Environmental and Experimental Botany 53 (2005) 135–138

Impact of hybridity on flavonoid spectrum of ber(Ziziphus mauritianaLamk.)

R. Bhargava∗, A.K. Shukla, N. Chauhan, B.B. Vashishtha, D.G. Dhandar

Central Institute for Arid Horticulture, 10th Km Milestone, Sriganganagar Road, Beechwal, Bikaner 334 006, Rajasthan, India

Accepted 18 March 2004

Abstract

Ber (Ziziphus mauritianaLamk.) demonstrates a rich biodiversity with respect to morphometeric and fruit characters. Muchof this has been generated on account of natural cross pollination. In depth studies have been conducted in different crops toevaluate the impact of hybridity on plant morphometric and yield parameters but very few studies were undertaken to evaluatethe impact on metabolite constitution. Accordingly, the present study was aimed at evaluating the foliar flavonoid spectrum oftwo hybrids developed at CIAH, Bikaner viz-a-viz their parents. The results demonstrated that some compounds encountered inparents were present in profiles of hybrids also. Similarly, some novel compounds were also found which might have producedas a result of new gene combinations in hybrid. Like wise some compounds present in parents were not found in hybrids. Thesecould be explained on the basis of epistatic phenomenon.© 2004 Elsevier B.V. All rights reserved.

Keywords:Flavanoid; Hybrid;Ziziphus mauritiana

1. Introduction

Among the various processes involved in introduc-ing genetic divergence in plant species, cross pollina-tion has contributed the most (Fernandes, 1942). It hasbeen observed that at time hybridity improves the ge-netic make up of plants, gives better quality and yieldof produce (Williams, 1959; Sharma et al., 1986).

Ber, (Ziziphus mauritianaLamk.) demonstrates arich genetic diversity. Majority of this has been devel-oped on account of natural cross pollination, becauseof self incompatibility (Godara, 1980). The selected

∗ Corresponding author. Tel.:+91-151-2250960;fax: +91-151-2250145.

E-mail address:rbciah@yahoo.co.in (R. Bhargava).

elite combination thus obtained got fixed on accountof availability of standardized vegetative multiplica-tion methods for this crop.

Although, an in depth studies have been conductedto understand the effect of hybridity on morphometricand associated parameters, very less is known aboutthe impact of hybridity on metabolite constitution ofhybrid plants. Flavonoids are secondary metaboliteswhich have gained popularity in plant biosystematicson account of being their conservative nature. It hasbeen proved time again that these compounds aid inspecies/cultivar identification (Kanzaki et al., 1997;Williams et al., 1997; Robards et al., 1997) as well asin assessing the phylogenetic relationship between theparents and hybrids (Bhargava, 1983; Bhargava et al.,1986). Accordingly, during present study attempt was

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136 R. Bhargava et al. / Environmental and Experimental Botany 53 (2005) 135–138

made to compare the foliar flavonoid spectrum of F1hybrids with their parents. The results thus obtainedconstitute the text of present investigation.

2. Material and methods

Two F1 hybrids [CIAH-H1, CIAH-H2] and theirparents cv. Seb and cv. Katha growing in the exper-imental ber block of CIAH, Bikaner constituted thematerial for present study. The distinguishing featuresof each germplasm are:

1. CIAH-H1: Semi-spreading growth habit, preco-cious and prolific bearer, early maturity, juicy andfairly tolerant to fruit rot.

2. CIAH-H2: Spreading growth habit, mid seasoncultivar, prolific bearer, less juicy as compared toCIAH-H1.

3. Seb: In Bikaner condition, it is mid-late in maturity,attractive fruit size, but susceptible to fruit fly andfruit cracking.

4. Katha: Mid to late in maturity, prolific bearer, in-festation of fruit fly is less in comparison to cv. Seb.

The fully matured leaves from 5-year-old plantswere used as the test material. The plants received uni-form cultural operations.

2.1. Extraction of flavonoids

For the extraction of flavonoid 2 g of mature leaf wasselected (in order to avoid any variation due to devel-opmental stage of leaf) and sample was fixed in 10 mlof methanol containing 1% HCl. The fixed sampleswere stored in dark, at room temperature and beforeanalysis they were macerated in mortar and pestle. Thewhole content was filtered and filtrate centrifuged at

Table 1Flavonoid spectrum of ber cultivars and hybrids

Cultivars Flavonoid spots

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Seb + + + + + + + + + +Katha + + + + + + + + + +Seb× Katha

(CIAH-H2)+ + + + + + + + + + + + + +

Seb× Katha(CIAH-H1)

+ + + + + + + + + +

10,000 rpm for 20 min at room temperature. The clearfluid was taken and evaporated to dryness in an ovenmaintained at 60◦C. Finally, the sample was taken in1 ml of methanol.

