Abstract Sequoia sempervirens is the tallest tree in the world, it has a low germination percentage, due to it the in vitro tissue culture has been used for its propagation. Even so, the multiplication technique development is still not efficient. This work aimed to the development of an efficient protocol for in vitro multiplication of S. sempervirens, using apical and basal stem explants, and four concentrations of kinetin and a culture medium with a low concentration of mineral salts to replace the use of the MS medium. Twenty axillary buds from an 18-years-old tree were established in vitro, after three subcultures, three experiments were established every six weeks. Kinetin (6- furfuryl- aminopurin) concentrations (0, 0.92, 1.85 y 3.71 µM) and two types of explants: apical and basal, were evaluated. The parameters evaluated were: height increase, over time growth, and number of axillary shoots emitted by explant type. The height increase for the three experiments was in apical explants (2.35, 4.26, and 4.78 cm) with 0 µM kinetin. The greatest increases in height of apical explants (2.35, 4.26 and 4.78 cm) were obtained with 0 μM of kinetin, in the three experiments. The greatest increases in height were in all evaluated treatments in the second and the fourth week. The greatest number of axillary shoots (2.11) were obtained in basal explants. The in vitro propagation protocol for S. sempervirens using apical and basal explants is efficient, the growth of the explants was successful with 12.38 mm of total nitrogen in culture medium.

Resumen Sequoia sempervirens (D. Don). Endl. es considerado el árbol de mayor altura en el mundo, con bajo porcentaje de germinación, por lo cual, se ha recurrido al cultivo in vitro para su propagación, pero los protocolos desarrollados para obtener brotes son poco eficaces. El objetivo del presente trabajo fue desarrollar un esquema eficiente de multiplicación in vitro de S. sempervirens utilizando explantes de tallo apicales y basales, cuatro concentraciones de kinetina, además de un medio de cultivo con baja concentración de sales para sustituir el uso del medio MS. Se establecieron in vitro 20 yemas axilares de una planta de 18 años, después de tres subcultivos fueron colocados tres experimentos consecutivos cada seis semanas. Se evaluaron cuatro concentraciones de kinetina (6-furfuril-aminopurina) (0, 0.92, 1.85 y 3.71 μM) y dos tipos de explantes: apicales y basales. Las variables evaluadas fueron: incremento de altura, crecimiento a través del tiempo, número de brotes axilares emitidos por tipo de explante. Los mayores incrementos en altura de explantes apicales (2.35, 4.26 y 4.78 cm) se obtuvieron con 0 μM de kinetina, en los tres experimentos. En las semanas dos y cuatro, se presentaron los mayores incrementos en altura en todos los tratamientos evaluados. La mayor cantidad de brotes se obtuvieron en explantes basales (2.11). El esquema de propagación in vitro de S. sempervirens fue eficiente con explantes apicales y basales, y el crecimiento de los explantes fue satisfactorio con 12.38 mM de nitrógeno total en el medio de cultivo.

Abstract The objective of this work was to evaluate the photosynthetic response of citrus varieties to the inoculation of isolates T30 and T3 of Citrus tristeza virus (CTV) characterized as having potential to provide cross-protection against severe CTV isolates in citrus. Citrus plants of 'Campbell' orange, 'Persian' lime, and 'Key' lime were subjected to inoculations by both isolates by patch. Noninoculated plants were used as the control. Symptom expression, photosynthetic and transpiration rates, and stomatal conductance were evaluated by IRGA. Stomatal density and size were evaluated by epidermal impressions. The CTV isolates were also evaluated and molecularly characterized by RT-PCR and Sanger sequencing. 'Campbell' orange and 'Persian' lime plants remained asymptomatic after the inoculations. The inoculation treatments with both CTV isolates did not reduce photosynthetic capacity, transpiration rate, and stomatal conductance, in comparison with the control. Stomatal density and size varied according to the citrus species. T30 and T3 do not affect the photosynthetic responses of 'Campbell' orange and 'Persian' lime; therefore, these isolates have the potential to provide cross-protection to citrus varieties against severe isolates of CTV.

Resumo O objetivo deste trabalho foi avaliar a resposta fotossintética de variedades de citros submetidas à inoculação dos isolados T30 e T3 de Citrus tristeza virus (CTV) caracterizados como tendo potencial para conferir proteção cruzada a citros contra isolados severos de CTV. Plantas de citros de laranja 'Campbell', limão 'Tahiti' e limão 'Galego' foram submetidas à inoculação de ambos os isolados por “patch”. Plantas não inoculadas foram utilizadas como controle. A expressão dos sintomas, as taxas fotossintética e de transpiração, e a condutância estomática foram avaliadas por IRGA. A densidade e o tamanho estomáticos foram avaliados por impressões epidérmicas. Os isolados de CTV também foram avaliados e caracterizados molecularmente por RT-PCR e sequenciamento de Sanger. As plantas de laranja 'Campbell' e limão 'Tahiti' permaneceram assintomáticas após as inoculações. Os tratamentos de inoculação com ambos os isolados de CTV não reduziram capacidade fotossintética, taxas de transpiração e condutância estomática, em comparação ao controle. A densidade e o tamanho estomáticos variaram de acordo com a espécie. T30 e T3 não afetam as respostas fotossintéticas de laranja 'Campbell' e limão 'Tahiti'; portanto, estes isolados têm potencial para promover proteção cruzada a variedades de citros contra isolados severos de CTV.

