Temporary Immersion System Improves Regeneration of in vitro Irradiated Recalcitrant Rice Embryogenic Calli

The development of gamma rays mutant rice lines would be a solution for introducing variability in already farmer using varieties. In vitro gamma (60Co) mutagenesis reduces chimeras and allows a faster selection of desired traits but requires laboratory process optimization. The objective of the present work was the in vitro establishment of a recalcitrant rice embryogenic calli, the determination of its sensitivity to gamma radiation (Co-60), sequencing MATK and Rubisco for identification purposes, as well as generation optimization. The radiosensitivity of embryogenic calli resulted in an LD50 of 110Gy, while the 20% lethal dose was 64Gy. All sequenced genes matched perfectly with already reported MATK and Rubisco O. sativa genes with a clear SNP that identifies the local variety related to the southeast Asia Region. Callus induction improves with an MS with 2mg/L 2,4D, and the regeneration was achieved with an MS medium with 3mg/L BAP and 0,5mg/L NAA. The optimized radiation condition was 60Gy with an 83% regeneration in a semisolid medium, allowing a balance between mutation and regeneration. When increased to 80Gy, the regeneration rate falls to 29%. An immersion system (RITA®) of either 60 or 120 seconds every 8hours allowed a systematic and homogeneous total regeneration of the recalcitrant line, in contrast with the semisolid medium that resulted in positive but irregular regeneration. Other well-known recalcitrant cultivars, CR1821, CR1113 also had an improving regeneration in the immersion system, demonstrating its potential use for recalcitrant materials. To our knowledge, this is the first report on using an immersion system to allow regeneration of gamma-ray mutants from recalcitrant rice materials.

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.

In Vitro Culture of Rosmarinus officinalis L. in a Temporary Immersion System: Influence of Two Phytohormones on Plant Growth and Carnosol Production

Emerging infectious diseases have become a major global problem with public health and economic consequences. It is an urgent need to develop new anti-infective therapies. The natural diterpene carnosol exhibit a wide variety of interesting antibacterial and antiviral properties, and it is considered a theoretical inhibitor of COVID-19 Mpro. However, this compound is present in the family Lamiaceae in low quantities. To obtain carnosol in concentrations high enough to develop pharmacological studies, we evaluated the efficiency of a micropropagation protocol of Rosmarinus officinalis using a solid medium and a temporary immersion system (TIS), as well as the effect of 6-benzylaminopurine (6-BAP) and α-naphthaleneacetic acid (NAA) on the growth of shoots. Moreover, we developed and validated an analytical method to quantify carnosol using the H-point standard additions method in the high-performance liquid chromatography diode array detector (HPLC-DAD). After 30 days of culture, TIS produced the maximum number of shoots per explant (24.33 ± 1.15) on a liquid medium supplemented with 6-BAP at 5.0 mg L−1. Next, we also evaluated the effect of immersion time and frequency for TIS. After 72 days of culture, the best results were obtained with an immersion cycle of 1 min every 12 h, yielding 170.33 ± 29.40 shoots. The quantification of carnosol on the samples was performed at a flow rate of 1.2 mL min−1 using binary isocratic mobile phase system 60:40 (v/v) 10 mM formic acid (pH 3.0) (A) and acetonitrile (B) on a reverse-phase column. The content of carnosol in the in vitro cultures was around 8-fold higher than in the wild plant. The present study represents an efficient alternative method to obtain carnosol for its pre-clinical and clinical development.

Plant Production and Leaf Anatomy of Mertensia maritima (L.) Gray: Comparison of In Vitro Culture Methods to Improve Acclimatization

Mertensia maritima is a commercially interesting herb with edible leaves and flowers, characterized by oyster flavor and taste. Plant propagation and traditional cultivation are challenging for this species. Therefore, the main purpose of the present study was to establish successful protocols aimed at ensuring oyster plant shoot propagation, rooting and in vivo acclimatization. Both micropropagation and rooting were tested, comparing the traditional in vitro solid substrate in jar vs. the liquid culture in a temporary immersion system (TIS) bioreactor (Plantform™). A Murashige and Skoog (MS) medium added with 4-µM thidiazuron (TDZ) and 1-µM α-naphthaleneacetic acid (NAA) was employed for micropropagation, while a half-strength MS medium supplemented with 4-µM indole−3-butyric acid (IBA) was used for rooting. Different acclimatization conditions in the greenhouse or in growth chamber were tested. Morphometric and microscopical analyses were performed on the oyster plant leaves at the propagation, rooting and acclimatization stages both in a jar and in a TIS. Micropropagation in a TIS allowed to obtain large shoots, while a great number of shoots was observed in the jar. M. maritima shoots rooted in TIS produced more developed roots, leaves with more developed waxy glands and well-formed stomata; moreover, the plants coming from the TIS showed the best acclimatization performances.