Critical vitrification steps towards survival of Garcinia hombroniana (Clusiaceae) shoot tips after cryopreservation / Etapas críticas de vitrificación para la supervivencia de ápices caulinares de Garcinia hombroniana (Clusiaceae) después de crioconservación

Abstract A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 h gave significantly higher survival (75 %) compared to 24 h preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana. Rev. Biol. Trop. 66(3): 1314-1323. Epub 2018 September 01.

Resumen Se desarrolló un protocolo de crioconservación in vitro para ápices caulinares de Garcinia hombroniana mediante la técnica de vitrificación, con cuatro etapas críticas: precultivo, carga de crioprotector, deshidratación con solución de vitrificación vegetal 2 (PVS2), y descarga. Ápices precultivados por 48 h sobrevivieron más (75 %) que los de 24 h (50 %) después de la crioconservación. El tratamiento con glicerol 1 M más sacarosa 0.4 M como solución de carga permitió mayor sobrevivencia (45.83 %) que los otros tratamientos (sacarosa 0.4 M + glicerol 2 M; sacarosa 0.4 M). Los ápices deshidratados con PVS2 por 25 min registraron la mayor sobrevivencia tras inmersión en nitrógeno líquido. El tratamiento gradual por 15 min con solución de PVS2 al 50 %, seguido por 10 min al 100 %, mejoró la sobrevivencia de ápices tras la crioconservación (41.67 %). El medio Murashige-Skoog (MS) con sacarosa 0.4 M produjo una sobrevivencia significativamente mayor (66.67 %) que MS con sacarosa 1.2 M (25 %) como solución de descarga. El contenido de agua disminuyó a lo largo del proceso de vitrificación desde 6.83 ± 1.66 g g-1 peso seco, en ápices frescos, hasta 2.93 ± 0.28 g g-1 peso seco después del tratamiento con PVS2. Se requiere de más investigación sobre cada etapa, incluyendo el medio de recuperación, para mejorar la tasa de sobrevivencia. Sin embargo, este estudio muestra el potencial de la vitrificación para la crioconservación de G. hombroniana.

Clonal cleaning and in vitro multiplication of Angelonia integerrima sprengel / Limpeza clonal e multiplicação in vitro de Angelonia integerrima sprengel

The objective of this study is to test stem apex sizes in the in vitro establishing of Angelonia integerrima in order to obtain explants without by fungi and bacteria contamination for further multiplications. The treatments consisted of different stem apex sizes (1.0, 3.0, 5.0, 7.0, 9.0 and 11.0 mm). At 45 and 90 days of cultivation, a count of contaminated explants and a count of shoots per explant formed were performed. In a second experiment, explants were cultivated in a medium containing different concentrations of benzylaminopurine (BAP) (0.0, 0.05, 0.10, 0.15 and 0.20 mg L-1). After 56 days of cultivation, the following variables were evaluated: shoot length, shoot fresh mass and number of shoots. During the explant establishment phase (45 days), only stem apexes with 1.0 mm in size were not contaminated. However, in the second subculture (at 90 days), only shoots from initial explants, with 7 mm in size or larger, were contaminated. Regarding multiplication, the presence of BAP showed a positive linear behavior for all variables. It is possible to obtain A. integerrima seedlings free of contamination in vitro by fungi and bacteria, using initial explants less than or equal to 5 mm. IBA provided a linear increment for the multiplication of this species.

O objetivo do trabalho foi testar tamanhos de ápices caulinares no estabelecimento in vitro de Angelonia integerrima, a fim de obter explantes sem contaminações por fungos e bactérias para posterior multiplicação. Os tratamentos consistiram de tamanhos de ápices caulinares: 1,0; 3,0; 5,0; 7,0; 9,0 e 11,0 mm. Após 45 e 90 dias de cultivo foi realizada a contagem de explantes contaminados e o número de brotos formados por explante. Em um segundo experimento, explantes foram cultivados em meio contendo diferentes concentrações de benzilaminopurina (BAP): 0,0; 0,05; 0,10; 0,15 e 0,20 mg L-1. Após 56 dias de cultivo foram avaliados: comprimento e massa fresca da parte aérea e número de brotos. Durante a fase de estabelecimento (45 dias), somente explantes com 1,0 mm não apresentaram contaminação, já no segundo subcultivo (aos 90 dias) somente brotações oriundas de explantes com tamanho inicial igual ou superior a 7 mm apresentaram contaminação. Com relação à multiplicação, a presença de BAP apresentou comportamento linear positivo para todas as variáveis analisadas. É possível obter mudas de A. integerrima livres de contaminações in vitro por fungos e bactérias, utilizando explantes iniciais menores ou iguais a 5 mm. O BAP proporcionou incremento linear para a multiplicação da espécie.