Micropropagation In Bioreactors Research Articles

Alstroemeria Micropropagation in a RITA® Temporary Immersion System.

The use of temporary immersion systems (TIS) for plant micropropagation is an efficient technique for plant production, and we have applied it for the production of alstroemerias. This method involves the cultivation of explants such as rhizomes and axillary buds in a nutrient medium to stimulate shoot growth. TIS offer advantages such as accelerated multiplication processes, uniform production, and cost reduction. This process has shown promise in meeting the growing demand for alstroemeria plants in the market. This chapter describes a specific protocol for temporary immersion bioreactor micropropagation of the "Albatroz" cultivar, with the potential for large-scale automation.

Phenotypic variation and epigenetic insight into tissue culture berry crops.

Berry crops, a nutrient powerhouse for antioxidant properties, have long been enjoyed as a health-promoting delicious food. Significant progress has been achieved for the propagation of berry crops using tissue culture techniques. Although bioreactor micropropagation has been developed as a cost-effective propagation technology for berry crops, genetic stability can be a problem for commercial micropropagation that can be monitored at morphological, biochemical, and molecular levels. Somaclonal variations, both genetic and epigenetic, in tissue culture regenerants are influenced by different factors, such as donor genotype, explant type and origin, chimeral tissues, culture media type, concentration and combination of plant growth regulators, and culture conditions and period. Tissue culture regenerants in berry crops show increased vegetative growth, rhizome production, and berry yield, containing higher antioxidant activity in fruits and leaves that might be due to epigenetic variation. The present review provides an in-depth study on various aspects of phenotypic variation in micropropagated berry plants and the epigenetic effects on these variations along with the role of DNA methylation, to fill the existing gap in literature.

In Vitro Propagation Strategies of Medicinally Important Berry Crop, Lingonberry (Vaccinium vitis-idaea L.)

Lingonberry (Vaccinium vitis-idaea L.) is a health-promoting small fruit crop rich in antioxidant metabolites that helps to reduce the incidence of degenerative diseases. Being heterozygous, lingonberries cannot preserve genetic characteristics through seed propagation. Conventional vegetative propagation, although it produces true-to-type plants, is not economically viable. In vitro propagation can multiply plants much faster than conventional methods. A liquid cultures system under a bioreactor micropropagation system is of significant importance to increase the multiplication rates of in vitro-produced shoots. Enhanced vegetative growth and variation in biochemical constituents are observed in micropropagated plants. Clonal fidelity, although it may be a serious problem for commercial micropropagation, can be verified efficiently by molecular markers. The current review provides detailed and updated information on lingonberry micropropagation along with conventional methods and their effects on morphological, molecular and biochemical characteristics in micropropagated plants, filling the gap in literature.

Biotechnological Potential of Endophytic Bacteria to Improve the Micropropagated Seedling of Variety RB92579 Sugarcane (<i>Saccharum officinarum L.</i>)

Endophytic bacteria may influence agricultural production in several ways, including promoting plant growth. Two experiments were conducted in order to evaluate the combination of endophytic bacteria from the Brazilian Northeast region aims at the commercial introduction of the inoculation of these bacteria in micropropagated sugarcane plants using a temporary immersion bioreactor. One experiment was done in tubes with sterile commercial substrate, and the other was done in pots with soil; both were installed in a greenhouse. A mixed inoculation was performed in six inoculated endophytic diazotrophic bacteria in micropropagated sugarcane plants, variety RB92579. In the experiment with soil, the mixed inoculation significantly increased the shoot dry matter of plants without the addition of nitrogen fertilizer. However, the accumulation of total-N in the tissues showed no significant differences between treatments with and without nitrogen fertilization. The evaluation of micropropagated seedlings showed no increases in the parameters tested. The results showed that the response of inoculation in temporary immersion bioreactor micropropagation is possible, and that the application of homologous strains may have contributed to a better response by the interaction of endophytic bacteria with sugarcane RB92579. Further studies should be conducted to improve the methodology, which indicates a great potential to optimize this process on a commercial scale.

