Daminozide Enhances Vigor and Steviol Glycoside Yield of Stevia (Stevia rebaudiana Bert.) Propagated in the Temporary Immersion Bioreactor

Stevia (Stevia rebaudiana Bertoni) contains sweet compound widely used as natural sweetener, steviol glycoside (SG). SG is a diterpenoid secondary metabolite synthesized from ent-kaurenoic acid, the same precursor of Gibberellin (GA). Therefore, in this study, a GA inhibitor, Daminozide (0, 10, 20 ppm) was used to block ent-kaurenoic acid conversion towards GA synthesis in attempt to increase SG content of stevia propagated in Temporary Immersion Bioreactor (TIB). Daminozide in 10 mg/L was observed to be the optimum concentration which increased biomass weight and SG content (stevioside and rebaudioside A) up to 40%. The treatment also increased transcripts accumulation of genes enrolled in SG biosynthesis, such as SrKA13H, SrUGT85C2, and SrUGT76G1, indicating SG pathway become more active due to the inhibition of GA pathway. Furthermore, the inhibition of GA was also indicated by the upregulated expression of GA biosynthesis gene (GA3ox) as the result of feedback regulation, and the downregulated expression of GA catabolism gene (GA2ox2) as the result of feed-forward regulation caused by inhibitor treatment.

An Efficient Method of Pennisetum x Advena ‘Rubrum’ Seedlings Production Using Temporary Immersion Bioreactor and Agar Cultures

The aim of this study was to propose an efficient method of Pennisetum x advena ‘Rubrum’ micropropagation. Agar cultures with MS medium supplemented with BAP in various concentrations (0.5 mg/L-2 mg/L) and a temporary immersion bioreactor system (TIS) with liquid medium MS with an addition of 1 mg/L BAP were used. For rooting ½ MS medium with different auxin combinations (IBA, NAA) and activated charcoal was utilized. The most efficient method turned out to be TIS which produced 36.9 new plants in four weeks. The seedlings were slender in shape, bright green in colour with no signs of hyperhydricity. The most suitable agar medium produced 19.5 new plants in an eight week period. Rooting should be carried on ½ MS supplemented with 0.5 mg/L IBA and 0.5 mg/L NAA with an 84% rooting rate. The addition of activated charcoal inhibited rooting.

Micropropagation of Raspberries (Rubus idaeus L.) in Liquid Media by Temporary Immersion Bioreactor in Comparison with Gelled Media

Temporary Immersion Bioreactor (TIB) is a suitable technique for large scale micropropagation of plant species. The aim of this work was to test the capacity of in vitro proliferation of the primocane-fruiting red raspberry cv Maravilla and floricane-fruiting red raspberry cv Willamette on gelled media compared to liquid media. The two varieties were cultured in vitro on two media, Murashige and Skoog 1962 (MS) and Driver and Kuniyuki walnut medium, 1984 (DKW), both supplemented with 0.5 mg/l 6-benzyladenine (BA). In the control cultures on gelled media the media were gelled with 5g/l Plant Agar, whereas for the cultures in liquid media Plantform bioreactors were used. After six weeks of in vitro culture we recorded the proliferation rates and lengths of the axillary shoots obtained in all the experimental treatments. The highest proliferation rate was 16 ± 2.03, obtained in cv. Willamette on gelled MS medium with 0.5 mg/l BA. The longest shoots (3.17 ± 0.32 cm) were obtained at cv. Maravilla on the DKW medium with 0.5 mg / l BA in the bioreactor. Our research highlighted that Rubus idaeus L. Maravilla and Willamette can be TIB propagated, although further research is needed to improve the efficiency of this method.

A novel temporary immersion bioreactor system for large scale multiplication of banana (Rasthali AAB—Silk)

Musa sp. cultivar Rasthali (Silk AAB) is a choice variety of the Asian sub-continent. Its production and sustenance are threatened by Fusarium wilt, which affects the livelihoods of small and marginal farmers. The use of quality planting material is one of the strategies to manage the disease. Availability of quality planting material for varieties other than Grand Naine is limited. Large-scale micropropagation using existing technologies is laborious and expensive. Temporary immersion bioreactor system is emerging as a potential advancement in the micropropagation industry. In this study, a cost-effective temporary immersion bioreactor (TIB) system has been developed and an efficient micropropagation method has been standardized. Explants cultured in TIB with 250 ml of culture medium in a 2-min immersion frequency of 6 h were found to be efficient for shoot proliferation and rooting. Its efficacy has been compared with the semisolid culture method. At the end of the 6th subculture, 1496 ± 110 shoots per explant were obtained in TIB. Chlorophyll, carotenoid, stomatal index, and the number of closed stomata were examined to determine the physiological functions of the plants grown in TIB and compared with semisolid grown plantlets. Plantlets grown in TIB were genetically stable and were confirmed using inter-simple sequence repeat (ISSR) markers. The multiplication of shoots in TIB was 2.7-fold higher than the semisolid culture method, which is suitable for large-scale production of planting material for commercial applications.

