María del Socorro Santos-Díaz

PEG-tolerant cell clones of chili pepper: Growth, osmotic potentials and solute accumulation

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 μM 2,4-d and 0.44 μM BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5–10% PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K+ was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3–3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.

IN VITRO ORGANOGENESIS OF PELECYPHORA ASELLIFORMIS ERHENBERG (CACTACEAE)

SummaryIn vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.

Lead, Chromium and Manganese Removal by in Vitro Root Cultures of Two Aquatic Macrophytes Species: Typha Latifolia L. and Scirpus Americanus Pers.

The ability of in vitro roots cultures of Typha latifolia and Scirpus americanus to remove metals was studied. Roots were cultivated on Murashige-Skoog medium with 15 μg L−1 Cr III, 60 μg L−1 Pb II or 1.8 mg L−1 Mn II. Adsorbed metal to root surface was removed by washing with 0.042% HNO3. T. latifolia roots were able to uptake 68.8 μg Pb g−1, 22.1 μg Cr g−1 and 1680 μg Mn g−1, while the S. americanus roots removed 148.3 μg Pb g−1, 40.7 μg Cr g−1 and 4037 μg Mn g−1. About 80–90% of Pb and Cr were absorbed in both cultures. On the contrary, the Mn removal was due mainly to an adsorption process (82–86%). In comparison to the T. latifolia cultures, S. americanus cultures were two fold more efficient to remove Pb and Cr, and three fold more efficient to remove Mn. Both plant species capture metals in the following order: Cr >Pb >Mn. This investigation confirms that in vitro roots cultures could be an alternative as a phytoremediation approach for contaminated water with heavy metals.

Induction of in vitro roots cultures of Thypha latifolia and Scirpus americanus and study of their capacity to remove heavy metals

We have established the conditions to obtain in vitro root cultures of Thypha latifolia and Scirpus americanus and have investigated their capacity to remove Pb(II), Mn(II) and Cr(III) from the culture medium. The best conditions for the in vitro culture growth were: an inoculum of 0.2 g of T. latifolia roots and 0.05 g of S. americanus roots (fresh weight), Murashige-Skoog medium and 2 mg L -1 of indolacetic acid. The T. latifolia and S. americanus root cultures were cultivated onto media containing Cr (15 µg L -1 ), Pb (60 µg L -1 ) or Mn (1.8 mg L -1 ). Both species were able to remove Pb and Cr near to 100% and 71-100% of Mn from the medium solution during the 6-8 days of experimentation. According to metal concentrations removed from the medium containing the growing root mass, the in vitro root culture of S. americanus can be considered as an accumulator for Pb (157.73 µg g -1 ), Cr (55.6 µg g -1 ) and Mn (5000 µg g -1 ).

Tissue culture of ornamental cacti

Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.

Fluoride bioaccumulation by hydroponic cultures of camellia (Camellia japonica spp.) and sugar cane (Saccharum officinarum spp.).

The ability of hydroponic cultures of camellia and sugar cane adult plants to remove fluoride was investigated. Plants were grown in a 50% Steiner nutrient solution. After an adaptation period to hydroponic conditions, plants were exposed to different fluoride concentrations (0, 2.5, 5 and 10 mg L(-1)). Fluoride concentration in the culture medium and in tissues was measured. In sugar cane, fluoride was mainly located in roots, with 86% of it absorbed and 14% adsorbed. Sugar cane plants removed 1000-1200 mg fluoride kg(-1) dry weight. In camellia plants the highest fluoride concentration was found in leaf. Roots accumulated fluoride mainly through absorption, which was 2-5 times higher than adsorption. At the end of the experiment, fluoride accumulation in camellia plants was 1000-1400 mgk g(-1) dry weight. Estimated concentration factors revealed that fluoride bioaccumulation is 74-221-fold in camellia plants and 100-500-fold in sugar cane plants. Thus, the latter appear as a suitable candidate for removing fluoride from water due to their bioaccumulation capacity and vigorous growth rate; therefore, sugar cane might be used for phytoremediation.

Comparative shotgun proteomic analysis of wild and domesticated Opuntia spp. species shows a metabolic adaptation through domestication.