Molecular approaches for monitoring clonal fidelity and epigenetic variation in in vitro-derived strawberry plants

The advancement of propagation techniques in vitro and their acceptance by commercial growers led to significant progresses in strawberry micropropagation worldwide. Bioreactor micropropagation in liquid media has promoted automation of strawberry micropropagation as a possible way to reduce labour and propagation cost. Production of plants genetically identical to the mother plant is a major concern in commercial strawberry production. DNA-based markers that allow direct comparison of different genetic material independent of environmental influences can efficiently monitor genetic stability of micropropagated strawberries. This review deals with progress of strawberry culture in vitro, genetic and epigenetic variations in micropropagated plants and the use of molecular markers for the assessment of these variations.

Molecular markers, clonal fidelity and antioxidant properties of micropropagated horticultural crops

In vitro propagation is practised all over the world and has now become a multibillion dollar industry for mass propagation of various plant species. The commercial use of micropagation represents one of the major success stories of utilizing tissue culture technology profitably. Fruits and vegetables contain relatively high levels of vitamin C, cellulose and pectin, and produce anthocyanins, which have important therapeutic values, including antitumor, antiulcer, antioxidant and anti-inflammatory activities. Tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has also occurred in various horticultural crops including berry crops. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of plant species in liquid culture systems. With the advent of plant tissue culture techniques, it is extensively employed to multiply true-to-type plants rapidly which provides year around production. Somaclonal variation which can be genetic or epigenetic, affects the use of tissue culture propagation negatively, in general. Plant cells when cultured in artificial environment make numerous genetic alterations and aberrations. The introduction of molecular biology techniques, such as DNA-based markers, allows direct comparison of different genetic material independent of environmental influences. This review describes the progress in depth of morphological and chemical characterization of micropropagated horticultural crops. It also describes the use of molecular markers to address fundamental and practical questions of in vitro culture, and the employment of molecular markers for the assessment of genetic fidelity, uniformity, stability and true-to-typeness among donor and micropropagated horticultural plants.

THE USE OF CONTINUOUS, TEMPORARY IMMERSION BIOREACTOR SYSTEM AND SEMISOLID CULTURE MEDIUM FOR THE PRODUCTION Of <i>Eucalyptus camaldulensis</i> CLONES

The plant micro-propagation in bioreactor systems is regarded as one way to reduce cost by automation and production scheduling. This research was carried out in order to obtain an efficient procedure for clone production of Eucalyptus camaldulensis on different types of bioreactor including continuous and temporary immersion bioreactor. To do so, the apical meristems (1 mm) and the apical meristems with adjacent tissue (2,5 mm) were used as initial explants. These tissues were cultured, for 60 days, in semisolid culture medium supplemented with 1 mg L-1 indole acetic acid (IAA) and 0.32 mg L-1 benzylaminopurine (BA). After 60 days, the meristems with adjacent tissue were transferred to a continuous immersion bioreactor and maintained in dark or light conditions. In order to verify the effect of the explant source on bioreactor multiplication, the explants subcultured from meristems multiplied in semisolid culture medium and the meristems multiplied in continuous immersion bioreactor were tested and maintained in dark conditions. After establishing this parameters, the multiplication experiments were carried out in continuous and temporary immersion and the multiplied explants were then rooted in MS medium supplemented with 0, 2, 4, 8 and 20 mg L-1 indole butyric acid (IBA) and kept in the dark or under controlled lighting conditions. After that, the rooting the plants were acclimatized in mist chamber. The meristem with adjacent tissue favored a greater number of buds/explants. The continuous immersion bioreactor in the dark provided higher shoots number and multiplication rate. The rooting was better on culture medium without auxin and kept in the dark for 15 days or the culture medium supplemented with auxin and maintained under light with 100% plantlet rooting. The Eucalyptus camaldulensis acclimatization was efficient, with high survival rate (76%). It was possible to establish the procedure for bioreactor micro-propagation of Eucalyptus camaldulensis large-scale clones.

