Lourdes Yabor

New Pineapple Somaclonal Variants: P3R5 and Dwarf

The Food and Agriculture Organization has highlighted pineapple as one of the most important tropical fruits. Since classical pineapple breeding is difficult, biotechnology has emerged as an attractive instrument. We obtained two new pineapple somaclonal variants derived from in vitro culture of cv. Red Spanish Pinar: P3R5 and Dwarf. The AFLP analysis revealed an existing genetic distance. So far 44 phenotype indicators selected due to their relation to a wide range of important agricultural, morphological and physiological processes have been evaluated. P3R5 differed from the donor in 19 variables (19/44; 43.18%), while Dwarf varied in 31 indicators (31/44; 70.45%). The number of shoots was significantly different among the three plant materials. Dwarf showed two shoots per plant while P3R5 and the donor did not form any shoots. We also observed that water use efficiency, chlorophyll b concentration, total chlorophyll concentration, transpiration rate, chlorophyll a concentration, thickness of leaf photosynthetic parenchyma, fruit mass with crown, content of free phenolics and superoxide dismutase specific activity were also very different among the three plant materials. The Euclidean distances of each somaclonal variant to the donor plant material taking into consideration the genotype (AFLP) and the phenotype evaluations were also calculated. Regarding the genotype information, P3R5 is separated from cv. Red Spanish Pinar by 2.83 units of Euclidean distance, and Dwarf by 3.00 units. However, the phenotype indicators revealed higher differences: 3.74 in P3R5 and 4.71 in Dwarf. To our knowledge, this is the first report of a comprehensive analysis of pineapple somaclonal variants.

Biochemical side effects of the herbicide FINALE on bar gene-containing transgenic pineapple plantlets

Pineapple is one of the most important tropical fruits and therefore intensive genetic improvement programs are being carried out in many countries, including Cuba. Our research team has previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. Herein, we report on the biochemical side effects of the herbicide FINALE® on these transgenic plantlets during hardening. Levels of aldehydes and chlorophylls, and peroxidase activity were recorded. The transformed clone studied here, not sprayed with FINALE®, showed the following side effects because of transgenesis only. Levels of malondialdehyde, other aldehydes, chlorophyll b, and total chlorophyll pigments decreased. The most remarkable biochemical differences between transgenic and non-transgenic plantlets after application of FINALE® follow. Levels of malondialdehyde and other aldehydes in transgenic material were not decreased by FINALE®, perhaps because these levels were already low as a result of transformation. FINALE® increased peroxidase activity in transgenic plantlets but such increase was higher in non-transgenic material. The herbicide increased contents of chlorophyll pigments (a, b, total) in transformed plantlets. However, as expected, non-transgenic plantlets decreased levels of chlorophylls (a, b, total) after application of FINALE®. The genetic transformation of pineapple with the bar gene not only conferred resistance to the herbicide FINALE®, but also promoted other biochemical changes.

Pineapple [ Ananas comosus (L.) Merr.]

A procedure for pineapple [Ananas comosus (L.) Merr.] genetic transformation is described, which involves temporary immersion bioreactors (TIB) for selection of transgenic plants. Success in the production of transgenic pineapple plants combines tissue culture factors. Firstly, the use of regenerable pineapple callus as starting material for transformation whose cells shown to be competent for Agrobacterium infection. Secondly, the used of filtered callus, resulting in homogeneously sized clusters, thereby increasing the contact between the cell surfaces and A. tumefaciens and releasing phenolic compounds which induce Agrobacterium virulence. Thirdly, regeneration of primary plants without selection pressure, that allowing a massive production of putative transgenic pineapples. Finally, we support that TIB technology is a powerful system to recover nonchimera transgenic plants by micropropagation with the use of an adequate selection agent.

Salinity induces specific metabolic changes in sugarcane shoot explants in temporary immersion bioreactors

There is a great demand of salt-tolerant sugarcane planting material in Cuba. Temporary immersion bioreactors (TIB) are effective to significantly increase sugarcane in vitro shoot proliferation rate from 1:4 in conventional containers to about 1:35. Sugarcane micropropagation in TIBs under NaCl stress may help screen mutants with salinity tolerance. We developed the experiment shown here to identify a NaCl concentration able to stress shoot in TIBs. At 30 days of culture initiation with different NaCl levels (0 - 200 mM), explant multiplication rate, shoot cluster fresh mass, and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. Content of soluble phenolics in the culture medium was also evaluated. Addition of NaCl decreased shoot multiplication rate and fresh mass. Other statistically significant differences were recorded but the most important were noted in the increased contents of carotenoids, malondialdehyde, other aldehydes and soluble phenolics in the plants, and in the soluble phenolics in the culture medium. This research may be useful for future experiments of in vitro selection of new sugarcane genetic materials with NaCl tolerance. Fifty percent of multiplication rate was reduced with 89 mM NaCl which can be used to stress shoots during micropropagation in TIBs and eventually detect mutants with salt tolerance.

Mineral composition of a transgenic pineapple clone grown in the field for 8 yr

Pineapple is economically among the most important tropical fruits worldwide. It is cultivated on more than one million hectares, and 24.8 million metric tons of fruit are produced annually with a gross production value approaching 9 billion US

Production of pineapple transgenic plants assisted by temporary immersion bioreactors

(FAOSTAT 2015; Ming et al. 2015). However, pineapple production is increasingly threatened by biotic and abiotic factors, including microbial pathogens and sunburn. To support the future production of pineapple by improving agronomic performance, there is increasing research effort for creating new varieties with increased microbial pathogen resistances and abiotic stress tolerance (Ogata et al. 2016; Rattanathawornkiti et al. 2016). Based on this prospect, we previously developed a protocol for pineapple genetic transformation, introducing foreign DNA into the pineapple genome. These were a chitinase gene originally from Phaseolus vulgaris; a DNA-sequence designated AP24 that codes for an anti-Phytophthora protein that destabilizes the fungal cell membrane and was cloned from Nicotiana tabacum; and the bar gene naturally found in different actinobacteria, encoding for resistance against the herbicidal compound phosphinothricin (Thompson et al. 1987). The genes were under control of the following promoters: OCS-35S CaMV-rice actin I, 35S CaMV, and maize Ubi1, respectively (Espinosa et al. 2002). While the intended function of these genes inserted into the pineapple genome are well known, unintended effects caused by the genetic transformation of pineapple remain to be characterized, before fruit or other parts of such genetically modified varieties can be considered and approved for consumption by humans or animals (Kuiper et al. 2001; Hossain 2016). While there are already abundant data on the extent or lack of unintended effects for GM crops cultivated mainly under moderate climatic conditions, like maize, soybean, potato, or other crops, data on unindended effects of genetically modified tropical crops, including pineapple, are scarce or do not exist at all. In previous studies, we have evaluated such transformed pineapple plants during hardening and 7 yr of field growth evaluating the third vegetative generation (Yabor et al. 2006; Yabor et al. 2008; Yabor et al. 2010; Yabor et al. 2016). The study presented here focused on the analysis of the mineral composition of transformed pineapple leaves and fruit after 8 yr of ex vitro development, analyzing the fourth vegetative generation of the same transgenic clone and controls. Minerals are an important factor to be considered for food quality because they play key roles in human and animal metabolisms (Fruton and Simmonds 1963; Pérez-Massot et al. 2013).