Towards genetic transformation of local onion varieties
Financial support: Secretaría de Ciencia y Tecnología, UNS, Agencia Nacional de Promoción Científica y Tecnológica (PICT 08-12671) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
Keywords: Agrobacterium tumefaciens, Allium cepa L., callus, zygotic embryos.
The aim of this work
was to explore the possibility of obtaining transgenic plants of onion
varieties cultivated in
On the frame of a good strategy directed to strengthen the genetic traits introduced by transformation, transgenes should be introduced in plant cultivars which are agronomically superiors and susceptible of being introduced in new plant breeding plans (Lydiate et al. 1995).
Eady et al. (2000), first reported an onion transformation protocol, based on immature zygotic embryos as target tissue. A drawback for this approach is that explant availability is limited to a short period during the year. Recently, Zheng et al. (2001) reported a reliable transformation protocol for onion and shallot (Allium cepa L.) using Agrobacterium tumefaciens as a transfection vector, and three-week old calli induced from mature zygotic embryos as target tissue. Since seeds are both easy to get and to preserve, for a successful onion transformation would be advantageous to use calli derived from mature zygotic embryos as target explant, because they can be used year-round (Zheng et al. 1998; Zheng et al. 1999).
the aim of this work was to explore the possibility of obtaining onion
transgenic plants from varieties cultivated in
zygotic embryos from three onion varieties of Valenciana, i.e.
Torrentina, Cobriza INTA and Grano de Oro, were in vitro cultivated
in a callus inductive medium during three culture periods, 40 days
each. The MSc was Murashige and Skoog (1962) which
also contained 1 mgL-1 2,4- dichlorophenoxiacetic acid,
0,1 mgL-1 6-bencilaminopurine, 30 gL-1 sucrose
and 8 gL-1 agar, pH 5,8. Thousand and fifty ME were cultivated:
662, 178 and 210 of the Torrentina, Cobriza INTA and Grano de Oro
varieties, respectively. The cultivation was carried out in Petri
dishes each containing 25 mL culture medium and 30 ME were sowed per
Petri dish. During the cultivation period, Petri dishes were kept
The transformation was carried out with either the Agl1 or the LBA 4404 strains of A. tumefaciens, both strains carrying a binary vector which contains the gene marker gus a (gus intron) and the NPTII gene for selection, both under the control of the 35 S promoter.
The Agl1 strain was cultivated in medium containing 5 gL-1 mannitol, 1 gL-1 glutamine, 5 gL-1 Triptone, 2.5 gL-1 yeast extract, 0.25 gL-1 KH2PO4, 0.1 gL-1 NaCl and 0.1 gL-1 MgSO4 7H2O at pH 7. One hundred mgL-1 carbenicilin and 50 mgL-1 kanamicin were added as selection antibiotics for the bacterium and plasmid, respectively. The LBA 4404 strain was cultivated in minimal medium (Lichtenstein and Draper, 1986) containing 100 mgL-1 rifampicin and 300 mgL-1 streptomicin as bacterial selection antibiotics and 50 mgL-1 kanamicin for plasmid selection.
transformation, groups of approximately 30 calli were cut into small
portions which were inoculated with 4 mL of the bacterial suspension
(OD600 0.5 – 0.7), allowing contact for 10 min. Then the
bacterial suspension was removed and a sterile absorbent paper was
used to eliminate the bacterial suspension excess by gently blotting.
The calli sections were then transferred to MSc medium. After four
days of co-cultivation, the calli were transferred into the selection
medium, i.e. MSc containing 10 mgL-1 geneticin and
300 mgL-1 cefotaxime. The callus portions showing growth
were then selected from each subculture and transferred into MSr (Murashige
and Skoog medium to which 1 mgL-1 of 6 - (γ, γ
Callus induction, during three to four months, was quite variable among varieties, being particularly low (37%) for Torrentina (Table 1). Calli were mainly of the compact type. Great variability in callus induction and plant regeneration among genotypes was reported previously (Tanikawa et al. 1998; Zheng et al. 1998; Zheng et al. 1999) and also in local varieties (Marinangeli et al. 2005).
the end of the selection period 342 portions of calli were transferred
to the regeneration medium, 73 came from the transformation with
Agl1 strain and 269 from the transformation with LBA
The fact of being able to recover calli of only one of the three varieties demonstrates the genotypic variability that this onion varieties exhibited in the tolerance to the antibiotic geneticin and, in this way, to the transformation mediated by A. tumefaciens. Zheng et al. (2001) demonstrated that both subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and even some varieties better than others.
different quantity of selected calli was also obtained from the transformation
with each one of the two A. tumefaciens strains. Probably this
may not be the result of a difference in the efficiency of the transgenes
transfer to the Torrentina variety, but of a higher aggressiveness
of the Agl1 strain which produced death of calli in the first stages
of the selection and high proportion of bacterial overgrowth (data
not shown). In fact, Zheng et al. (2001) found not
differences in onion transformation efficiency working whit the super-virulent
EHA 105 and the ordinary LBA
Forty two per cent of selected calli evaluated through the glucuronidase expression, presented extensive blue stained areas or were completely blue. On the other hand, the tissues evaluated at the end of the regeneration period did not presented neither partial nor total blue coloration.
The production of a high proportion of calli which were unable to express or just partially express the gus gene, could be the result of a low selection pressure which could otherwise produce undesirable transgenic chimerical plants. This limitation could probably be removed by using a higher concentration of the selection agent geneticin from the beginning of selection or even in later culture transfers. Zheng et al. 2001 proved that geneticin was not successful as selective agent in ME derived calli transformation. On the other hand, he proved hygromicin as effective selective antibiotic.
Although it was not possible to recover transgenic plants yet, this work clearly represents an advance in the endeavour of obtaining a genetic transformation from a local variety of onion, as it was shown by recovering calli exhibiting a marked glucuronidase expression.
Differences in sensitivity to geneticin among varieties and poor in vitro plant regeneration rate shown by this species would be the main limiting steps in the whole process of obtaining transgenic plants of onion local varieties.
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