Fernando Pliego-Alfaro

Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene

Strawberry (Fragaria ×ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.

Evidence for a positive regulatory role of strawberry (Fragaria×ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance

Knowledge of the molecular basis of plant resistance to pathogens in species other than Arabidopsis is limited. The function of Fa WRKY1, the first WRKY gene isolated from strawberry (Fragaria x ananassa), an important agronomical fruit crop, has been investigated here. Fa WRKY1 encodes a IIc WRKY transcription factor and is up-regulated in strawberry following Colletotrichum acutatum infection, treatments with elicitors, and wounding. Its Arabidopsis sequence homologue, At WRKY75, has been described as playing a role in regulating phosphate starvation responses. However, using T-DNA insertion mutants, a role for the At WRKY75 and Fa WRKY1 in the activation of basal and R-mediated resistance in Arabidopsis is demonstrated. At wrky75 mutants are more susceptible to virulent and avirulent isolates of Pseudomonas syringae. Overexpression of Fa WRKY1 in At wrky75 mutant and wild type reverts the enhanced susceptible phenotype of the mutant, and even increases resistance to avirulent strains of P. syringae. The resistance phenotype is uncoupled to PATHOGENESIS-RELATED (PR) gene expression, but it is associated with a strong oxidative burst and glutathione-S-transferase (GST) induction. Taken together, these results indicate that At WRKY75 and Fa WRKY1 act as positive regulators of defence during compatible and incompatible interactions in Arabidopsis and, very likely, Fa WRKY1 is an important element mediating defence responses to C. acutatum in strawberry. Moreover, these results provide evidence that Arabidopsis can be a useful model for functional studies in Rosacea species like strawberry.

Antisense Down-Regulation of the FaPG1 Gene Reveals an Unexpected Central Role for Polygalacturonase in Strawberry Fruit Softening

The strawberry (Fragaria × ananassa ‘Chandler’) fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening.The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria x ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.

SummaryAn efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.

Somatic embryogenesis in mango (Mangifera indica L.)

The mango Mangifera indica L. is considered to be one of the most important fruit crops of the world (Fig. 1). Its annual production is exceeded only by Musa (bananas and plantains), citrus, grapes and apples (FAO Production Yearbook, 1990). Currently, it is the most important fruit crop of Asia. Mango is in the Anacardiaceae family, which includes several other tropical and subtropical tree species of economic importance, e.g., cashew Anacardium occidentale, pistachio Pistachia vera, and several Spondias species, including S. cytherea, S. mombin and S. purpurea. The center of diversity for the genus Mangifera is in southeast Asia, with the greatest number of species being found on the island of Borneo. According to Mukherjee (1985), there are 39 currently recognized Mangifera species. The mango is the most widely cultivated species within the genus, and has a natural distribution throughout outheast Asia. It is found in forests as far west as the Indo-Burman region (Assam). There appear to be two distinct geographical races of the species: a polyembryonic race that occurs in the tropical rainforest of southeast Asia and a monoembryonic race that is associated with India. Some taxonomists have suggested that the mango originated in India, because of the ancient association between Indian culture and religion with this fruit. It has also been noted that the word for mango used throughout Asia is derived from the Tamil “maanga”.

Effects of CO2 and sugars on photosynthesis and composition of avocado leaves grown in vitro

The effects of micropropagation conditions on avocado (Persea americana Mill.) have been measured in leaves and plants cultured in vitro. The consequences of the type and concentration of sugar in the medium and of carbon dioxide concentration in the atmosphere on the rates of photosynthesis and amounts of ribulose 1,5-biphosphate carboxylase-oxygenase (EC 4.1.1.39; Rubisco) and total soluble protein (TSP) were measured. At the highest sucrose supply (87.6 mM), Rubisco content was substantially decreased in leaves, and even more when elevated CO2 (1 000 μmol · mol-1) was supplied. Maximum photosynthetic rate (P(max)) was significantly decreased when plants developed in high sucrose and elevated CO2. However, Rubisco concentration was significantly greater when glucose was supplied at the same molar concentration or when the concentration of sucrose was small (14.6 mM), and no differences were observed due to the CO2 concentration in the air in these treatments. The ratio of Rubisco to total soluble protein (Rubisco/TSP) was dramatically decreased in plants grown in the highest concentration of sucrose and with elevated CO2. Leaf area and ratio of leaf fresh weight/(stem + root) fresh weight, were greater in plants grown with CO2 enriched air. However, upon transplanting, survival was poorer in plants grown on low sucrose/high CO2 compared to those grown on high sucrose/high CO2.

