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Somaclonal variation incarotene content of carrot(Daucus carota L.)Nathalie Pawlicki a , Rajbir Singh Sangwan a &Brigitte Singh Sangwan-Norreel aa Université de Picardie, Faculté des Sciences,laboratoire Androgenèse et Biotechnologie , 33 rueSaint-Leu, F-80039 , Amiens CedexPublished online: 27 Apr 2013.

To cite this article: Nathalie Pawlicki , Rajbir Singh Sangwan & Brigitte SinghSangwan-Norreel (1993) Somaclonal variation in carotene content of carrot(Daucus carota L.), Acta Botanica Gallica: Botany Letters, 140:1, 17-22, DOI:10.1080/12538078.1993.10515563

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Acta bot. GaUica, 1993, 140 (1), 17-22.

Somaclonal variation in carotene content of carrot (Daucus carota L.)

by Nathalie Pawlicki (1), Rajbir Singh Sangwan and Brigitte Singh Sangwan-Norreel

Univer&ile de Picardie, Faculte de& Science&, laboratoire Androgenese et Biotechnologie, 33 rue Saint-Leu, F -80039 A mien& Cedex (1) Pre&ent adreu : The Swedi&h University of Agricultural Sciences, Department of Horticultu­ral Science, Box 55, S-23053 Alnarp

INTRODUCTION

(Manuscril ret;u le 6 janvier 1992; accepte le 11 mai 1992)

Summary.- Carrot plants have been regenerated by somatic embryogene­sis from different explants Issued from aseptic seedlings. The petiole seg­ments are the more embryogenic explants. Spectrophotometric analyses of carotene extracts In taproots of these carrots have led to the identification of some plants with significantly higher content than the control plants grown from seed in varieties 3024C, Tantal, 29026, Red Core Danvers, Karaf and 6ottex, out of 9 analysed.

Resume.- Des plantas de carone ont ate regenerees par embryogenese so­matique ~ partir de dilferents explants pre laves sur des germinations steriles. Las fragments de petioles se revelent Atre las explants las plus embryogenes. Des analyses spectrophotometriques des carotenes dans las pivots de cas plantas ont permis d'ident~ier des plantas surproductrices chez 6 vari9tes (3024C, Tantal, 29026, Red Core Danvers, Karaf et 6ottex) sur las 9 etu­diees, par rapport aux analyses de plantas temoins issues de semis.

Key words : carrot - carotenes - somatic embryogenesis - somaclonal varia­tion.

Vitamin A is mainly obtained from carotenes of vegetables and fruits. Car­rot (Daucus carota L.) is considered as one of the major source of carotene. Large diversity in pigmentation and to­tal carotenoid content of wild and culti­vated carrot taproots have been repor­ted (Banga, 1962). The inheritance of

taproot color in carrot appears to he complex ; although inhibitor genes (Buishand and Gahelman, 1979) and pigment enhancing genes (Kust, 1970) as well as genes that control the synthe­sis of specific pigments (Laferriere and Gahelman, 1968) have been described, little is known about the action of these genes on the biosynthetic pathway. It has also been found that environmental

© Societe botanique d6 France 1993. ISSN (en cours).

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conditions such as photoperiod, soil type, pH and fertilizer levels, largely influence the synthesis and the accumu­lation of carotenes in taproots (Aubert, 1981).

The cultured carrot has been ob­tained mainly through classical bree­ding, and genetic selection has created new varieties with high carotene con­tents (Simonet al., 1989). Although the role of somaclonal variations in plant breeding needs to be defmed further, there is reason to believe that they are important in adding new variations to the existing genetic resources (Larkin and Scowcroft, 1981). Also, the hyper­production of secondary metabolites in plants regenerated by in vitro cultures (Fujita, 1988 ; Herouart et al., 1988) has prompted us to use in vitro somatic embryogenesis in carrot to obtain plants wit~ variations in carotene contents.

MATERIALS AND METHODS

CuHure methods Seeds of 9 varieties of carrot varying in the co·

lor of their taproots (Table 1) were soaked in tap water

for approximatively 30 minutes, surface sterilized in hy· drogen peroxide 110 vols (1-1,0

