Agaves are plants with multiple possibilities of use and are naturally tolerant to low water availability conditions and high temperatures. This makes them species of great interest in the context of the necessary substitution of crops due to climate change. Unfortunately, the overexploitation of wild specimens has endangered many species of the genus that have not been domesticated or cultivated intensively. In vitro mass culture and propagation techniques have emerged as a very efficient option to produce agave plants that can be used without damage to the natural populations. A protocol is presented here for the in vitro micropropagation of agaves in a two-stage process. In the first step, clusters of slightly differentiated shoots are generated from stem segments cultivated on a semisolid medium added with cytokinin. In a second step, these shoot clusters are cultured in temporary immersion bioreactors where they grow and complete their differentiation, and then the shoots are rooted and transferred to soil. This protocol has been successfully applied to several threatened species of the Agave genus.

The genus Musa sp. contains commercially important fleshy fruit-producing plants, including plantains and bananas, with a strong potential for providing food security and sources of revenue to farmers. Concerns with the quality of vegetative tissues along with the possibility of the transmission of phytopathogens makes the availability of healthy plantlets limited for farmers. Micropropagation of plantains offers an alternative to producing large numbers of plantlets. However, conventional methods of micropropagation have high production costs and are labor-intensive. Recently, the temporary immersion bioreactor (TIB) has emerged as an alternative to conventional micropropagation (CM) methods. Our work utilized SEM microscopy (scanning electron microscope) and molecular and biochemical tools (qRT-PCR and ICP-OES) to characterize and compare the morphological properties, elemental composition, and photosynthetic gene expression of plantains cultured on TIB. Additionally, morphological features of growth and propagation rates were analyzed to compare outputs obtained from TIB and CM. Results showed higher growth and multiplication rates for plantlets cultivated in TIB. Gene expression analysis of selected photosynthetic genes demonstrated high transcript abundance of phosphoenolpyruvate carboxylase (PEPC) in plantain tissues obtained by TIB. Elemental composition analysis showed higher content of iron in plantains grown in TIB, suggesting a potential correlation with PEPC expression. These results demonstrate that micropropagation of Musa sp. via the liquid medium in TIB is an efficient and low-cost approach in comparison with solid media in CM.

The production of blueberries for fresh and processed consumption is increasing globally and has more than doubled in the last decade. Blueberry is grown commercially across a variety of climates in over 30 countries. The major classes of plants utilized for the planting and breeding of new cultivars are highbush, lowbush, half-high, Rabbiteye, and Southern highbush. Plants can be propagated by cuttings or in vitro micropropagation techniques. In vitro propagation offers advantages for faster generation of a large number of disease-free plants independent of season. Labor costs for in vitro propagation can be reduced using new cultivation technology and automation. Here, we test and demonstrate successful culture conditions and medium compositions for in vitro initiation, multiplication, and rooting of the Southern highbush cultivar \'Blue Suede™\' (Vaccinium hybrid).

Centella asiatica (L.) Urban is an important pharmacopoeial plant used not only in medicine but also in cosmetology. C. asiatica agitated shoot cultures were established to study the influence of ethephon, methyl jasmonate, l-phenylalanine (Eth 50 µM, MeJa 50 µM, L-Phe 2.4 g/L of medium, respectively; seven variants of the supplementation) on the accumulation of secondary metabolites: the main centellosides (asiaticoside and madecassoside) and selected phenolic acids, and flavonoids in the biomass. Microshoots were harvested two and six days after the supplementation. Secondary metabolites were analyzed in methanolic extracts by UPLC-MS/MS (centellosides) and by HPLC-DAD (phenolics). In comparison with the reference cultures, the concentrations of individual secondary metabolites increased as follows: centellosides up to 5.6-fold (asiaticoside), phenolic acids up to 122-fold (p-coumaric acid) and flavonoids up to 22.4-fold (kaempherol). The highest production increase of individual compounds was observed for different variants of supplementation. Variant C (50 µM MeJa), the most optimal for centellosides and flavonoid accumulation, was selected for the experiment with bioreactors. Bioreactor Plantform™, compared to RITA® system and agitated cultures, appeared to be the most advantageous for secondary metabolites production in C. asiatica shoot cultures. The phenolic acid, flavonoid, centelloside, and total secondary metabolite productivity in Plantform™ system is 1.8-fold, 1.7-fold, 2.8-fold, 2.1-fold, respectively, higher than in MeJa elicitated agitated cultures, and 4.3-fold, 7.3-fold, 12.2-fold, 7.2-fold, respectively, higher than in control agitated cultures.