ABSTRACT: A generalized concept in the Citrus genus is that highly polyembryonic varieties produce only a small number of hybrids. Small zygotic embryos congregate primarily near the micropyle, while nucellar embryos organize near or away from the micropyle. In the present study, the authors determined the number of polyembryonic seeds, embryos per seed, and the largest embryo (LE) position in five citrus cultivars: C35 citrange, Volkamer lemon, Amblycarpa mandarin, Minneola tangelo, and Valencia orange. The percentage of nucellar seedlings obtained exclusively from the LE per seed was then calculated. The polyembryony percentage varied largely between genotypes, from 65 to 98 %, and the mean number of embryos ranged from 2.9 to 4.6. The chalaza contained up to 87 % of the LE. Out of 30 primers, 17 Simple sequence repeats [SSRs] (AG14, ATC09, CAT01, CCSM147, CCSM18, CCSM13, CCSM4, F2, F4, F6, GT03, TAA41, TAA45, TAA1, F7, F11 and TAA52) identified nucellar plants identical to the female parent (genetic similarity index [GSI] value ≥ 0.95). This study establishes for the first time the relationship between the sexual or asexual origin of seedlings derived from LE embryos isolated from seeds and the SSR primers described above. While the five citrus cultivars had high polyembryony levels, 30 % of the resulting plants differed from the female parent in C35 Citrange, 45 % in Volkamer lemon, 15 % in Amblycarpa mandarin, 15 % in Valencia orange, and 45 % in Minneola tangelo. The largest seedling is not always nucellar: in the five citrus species studied, the LE produces 55 to 85 % of the nucellar embryos.

ResumenLas hojas de Phyllonoma laticuspis se utilizan en Carrizal de Bravo, Gro. México, para curar lesiones de la piel como heridas, secuelas de viruela y afecciones de la diabetes mellitus tipo 2. Los extractos orgánicos de hojas, mostraron efectos antibacterianos. La gran demanda de P. laticuspis como planta medicinal, ha disminuido sus poblaciones naturales, y no existen reportes de la propagación de la especie. Por lo tanto, el propósito del presente trabajo fue evaluar la propagación vegetativa de la especie a través de estacas y acodos, y su propagación sexual en una población conservada. En la propagación por estacas y acodos se aplicaron concentraciones de 1 000, 4 000 y 6 000 ppm de un enraizador comercial con ácido indolbutírico (AIB), y un tratamiento control sin AIB. La germinación se evaluó en luz y oscuridad mediante el uso de lotes de semillas recién recolectadas y lotes de semillas almacenadas por tres meses a 4 °C y a 24 ± 2 ºC. Todos los experimentos se realizaron en un diseño completamente al azar. Las estacas no desarrollaron raíces en ninguna de las concentraciones, mientras que el 100 % de los acodos enraizaron, produciendo raíces vigorosas con la concentración de 4 000 ppm de AIB, a los cuatro meses de la aplicación. Con respecto a la germinación bajo condiciones de luz y oscuridad, las semillas recién recolectadas germinaron más del 60 %, mientras que las semillas almacenadas a 4 ºC menos del 20 % y las almacenadas a 24 ± 2 ºC cerca del 50 %. No hubieron diferencias significativas entre la germinación en luz y oscuridad, por lo que se consideraron como semillas fotoblásticas indiferentes. El contenido de humedad de 13.5 % y el comportamiento de la germinación en respuesta al almacenamiento en frío, sugieren la cualidad de semillas de tipo intermedias. La propagación de P. laticuspis para la producción de follaje a corto tiempo, puede hacerse por acodos en poblaciones con alta densidad de plantas adultas como fuente de material vegetal y para la restauración de áreas alteradas en la misma localidad. Mientras que la producción de follaje a mediano plazo y la conservación de la variabilidad de la especie, se puede lograr con la obtención masiva de plántulas a través de semillas.

Abstract:Phyllonoma laticuspis leaves are used in Carrizal de Bravo, Guerrero, Mexico, to heal skin lesions such as injuries and smallpox sequelae and to treat diabetes mellitus type 2, and organic extracts of these leaves have been reported to exert antibacterial effects. High demand of P. laticuspis as a medicinal plant has decreased its natural populations, and propagation of the species has not yet been reported. Therefore, the purpose of this study was to assess the vegetative propagation of the species through cutting and air layering, as well as its sexual propagation in a preserved population. For this, concentrations of 1 000, 4 000 and 6 000 ppm of a commercial root enhancer, with indole butyric acid (IBA), and a control treatment without IBA, were applied to the cuttings and air layers. Germination was evaluated under light and dark conditions using lots of freshly collected seeds and lots of seeds that had been stored for three months at 4 °C or 24 ± 2 °C. All experiments were performed in a completely randomized design. The cuttings did not develop roots in any concentration, whereas 100 % of the air layers rooted, displaying vigorous roots in the presence of 4 000 ppm IBA, after four month of treatment application. Regarding germination, more than 60 % of the freshly collected seeds germinated, whereas less than 20 % of the seeds stored at 4 °C, and close to 50 % of the seeds stored at 24 ± 2 °C germinated under light and dark conditions. No significant differences were observed between light and dark conditions, so they were categorized as indifferent photoblastic seeds. The observed moisture content of 13.5 % and germination behaviour as the response to cold storage, suggest that the resultant seed quality was intermediate. P. laticuspis propagation for short-term foliage production can be carried out in air layers, in populations with a high density of adult plants as a source of plant material and for the restoration of disturbed areas, in the same locality. On the other hand, large-scale seedling production, medium-term foliage production and preservation of species variability can be achieved using seeds. Rev. Biol. Trop. 65 (1): 9-19. Epub 2017 March 01.

