In Vitro Propagation of Agave guiengola Gentry Using Semisolid Medium and Temporary Immersion Bioreactors

Agave guiengola Gentry is an endemic plant from a very small locality in Oaxaca,Mexico.Its conservation status is fragile and can rapidly worsen.Because of its scarcity,this agave has been used solely for ornamental purposes,but it could have other uses if more plants were available.In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium(MS)as well as basal medium supplemented with cytokinins 6-Benzylaminopurine(BA)or 6-(γ,γ-Dimethylallylamino)purine(2iP).The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^(–1) BA,obtaining a mean of 3.7 shoots per explant.Other interesting responses were observed,such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid(2,4-D);root induction without Plant Growth Regulators(PGR);and generation of shoot clusters.These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors,obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies.All new plants rooted and survived the transfer to soil.This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation,economical purposes,or to carry out studies in this species to assess its full potential,avoiding exploitation from wild plants.

Design and Construction of a Pneumatic Temporary Immersion Bioreactor System for the Multiplication of <i>Ananas comosus</i>var. Trujillana Red

The present work initially identified the design parameters of a temporary immersion bioreactor to later scale it to a complete system for the in vitro multiplication of Ananas comosus var. Trujillana Red. Thus, a low-cost pneumatic temporary immersion bioreactor system was designed and built with 24 tanks of 2 L each. The automation of the system was designed and implemented by means of a timer circuit whose design parameters were: duration of the propagation process, which depends on the multiplication period of the crop and is an open variable, which means that the operator decides when to turn off the system;the duration of each dive, which for reasons of complexity of the algorithm was standardized as one minute;immersion frequency, which was programmed for intervals of 1, 2, 3, 4, 5, 6, 7, 8 hours respectively and duration of aeration, which from a test run times of 0.20 were chosen, 30, 40, 50, 60, 70, and 80 seconds that correspond to the time of delivery of compressed air;additionally, the multiplication rate of Ananas comosus var. Trujillana Red in the immersion system which was 6.5 times per propagative unit inoculated in thirty days.

High Efficiency of Propagation for Sagittaria sagittifolia Using a Temporary Immersion Bioreactor System

Although several studies have reported on the propagation of the Sagittaria sagittifolia to date, none of these methods have efficiently achieved the mass production of these plants. The present study aimed to investigate the propagation and growth of S. sagittifolia using a temporary immersion bioreactor system （TIBS） compared with conventional semi-solid and liquid culture. The effect of different immersion frequencies and immersion times together with supplementation of various plant growth regulators to the Murashige and Skoog （MS） medium was evaluated on shoot proliferation and plant growth. The results showed that the higher immersion frequency （every 6 h） and shorter immersion time （3 min and 10 rain） in medium containing 4 mg/L BA and 0.1 mg/L NAA produced the highest multiplication rate （23）, which are significantly higher than conventional semi-solid （3.6） and liquid （4.5） method, and the best plant growth parameter. While, the lower immersion frequency and longer immersion time （30 rain every 12 h and 60 min every 24 h） induced vitrification and pollution rate in shoot tips 16.6% and 19%, 42% and 37%, respectively. There is distinct decrease in pollution rate （8.3%） in TIBS （10 min every 6 h） compared with the conventional semi-solid and liquid cultures. Medium containing 4 mg/L BA and 0.5 mg/L NAA using 10 min immersion every 6 h showed satisfaction at the rooting stage, with high shoot proliferation rate （21.6）, 100% rooting and 94% plant survival. Therefore, applying TIBS in S. sagittifolia is an efficient method for scaling up the production of plantlets with high quality seedlings.

Rapid Propagation of Virus-free Sugarcane Plantlets via Temporary Immersion Bioreactor System

By employing temporary immersion bioreactor system(TIBs),we studied virus-free culture of seedlings from sugarcane varieties ROC16 and ROC22,from medium recipe,inoculation amount,sucrose concentration,and variety difference. The results showed,using this method,that proliferation rate of ROC16 improved by 40 times,per flask generated about 800 plantlets; of ROC22 improved by 30 times,per flask generated about 400-600 plantlets. The results provided basis for using TIBs in rapid propagation of plantlets via tissue culture.