Corrigendum: Bioreactors and molecular analysis in berry crop micropropagation—A review

Debnath, S. C. 2011. Bioreactors and molecular analysis in berry crop micropropagation - A review. Can. J. Plant Sci. 91: 147-157. While berry fruits have long enjoyed huge popularity among consumers, tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has occurred. Of particular significance has been the evolution of the technology permitting multiplication of berry plants in bioreactors containing liquid media. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of berry plants in liquid culture systems. Clonal fidelity can be a serious problem, and molecular strategies have been developed in order to reduce the variation to manageable levels....

GERMPLASM CHARACTERIZATION, PROPAGATION AND MAINTENANCE OF HEALTH-PROMOTING WILD BERRIES AND MEDICINAL PLANT ROSEROOT (RHODIOLA ROSEA L.)

Berry crops include, but are not limited to the members of the genera: Fragaria (strawberry; Rosaceae), Rubus (brambles: raspberry and blackberry; Rosaceae), Vaccinium (blueberry, cranberry and lingonberry; Ericaceae) and Ribes (currant and gooseberry; Grossulariaceae). Berry fruits contain relatively high levels of vitamin C, cellulose, and pectin, and produce anthocyanins which have important therapeutic values, including antitumor, antiulcer, antioxidant and anti-inflammatory activities. Blueberry, cranberry and lingonberry are three Vaccinium fruit crops of economic importance which have been domesticated in the twentieth century. Cloudberry (Rubus chamaemorus L., family Rosaceae) is a less know small fruit of medicinal importance. The roseroot (Rhodiola rosea L.), also called golden root or arctic root, is used as an adaptogen in herbal medicine and is valued for its ability to enhance human resistance to stress or fatigue and promote longevity. There is a pressing need to develop reliable methods for conserving, maintaining and identifying berry germplasm and for assessing genetic biodiversity for practical breeding purposes and proprietary-rights protection. The introduction of molecular biology techniques, such as DNA-based markers, allows direct comparison of different genetic material independent of environmental influences. This paper presents the progress in-depth of various aspects of molecular diversity analyses in wild berry species and cultivars collected from Canada, Europe and USA. Inter simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD), expressed sequence tag-polymerase chain reaction (EST-PCR) and EST-simple sequence repeat (SSR) markers detected a sufficient degree of polymorphism to differentiate among wild clones and cultivars, making these technologies valuable for cultivar identification and for the more efficient choice of parents in berry breeding programs. Tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has also been occurred. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of berry plants in liquid culture systems. The paper describes the progress in-depth of various aspects of molecular characterization of berry and roseroot germplasm, their propagation and maintenance, and on the employment of molecular markers for the assessment of genetic fidelity, uniformity, stability and trueness-to-type among donor plants and tissue culture regenerants.

STRATEGIES TO PROPAGATE VACCINIUM NUCLEAR STOCK AND USE OF MOLECULAR MARKERS FOR CLONAL FIDELITY

Significant progress in plant tissue culture, resulting in great advances in micropropagation, has occurred with Vaccinium species. The in vitro morphogenesis is highly dependent on plant growth regulators and media used for culture, which is again genotype specific. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon better understanding of physiological and biochemical responses of plants to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of berry plants in liquid culture systems. Increased branching, vigorous vegetative growth and change in biochemical components are noted in micropropagated Vaccinium plants acclimatized under ex vitro condition. Clonal fidelity can be a serious problem and strategies have been developed in order to reduce the variation to manageable levels. Molecular markers have been introduced in tissue culture research and can potentially be used in various facets of pertinent studies with berry crops. This review describes the progress in-depth of various aspects of Vaccinium culture in vitro, characterization of micropropagated plants for morphological characters, anthocyanin contents and antioxidant activity, and on the employment of molecular markers in these plants for the assessment of genetic fidelity, uniformity, stability and trueness-to-type among donor plants and tissue culture regenerants.

