Nuria Alburquerque

Agrobacterium -mediated transformation of apricot ( Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.

Apricot flower bud development and abscission related to chilling, irrigation and type of shoots

Abstract Flower bud drop dramatically affects productivity in some apricot cultivars. The influence of different chilling and irrigation conditions on flower bud development and drop of the apricot cv. ‘Guillermo’ was studied. Flower buds from trees grown in three places, with different winter conditions, were collected periodically to compare their developmental stage. Slight differences in the developmental pattern were observed between the two coldest places. Just before bloom, flower buds from the warmest place were found to be more delayed. Also, flower bud development was compared in three irrigation treatments. The lack of autumn irrigation induced a slow development, but when irrigation was reinstated, the flower bud development was hastened. The suppression of irrigation in winter did not have an apparent effect on flower bud development compared with the more irrigated treatment. In flower buds of short shoots no important abnormalities were found, but they were in the analysed flower buds of long shoots. In ‘Guillermo’ cultivar a serious drop was recorded in every location or treatment. However, no correlation was found between accumulation of chill unit or irrigation treatments and flower bud drop or fructification. In long shoots, the heavy flower bud drops (more than 90%), together with the high number of malformed flower buds, indicate that they do not produce an acceptable fruit set in this apricot cultivar.

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of nptII-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20μM paromomycin was used, although up to 40 μM streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7 μM kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.

Consequences to fertilization of the developmental stage of apricot ovules at anthesis

Summary Flowers at anthesis from the apricot cvs Palstein and Goldrich were examined to determine their ovule maturity stage. Further, flowers from both cultivars were pollinated and collected after 2000 and 3000 growing degree hours (GDH) in two consecutive years. Numbers of fertilized ovules were recorded in those flowers. Ovules of ‘Palstein’ from flowers at anthesis showed predominantly an embryo sac at the eight nuclei stage of development while embryo sacs in ‘Goldrich’ were mainly in a four nuclei stage. After 2000 GDH from pollination 90% of viable ovules were fertilized in ‘Palstein’ whereas only 37.5% of them were fertilized in ‘Goldrich’. After 3000 GDH, Goldrich presented the same percentage of fertilized ovules that was recorded after 2000 GDH. Non-fertilized ovules did not degenerate and developed normally in pollinated and non-pollinated flowers from both cultivars. Further, non-fertilized pollinated flowers were not different from non-pollinated flowers in the development of the embryo sac, indicating that the chances for fertilization would not be increased by pollination in apricot. We suggest that the lack of fertilization in ‘Goldrich’ is due to degeneration of the male gametophyte, which would have a limited life, not long enough for the embryo sacs to develop to maturity.

Protective effect of three brown seaweed extracts against fungal and bacterial diseases of tomato

The effect of extracts of the brown algae Cystoseira myriophylloides, Laminaria digitata, and Fucus spiralis against the tomato pathogens Verticillium dahliae and Agrobacterium tumefaciens was evaluated in vitro and in the greenhouse. A significant inhibition of growth was observed only with methanolic seaweed extracts (MSE). Disease resistance was assessed in the greenhouse against Verticillium wilt using spray application of aqueous seaweed extracts (ASE) on the whole plant or using seed imbibition. Both methods significantly reduced disease severity whatever the algal species, though protection observed after seed treatments was higher than that observed after spray treatment. Spray application of ASE from C. myriophylloides and F. spiralis also resulted in significant reduction of Crown gall disease caused by the bacterial pathogen A. tumefaciens. ASE-treated plants had significantly higher levels of activity of defense enzymes polyphenol oxidase and peroxidase compared to the control. ASE did not inhibit mycelium growth of V. dahliae or development of A. tumefaciens in vitro; it is therefore suggested that induced resistance is probably the main mechanism of protection afforded by ASE.