2) for 15 minutes, rinsed

with sterile distilled water, and placed in a 7 % (w/v) calcium hypochlorite solution. After 20 minutes, the seeds were extensively rinsed and aseptically germi· nated on G1 medium [haW-strength (Murashige and Skoog, 1962) inorganic salts and half-strength (Nitsch and Nitsch, 1965) vitamins] containing 0.5% (w/v) su· erose and 0.8 % (w/v) Dnco Bacto agar for 3-4 weeks. Petioles, cotyledons, hypocotyls and roots of the asep­tic seedlings were excised into sections of 1.0 em and cultured on CN medium [Lin and Staba (1961) macro· salts, Nitsch and Nitsch (1965) microsalts and vitamins) supplemented with 2 % (w/v) sucrose and 0.8 % (w/v) agar (pH 5.7). To induce embryogenic calli, 4,51JM of 2,4-dlchlorophenoxyacetic acid (2,4·D) was supplied to the medium (CN1 medium) during 4 weeks. For the development of somatic embryos into plantlets, the ex· plants were transferred to a new medium devoided of the auxin. The cultures were incubated in a culture room at 27• ± t•c with a 16 h photoperiod (30 !JE m·•.s·') provided by fluorescent light. The roo­ted plantlets were acclimatized in a greenhouse (Phy· totron, Gil-sur-Yvette, France) at 22•c under a photo· period of 16 h. AHer 3 months, the plants were harves· ted, and the taproots were used for carotene analyses.

Carotene analysis Cross-sections (5-1 0 g) taken from the middle

part of the taproots were lyophilised and blended with 30 ml of hexane-acetone (713), overnight under nitro­gen atmosphere to minimize oxidations. To extract ca­rotenes, saponnied samples were homogenized for 5 minutes in 70 ml of hexane and concentrated in a

Table 1.- Characteristics of the different varieties studied in Daucus carota L. Tableau 1.- Caracteristiques des dnferentes variates etudiees chez Daucus carota L.

Variety Source Color of Dry matter Carotene content*

(abbreviations) the taproots (%) (mglg dry matter)

3024C (30C) As grow Dark orange 11.17 551.4 ± 1.70 Tantal (TA) Clause Dark orange 9.99 489.8 ± 1.06

29028 (298) As grow Orange 11.23 340.9 ± 1.64 Red Core Danvers (RC) As grow Orange 11.82 258.3 ± 1.60

De Chantenay (DC) Vilmorin Orange 10.43 330.8 ± 1.84 Nanco (N) Vilmorin Orange 11.65 446.0 ± 1.34

Karaf (K) As grow Light Orange 11.61 58.1 ± 2.36 Boltex (80) Clause Light Orange 10.44 116.3 ± 1.62

Scarla (SC) Clause Light Orange 11.44 55.6 ± 1.96

* : Harvest of the plants 3 months after the sowing in greenhouse. Data, given as mean ± S.D., are average values of 1 0 plants.

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N. PAWLICKI, R.S. SANGWAN AND B.S. SANGWAN-NORREEL 19

rotary evaporator. Residue was extracted w~h petro­leum ether and chromatographed on an aluminium oxide column. A small amount of anhydrous sodium sulfate was placed on the top of the column to remove excess water. The whole a-, B· and ..,..carotenes was eluted from the column by petroleum ether. Absorption at 450 nm was determined w~h a H~achi U-2000 spectro­photometer, and the concentration of pigments was cal­culated with E"" value equal to 2600 (Davies, 1965). The data (mg/g dry matter), given as average values from 10 replicate analyses, were compared to those obtained lor taproots of plants grown from seed in green­house using the t table from the Student test w~h P = 0.05 (Schwartz, 1980).

RESULTS

Regeneration of carrot plants from somatic embryos

The appearance, as well as the number of somatic embryos produced on the explants cultured on the embryo­genic medium, vary according to the va­rieties (Table 2). For the most part of the varieties, somatic embryos appea­red after 3 or 4 weeks, but for 3024C the delay was longer (8 weeks), and the production of somatic embryos weaker. This variety, whose its selection was car­ried out from seeds harvested in Asia, germinated with difficulty in fields, and

the flowering was previous to those of other varieties (F. Guyot, personal com­munication). Likewise, differences were noted according to the cultured explants (data not shown). Petiole segments gave the best percentage of somatic embryo­genesis, while with root segments very few somatic embryos were produced. As to cotyledon and hypocotyl segments, their efficacity to produce somatic em­bryos depended of the varieties.

After 3 months of acclimatization in the greenhouse, the plants issued from somatic embryos varieties were harvested. The taproots did not look like those issued from seed. Indeed, all the roots, formed under in vitro condi­tions, were tuberized to give several lit­tle intermingled taproots (Fig. 1). Ne­vertheless, these particular taproots had the specific orange color of carrots due to the presence of carotenes.

Carotene contents after somatic em­bryogenesis

For the determination of carotene contents 25 to 40 plants derived from somatic embryos, for each variety, were separately analysed and compared to

Variety Appearance of SE(a) % of

(weeks) embryogenesis (b)

3024C 8 46.25 c Tan tal 4 79.20 a

29028 4 73.30 a Table 2.- Regeneration of somatic

Red Core Danvers 3 85.00 a embryos on CN1 medium from

De Chantenay 3 85.80 a petiole segments of aseptic seedlings.

