Low CO2 Levels Are Detrimental for In Vitro Plantlets through Disturbance of Photosynthetic Functionality and Accumulation of Reactive Oxygen Species

Photosynthesis of plantlets in tissue culture containers is not considered important, compared to photosynthesis of ex vitro plants, due to the exogenous source of carbohydrates present in tissue culture media. However, CO2 starvation can generate a burst of reactive oxygen species (ROS). We examined this phenomenon in tissue culture, since CO2 levels may become very low during the light period. The research was carried out with lily scales, regenerating adventitious bulblets, and with Arabidopsis seedlings. CO2 starvation was achieved by placing a small vial of concentrated KOH solution in the culture container. CO2 removal reduced the growth of regenerated lily bulblets by 33% or 23%, with or without sucrose in the medium, respectively. In Arabidopsis seedlings, CO2 removal decreased growth by 50% or 78% in the presence or absence of sucrose in the medium, respectively. Therefore, the addition of sucrose as a replacement for photosynthesis resulted in only partial recovery of growth. Staining with nitroblue tetrazolium (NBT) showed little to no ROS in ex vitro growing seedlings, while abundant ROS were detected in seedlings grown under in vitro CO2 starvation. Seedlings grown under normal tissue culture conditions (no CO2 withdrawal) showed low levels of ROS. In lily tissue culture, CO2 starvation decreased the maximum quantum efficiency of photosystem II (Fv/Fm) from 0.69 to 0.60, and in Arabidopsis from 0.76 to 0.62. Fv/Fm of ex vitro lily and Arabidopsis seedlings was 0.77 and 0.79, respectively. This is indicative of a disturbance in photosynthesis functionality and the occurrence of in vitro stress under reduced CO2 concentrations. We conclude that poor growth, in the absence of CO2, was partly due to strongly reduced photosynthesis, while the detrimental effects were most likely due to a burst of ROS.

Structural Adaptation and Antioxidant Response of Guarianthe bowringiana (O’Brien) Dressler & W. E. Higgins (Orchidaceae Juss.) Seedlings During Ex Vitro Acclimatization

Guarianthe bowringiana is one of the oldest samples cultivated at NBG’s orchid unit glasshouses since 1970s. An efficient protocol for asymbiotic in vitro seed germination of G. bowringiana has previously been established. Given that acclimatization is a crucial step in micropropagation, this study assesses the structural adaptation and antioxidant response of G. bowringiana seedlings during ex vitro acclimatization to ex vitro conditions. The leaf surface micromorphology of the G. bowringiana juvenile plants propagated in vitro from seeds as well as the leaves of adult plants cultivated in glasshouse were analyzed using scanning electron microscopy. The levels of lipid peroxidation (TBARS level), superoxide dismutase (SOD) activity, and the photosynthetic activity were monitored for seven days from the transfer of seedlings from the in vitro cultivation vessels as they are markers indicating the response of the leaves of in vitro propagated G. bowringiana plants to oxidative stress during the early stages of acclimatization to ex vitro conditions. During the initial 2 days of the monitored acclimatization period (0–7 days), the level of photosynthetic pigments (chlorophyll a , b , and carotenoid content) increased, followed by an insignificant increase during the successive period (by the seventh day) of acclimatization. At the same time, the level of the tested antioxidant enzyme (SOD) exhibited an increasing trend throughout the acclimatization period. The SOD activities in the leaves of G. bowringiana seedlings were significantly affected when they were transferred from in vitro to ex vitro conditions due to drought stress. Thus, it was revealed that in the early stages of acclimatizing to the altered environments, G. bowringiana seedlings exhibited a rapid increase in photosynthetic pigments, superoxide dismutase activity, and lipid peroxidation levels after being transferred to ex vitro conditions. Comparison of the leaf micromorphologies of G. bowringiana plants grown under in vitro and those grown under ex vitro conditions revealed that leaf development had undergone significant changes during acclimatization to the altered conditions. In vitro to ex vitro transfer leads to a transient decrease in photosynthetic parameters.

Efeito de estimulantes comerciais na aclimatização de orquídeas Cattleya caulescens (Lindl.) Van den Berg (Orchidaceae)

As orquídeas são vegetais pertencentes à família Orchidaceae, que por apresentarem características marcantes, como o seu formato, tamanho e diferentes colorações das flores. Atualmente nota-se uma expansão em sua comercialização principalmente do gênero Cattleya caulescens, devido o interesse desde colecionadores até população em geral. Entretanto, é necessário métodos de propagação eficientes para a propagação da espécie, em larga escala, como por exemplo o método de micropropagação (cultivo in vitro). Porém, estudos com relação ao cultivo ex vitro, e utilização de fitoestimulantes comerciais é incipiente. Sendo assim, o objetivo deste trabalho foi analisar o desenvolvimento das plântulas, sob o efeito de produtos bioestimulantes, composto por 4 tratamentos e 9 blocos, sendo: (T1) – extrato de algas marinhas de Kappaphicus alvarez a 1,5% 150 mL de extrato para 850 mL de água; (T2) – Forth® 5 ml L-1; (T3) – Seacrop® a 1 ml L-1 e (T4) - cloridrato de Tiamina a 500 mg L-1. As plântulas selecionadas foram lavadas em água corrente e colocadas para secar em jornal por dois dias. Logo após colocadas em imersão por cinco minutos em cada solução nutritiva correspondente e plantadas em potes de polietileno. O Delineamento utilizado foi de Blocos Casualizados (DBC) e submetido ao teste de Tukey a 5% de probabilidade para comparação de médias. Sendo observado o Tratamento T3 (Seacrop®) foi o que proporcionou melhor enraizamento em comparação aos demais como também melhor resultado de altura de plantas, número de folhas e de tamanho de raízes sendo o mais indicado para aclimatização de plântulas de orquídeas.

