The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 μM was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.

Optimization of plantain (Musa AAB) micropropagation by temporary immersion system

Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.

/pdf/morphological-and-physiological-responses-of-proliferating-234bqawhgq.pdf

Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments

Although several studies have been reported on the micropropagation of the pistachio and its rootstocks, to date none of them had been efficient on the mass production of these plants in bioreactor systems. Thus, the micropropagation of juvenile pistachio shoot tips and nodal buds was investigated in a temporary immersion bioreactor system (RITA®) and on a conventional semi-solid medium. Among the tested immersion conditions, immersion for 24 min every 16 h reduced vitrification and improved proliferation in the pistachio. Interactions were evident in immersion time and frequency in nodal segments. Nodal buds were better than shoot tips as the highest multiple shoot formation was recorded in MS medium containing 4 mg L−1 BA and 0.1 mg L−1 GA3 in RITA®. Although shoot tip necrosis (STN) was observed in shoots proliferated on semi-solid MS medium, such a symptom did not occur in shoots sprouted in the RITA®. Additionally, these optimized conditions were applied to nodal buds of mature male pistachio ‘Atli’ and Pistacia rootstocks (P. khinjuk Stocks and P. atlantica Desf.), and the micropropagation in the bioreactor system, in comparison to the semi-solid medium, was also improved. Furthermore, in vitro rooting of pistachio plantlets, despite the lower range (27.5 %), was also achieved in RITA®. However, rooting was better on semi-solid medium for all tested species (ranged between 50 and 70 %). The results of this study showed that RITA® could be used for the mass propagation of pistachio and its rootstocks, as well as for other woody plant species.

Micropropagation of the pistachio and its rootstocks by temporary immersion system

A temporary immersion bioreactor system (TIB system) provides a convenient and efficient way to propagate plant material in vitro while requiring significantly lower labor input than conventional methods. The applicability of a TIB system for adventitious shoot regeneration from strawberry leaf explants was studied. Five commercial cultivars, i.e. Bounty, Jonsok, Korona, Polka, and Zephyr, were propagated in regeneration medium in commercially available TIB bioreactors (RITA®) and, for comparison, on the same medium solidified with agar. The TIB system proved to be well suited for shoot propagation and for subsequent subculture of the developing plantlets. Regeneration frequencies were 70±8 to 94±2% and 83±5 to 92±3% in the TIB system and on semi-solid medium, respectively. The labor time taken by the TIB system was less than half of the time required for handling plant material for cultivation on semi-solid medium. This system thus provides a convenient method that could be adopted for commercial in vitro propagation or for regeneration of transgenic strawberry cultivars.

https://bioone.org/journals/in-vitro-cellular-and-developmental-biology---plant/volume-41/issue-6/IVP2005714/SHOOT-REGENERATION-FROM-LEAF-EXPLANTS-OF-FIVE-STRAWBERRY-FRAGARIAANANASSA-CULTIVARS/10.1079/IVP2005714.pdf

Shoot regeneration from leaf explants of five strawberry (fragaria×ananassa) cultivars in temporary immersion bioreactor system

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.

In vitro propagation of North American ginseng ( Panax quinquefolius L.)

Crescentia cujete L. is a widely distributed medicinal tree with a diverse range of phytochemicals used as medicinal compounds. Seedlings of wild-harvested C. cujete were established in vitro and used as the starting material for the establishment of axenic cultures. Shoots were proliferated from nodal segments and were maintained over a period of more than 2 years by sequential subculture on a medium containing 1.0 µmol l−1 kinetin. De novo regeneration was induced on petiole sections cultured onto a medium containing thidiazuron in combination with 2,4-dichlorophenoxyacetic acid. Axenic cultures were also used to test the efficiency of three different cultivation systems for production of biomass of C. cujete. Growth of plantlets in a temporary immersion bioreactor resulted in significant increases in biomass, leaf number, shoot height and transplant efficiency. Plantlets grown in the bioreactors were acclimatized under greenhouse conditions. Together, these experiments have established optimized parameters for propagation and growth of C. cujete plantlets in a sterile controlled environment for biochemical characterization and production of high-quality medicinal products.

In vitro Culture and Temporary Immersion Bioreactor Production of Crescentia cujete

Plastics have become an ever-increasing environmental pollution problem since high amounts of them are dumped every year into the environment. To remediate this problem, many proposals attempt to produce new plastics with the same sound characteristics as the old ones, but with an increased biodegradability to remain only a few months in the environment before they can be turned into safe non-polluting natural products by microbes. One example of these materials is the polylactic acid-based plastic (PLA), which is presented as a biodegradable-compostable material but usually with a warning that they perform in this way under particular conditions. In this work, we evaluate the biodegradability of PLA and other plastics in a composting system under normal atmospheric conditions with an average temperature around 24 ºC in order to establish its compostability in typical conditions of tropical countries. Results obtained by monitoring the integrity and weight of plastic pieces and NMR over time reveal that none of the materials was biodegraded after 31 weeks (∼7 months) under the studied conditions. On the other hand, amplicon sequencing of the 16S rRNA showed the presence of bacterial groups identified previously on the degradation of biodegradable plastics (e.g., Methylotrophs and Actinobacteria) and are aligned with previous studies that conclude that plastics considered as biodegradable influence the soil microbiota. This work contributes to establishing which plastic materials should be classified as biodegradable in prevailing conditions in tropical countries. 

Rosaura M. Romero

Papers

In vitro Culture and Temporary Immersion Bioreactor Production of Crescentia cujete

Biodegradation of Plastics at Home Composting Conditions