D.K. (1963). The chromosome number of the palilioniod legumes. Unpublished graduate student special report Botany Department, Iowa State University, Ames. U.S.A. Galasso, et al (1997) Ann Bot. 79: Goldblatt, P. (1981 Advances in Legume Systematics. Ann. Missouri Bot. Gard Graham, A. and Tomb, A.S. (1977). Lloydia 40: Hutichinson, J. ( 1964). The genera of Flowering Plants Vol I, Oxford University Press. Ignacimuthu, S. and Babu, C.R. (1988).. Cytologia (Tokyo) 53 (3) : Jones, W.E., et al (1998). Ann. Bt. 82 (2): Lackey, J.A. (1977).. Bot. J. Linn. Soc. 74 : Lanerayburn, A., et al (1997). Ann. Bot. 80 (3): Le Coq et al (1992). Genome (Ottawa) 35 (4) : Lewis, W.H. (1974).Lloyds 37: Mukherjee, S. and Sharma, A.K. (1986). Perspectives in Cytology and Genetics. (Manna, G. K. and Sinha, U. eds.) 5: Mukherjee, S. and Sharma. A.K.(1990) Proc. Indian Acad. Sci. ( Plant Sci ) 100 (1) : 1-6. Mukhopadhyay, S. and Sharma, A.K. (1987). Cytologia, 52 : Nandini, A.V. et al (1997). Bot. J. Linn.Soc. 125 (4): Naranjo, C. A., et al.(1998). Ann. Bot. 82: Narayan, R. K. J. and Rees,H. Chromosoma (1976) 54: Narayan, R.K.J. (1982) Evolution 36: Narayan, R.K.J. (1985) In. Kew Chromosome Conference- 2. P.E.Brandham and M..D.Bennet (eds) George Allen and Unwin(Lond.).pp Narayan,R.K.J.(1987) Pl.Syst.Evol.157: Narayan,R.KJ.(1988) Evol. Trends in Plants 2: Palomino, G., Sousa, M.S. (2000). Ann. Bot. 85 (1): Parida, A. et al.(1990). Genetica 82 (2) : Phillips, L.L and Strickland, M.A. (1966) Can. J. Genet. and Cytol Polhill R.M.N. and Raven P.H. (1981). In Advances in Legume Systematics, (ed. R.M Polhill and P.H.Raven). Royal Botanic Gardens, Kew. Price,H.J and Bachmann.K (1975) Amer. J. Bot. 62: Raven, P.H. (1977). Lloydia, 40: Rees,H. and Hazarika,M,H.(1967) Chromosome Today 2: Rees,H and Jones,R,N (1967) Nature(Lond) 216: Sharma, A.K. and Mukhopadhyay, S. (1984). Proc. Indian Acad. Sci. (Plant Sci.) 93: Taubert, P. (1894). Leguminosae. In Die Naturlichen Pflanzen- familien. (A Engler and K. Pranti, eds.) vol III, 3: Wilhelm Engelmann, Leipzig. Van t Hof., J. (1965).. Exp. Cell. Res. 39 : Watson, E.M. (1987). Genome 29 (2) :

SEEDCOAT SEM STUDIES OF SELECTED TAXA OF THE TRIBE PHASEOLEAE (FABACEAE)

Legume Res., 28 (4) : 235-243, 2005 SEEDCOAT SEM STUDIES OF SELECTED TAXA OF THE TRIBE PHASEOLEAE (FABACEAE) Baisakhi Bandyopadhyay, Subhas Chandra Santra and Mashahiro Kato [kparlment of Environmental