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.

Temporary Immersion System for Production of Biomass and Bioactive Compounds from Medicinal Plants

The cultivation of medicinal plants and the production of bioactive compounds derived from them are of fundamental importance and interest, not only at the pharmacological level but also in nutraceutical and cosmetic industries and in functional foods, as well as plant protection in agriculture. In order to respond adequately to the increased demands of the global market from a quantitative and qualitative point of view and to guarantee environmental sustainability of the productions, it is necessary to resort to innovation tools, such as tissue culture in vitro technology. Nowadays, it is well known that the cultivation through the Temporary Immersion System (TIS) in a bioreactor has considerable advantages both for the in vitro mass production of the plants and for the production of secondary metabolites. The present review focuses on the application of TIS during the last two decades to produce biomass and bioactive compounds from medicinal plants. Indeed, almost one hundred papers are discussed, and they particularly focus on the effects of the culture system, vessel design and equipment, immersion time and frequency, and substrate composition for 88 medicinal species in TIS bioreactor culture.

Microtubers production by using Temporary Immersion System (TIS) bioreactor to potato varieties

As the national potatoes growth has become lower from year to year, it is now known that the problems came from the lack of certified potato seed varieties and the minimum access to sophisticated technology to make a good potato variety. The solution that can be made is to utilize microtubers as an efficient factor. The purpose of this research is to find the most effective method in micro tubers cultivation in vitro by comparing the conventional tissue culture method and the Temporary Immersion System (TIS) bioreactor method to four different potato varieties (Granola L., Dayang Sumbi, Atlantic Malang, Maglia). This research uses a split-plot design with a completely randomized design by using two factorial. The result of this study shows that the microtubers in the multiplication and production step using the TIS bioreactor method has a higher average compared to the conventional tissue culture method. As the various details, Dayang Sumbi has the highest parameter such as most sprouts, primer roots, diameter, wet weights, and fastest time growth. Granola L. excel in planlet height and most tubers. Atlantic Malang in the most multiplication and nodus. Meanwhile Maglia excel in most leaves.

In Vitro Propagation of Easter Island Curcuma longa from Rhizome Explants Using Temporary Immersion System

Curcuma longa (C. longa) is widely known for its medicinal properties. However, the potential overexploitation of this plant raises doubts about its long-term survival on Rapa Nui. Micropropagation using a temporary immersion system (TIS) could be the basis for developing a cost-effective and highly productive method of large-scale cultivation of this plant. Our objective was to develop and refine the in vitro multiplication system for mass propagation of C. longa, and thus help restore the fragile ecosystem of Rapa Nui. Three parameters were evaluated: number of explants per flask, flask capacity, and LEDs spectrum. For each parameter evaluated, four aspects were analyzed: fresh weight per plant, number of shoots, percentage of non-sprouting explants, and the proliferation rate. The use of 30 explants per two-liter flask results in more plants with high fresh biomass than other configurations. In addition, LEDs with a red:blue ratio of 2:1 provided the best lighting conditions for in vitro propagation and positively affected C. longa proliferation and rooting. Therefore, our results show that 30 explants per two-liter flask and an LED source with a red:blue ratio of 2:1 allow a higher number of C. longa plants to be obtained using TIS.

The Effect of Sucrose Supplementation on the Micropropagation of Salix viminalis L. Shoots in Semisolid Medium and Temporary Immersion Bioreactors

The effect of sucrose concentration on the micropropagation of axillary shoots of willow was investigated. The following factors were examined: the culture system (semisolid medium in glass jars versus liquid medium in temporary immersion bioreactors), the type of explant (apical and basal sections), the frequency of immersion, and CO2 enrichment. Shoots and leaf growth were significantly higher in RITA® bioreactors than in the jars for all the sucrose treatments. Apical or basal sections of willow cultured in bioreactors under high light intensity (150 µmol m−2 s−1) and ventilated six times a day with CO2-enriched air were successfully proliferated without sucrose, whereas shoots cultured in jars did not proliferate well if sucrose concentration was 0.5% or lower. More roots were formed when sucrose was added to the medium. Shoots cultured in bioreactors were successfully acclimatized irrespective of the sucrose treatment and the root biomass when transferred to ex vitro conditions. This is the first report of photoautotrophic willow micropropagation, our results confirm the importance of proper gaseous exchange to attain autotrophy during in vitro propagation.

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.