Tissue culture of ornamental pot plant: A critical review on present scenario and future prospects

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

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.

The mode of action of thidiazuron: auxins, indoleamines, and ion channels in the regeneration of Echinacea purpurea L.

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.

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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

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

Induction of petiole explants with thidiazuron resulted in the development of somatic embryos originating from the epidermal cells of African violet petioles. Orientation of the petiole explants and auxin transport were found to be a crucial factor in regulation of the embryogenic response. The current study was designed to assess the effect of thidiazuron on the induction of somatic embryogenesis and the role of ion transport in the thidiazuron-induced morphogenesis. Incorporation of the agonists of calcium channels, (S)-Bay K8644, into the thidiazuron-medium changed the pattern of somatic embryogenesis such that an embryogenic callus formed across the tissue and subsequently embryos formed on the callus. Antagonists of calcium movement, diltiazem and verapamil, inhibited the embryogenic response. Lidocaine, an antagonist of sodium channels, redirected the morphogenic response with root formation at lower concentrations and callus proliferation on explants cultured with higher amounts of the inhibitor. These data provide evidence that the transport of calcium and sodium play an important role in cell competence and thidiazuron-induced somatic embryogenesis.

Auxin, calcium and sodium in somatic embryogenesis of african violet (saintpaulia ionantha wendl. cv. benjamin)

The development of an in vitro regeneration system that utilizes a plant growth regulator having cytokinin activity for the induction of de novo shoots or somatic embryos on explants of phytopharmaceutical plants is provided. Transfer of the regenerated shoots or somatic embyros into a solid or liquid medium with no plant growth regulators results in the rapid and prolific growth of viable plantlets. The method and its modifications are intended for application to all phytopharmaceutical plants, in particular St. John's wort (Hypericum perforatum cv. Anthos), Huangqin (Scutellaria baicalensis), Echinacea sp., feverfew (Tanacetum parthenium), garlic (Allium sp.) and the like.

Micropropagation of phytopharmaceutical plants

Summary Recent research has reported the presence of melatonin (N-acetyl-5-methoxytryptamine), a mammalian indoleamine neurohormone, in higher plants, indicating that melatonin may be an important metabolic regulator that has been highly conserved across biological kingdoms. Melatonin is synthesized from tryptophan in the mammalian pineal gland and a similar biosynthetic pathway was recently described in St. John’s wort shoot tissues, wherein radiolabel from tryptophan was recovered in serotonin and melatonin as well as indoleacetic acid. There is growing information describing melatonin control of physiological processes in mammals, yeast, and bacteria, including diurnal responses, detoxification of free radicals, and environmental adaptations. However, at the current time, there is no known specific role for melatonin in plant physiology. Alterations in melatonin concentrations in plant tissues have been shown to affect root development, mitosis, and mitotic spindle formation. The recent advancements in melatonin research in plants and some directions for important areas of future research are reviewed in this article.

Praveen K. Saxena

Papers

In vitro Culture and Temporary Immersion Bioreactor Production of Crescentia cujete

Auxin, calcium and sodium in somatic embryogenesis of african violet (saintpaulia ionantha wendl. cv. benjamin)

Micropropagation of phytopharmaceutical plants

Invited review: melatonin: a potential regulator of plant growth and development?