2.2. Analysis of flavonoids

The flavonoids were separated on TLC plates coatedwith 0.6 mm thick layer of cellulose. An aliquot of10�l was loaded on one corner of the plate. The platewas first developed with 2% formic acid and plates,after rotating at 90◦, in solvent containing amyl alco-hol:acetic acid and water in the ratio of 10:6:5. Theplates, after air drying, was view for flavonoids spotas under:

1. without any spray;2. after exposure to vapours of ammonia under UV;3. after spray with 1% methanolic AlCl3 under UV;4. after spray with 1% methanolic NaOH under UV.

The individual chromatogram were developed bytreatments as listed above and spots visible weremarked. Each chromatogram was overlapped and thecommon spots were identified and marked. Thus, bypooling all the spots developed by individual treat-ment, the pooled chromatograms was prepared foreach genotype. Based on the Rf values the spots werenumbered and master chromatogram was preparedfor comparison of cultivars.

3. Results and discussion

The foliar flavonoid spectrum of cvs. Seb, Katha andtheir hybrids (CIAH-H1 and CIAH-H2) is presentedin Table 1 and depicted inFig. 1. Perusal of table

R. Bhargava et al. / Environmental and Experimental Botany 53 (2005) 135–138 137

Fig. 1. Master chromatogram showing position of flavanoid spots.

and pooled chromatogram reveals that a total of 23flavonoids spots were present. Among these spot nos.11, 12 and 14 are present in all the cultivars. It wasfurther observed that cultivar Seb has a total of 10flavonoid spots of which spot nos. 5, 8, 19, 20 and 23are unique to its spectrum. Similarly, the flavonoidsspectrum of cultivar Katha demonstrate a total of 10spots of which spot nos. 7, 9, 10, 16 and 21 are typicalto its profile.

Perusal of flavonoids spectrum of CIAH-H1 revealsthe presence of a total of 10 spots. Of these spotnos. 4 and 18 are unique to this hybrid. Similarly, theflavonoids spectrum of CIAH-H2 shows the presenceof a total of 14 spots. Of these spot nos. 1, 2, 3, 6 and13 are unique to this hybrid taxon.

Comparison of flavonoid spectrums of Seb andKatha reveals that spot nos. 5, 8, 19, 20 and 23 arepresent only in cv. Seb where as spot nos. 7, 9, 10, 16and 21 were present in cv. Katha only. These flavo-noids demonstrate that they have significant role toplay in assessing the interrelationship of hybrid lines.

The foliar flavonoids spectrum of CIAH-H1 revealsthe presence of 10 spots perusal of the spectrum re-

veals that it has spot nos. 5 and 8, which are typical ofcv. Seb, and spot nos. 9, 10 and 21, which are typicalof cv. Katha. Thus, the above results indicates that theflavonoid profile of hybrids demonstrate the presenceof typical spots of parents.

Perusal of foliar flavonoids spectrum of CIAH-H2reveals that it has a total of 14 foliar flavonoids spots.Out of these, it contains spot nos. 5 and 19, which aretypical of cv. Seb, and spots 9 and 21 that are typicalof cv. Katha. Thus, out of five typical spots of cv. Sebonly two are present in the flavonoid profile of hybrid.Similarly, out of five typical spots of cv. Katha twoviz. spot no. 9 and spot no. 21 were encountered inthis hybrid.

These results are similar to those obtained byAlston and Turner (1962)in Baptisia, Iiyama andGrant (1972)in Avena,Murray and Williams (1973)in Briza media andKohli and Denford (1977)inPotestilla andBhargava (1983)in Narcissus.

Another interesting feature encountered in thisgroup is appearance of novel flavonoids. The spotnos. 1, 2, 3, 6, 13, 17 in CIAH-H2 and spotnos. 4, 17 and 18 in CIAH-H1 illustrate this.

138 R. Bhargava et al. / Environmental and Experimental Botany 53 (2005) 135–138

Presence of novel compounds have also beendemonstrated byBhargava et al. (1986), Bhargavaet al. (1988), Sharma et al. (1989), origin of newcompounds through hybridity can be explained onthe basis of new gene combinations leading to novelexpressions.

In addition to above, some compounds present inparents were not found in the hybrids. This is illus-trated by the fact that CIAH-H2 do not show spot nos.7, 8, 10, 16, 20, 22 and 23 and CIAH-H1 do not showspot nos. 7, 12, 14, 16, 19, 20 and 23. The disappear-ance of spots from the hybrids can be explained on thebasis of epistatic phenomenon, i.e. the masking effectof one gene over other.

The impact of hybridity on flavonoids spectrumhave been documented in the present paper. Attemptswill be made to strengthen the study by use of othermolecular markers.

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