Micropropagation of adult avocado

A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock. Cultures were initiated from basal shoots obtained after pruning a tree back to ground level. Buds sprouted in Murashige and Skoog solid medium with macroelements at half strength and a 1.3 μM benzyladenine supplement. To induce proliferation, shoots were cultured for 2 weeks in liquid medium in a rollordrum with the Gamborg salt formulation and 1.3 μM benzyladenine, followed by 6 weeks in double phase conditions (solid medium with a layer of liquid medium on the top) using the same salt formulation and two different benzyladenine supplements, 2.8 μM in the solid phase and 0.4 μM in the liquid phase. Ninety percent of the shoots rooted after a 3-day culture in liquid medium in a rollordrum with MS macroelements at 0.3× and 4.9 μM indolebutyric acid, followed by transfer to solid medium in the absence of auxin but with 1 g l−1 activated charcoal. Survival rate during the acclimatization in the greenhouse was 70%.

Agrobacterium cells as microprojectile coating: a novel approach to enhance stable transformation rates in strawberry

The effect of combining Agrobacterium tumefaciens infection and biolistic bombardment on the transformation of strawberry (Fragaria × ananassa Duch.) cv. Chandler, was evaluated. Bombarding leaf explants with uncoated gold particles followed by Agrobacterium infection did not improve transformation, and yielded similar percentages of shoot regeneration in the presence of kanamycin in bombarded and non-bombarded explants (7.2%). In a novel approximation, gold particles were coated with Agrobacterium cells and used to bombard leaf explants. Helium pressures of 4.5, 6.2 and 7.6 MPa and target distances of 3 and 9 cm were tested. An average of 96.2% of the explants showed β-glucuronidase (GUS) expression 15 d after bombardment, in comparison with 26.6% in explants bombarded with gold particles coated with the plasmid pGUSINT or 58.3% in non-bombarded Agrobacterium-infected explants. After 25 weeks of culture, the highest transformation frequency was obtained using a 6.2 MPa helium pressure and 3 cm target distance, yielding 69% kanamycin-resistant explants and a final transformation fre-quency of 20.7%. These values were 4.5 times higher for kanamycin-resistant explants (69% with biolistic vs 16% with Agrobacterium infection) and 2.9 times higher for transformation frequency (20.7 vs 7%,) compared with those obtained with standard Agrobacterium transformation procedures (Barcelo et al. 1998, Plant Cell, Tiss. Org. Cult. 54, 29–36). More than 15 independent transgenic plants obtained by the Agrobacterium-coated particle system were acclimatized and confirmed as transgenics by GUS activity and PCR. Segregation analysis of kanamycin resistance has been performed in seven independent lines, three of which contained a single insertion of the T-DNA.

A convenient protocol for extraction and purification of DNA from Fragaria

SummaryIn this paper we describe a simple and efficient DNA extraction protocol for Fragaria species, a highly recalcitrant genus due to the large amount of polyphenols and polymeric carbohydrates present in strawberry tissues. The protocol yields a high quality DNA that can be amplified by polymerase chain reaction and digested with restriction endonucleases.

Variations in storage protein and carbohydrate levels during development of avocado zygotic embryos

Variations in carbohydrates and proteins were monitored during avocado (Persea americana Mill.) zygotic embryo development and correlated with growth parameters in order to define specific markers characterizing distinct embryogenic phases. Hexose (glucose and fructose) levels were initially high and declined as embryo development advanced reaching the lowest levels in completely mature embryos. Sucrose and starch evolution showed an opposite trend with a progressive increase during embryo growth. The beginning of the maturation phase could be identified by a switch in the carbohydrate status from high hexose/sucrose ratio to low hexose/sucrose ratio. Storage protein accumulation began at early cotyledonary stages (7–8 mm), increasing significantly in the maturation phase where they represented 83% of total proteins. Mature embryos (38–40 mm) contained albumins, globulins and glutelins, albumins being the predominant and most heterogeneous fraction. Storage protein accumulation occurred in a sequential and specific way suggesting a possible role as indicators of embryo development. The complete maturation stage could be characterized by the synthesis and accumulation of a 49 kDa albumin.