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Effects of Flurprimidol on the organogenic ability of garlic root-tips using a one
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step in vitro system
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Effects of FLP on garlic in vitro shoot regeneration
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Nieves Martín-Urdíroz, José Garrido-Gala, Jesús Martín, Xabier Barandiaran.
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Authors:
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J. Garrido-Gala, J. Martín, X. Barandiaran.
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Departamento de Microbiología.
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Facultad de Ciencias, Universidad de Córdoba.
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Campus de Rabanales C6
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14071 Córdoba, Spain
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Corresponding author:
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Nieves Martín-Urdíroz
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Allium Laboratorios S.L.
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C/ Gabriel Ramos Bejarano, parc. 110D, puerta I.
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Polígono Industrial “Las Quemadas”.
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14014 Córdoba, Spain.
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Telephone: +34 957 326473
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Fax: +34 957 326473
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e-mail: [email protected]
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KEYWORDS
Allium sativum, genetic variability, gibberellin synthesis inhibitor, light effects,
organogenesis, plant growth regulation.
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ABSTRACT
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The influence on garlic regeneration of different flurprimidol concentrations and
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its possible interactions with light has been studied
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regeneration system . The number of shoots produced by regenerative calli significantly
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increased when low concentration of growth retardant was used. In this conditions the
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number of garlic shoots regenerated per gram of callus overpass those obtained in
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previous works revealing this growth regulator could be used to optimize garlic
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regeneration and micropropagation.
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using a one step in vitro
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ABBREVIATIONS
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2,4-D: 2,4-dichlorophenoxyacetic acid, 2iP: 6---(dimethylallylamino)-purine
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BAP: 6-benzilaminopurine, FLP: Flurprimidol, IAA: indole acetic acid, NAA:
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naphtalenacetic acid.
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INTRODUCTION
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Flurprimidol (-(1-methylethyl)--[4-(trifluoromethoxy) phenyl]-5-pyrimidine-
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methanol) is a gibberellin biosynthesis inhibitor that retards shoot growth on a variety of
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plant species at different concentrations (Ronco 1999, Pobudkiewicz et al. 1997). The
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mode of action involves complexing of the growth retardant with cytochrome P450-
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dependent enzymes in the metabolic pathway for gibberellins (Sperry et al. 1999).
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In general, plant growth retardants are used in agronomic and horticultural crops
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in order to reduce the shoot length of plants without lowering plant productivity and
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without changing developmental patterns or being phytotoxic (Rademacher 2000). In
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ornamental practice (Swainsona formosa), growth retardants are applied to improve
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flower productivity by reducing the vegetative growth (Hamid et al. 1997). In addition,
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some gibberellin biosynthesis inhibitors such as paclobutrazol have fungicidal side
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activities (Rademacher, 2000).
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Concerning the use of growth retardants in in vitro culture, it has been mainly
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tested to study the regeneration of shoot buds from callus. Ezura et al. (1995) showed
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that callus derived from mutants of Arabidopsis thaliana which have reduced levels of
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endogenous bioactive gibberellins, regenerated shoot buds more readily than those
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callus derived from wild-type controls. In addition, exogenous gibberellins reduced and
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exogenous paclobutrazol increased the frequency of shoot bud regeneration from wild-
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type callus.
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Garlic shoot regeneration from callus has been reported in several studies (i.e.
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Haque et al., 1997; Myers et al., 1998; Barandiaran et al., 1999a, 1999c; Zheng et al.,
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2003), commonly the growth regulators used have been auxins (NAA, 2,4 D, IAA and
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others) and cytokinins (BAP, 2iP and others). In garlic, the group of growth retardants
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has rarely been used for in vitro processes. Kim et al. (2003) described that CCC
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(chlorocholine chloride) and other growth retardants promoted induction and growth of
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bulblets of garlic in liquid cultures. However, some studies has been made in order to
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evaluate their effects on field cultivated plants (Souza et al., 1992a, 1992b; Resende et
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al., 1994, 1999).
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In this work we determine the influence of FLP on in vitro callus formation and
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shoot regeneration of garlic root-tips cultured under different light conditions. Culture
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in light or in the dark from the beginning of the culture has been shown to exert
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considerably effects in garlic shoot regeneration (Martín-Urdíroz et al., 2003). On the
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other hand, in vivo gibberellin biosynthesis pathways are regulated by light (Kamiya et
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al., 1999) so the interaction of this factor with the presence or absence of a gibberellin
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biosynthesis inhibitor could have significant consequences in the in vitro organogenic
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process.