Development of an in vitro Microtuberization and Temporary Immersion Bioreactor System to Evaluate Heat Stress Tolerance in Potatoes (Solanum tuberosum L.)

High temperature (heat) stress reduces tuber yield and quality of potatoes. Screening potatoes for heat tolerance is increasingly important, considering the climate change scenario and expansion of potatoes to countries where heat stress is an issue. In vitro screening for tolerance to abiotic stresses offers several advantages, including quick evaluation of numerous genotypes (clones) in reduced space, controlled environmental conditions (temperature and photoperiod), and free from confounding variables inherent to greenhouse and field conditions. In this study, we explored the feasibility of using a temporary immersion bioreactor system for heat tolerance screening of potatoes. We determined the best hormone-free microtuberizing media for this system (MSG with 8% sucrose) to enhance microtuber number and size. Comparisons of microtubers produced at 30°C as heat treatment, with 16°C as normal condition, allowed to identify heat tolerant and susceptible potato clones. The use of bioreactors allowed distinguishing well-formed (non-deformed) from deformed microtubers. Heat stress increased the total biomass of plant tissues in all the clones. However, the effect of heat stress on microtuber number and weight varied among the clones. Incubation at 30°C decreased the weight and number of non-deformed microtubers in all the clones except for Reveille Russet in which the weight of non-deformed microtubers was significantly increased and the count of non-deformed microtubers was not affected. The potato variety Reveille Russet, which was selected under high-temperature field conditions in Texas, had many non-deformed microtubers per explant and the highest microtuber weight among four clones evaluated under heat stress. We described a faster and reliable in vitro microtuberization system for abiotic stress tolerance screening, identified Reveille Russet as a promising heat-tolerant potato variety, and confirmed Russet Burbank and Atlantic as susceptible heat-tolerant checks.

Agronomic Performance of Temporary Immersion Bioreactor – Derived Potato Microtubers in a Peruvian Low Input Cropping Agriculture System

Potato cultivation is limited by a lack of access to quality propagation material. The application of micropropagation techniques combined with the diagnosis and sanitation of the main pathogens of the crop, has contributed to increased production efficiencies. In this regard, the use of temporary immersion bioreactors (TIBs) has improved the quality of microtubers micropropagated along with savings in costs of production. With the final goal of applying these technologies for commercial production, the current study investigated the agronomic performance of Peruvian Canchan potato microtubers derived from TIBs (basic agamic seed 1 and 2) under low-input agro-technology in the coastal zone of Peru. The results indicated that following 75 d of growth, plants derived from microtubers produced in TIBs displayed slower vegetative growth than those from conventional tubers. However, at harvest, these differences were no longer apparent. Although plants raised from conventional tubers produced the highest fresh mass of tubers, significantly more propagules were produced by plants regenerated from basic agamic seed 1 and 2 derived from micropropagation in liquid media. These results demonstrate that much more planting material (seed tubers) can be obtained from microtubers in the field (basic agamic seed 1) than from the conventional commercial seed tubers.

Characteristics and yield of basic seeds of native potato (Solanum goniocalyx) obtained in temporary immersion bioreactor under greenhouse conditions

The characteristics and yield of basic seeds of two native Peruvian potato varieties "Peruanita" and "Amarilla Tumbay" (Solanum goniocalyx), obtained in temporary immersion bioreactors under greenhouse conditions, were evaluated in order to obtain quality seeds in a continuous and sustainable way to guarantee farmers' production and to meet national demand. For this purpose, microtubers and cuttings obtained by temporary immersion bioreactors (TIB) were cultivated in an area of 240 m2 at 3.201 masl, a completely randomized block design was applied with a bifactorial arrangement (varieties x propagules) of 4 treatments and 3 repetitions. The variables evaluated were: survival (SP) and plant height (AP) at 30 days, number of tubers per plant (NT), tuber diameter (DT), tuber weight (PT), tuber size (TT), tuber skin color (CP) and yield in kg per 14.05 m2 (R). Tubers produced from the microtubers stood out from the cuttings in yield for basic seed, presenting an R=26.89 ± 0,483 kg.14.05 m-2, PT= 0,12 ±0,0036 kg and a TD= 22.59 ±0,451 mm; being the variety "Peruanita" the most optimal in number and weight of tubers per plant for basic seed. The basic seed coming from microtubers was obtained in 5 months, being an alternative to guarantee a constant production, allowing to take care of the demand of farmers and to lower the costs of production.