The objetives of this work were to evaluate the effect of the treatment with chemical products and the humidity content for conservation of seeds of 'Cleopatra' mandarin (Citrus reshni Hort ex Tan). Seeds with 14.7% of moisture content were treated with four fungicides and 8-hydroxyquinoline and stored at 4±2°C for one year. Three groups of seeds with 5.0, 14.7% and 26.8% of moisture content were stored as above. Germination and emergence were evaluated at the beginning of the two experiments, at six and twelve months of storage. In the test with chemical products, storage of seeds with 14.7% of moisture content was possible only for six months, with higher percentages of germination and emergence (59.3% and 54.0%, respectively), while in seeds without chemical treatment percentages of germination and emergence were about 33.3% and 27.3%, repectively. As far as the moisture content of the seed is concerned, at six months of storage the highest seedlings emergence (63,6% and 58.0%) were obtained, in seeds with 26.8% and 5% of moisture, respectively. Twelve-months-seeds with 5% of moisture content caused 75.6% of emergence.

Los objetivos del trabajo fueron evaluar el efecto del tratamiento con productos químicos y contenido de humedad en la conservación de semillas de mandarino 'Cleopatra' (Citrus reshni Hort ex Tan). Semillas con 14,7% de humedad tratadas con cuatro funguicidas y 8-hidroxiquinoleina, fueron almacenadas a 4±2°C durante un año, al igual que tres lotes de semillas con 5,0%, 14,7% y 26,8% de humedad, tratadas con y sin 8-hidroxiquinoleina. Se evaluó la germinación y emergencia al inicio del experimento, a los seis y 12 meses de almacenamiento. En la prueba con productos químicos, el almacenamiento de semillas con 14,7% de humedad sólo fue viable hasta los seis meses, con mayores porcentajes de germinación y emergencia (59,3% y 54,0%, respectivamente), mientras que, en las semillas sin tratamiento químico fue de 33,3% y 27,3%, respectivamente. En relación con el contenido de humedad de las semillas, a los seis meses de almacenamiento se obtuvo mayor emergencia de plántulas (63,6% y 58,0%) en semillas con 26,8% y 5%, mientras que a los 12 meses, las semillas con 5%, de humedad tuvieron 75,6% de emergencia.

Os objetivos deste trabalho foram avaliar a freqüência da poliembrionia e indentificar os seedlings zigóticos e nucelares de Citrus volkameriana usando RAPD. Cultivaram-se in vitro 25 sementes poliembriônicas e 8 monoembriônicas, por seis meses. Extraiu-se o DNA dos seedlings e usou-se em combinação com cinco primers RAPD para identificar a origem zigótica e nucelar das plântulas. Os resultados mostraram que as condições ambientais do ano afetaram significativamente (P<0,05) as características morfológicas dos frutos e o número de embriões por semente. Os índices de sementes poliembriônicas variaram nos três anos de avaliação (30,9%, 44,8% e 54,4%). As características morfológicas não se correlacionaram com a poliembrionia. A cultura in vitro possibilita que todos os embriões de cada semente cresçam, favorecendo os seedlings identificados como zigóticos. Nas sementes poliembriônicas e monoembriônicas, 25,9% e 87,5% dos seedlings, respectivamente, originaram-se sexualmente. Nem todos os seedlings zigóticos foram produzidos por embriões localizados no micrófilo das sementes poliembriônicas.

The objectives of this work were to evaluate the frequency of polyembryony, and to identify zygotic and nucellar seedlings of Citrus volkameriana using RAPD. Twenty-five polyembryonic and eight monoembryonic seeds were cultivated in vitrofor six months. DNA from seedlings was extracted and used in combination with five RAPD primers to identify zygotic or nucellar origin of the seedlings. Environmental conditions of the year affected significantly (P<0.05) the morphological characteristics of fruitsand the number ofembryos per seed. Polyembryonic seeds ranged from 30.9%, 44.8% to 54.4% over three years. Morphological characteristic was not correlated with polyembryony. In vitro culture enable all embryos of each seed to grow, favoring the percentage of seedlings identified as zygotic. In polyembryonic and monoembryonic seeds, 25.9% and 87.5% of the seedlings, respectively, were sexually originated. In polyembryonic seeds, not all zygotic seedlings were produced by small embryos located at the micropyle.