José Carlos Lorenzo

Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems

Abstract A procedure for the mass propagation of pineapple plants (Ananas comosus L. Merr) using a temporary immersion technique is described. This procedure involved three distinct phases in the automated temporary immersion system: shooting, bud differentiation and elongation. To establish this protocol, we used in vitro shoots obtained from established liquid culture as starting materials. Three culture methods (solid, liquid and temporary immersion) were compared. Temporary immersion increased the multiplication rate and fresh and dry weight after 42 days. Conventional micropropagation (liquid medium) and temporary immersion were compared in combination with paclobutrazol. Paclobutrazol promoted the formation of compact bud clusters with limited leaf development. The highest multiplication rate (106) was found when ex-plants were cultured in shooting medium (MS+2.1 mg/l BA+0.3 mg/l NAA) supplemented with 1 mg/l PB for 7 weeks. A 10-l temporary immersion bioreactor was used to test two approaches during elongation stage: reduction of the shoot-formation period or decrease of the initial number of explants. The highest number of competent and uniform plants (191.8 plant/l) was achieved when shoots were cultured for 4 weeks in shooting medium supplemented with PB.

Sugarcane shoot formation in an improved temporary immersion system

A new protocol was established for sugarcane cv. C-1051-73 shoot formation in a temporary immersion system. The two-step protocol involves shoot formation in 50 ml of culture medium per explant and 1.0 mg l-1 paclobutrazol for 30 days followed by shoot elongation by exposure to 1.0 mg l-1 gibberellic acid for 15 days. The multiplication rate was doubled in comparison with the conventional micropropagation protocol (Jiménez et al., 1995) and the cost has been reduced by 46%. Three additional sugarcane varieties have been micropropagated according to this new protocol and results are comparable. Temporary immersion-derived plants have also been compared with conventionally propagated plants in sugarcane fields for more than 9 months and their agricultural indicators of performance are similar.

Sugarcane micropropagation and phenolic excretion

Sugarcane shoot formation was followed using a temporary immersion system. Plant fresh weight, plant dry weight, shoot number and phenolic excretion to the culture medium were recorded during shoot formation. Shoot number increased for 30 days of culture but formation of new shoots was greatly reduced from 31 to 40 days. Phenolic excretion also increased during the first 20 days of culture (gallic acid represented 82% total phenolics) and decreased during the last 10 days (31–40 days of culture). The most intensive period of phenolic excretion (11–20 days) preceded the most intensive period of shoot formation (21–30 days). The same relationship does not seem to exist between the accumulation of fresh and dry weights. Subculture onto fresh medium at the beginning of proliferation (10 days after culture initiation) was detrimental to shoot formation in the subsequent period (11–20 days). However, such a detrimental effect could be avoided if gallic acid was added to the medium. Addition of cysteine to the culture medium reduced both excretion of phenolics and shoot formation but not fresh weight. The use of temporary immersion systems, the increase of culture medium volume per initial explant and the addition of paclobutrazol promoted both phenolic excretion and sugarcane shoot formation. Results presented here indicate a relationship between phenolic excretion and shoot formation but not with accumulation of plant weight.

Field performance of temporary immersion bioreactor-derived sugarcane plants

SummaryThe temporary immersion bioreactor has been found to be an important tool for sugarcane micropropagation, allowing higher shoot formation rates and cost reduction. This research was conducted to demonstrate the agricultural value of temporary immersion bioreactor-derived sugarcane plants. The experiment was carried out for about 2 yr to study the field performance of these plants. Two control treatments were also evaluated representing the conventional forms of micro- and macropropagation. Growth of sugarcane stools, first ratoon and the use of micropropagated plants for macropropagation were recorded. Some botanical and chemical characteristics were evaluated. Differences among propagation systems were only found in the first 6 mo. of field growth, regarding the stem length and diameter. Such differences disappeared with the course of the experiment.

