Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets.

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (µ) was 0.11 d-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens.

Germinação in vitro de Hyptis leucocephala Mart. ex Benth. e Hyptis platanifolia Mart. ex Benth. / In vitro germination of Hyptis leucocephala Mart. ex Benth. and Hyptis platanifolia Mart. ex Benth

Hyptis leucocephala e Hyptis platanifolia (Lamiaceae) são espécies aromáticas endêmicas do semiárido nordestino e possuem grande importância econômica devido ao seu potencial medicinal. O presente trabalho teve por objetivo obter o protocolo para o estabelecimento inicial in vitro das espécies H. leucocephala e H. platanifolia. O meio de cultura WPM promoveu a maior porcentagem de germinação (96,81%) para H. leucocephala, porém não diferiu estatisticamente dos resultados obtidos no MS½ na última semana de cultivo. Para a germinação de sementes de H. platanifolia, o MS½ foi o meio que proporcionou a maior porcentagem (40,83%). O tipo de esterilização não interferiu na germinação in vitro para as duas espécies. Para a espécie H. platanifolia, quando se utilizou o fechamento dos tubos de ensaio com tampão de algodão, a hiperidricidade foi eliminada; porém, a porcentagem de germinação das sementes foi menor. Avaliando-se o tipo de meio de cultura no crescimento in vitro de H. leucocephala observou-se que este fator não influenciou no número de brotações, nem o comprimento de parte aérea e da matéria seca de raiz. O meio MS proporcionou incremento para o número de folhas, matéria seca da parte aérea, e comprimento da maior raiz, enquanto o meio MS½ foi o que promoveu maior número de raízes. O meio de cultura ideal para a germinação de H. leucocephala e H. platanifolia foi o MS½, podendo ser esterilizado com hipoclorito de sódio, enquanto que o MS foi o melhor meio de cultura para o crescimento in vitro de H. leucocephala.

The Hyptis leucocephala and Hyptis platanifolia (Lamiaceae) are aromatic species, endemic to the Brazilian Semiarid northeast, and they have great economic importance becaude of their pharmacological potential. This study aimed to obtain the protocol for the in vitro establishment of H. leucocephala and H. platanifolia plants. The WPM promoted higher germination percentage (96.81%) for H. leucocephala, but there was no difference between the results obtained in ½MS, in the last week of cultivation. For the germination of H. platanifolia, ½MS was the one that provided the highest percentage (40.83%). The type of sterilization did not affect the in vitro germination of both species. For the H. platanifolia species, when we closed the tubes with a cotton plug, hyperhydricity was eliminated, but the percentage of seed germination was the lowest one. Evaluating the type of culture medium on the in vitro growth of H. leucocephala, it did not influence the number of shoots, length of shoot or root dry matter. The MS medium promoted an increase of the number of leaves, dry shoot and longest root length, and the ½MS medium was more suitable for root induction. The optimal culture medium for the germination of H. leucocephala and H. platanifolia is ½MS, as it can be sterilized with sodium hypochlorite, while the MS was the best culture medium for the in vitro growth of H. leucocephala.

Propagação vegetativa de Hyptis leucocephala Mart. ex Benth. e Hyptis platanifolia Mart. ex Benth. (Lamiaceae) / Vegetative propagation of Hyptis leucocephala Mart. ex Benth. and Hyptis platanifolia Mart. ex Benth

O gênero Hyptis (Lamiaceae) apresenta metabolismo especial de notável variabilidade, possuindo predominância de óleos essenciais, que têm muito valor junto a diversas comunidades que utilizam essas plantas pelas propriedades terapêuticas. Diversos estudos com esse gênero têm demonstrado que os óleos essenciais apresentam propriedades antimicrobiana, antifúngica, citotóxica, antiinflamatória, anti-HIV e inseticida, entretanto, poucos são os estudos voltados para a exploração sustentável dessas espécies. O objetivo desse trabalho foi avaliar o efeito de diferentes tipos de estaca e diferentes concentrações de ácido indolbutírico (AIB) sobre a taxa de sobrevivência, crescimento e enraizamento de estacas de Hyptis leucocephala Mart. ex Benth. e Hyptis platanifolia Mart. ex Benth., espécies endêmicas do semi-árido nordestino cujo valor medicinal tem sido amplamente comprovado. O experimento teve duração de quatro meses. Foram utilizadas estacas apicais, médio-apicais, médio-basais e basais tratadas com soluções de 0, 1000, 2000 e 4000 mg L-1 de ácido indolbutírico (AIB), durante 5 minutos. A utilização de estacas apicais, médio/apicais e médio/basais induzidas com 2000 e 4000 mg L-1 de AIB possibilitaram melhores resultados para propagação vegetativa de Hyptis leucocephala. Para Hyptis platanifolia os melhores resultados foram obtidos com a utilização de estacas médio/basais e basais tratadas com AIB na concentração de 2000 e 4000 mg L-1.

The genus Hyptis (Lamiaceae) has a special metabolism with remarkable variability and predominance of essential oils of great value to the various communities that use these plants due to their therapeutic properties. A number of studies on this genus have demonstrated that its essential oils present antimicrobial, antifungal, cytotoxic, anti-inflammatory, anti-HIV and insecticidal activities. However, few studies have focused on the sustainable exploitation of these species. The aim of this study was to evaluate the effect of different types of cuttings and different concentrations of indolebutyric acid (IBA) on the survival, growth and rooting rates of cuttings from Hyptis leucocephala Mart. ex Benth. and Hyptis platanifolia Mart. ex Benth., species endemic to the semi-arid region of Northeastern Brazil and with largely proven medicinal value. The experiment lasted four months. Apical, middle-apical, middle-basal and basal cuttings were treated with 0, 1000, 2000 and 4000 mg L-1 IBA solutions for 5 min. The use of apical, middle-apical and middle-basal cuttings induced by 2000 and 4000 mg L-1 IBA led to the best results for vegetative propagation of H. leucocephala. For H. platanifolia, the best results were obtained using middle-basal and basal cuttings treated with IBA at 2000 and 4000 mg L-1.