A Comparative Analysis between Totipotency and Growth Environment Conditions of the Donor Plants in Tissue Culture of Zea mays L

Immature embryos of four cultivars of maize (Zea mays L.), two inbreds and their simple hybrid in direct and inversed version were cultured by the atrophic tissue method (Torné et al. 1984) in a medium containing 2,4-D in order to obtain totipotent callus. With this method, we cultivated embryos of 14 To 22 days postpollination for three consecutive years. Each experiment was done with 50 embryos. The in vitro conditions were the same for all the experiments and for all the years. The only variable was that the donor plants were grown in the field under natural conditions. A simple statistical analysis was carried out in order to compare. I) The differences in totipotency observed: a) within the same year, demonstrating significant differences at the same age between some inbreds and hybrids, and also with hybrids at certain ages; b) comparing the three years, showing significant differences in some of the hybrids at the same age. II) The differences in embryo development comparing the three years; embryos of the same age are significantly different in size according to the year of experimentation. We also observed that size of direct and inverted hybrid embryos is significantly different especially in the hottest year. It is concluded that the same embryo size does not give the same totipotency and that this depends on the growth environment conditions of the donor plants.Peer reviewe

Effect of Exogenous Arginine, Ornithine, Methionine and γ-Amino Butyric Acid on Maize (Zea Mays L.) Embryogenesis, and Polyamine Content

The influence of four exogenous amino acids related to polyamine metabolism (γ-aminobutyric acid, arginine, methionine and ornithine) on maize (Zea mays L.) somatic embryogenesis was investigated. The endogenous polyamine contents of the treated calli were analyzed, and arginine and ornithine decarboxylase activities were determined. An established embryogenic callus (Type 1) of the inbred W64Ao2 was used. The endogenous polyamine content of calli was increased by addition of all four amino acids tested and the levels of spermidine plus spermine were higher than those of putrescine in all cases. Upon the addition of 2 mM arginine to the culture medium a 25% increase in embryogenesis was observed. Moreover, arginine decarboxylase (ADC) activity was significantly improved and ornithine decarboxylase (ODC) activity was also raised. The addition of 1.5 mM ornithine also increased embryogenic callus production, and ODC and ADC activities. Nevertheless, this increase was not as marked as in the case of arginine. This study indicates that the addition of amino acids, which are precursors of polyamine synthesis (especially Arg but also Orn), may be used to improve the rate of embryogenic callus production in an auxin-established maize culture system. A possible explanation of this effect is also dicussed.Peer reviewe

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Research work was supported by a grant from CICYT: BIO 088-0215.Peer reviewe

Somatic embryogenesis in Araujia sericifera

This work presents the preliminary results of in vitro studies with Araujia sericifera, which is cultivated for ornamental purposes. Immature seeds from wild plants were used to start the cultures. Somatic embryos and friable embryogenic calluses were obtained from white cotyledons in media containing naphthaleneacetic acid and benzyladenine or 2,4-dichlorophenoxyacetic acid. Plants were regenerated from these somatic embryos. Cell suspensions obtained from friable calluses cultured in M1 modified medium showed a considerable growth capacity. The packed cell volume was doubled in about 15 days of culture at the exponential phase. the results obtained may be used to design further experiments with the aim of improving somatic embryogenesis.This work was supported by a grant from CICYT (Bio 88-0215).Peer reviewe

Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.This work has been supported by a grant from DGICYT PB-92-0087.Peer reviewe

Methods of obtaining maize totipotent tissues. II. Atrophic tissue culture

Placing Zea mays L. immature embryos of different cultivars in modified Murashige and Skoog (MS) medium with 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and in light, anomalous germinations were produced and the so-called 'atrophic tissues' resulted. These cultivated in the same medium produced two types of totipotent formations: meristematic and yellowish-white callus, both proved capable of plant regeneration. The differentiation medium used contained 0.25-0 mg/l of 2,4-D plus 1 mg/l naphthaleneacetic acid (NAA) and 0.05 mg/l 6-(γ,γ-dimethylalylamino) purine (2iP). This is the method at present used in our laboratory for all in vitro maize cultures.Peer reviewe

Regeneration of plants from mesocotyl tissue cultures of immature embryos of Zea mays L

Callus cultures have been established from mesocotyl tissues taken from immature embryos of one experimental hybrid homozygous fl2. They were initiated and maintained on a modified Murashige and Skoog medium. Regeneration of Complete plants from these calluses was accomplished by a subculture of the callus on a medium containing 0.25 mg 2,4-D and NAA for 40 days. Afterwards they were transformed to a medium containing NAA and 2iP free of 2,4-D.Peer reviewe

Methods of obtaining maize totipotent tissues. I. Seedling segments culture

From nine cultivars of Zea mays L. through in vitro culture of segments of seedlings from matured seeds, we tried to regenerate plants. The following factors were taken in consideration: (1) type of seed germination; (2) excision zone; (3) position of the inoculum in the medium. In order to obtain callus, a Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic a acid (2,4-D) was used. To induce the differentiation the 2,4-D concentration was reduced, and naphthaleneacetic acid (NAA) and 6-(γ,γ-dimethylallylamino)purine (2iP) were added. Plant regeneration took place in only one of the cultivars: hybrid H-113, and always from inoculi that contained the meristem of the seedling node. Neither the effect of the type of germination nor the position of the inoculum in the medium was conclusive for the obtention of callus and plant differentiation. A very interesting point of this work is that the totipotent maize tissue structure obtained is very different from any previously described. We have designated it 'meristematic callus'.Peer reviewe

Effect of DL-Alpha-Difluoromethylornithine Pretreatments in Maize Callus Differentiation

The effect of pretreatments with DL-alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of the ornithine decarboxylase (ODC) activity, in plant differentiation, polyamine (PA) and amino acid contents of maize callus cultures was investigated. This study indicates that DFMO pretreatments can be used to improve regenerative response from maize callus cultures. These findings may also be useful in other recalcitrant cultures.Peer reviewe

Rice (Oryza s.) transformation by the maize transglutaminase (TGZ21) gen

Trabajo presentado en el 5th International Meeting on Biotechnology, organizado por BioSpain en septiembre de 2010