Media derived from brown seaweeds Cystoseira myriophylloides and Fucus spiralis for in vitro plant tissue culture

In the present study, the effect of seaweed extract (SE) from Fucus spiralis (Fs), Cystoseira myriophylloides (Cm) and Laminaria digitata (Ld) on in vitro plant tissue culture was examined. Combination of 25 % of SE from Cm with 25 % of MS medium increased adventitious shoot regeneration from Nicotiana benthamiana leaf discs explants by 620 %, when compared to the conventional regeneration medium. Similarly SE from Fs and Ld enhanced regeneration by about 500 %. However, when increasing SE to 50 %, only Cm significantly enhanced shoot regeneration. The effect of SE was also evaluated on in vitro micropropagation of N. benthamiana, grape, plum and apricot by assessing shoot length, number of leaves and internodes. When used alone but at lower concentrations (2.5 and 12.5 %), SE from Fs and Cm resulted in at least the same efficacy as MS alone for micropropagation of N. benthamiana shoots. However, for micropropagation of grapevine, plum and apricot woody plants, a combination of 50 % of SE from Cm or Fs with 50 % of their conventional micropropagation media was necessary. Rooting was also enhanced in N. benthamiana and grapevine, and was correlated with their higher concentrations of indole acetic acid when compared to SE from Ld. This finding, in addition to mineral analysis data, suggests that SE of Fs and Cm contain necessary nutrients and growth regulators to allow their use as medium for in vitro plant culture.

New Transformation Technologies for Trees

Application of transgenic techniques to trees has emerged as a powerful tool for their genetic improvement. However, for some recalcitrant or transformation time-consuming species transgenic research should be strengthened and further efforts will be necessary to improve regeneration and transformation efficiencies. This review focuses on the recent advances and techniques for genetic manipulation that can be applied to obtain transgenic trees with enhanced biosafety. After selection of transformed plants, marker genes presence becomes useless and undesirable. For generation of marker-free transgenic plants or resolving complex transgene integration structures, several methodologies based on site-specific recombination have been developed. Precise homologous recombination-mediated integration of a DNA sequence of interest at a particular site within a genome is the ultimate tool for genetic engineering. Genome editing allows a much more precise manipulation of tree genomes. Expression of multiple transgenes is often required for engineering metabolic pathways. Recent progress has made powerful techniques available and gene stacking can be achieved in trees by two methodologies: co-transformation and serial transformation. The application of technologies based on small RNAs allowing silencing target genes (RNA interference, artificial microRNAs, and artificial trans-acting siRNAs) is of particular interest to produce directed down-regulation of target genes in tree species. Mixing of genetic materials between species that cannot hybridize by natural means is one of the major criticisms to transgenic crops. Cisgenesis and intragenesis were developed as alternatives to transgenesis.

Semisynthetic Triterpenes Derived from Euphorbia officinarum as Plant Growth Promoters and Inducers of Disease Resistance

This study evaluated the effect of application of the semisynthetic triterpenes 3β-acetoxy-norlup-20-one (F4) and 3-chloro-4α,14α-dimethyl-5α-cholest-8-ene (F6) triterpene derivatives from Euphorbia officinarum on the growth of tomato seedlings under normal conditions and when challenged with the pathogens Verticillium dahliae and Agrobacterium tumefaciens. Foliar spray of F4 and F6 significantly improved growth rate, fresh weight, dry weight, and leaf area. In addition, they enhanced several physiological parameters including photosynthetic pigments, proline content, and nitrate reductase activity. Moreover, they induced H2O2 accumulation and increased the activity of several antioxidant enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase. They also enhanced disease resistance against V. dahliae and A. tumefaciens. These results suggest that the two semisynthetic triterpenes represent new plant growth regulators and inducers of plant disease resistance.

Towards the valorization of the invasive seaweeds Caulerpa cylindracea and Asparagopsis taxiformis in the Mediterranean Sea: applications for in vitro plant regeneration and crop protection

Caulerpa cylindracea and Asparagopsis taxiformis are both invasive seaweeds representing a major threat to indigenous species and the native ecosystems of certain regions of the Mediterranean Sea. This work examines the possibility of adding value to this abundant biomass in the fields of in vitro culture and plant protection. Results showed that using a 10% aqueous extract from C. cylindracea and A. taxiformis to in vitro culture media enhanced the regeneration percentage of apricot hypocotyl slices, as well as the diameter of explants and the number of regenerating buds per explant. The combination of extracts from these two seaweeds with an increasing concentration of conventional plant growth regulators further significantly enhanced most of these parameters. In addition, the data showed that soaking seeds of Nicotiana benthamiana in a 1.5% solution of aqueous extracts of C. cylindracea resulted in seedlings with reduced disease severity, when inoculated with the plum pox virus. These observations demonstrated that it would be possible to transform and perhaps manage the biomass of these invasive algae, which could pave the way to multiple valorizations.