UNLABELLED The Opuntia genus is widely distributed in America, but the highest richness of wild species are found in Mexico, as well as the most domesticated Opuntia ficus-indica, which is the most domesticated species and an important crop in agricultural economies of arid and semiarid areas worldwide. During domestication process, the Opuntia morphological characteristics were favored, such as less and smaller spines in cladodes and less seeds in fruits, but changes at molecular level are almost unknown. To obtain more insights about the Opuntia molecular changes through domestication, a shotgun proteomic analysis and database-dependent searches by homology was carried out. >1000 protein species were identified and by using a label-free quantitation method, the Opuntia proteomes were compared in order to identify differentially accumulated proteins among wild and domesticated species. Most of the changes were observed in glucose, secondary, and 1C metabolism, which correlate with the observed protein, fiber and phenolic compounds accumulation in Opuntia cladodes. Regulatory proteins, ribosomal proteins, and proteins related with response to stress were also observed in differential accumulation. These results provide new valuable data that will help to the understanding of the molecular changes of Opuntia species through domestication. BIOLOGICAL SIGNIFICANCE Opuntia species are well adapted to dry and warm conditions in arid and semiarid regions worldwide, and they are highly productive plants showing considerable promises as an alternative food source. However, there is a gap regarding Opuntia molecular mechanisms that enable them to grow in extreme environmental conditions and how the domestication processes has changed them. In the present study, a shotgun analysis was carried out to characterize the proteomes of five Opuntia species selected by its domestication degree. Our results will help to a better understanding of proteomic features underlying the selection and specialization under evolution and domestication of Opuntia and will provide a platform for basic biology research and gene discovery.

Clonal propagation of Turbinicarpus laui Glass & Foster, a cactus threatened with extinction

Summary: This work describes the in vitro propagation of Turbinicarpus laui, a Mexican cactus considered as threatened with extinction. In vitro cultures were initiated using seeds. Five shoots per explant of T. laui were obtained on MS medium with 2.2 μM benzyladenine and two shoots on medium with 4.4 μM benzyladenine (P < 0.05) at 40 days. Quality of shoot was recorded as percentage of callus and hyperhydricity. The presence of callus was similar in both media at 20 days, but a reduction was observed at 40 days. A higher number of shoots with low hyperhydricity was obtained on medium with 4.4 μM benzyladenine. The development of shoots (measured as increase on epicotyl length, and fresh and dry weight after 6 weeks) was similar in both media. A continuous increase (15 to 20%) of new shoots was observed after subcultures and in about 1 year 1,200 well defined shoots of T. laui were obtained. Rooting of T. laui shoots was observed on media MS containing 1% polyethyleneglycol and 1% activated charcoal and the survival of the plants to soil was 80%. The first flowering was observed after 2 years in greenhouse conditions.

Removing Heavy Metals by In Vitro Cultures

In vitro roots cultures of Typha latifolia and Scirpus americanus aquatic plants have the capacity to remove Pb (II), Mn (II), and Cr (III) from the culture medium. Both species remove Cr and Pb by an absorption process, while Mn is mainly adsorbed to the root surface. This chapter describes a protocol for the establishment of in vitro roots cultures (nontransformed) from T. latifolia and S. americanus, and the procedure for the uptake analysis of Pb (II), Mn (II), and Cr (III) by roots.

Effect of coconut water, darkness and auxins on morphogenesis of Ariocarpus kotschoubeyanus (Cactaceae)

Summary: This study describes a method to micropropagate Ariocarpus kotschoubeyanus, using coconut water and darkness conditions. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium (MS) with 2 mg/l zeatin. The calli generated were transferred to medium containing 5 ml, 10 ml or 15 ml of coconut water per litre. After 84 days, five green shoots per callus were generated under photoperiod but 14 well defined shoots were produced in darkness. The shoots were cultivated on media with activated charcoal and polyethyleneglycol to reduce callus and hyperhydricity, respectively. To induce rooting the effect of the auxins indolacetic acid and indolbutyric acid (1 mg/l) were tested. The roots type II (20 to 30 mm) and type III ( > 30 mm) were observed after 120 days onto media containing auxins but not in control medium. The regenerated plants were transferred to horticultural earth, peat moss and sand (30:30:40) and the survival of the plants in soil was 80%. The plant adaptation was complete and the first flowering was observed after 1 year in greenhouse conditions. The procedure developed is an alternative to the successful propagation of A. kotschoubeyanus and reduces the risk of extinction of this species.