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MATERIALS AND METHODS
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Plant material
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Axenic root-tips were obtained from in vitro garlic plants cultured following the
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protocol of Barandiaran et al. (1999b). The accessions used were chosen to include
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three general groups of garlic: E213072 (red type), E432095 (chinese type) and
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E112054, E111300 (white type). They were kindly provided by the Garlic Germplasm
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Bank of Cordoba (C.I.F.A.) (curator: F. Mansilla). Accession numbers corresponds to
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those used in the European Allium Database (EADB).
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Root culture
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Root-tips (1 cm long) were excised from micropropagated plants under aseptic
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conditions. Explants were cultivated in Petri dishes containing 25 ml of four different
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media based on A3 medium used by Barandiaran et al. (1999a). A3 medium consisted
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in B5 medium (Gamborg et al., 1968) with 0.03 mg/l 2,4 D, 2 mg/l NAA and 3 mg/l
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BAP. In addition, this medium (A3) was supplemented with different concentration of
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FLP: 0, 0.3, 1 and 5 mg/l conforming the media A3 (control), F0.3, F1 and F5,
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respectively. All these media were supplemented with 3% sucrose and 0.9% Bactoagar.
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Media were adjusted to pH 5.7 prior to autoclaving. No media change or refreshment
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were applied along the culture period. Cultures were incubated in a growth chamber at a
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temperature of 25o  2º C for two months. Light conditions applied to the cultures were
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different: one set was subjected to a photoperiod 16/8 hours using Grolux fluorescent
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lights (55 mol m-2s-1) while other set of cultures were incubated continuously in the
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dark.
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Experimental design and statistical analysis
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The experimental design was completely randomised, with 32 treatments
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(genotype/medium/light condition) and 3 replications per treatment. One replication
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consisted of 10 Petri dishes with 3 explants each.
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Data recorded after 8 weeks of culture were callus formation percentage,
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organogenic callus percentage and number of shoots per organogenic callus. Analyses
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of variance (ANOVA) and the comparison of means (LSD) were analysed with
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Statistix.
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RESULTS AND DISCUSSION
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ANOVA test of the results obtained in callus formation showed highly
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significant differences among media (df= 3; F= 89.34; P 0.001). Media F0.3 and F1
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showed the best results in callus formation, with results ranging from 39 to 71%,
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although they were no significatively differents of the results obtained in medium A3
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(control). Callus formation rate did not show significant differences among light
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conditions and genotypes (Fig.1).
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Respect to shoot regeneration, ANOVA test showed significant differences for
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organogenic callus percentage among genotypes (df= 3; F= 9.39; P 0.001), media (df=
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3; F= 28.2; P 0.001) and light conditions (df= 1; F= 11.3; P 0.01) (Fig. 2). On F5
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medium, none genotype was able to regenerate shoots, probably due to a toxic effect of
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flurprimidol. In general, the organogenic callus percentage was higher in light than in
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the dark. Exceptionally, Red type accession presented a percentage higher in the dark
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than in light when cultured on F0.3 medium (Fig. 2). In these conditions, it was found
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an interaction genotype-medium: Red type accession showed an increase in the
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percentage of callus that underwent shoot regeneration when a low concentration of
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FLP was used, while the remaining accessions decreased their organogenic callus
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percentage.
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ANOVA test showed significant differences for the trait shoots per organogenic
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callus among genotypes (df= 3; F= 4.41; P 0.01) and media (df= 3; F= 9.7; P 0.001).
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White type accessions regenerated the highest number of shoots (5.2 and 3) while the
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Red type accession showed the lowest value (1.4 shoots/organogenic callus).
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Concerning the media, the presence of a low concentration of FLP (F0.3) significantly
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improved the production of shoots (6.1 shoots per organogenic callus) while lower or
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higher concentrations produced worse results (2.9, 2.5 and 0, respectively) (Fig. 3).