Rapid Propagation of Three Herbal Species in a Newly Developed Temporary Immersion System

[ Objeelive] This study aimed to develop a rapid propagation method in a novel temporary immersion bioreactor system （TIS） for herbal plantlets com- pared with solid culture method. [ Method ] Three herbal species, including Dendrobium candidum Wall. Ex IJndl （D. candidum）, Anoectochilus roxburghii （Wall.）Lindl. （A. roxburghii） and Lilium davidii var. unicolor （L. davidii）, were used and tested by TIS against solid culture method. [Result] When the two culture methods were compared, the multiplication rate of D. candidum in TIS was found to be 1 ： 24.71, which was 6.55 times to those of solid culture. The multiplication rate of A. roxburghii was higher than those of the solid culture, but the plantlets was poorer than those of solid culture at the last phase in bioreactor culture, under the culture condition tested. The multiplication rate in TIS ofL. davidii was 1：17.23 whilst the rate was only 1：4.45 on solid culture, resulting larger bulbs than those in the solid culture. [ Conclusion] The TIS designed in our study could provide a potential mean for industrial production of plandets. However, the parameters vary greatly among different species, and it is to be optimized according to plant species.

TIB for Micropropagation and the Relationship between Anthocyanins and Chlorophyll of Strawberry Seedlings

In this study, we employed a temporary immersion bioreactor(TIB)system for the micropropagation of strawberry seedlings. The TIB method and the conventional method for the micropropagation of strawberry seedlings were compared in terms of explant propagation coefficient, seedling fresh weight,contents of anthocyanins and chlorophyll, and photosynthetic characteristics. The results showed that an inoculation density of 40 explants/L was suitable for the micropropagation of ’Benihope’ strawberry seedlings in the TIB. The propagation coefficient, fresh weight, photosynthetic rate, stomatal conductance, transpiration coefficient, and the contents of total anthocyanins and chlorophyll of seedlings micropropagated in the TIB were significantly higher than those of the seedlings micropropagated by the conventional method. In conclusion, the TIB method was superior to the conventional method in the micropropagation of strawberry seedlings.The negative reciprocity did not occur between anthocyanins and chlorophyll in strawberry seedlings cultured under forced ventilation such as TIB.

Simple Protocol for the Micropropagation of Teak(Tectona grandis Linn.)in Semi-Solid and Liquid Media in RITA^(█) Bioreactors and ex Vitro Rooting

In Latin America the forestry of exotic species such as teak has been increasing in recent decades, due to their advantages in wood quality, rapid growth;and the relative ease of producing clones and their multiplication with respect to native species. Therefore, there is great interest in developing larger-scale propagation strategies that reduce costs and intensive manual labor. Culture in liquid media with temporary immersion and the semi-automation of the system has raised expectations for large-scale micropropagation. We report a protocol for teak, which reuses the primary explants in several culture cycles in semi-solid medium to produce nodal explants for the multiplication phase in temporary immersion bioreactors (RITA&reg;). The control of factors such as cytokinin concentration, explants density, immersion frequencies and culture duration was analyzed. The number of shoots increased with 0.5 mg&middot;l-1 of BA (6-Benzyladenine), alone or in combination with 0.5 mg&middot;l-1 of Kinetin, with 2 daily immersions of 1 minute each;however, these shoots showed a high degree of hyperhydricity. When 0.05 mg&middot;l-1 of BA was used with 1 immersion of 1 minute every 2 days, the hyperhydricity decreased. Although the number of shoots was lower, they showed good length to be used during multiplication and rooting ex vitro. Our results suggest that teak micropropagation can be simplified in two phases in vitro, the establishment and multiplication;followed by rooting ex vitro and acclimatization. This would imply a reduction in production costs, since most of the multiplication would take place in RITA&reg;containers.