Propagation Strategies and Genetic Fidelity in Strawberries

While strawberries have long enjoyed huge popularity among consumers, tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has occurred. The in vitro morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, which is again genotype specific. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon a better understanding of physiological and biochemical responses of plants to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of strawberry plants in liquid culture systems. Clonal fidelity can be a serious problem and molecular strategies have been developed in order to reduce the variation to manageable levels. The article describes the progress in-depth of various aspects of strawberry propagation in vitro on semi-solid gelled media and in liquid media using bioreactors, for their improvement and for commercial production. The article also focuses on the employment of molecular markers in micropropagated plants for the assessment of genetic fidelity, uniformity, stability, and trueness-to-type among donor plants and tissue culture regenerant.

Morphological and molecular analyses in micropropagated berry plants acclimatized under ex vitro condition

Debnath, S. C., Vyas, P., Goyali, J. C. and Igamberdiev, A. U. 2012. Morphological and molecular analyses in micropropagated berry plants acclimatized under ex vitro condition. Can. J. Plant Sci. 92: 1065–1073. Berry crops include, but are not limited to, the members of the genera Fragaria (strawberry; Rosaceae), Rubus (brambles: raspberry and blackberry; Rosaceae), Vaccinium (blueberry, cranberry and lingonberry; Ericaceae) and Ribes (currant and gooseberry; Grossulariaceae). While berry fruits have long enjoyed huge popularity among consumers, tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has occurred. The in vitro morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, which is again genotype specific. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing the cost of propagation, optimal plant production depends on better understanding of physiological and biochemical responses of plants to the signals of the culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of berry plants in liquid culture systems. Increased branching, vigorous vegetative growth and change in biochemical components are often noted in micropropagated plants acclimatized under ex vitro condition. Clonal fidelity can be a serious problem and strategies have been developed to reduce the variation to manageable levels. Molecular markers have been introduced in tissue culture research and can potentially be used in various facets of pertinent studies with berry crops. This paper describes in depth the progress of various aspects of berry propagation in vitro, the characterization of micropropagated berry plants for morphological characters, and the employment of molecular markers in these plants for the assessment of genetic fidelity, uniformity, stability and trueness-to-type among donor plants and tissue culture regenerants.

Bioreactors and molecular analysis in berry crop micropropagation – A review

Debnath, S. C. 2011. Bioreactors and molecular analysis in berry crop micropropagation – A review. Can. J. Plant Sci. 91: 147–157. While berry fruits have long enjoyed huge popularity among consumers, tremendous progress in plant tissue culture, resulting in great advances in micropropagation, has occurred. Of particular significance has been the evolution of the technology permitting multiplication of berry plants in bioreactors containing liquid media. Although automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost, optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of berry plants in liquid culture systems. Clonal fidelity can be a serious problem, and molecular strategies have been developed in order to reduce the variation to manageable levels. Molecular markers have been introduced to tissue culture research and can potentially be used in various facets of pertinent studies with berry crops. The paper focuses on bioreactor systems combined with semi-solid media used for in vitro culture of berry crops, cultivation of micropropagules and employment of molecular markers in micropropagated plants for the assessment of genetic fidelity, uniformity, stability and trueness-to-type among donor plants and tissue culture regenerants. The pertinent literature is reviewed and the relative merits and shortcomings of the various molecular markers applied are presented with an emphasis on the nature of tissue culture-induced variation.

BERRY BIOREACTOR MICROPROPATION AND MOLECULAR ANALYSIS FOR GENETIC FIDELITY

An improved understanding of the important role of dietary fruit in maintaining human health has led to a dramatic increase in global berry demand and production in recent years. While berry fruit crops have long enjoyed huge popularity among consumers, tremendous progress in plant tissue culture and berry crop micropropagation has occurred. Of particular significance has been the evolution of the technology permitting multiplication of berry plants through bioreactor micropropagation. Automation of micropropagation in bioreactors has been advanced as a possible way of reducing propagation cost. However, optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of liquid culture microenvironment. Clonal fidelity can be a serious problem and strategies have been developed in order to reduce the variation to manageable levels. Molecular markers such as RAPDs, RFLPs, AFLPs, DAFs, SCARs, SSRs and ISSRs have been introduced in tissue culture research and can potentially be used in various facets of pertinent studies with berry crops. This review focuses on the bioreactor systems used for in vitro culture of berry crops and employment of molecular markers in micropropagated berry plants for the assessment of genetic fidelity, uniformity, stability, and true-to-typeness among donor plants and tissue culture regenerants. The relevant literature is reviewed and the relative merits and shortcomings of the various molecular markers applied are presented with an emphasis on the nature of tissue culture-induced variation.