Temporary Immersion Bioreactors (TIBs) are used for increasing plant quality and plant multiplication rates. These TIBs are actioned by mean of a pneumatic system. A failure in the pneumatic system could produce severe damages into the TIB. Consequently, the whole biological process would be aborted, increasing the production cost. Therefore, an important task is to detect failures on a temporary immersion bioreactor system. In this paper, we propose to approach this task using a contrast pattern based classifier. We show that our proposal, for detecting pneumatic failures in a TIB, outperforms other approaches reported in the literature. In addition, we introduce a feature representation based on the differences among feature values. Additionally, we collected a new pineapple micropropagation database for detecting four new types of pneumatic failures on TIBs. Finally, we provide an analysis of our experimental results together with experts in both biotechnology and pneumatic devices.

Developing scale-up system and automation of micropropagation in a bioreactor has been a possible way of cost reduction and intensive manual handling. We report a comparison between the results of experiments aimed at improving carnation micropropagation using new bioreactor according to Temporary Immersion Bioreactor (TIB) and solid culture. By applying different levels of BAP, at the concentration of 3 mg L-1, we observed 14.3 new shoots in TIB, but the number of new shoots on solid medium reached to 5.7 at the same treatment. Our results also showed that with 3 mg L-1 BAP in TIB, the initial fresh weight of plant material increased from 10 g to 450 g after 15 days. It is concluded that TIB showed more than 10 times shoot production of carnation. Shoot elongation and rooting induction was successfully stimulated in TIB by applying 1 mg L-1 IBA. Rooting of proliferated plantlets from TIB and solid culture were successfully happened, and led to highest number of roots (4.6 cm) and highest length of roots (6.87 cm) in TIB. More than 90% of plantlet was acclimatized to ex vitro. Our results suggested that mass production of carnation shoots in our simple TIB, with effective result, can be considered as a critical first step toward large scale production of carnation.

BACKGROUND: Thapsigargin and nortrilobolide are sesquiterpene lactones found in the Mediterranean plant Thapsia garganica L. Thapsigargin is a potent inhibitor of the sarco/endoplasmic reticulum calcium ATPase pump, inducing apoptosis in mammalian cells. This mechanism has been used to develop a thapsigargin-based cancer drug first by GenSpera and later Inspyr Therapeutics (Westlake Village, California). However, a stable production of thapsigargin is not established.
RESULTS: In vitro regeneration from leaf explants, shoot multiplication and rooting of T. garganica was obtained along with the production of thapsigargins in temporary immersion bioreactors (TIBs). Thapsigargin production was enhanced using reduced nutrient supply in combination with methyl jasmonate elicitation treatments. Shoots grown in vitro were able to produce 0.34% and 2.1% dry weight of thapsigargin and nortrilobolide, respectively, while leaves and stems of wild T. garganica plants contain only between 0.1 and 0.5% of thapsigargin and below detectable levels of nortrilobolide. In addition, a real-time reverse transcription PCR (qRT-PCR) study was performed to study the regulatory role of the biosynthetic genes HMG-CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS), epikunzeaol synthase (TgTPS2) and the cytochrome P450 (TgCYP76AE2) of stem, leaf and callus tissues. Nadi staining showed that the thapsigargins are located in secretory ducts within these tissues.
CONCLUSIONS: Shoot regeneration, rooting and biomass growth from leaf explants of T. garganica were achieved, together with a high yield in vitro production of thapsigargin in TIBs.

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic-driven TIB (Hy-TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move the media that is a characteristic of other TIB designs. The advantage of utilizing low volumes of gas mixtures (that are more expensive than air) is shown by a doubling of the growth rate of plant root cultures under elevated (40%) oxygen in air, and with CO2 supplementation showing improved phototrophic and photomixotrophic growth of seedless watermelon meristem cultures. The development of this bioreactor system involved overcoming contamination issues associated with utilizing very low gas flow rates and included utilizing microchip pressure sensors to diagnose unexpected changes in internal bioreactor pressure (± 20 Pa ∼0.0002 atm) caused by flexing of non-rigid plastic bag vessels. The overall design seeks to achieve versatility, scalability and minimum cost such that bioreactor technology can play an increasing role in the critical need to improve plant productivity in the face of increasing demand for food, reduced resources, and environmental degradation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:337-345, 2016.