Optimization of in vitro rooting and ex vitro adaptation conditions of Melissa officinalis L. microshoots during clonal micropropagation

Melissa officinalis L. is a perennial herbaceous essential and medicinal plant widely used in pharmacology, perfumery and cosmetics, as well as in alcoholic beverage and food industries. The low content of essential oil in lemon balm raw material determines the selection work aimed at creating high-oil cultivars. The use of clonal micropropagation method in vitro will increase the efficiency of this process and accelerate promising breeding samples multiplication. The aim of our research was to study the influence of cultivation conditions and cultivar on the M. officinalis in vitro rhizogenesis and ex vitro adaptation. It was found that the maximum frequency of shoot rooting (up to 93.3%) in cultivars ‘Citronella’ and ‘Sobornaya’ was on MS culture medium supplemented with 0.5 mg/l NAA. The number of roots was 10.1 and 13.6 pcs. per shoot, respectively. The highest rates of root formation for the cv. ‘Crimchanka’ was found on a culture medium supplemented with 1.0 mg/l IAA (8.7 roots per shoot). The mixture of peat, sand and perlite (2:1:2) as a substrate provided up to 93% of adapted ex vitro lemon balm microplants. The presented studies were used to develop a technique for clonal micropropagation of M. officinalis.

Ipomoea batatas (L.) Lam. cultivation in the conditions of light culture in vitro and ex vitro

Relevance. Currently, food products that include prebiotics, in particular, inulin, are particularly popular. Interest in this substance is justified by its valuable properties – it is a good immunomodulator, cleanses the body of toxins, radionuclides, "bad" cholesterol, promotes the assimilation of useful trace elements necessary for human life. Inulin is contained in plants such as jerusalem artichoke, chicory, as well as in sweet potatoes, the popularity of which is increasing every year. However, sweet potato plants are afraid of cold and frost-resistant. Therefore, the creation of new varieties and hybrids that are resistant to low temperatures is an urgent problem. Cellular biotechnology is aimed at solving this problem using methods of clonal microreproduction, cell selection, somatic hybridization, etc. For rapid reproduction and obtaining high-quality planting material, biotechnology methods are used, in particular, clonal micro-propagation. However, in this technology there are difficulties associated with poor adaptation of microclones to ex vitro conditions. This fact introduces an additional requirement for the selection of optimal rooting modes in vitro and ex vitro adaptation of microclones.Material and methodology. The aim of the work was to study the influence of cultivation conditions on in vitro rooting and ex vitro adaptation of I. batatas (L.) microclones. The object of the study was sweet potato microgears propagated in vitro. I. batatas micro-gears were cultured in vitro on a Murashige-Skug medium, differing by the type of auxins. The influence of red (R) and far red (FR) light on the shoots rooting in vitro and the adaptation of microclones ex vitro was studied.Results. It has been experimentally established that the cultivation of micro-gears on a medium containing indolyl butyric acid at a concentration of 0.5-1 mg/l and under conditions of illumination by LED lamps of red and far red light in equal amounts leads to the production of microclones with a well-developed root system and vegetative biomass. The use of an aeroponic installation at the last stage of clonal micro-propagation makes it possible to obtain high-quality planting material that can adapt well to open ground conditions.

Regeneration from leaf explants of steppe cherry (Prunus fruticosa Pall)

The article represents data on morphogenesis from leaf explants of three steppe cherry genotypes, as well as the degree of somaclonal variability at in vitro and ex vitro stages, and in the field. It was revealed that a content of 6-benzylaminopurine, 4.43 μM, in combination with auxin, 0.5–0.6 μM, stimulates in the light the direct organogenesis in the tissues of the leaf base. This reaction was observed from 16.7 to 75.0% of explants, depending on the genotype. An equal 6-benzylaminopurine - auxin ratio (1: 1) led to the callus along with microshoots. Depending on the genotype, up to 30.0% of explants had such a mixed type of organogenesis. The mitotic index value in the apical leaflets differed depending on the day time. At the stage of micropropagation itself, an increase of the mitotic index was observed from 10 to 16 hours; at the stage ex vitro, no significant differences in the mitosis frequency were revealed within this time interval. No significant differences were found between the level of the mitotic index for plants obtained directly from leaves and those from buds. The mitosis passed without disturbances. No phenotypic changes in plant habit, shape and color of leaves were found.