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Other trait studied directly related with the number of shoots per organogenic
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callus was the number of shoots regenerated per gram of callus. Root tips from E111300
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(White type accession) cultured in F0.3 medium and incubated in light produced up to
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350 shoots/g organogenic callus in 2 months. This result improves those obtained by
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Robledo-Paz et al. (2000) (170 shoots/g) and Martín-Urdíroz et al. (2003) (250
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shoots/g). In general, the culture in light presented positive effects on organogenic
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process as it is shown in Figure 4, confirming the results obtained by Martín-Urdíroz et
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al. (2003).
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The data obtained in these experiments show that a low concentration of FLP
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reduces organogenic callus percentage (except for Red type accession), although
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increasing the number of shoot per organogenic callus suggesting that these two events
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are carried out by different pathways. Similar effects were observed on in vitro culture
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of Alstroemeria x hybrida “Juanita” (Podwyszynska et al., 1998) and cucumber
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(Konstas et al., 2003), in which flurprimidol increased the number of shoots. Moreover,
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it has been reported that FLP reduces in vitro shoot multiplication on some other plants
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as moth bean (Upadhyaya et al., 1989). In fact, it has described that gibberellic acid
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(GA3) inhibits shoot formation via inhibition of the meristemoid initiation but is
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required for shoot development once meristemoids are formed (Jarret et al., 1981). This
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affirmation might explain why FLP increased the number of shoots produced in garlic
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calli. Moreover, it has been described that light inhibits stem elongation during
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photomorphogenesis (McNellis et al., 1995) and other work (Ait-Ali T. et al., 1999) has
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shown a rapid and reversible decrease of GA1 content in the apical shoot of etiolated
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pea seedlings upon light irradiation. Flurprimidol as light also reduces gibberellin tissue
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content (Rademacher, 2000), so the stem elongation inhibitory effect associated with
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gibberellin reduction could be related with the positive effects in the shoot
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multiplication rate shown by flurprimidol by a similar way to the increase in the
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production of shoot meristems or “multi-branching” derived of the release of apical
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dominance following decapitation or cytokinin application (Sachs et al., 1964, 1967;
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Bangerth, 1994; Li et al., 1995).
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Considering the whole regeneration process, the productivity of shoots using
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medium with 0.3 mg/l FLP was higher than the control medium only for Red type
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accession. In fact, it was higher than the productivity obtained using medium B4 that
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has been described as the best medium for that genotype (Martin-Urdíroz et al. , 2003).
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This genotype-dependent behaviour might be due to the different levels of endogenous
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gibberellins produced by each genotype.
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Shoots regenerated in media containing FLP were well-developed, and no
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hyperhydricity was observed. They were cultured for aditional 2 months as previously
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described (Barandiaran et al., 1999b), in which rooting and bulbing were achieved.
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Resulting plantlets were succesfully acclimated and transferred to soil pots. Plant
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development was comparable to field cultivated garlic.
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This work is the first describing the use of FLP on garlic in vitro culture. As it
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has been shown, moderate amounts of FLP can be included in garlic tissue culture
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media in order to increase the final shoot production per explant. However, further
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investigations are needed to optimize the concentration of FLP used in this one-step
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regeneration system for the different genotypes.
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ACKNOWLEDGEMENTS
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This work was partially funded by the national project (INIA, RF00-014) and the
EU project (QLK1-CT-1999-00498).
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FIGURES
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Fig. 1. Histogram showing the percentage of callus formation in each treatment studied.
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Fig. 2. Histogram showing the percentage of callus that underwent shoot regeneration in
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each treatment.
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Fig. 3. Histogram showing the number of shoots produced per callus that underwent
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shoot regeneration on each treatment.
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Fig. 4. Root-tips from plantlets cultivated on F0.3 medium in light (left) and in dark
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culture (right).
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Fig. 1.
90
80
70
60
50
40
30
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10
0
E111300
E213072
E432095
E112054
Light Dark Light Dark Light Dark Light Dark
A3
F0.3
F1
F5
2
3
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Fig. 2.
70
60
50
E111300
E213072
E432095
E112054
40
30
20
10
0
Light Dark Light Dark Light Dark Light Dark
A3
F0.3
F1
F5
5
6
7
8
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Fig. 3.
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16
14
12
10
8
6
4
2
0
E111300
E213072
E432095
E112054
Light Dark Light Dark Light Dark Light Dark
A3
F0.3
F1
F5
2
3
Fig. 4.
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10
20
1
2
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