Artificial endosperm of Cleopatra tangerine zygotic embryos: a model for somatic embryo encapsulation

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype-derived plants in a short time. In this work, a range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. Different complexing ions in the form of alginate capsules, zeolite as an ion exchanger and the relationship between capsule-nutrient gel on germination of zygotic embryos, were evaluated. Artificial endosperm assays showed that abscisic acid (1 μM) and mannitol (0.25 M) delayed germination and conversion of zygotic embryos, whereas amino acid supplements (proline, glutamic acid and arginine) accelerated the conversion process. An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants.

Field performance of new in vitro androgenesis-derived double haploids of barley

Although barley is extensively grown in the Peruvian highlands as a food crop, agricultural and quality performance have historically been poor. A double-haploid technique was used to obtain barley varieties better suited to this environment. Three crosses were constructed: Ya/LM94, B16/LM94 and B12/LM94. From all crosses, F1 anthers were cultured in vitro to produce double-haploid lines. At DH6, promising materials were evaluated in the Peruvian highlands, along with their parents and two commercial controls (PPch, UNA80). Expert agronomic criteria for the ideal barley cultivar for this region were defined and Euclidean distance was used for simultaneous selection of the recorded agricultural traits. Ya/LM94-PC27 exhibited the shortest Euclidean distance to the expert criteria (0.41), higher yield than controls, and low plant height. Periods from first rain to flowering and maturity suited growth during the rainy season. Ya/LM94-PC27 was resistant to stripe rust, and its grain protein content suited the malting-brewing industry. Hectoliter and 1,000-grain masses met or surpassed industry requirements.

EFFECT OF SUCROSE, INORGANIC SALTS, INOSITOL, AND THIAMINE ON PROTEASE EXCRETION DURING PINEAPPLE CULTURE IN TEMPORARY IMMERSION BIOREACTORS

SummaryAlthough pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations of this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore, we decided to modify the culture medium composition of this phase looking for an increase in protease excretion. Four independent experiments were performed to evaluate the effects of different levels of sucrose (0–350.4 mM), inorganic salts [0–200% Murashige and Skoog (MS) salt strength], inositol (0–2.20 mM), and thiamine (0–1.2μM). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. Specific protease activity, the most important indicator recorded, was highest with 262.8 mM sucrose, 100% MS salt strength, 0.3 μM thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 mM sucrose, 100% MS salt strength, 0.55 mM inositol, 0.3 μM thiamine) are not always adequate for protease excretion.

Use of culture-derived Fusarium oxysporum f. sp. cubense, race 1 filtrates for rapid and non-destructive in vitro differentiation between resistant and susceptible clones of field-grown banana

Banana and plantain are among the most important food crops in developing countries but production is threatened by increasing virulent forms of Fusarium oxysporum f. sp. cubense. Chemical control is not economically effective and,therefore, breeding programs are necessary. Traditional field studies of new genotype resistance to this disease are time-consuming and destructive. Therefore,we developed a rapid and non-destructive procedure to differentiate field-grown banana resistant from susceptible clones. This procedure implicates application of culture filtrates of Fusarium oxysporum f. sp. cubense race 1 onto banana leaves. The relationship between duration of the fungal in vitro incubation, and the fungal culture fresh mass, the culture filtrate absorbency, and the Gross Michel (susceptible cultivar)leaf lesion area (after application of the culture filtrate) were similar and at 24day-incubation the highest values of the recorded indicators were observed. A comparison between Gross Michel and FHIA-01(resistant) was also performed. The most relevant differences between cultivars were observed at 48 hours after application of the culture filtrate, and in the middle-aged leaves. The position of the culture filtrate application in the leaf limb (distal, middle, proximal) was not determinant. A wider comparison among banana cultivars confirmed previous results informed by other researchers using different systems to study this plant-fungus interaction. Such a confirmation validates the effectiveness of the procedure described here to select rapid and non-destructively banana resistance to this disease at field level.

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.