Nanco 3 80.80 a Tableau 2.· R~g~neratlon d'embryons

Karaf 4 77.00 a somatlques sur Ia milieu CN1 a partir de fragments de ~-

Boltex 3 80.00 a tiole issus de germinations

Searl a 3 62.20 c st~riles.

a : Delay of appearance of somatic embryos. b : Values wHh different suffix letter are slgnHicantly different at P = 0.05.

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20 ACTA BOTANICA GALLICA

those of control plants. For each har­vested plant, 10 replicate analyses were made. For the most part of the in vitro plants, similar data were obtained com­pared to plants grown from seeds, but in some varieties differences were no­ted for 1 or 2 plants (Fig. 2). For the varieties Nanco and Scarla, the caro­tene contents were always identical in roots of plants regenerated from soma­tic embryos and those grown from seeds. The statistical analysis did not show si­gnificant differences at the 5 % level. For the variety De Chantenay, one plant, out of 35 analysed, showed a significant lower carotene content (290 pglg dry matter). For the six other varieties studied (3024C, Tantal, 2902B, Red Core Danvers, Karaf and Boltex), the taproots of 1 or 2 plants derived from somatic embryos had higher caro­tene levels than plants grown from seeds (the differences observed were signifi­cant at a 5 % level). Particularly, the variety Karaf showed an exceptional va­riation : the carotene content of one plant regenerated from one somatic em­bryo was 11 times higher (652 pglg dry

Fig. 1.- Taproot of carrot regenerated alter somatic embryogenesis.

Fig. 1.- Pivot d'une plante de carotte regllnllree apres embryogenese somatique.

matter) in comparison with the control plant (58.1 pg/g dry matter).

DISCUSSION

In comparison with indirect orga­nogenesis, somatic embryogenesis is of­ten described as an excellent technic to obtain clones for the vegetative multi­plication of plants (Sharp et al., 1982). But, some studies on the regeneration of plants from somatic embryos in maize (Armstrong and Phillips, 1988) or in cu­cumber (Custers et al., 1990) suggested that somatic embryogenesis can he a source of variability at a genotypic le­vel, and consequently at a phenotypic level of the regenerated plants. In our study, variations in carotene production in carrot (Daucus carota L.) plant li­nes derived through somatic embryoge­nesis was obtained. However, it is known whether the observed variation is genetic or epigenetic. We are presen­tly investigating these possibilities in more details by determining the chro­mosome numbers and by examining the

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N. PAWLICKI, R.S. SANGWAN AND B.S. SANGWAN-NORREEL 21

700

1600 C;-500 ""' .,. 0:0400 ~

~ ~ 300 0 u

:!! 200 Ill

8 100

0 30C TA 29B RC cc N K BO sc

Varieties

Fig. 2.- Carotene contents in taproots of carrot plants from 9 varieties obtaines by somatic embryogenesis and seeds. Only the more significant resuHs for 1 plant are given (average of 10 replicate analyses). For a same variety, common letter indicate no significant differences at P = 0.05 (test t).

Fig. 2.- Comparaison des teneurs en carot~nes dans les pivots de plantas de 9 varietlls de carone issues de semis ou rllgllnllrlles par embryogen~se somatique. Seuls les rllsuHats les plus signicatlfs pour une plante sont donnlls (moyenne de 10 analyses). Pour une mArne varillt6, une mArne lenre indique des r6suHats non significatifs a P = 0,05 (test t).

genetic stability of regenerated plants through isozyme patterns (Orton, 1985). Stress induced by tissue culture may also alter carotene contents in the rege­nerated plants. In the litterature, it seems that some somaclonal variations observed in regenerated plants after somatic embryogenesis were physiologi­cal, and probably induced by the use of some constituents of the culture me­dium which could modify the sequence of genes in the ontogeny of plants (Phillips et al., 1990). Altered pigmen­tation and carotene contents in calli of carrot had reported (Naef and Turian, 1963 ; Sugano et al., 1971 ; Mok et al., 1976). For Mok et al. (1976), the syn-

thesis of carotenes in cultured cells was dependent of the components of the me­dium. Studying the effects of hormones on carotenogenesis, these authors shown that kinetin inhibited, 2,4-D enhanced, and gibberellic acid (GA3) had no ef­fect on the synthesis of carotenes. In our investigations, it was possible that 2,4-D, supplemented to the medium to induce somatic embryogenesis, stimula­ted the synthesis of carotenes.

Acknowledgements - This work was partly supported by the "Centre de Valorisation des Glucides de Picar­die" (CVG) and by "Minist~re de Ia Recherche et de Ia Technologle" (MAl). The seeds were kindly supplied by Asgrow France and Vilmorin France.

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