Efficiency of liquid culture systems over conventional micropropagation: A progress towards commercialization

The most common methods of micropropagation involve the proliferation of shoots via a semi solid system. While such semi solid systems have been moderately to highly successful in terms of multiplication yields, it has become increasingly important to improve productivity and reduce the time taken to multiply commercially important material. Micropropagation by conventional techniques is typically a labor intensive time taking means of clonal propagation. To overcome this, the use of shake cultures utilizing liquid culture medium has been promoted. The liquid medium allows the close contact with the tissue which stimulates and facilitates the uptake of nutrients and phytohormones, leading to better shoot and root growth. Continuous shaking promotes lesser expression of apical dominance which generally leads to induction and proliferation of numerous axillary buds. Further, with in the shake culture conditions, the growth and multiplication rate of shoots is enhanced by forced aeration, since continuous shaking of medium provides ample oxygen supply to the tissue which ultimately leads to their faster growth. Bioreactor provides a rapid and efficient clonal propagation system utilizing liquid medium to avoid intensive manual handling. Automation of micropropagation in bioreactors has been advanced by several authors as a possible way of reducing cost of micropropagation. Micropropagation in bioreactors for optimal plant production depends upon better understanding of physiological and biochemical responses of plant to the signals of culture microenvironment and an optimization of specific physical and chemical culture conditions to control the morphogenesis of plants in liquid culture systems.

PROPAGATION OF PLANTS IN BIOREACTORS: PROSPECTS AND LIMITATIONS

Bioreactors provide a rapid and efficient plant micropropagation system but as yet are not fully exploited commercially for agricultural crops and forest trees. The culture system was applied to herbaceous and woody plant species utilizing liquid media to overcome intensive manual handling. Large-scale liquid cultures were used for propagation through organogenesis or somatic embryogenesis pathways depending on the species. Bioreactors have several advantages over agar cultures with a better control of the culture conditions. Direct contact of the plant tissue with the medium, optimal nutrient and growth regulators supply, continuous aeration and circulation, filtration of the medium for controlling exudates and contamination and the production of clusters of buds, meristems or protocorms for automated dispensing. The major issue imposed by liquid media in bioreactors is the phenomenon of hyperhydricity, morphogenic shoot and leaf malformation, due to the continuous immersion of the tissue in the medium. The malformations are manifested in glossy hyperhydrous leaves with distorted anatomy. The very nature of liquid tissue culture is an imposing stress to which the plants respond to the environmental signals in developmental aberration. The submerged tissue was found to exhibit oxidative stress symptoms, with elevated levels of reactive oxygen species that were associated with a change in anti-oxidant enzyme activity. These changes greatly affected the anatomy and physiology of the plants and their survival after transplanting. Two major solutions for malformation control were proposed: the use of growth retardants to control rapid proliferation and temporary immersion bioreactors. Growth retardants reduced water uptake during cell proliferation, decreased vacuolation and rapid growth, shortened stems and inhibited leaf expansion, inducing the formation of clusters. In tuber, bulb and corm producing plants growth retardants were found to enhance storage organs formation. Temporary immersion bioreactors were used to provide an improved aeration system to reduce hyperhydricity, by shortening plant duration in the liquid phase. The frequency of immersion and the medium composition improved plant development. Future prospects of micropropagation in bioreactors for optimal plant production will depend on both basic and applied studies. The former contributing to our understanding of plant responses to microenvironment signals to overcome the limitations of liquid cultures and the latter to provide specific culture manipulation to control the morphogenesis of plants in vitro improving survival ex vitro.