Ornamental Chrysanthemums: Improvement by Biotechnology

The in vitro tissue culture and micropropagation of chrysanthemums, important floricultural (cut-flower) and ornamental (pot and garden) plants, have been well studied. An increase in genetic transformation studies aimed at improving aesthetic and growth characteristics of the plants has been hampered by low transformation efficiencies and genotype dependence of protocols. As a result chrysanthemum regeneration studies have once again emerged as an essential complement of transformation studies. This review highlights the impact that biotechnology has had on the improvement of chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.     

Chilli peppers — A review on tissue culture and transgenesis

Biotechnology techniques involving plant tissue culture and recombinant DNA technologies are powerful tools that can complement conventional breeding and expedite Capsicum improvement. The rate of progress in Capsicum is relatively slower than other members of Solanaceae because of its high genotypic dependence and recalcitrant nature. Capsicum is a recalcitrant plant in terms of in vitro cell, tissue and organ differentiation, plant regeneration and genetic transformation which makes it difficult to apply recombinant DNA technologies aimed at genetic improvement against pests, diseases and abiotic stress. Despite this, application of tissue culture and genetic transformation have led to significant development in chilli pepper plants, and studies are underway to achieve the targets of pre-harvest improvement and post-harvest characterization for value addition to this crop. This review presents a consolidated account of in vitro propagation and focuses upon contemporary information on biotechnological advances made in Capsicum.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and agronomic performance of regenerated chili pepper (C<Emphasis Type="Italic">apsicum</Emphasis> spp.) plants from commercially important genotypes

The application of modern biotechnology for improvement of chili pepper productivity requires an efficient in vitro plant regeneration protocol. In this study, a reliable protocol was developed for the in vitro regeneration of four types of chili, Capsicum annuum var. annuum (Jalapeño and Serrano), C. annuum var. glabriusculum/aviculare (Piquin), and C. chinense (Habanero) by direct organogenesis using three different explants (cotyledon, hypocotyls, and embryo) and three induction media. All evaluated culture media promoted the formation of adventitious shoots. When embryos or hypocotyls were used as explants, morphologically normal adventitious shoots developed, while culturing cotyledons resulted in nonelongating rosette-shaped shoots. The highest in vitro regeneration efficiency (14.6 shoots per explant) was achieved when Habanero chili hypocotyls were grown on Murashige and Skoog medium containing 1.7 μM indole-3-acetic acid and 22.2 μM N⁶-benzyladenine. This regeneration rate is higher than that obtained in previous reports. Regenerated plants were ready to be transferred to the greenhouse 13 wk after the explant culture. An evaluation carried out under greenhouse conditions showed differences in agronomic performance between in vitro regenerated plants and plants developed from seeds with the magnitude of the differences depending on the genotype being studied.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R ₀ and R ₁ transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level. Collaborator via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems     

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.     

Plant tissue culture. Biotechnology. Milestones

Summary The progress in the development of the technologies of plant tissue and cell culture over the past four decades has been remarkable. This article covers my personal reflections on the various topics and is based on my involvement in the field during that period. There are three fundamental technologies which constitute most of what is referred to as plant in vitro technologies or tissue culture. The origin and some of the key persons involved in the development of each of these procedures will be discussed. The technology that is most common is growing plant tissue on gel-solidified nutrient media. That technology is being used in the most vital procedures, namely the regeneration of plants from cultured cells. The culture of plant cells in liquid suspension was developed very shortly after that, and has become a very effective technology for plant regeneration by somatic embryogenesis. The method of meristem culture arose out of a need for developing plants that were virus-free. In many species the technique is now being used to produce virus-free crop plants. Another important technology is the culture of anthers and microspores for producing haploid and homozygous plants. Included with plant tissue culture is the development of the plant protoplast and cell fusion technologies for the production of new plant hybrids. The final aspect of the development concerns the integration of tissue culture with molecular genetics, which has developed into the rapidly expanding field of biotechnology.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Sepecial symposium: In vitro plant recalcitrance in vitro plant recalcitrance: An introduction

Summary In vitro recalcitrance is the inability of plant cells, tissues and organs to respond to tissue culture manipulations. With respect to plant regeneration, recalcitrance can be a major limiting factor for the biotechnological exploitation of economically important plant species and it can also impair the wider application of in vitro conservation techniques. This first paper introduces a compilation of Symposium papers, collectively entitled “Do we understand in vitro plant recalcitrance?”, presented at the 1999 Congress of the Society for In Vitro Biology. The Symposium reviewed recalcitrance in the context of genetic predeterminism, molecular markers and gene expression patterns, whole and explant physiology, stress physiology, habituation, neoplastic progression and plant cancer. The symposium contributors present fundamental and applied investigative approaches which have the potential to enhance our current understanding of in vitro recalcitrance and to assist in overcoming the problems associated with nonresponsive plant cultures. This introductory paper presents the general concept of recalcitrance in relation to whole-plant and explant physiology and considers basic aspects of tissue culture manipulations in the context of recalcitrance problems.     

Factors Affecting Plant Regeneration from Tissue Cultures of Chinese Leymus (Leymus chinensis)

Chinese leymus (Leymus chinensis Trin.) is a perennial grass of the Gramineae, which is widely distributed in China, Mongolia and in Russian-Siberian. In order to explore the potential of biotechnology for genetic improvement of this forage grass, an efficient tissue culture system was established and the factors affecting plant regeneration were evaluated. Immature inflorescence segments 3–5 mm in length from eight accessions were cultured on N6 medium supplemented with 2.26–22.60 µM 2,4-D. The callus induction frequency ranged from 72.11 to 82.19%. Shoots were differentiated from the calli on N6 medium containing 4.65 µM kinetin and 4.44 µM BA. Viable regenerants were developed on hormone-free medium. Normal plants were obtained after natural vernalization in the field. The plant regeneration frequency in Chinese leymus was associated with different genotypes and different combinations of growth regulators in medium. The concentration of 2,4-D in the callus induction medium had a strong effect on successive plant regeneration. Relatively higher concentrations of 2,4-D (i.e., 9.04 and 22.60 µM) were more favorable to the plant regeneration than lower ones (i.e., 2.26 and 4.52 µM). This is the first report on plant regeneration in vitro in L. chinensis.     

Biotechnology Applications for Sugar Beet

Sugar beet (Beta vulgaris L.) is an important industrial crop, being one of only two plant sources from which sucrose (i.e., sugar) can be economically produced. Despite its relatively short period of cultivation (ca. 200 years), its yield and quality parameters have been significantly improved by conventional breeding methods. However, during the last two decades or so, advanced in vitro culture and genetic transformation technologies have been incorporated with classical breeding programs, the main aim being the production of herbicide-and salt-tolerant, disease- and pest-resistant cultivars. Among the many applications of in vitro culture techniques, sugar beet has benefited the most from haploid plant production, protoplast culture, and somaclonal variation and in vitro cell selection. Several genetic transformation technologies have been developed, such as Agrobacterium-meditated, PEG-mediated, particle bombardment, electroporation, sonication and somatic hybridization, the first two being the most successful. Development of herbicide- and salt-tolerant, virus-, pest/nematode-, fungus/Cercospora- and insect-resistant sugar beet has been demonstrated. However, only herbicide-tolerant varieties have been approved for commercialization but not yet available in the marketplace; rhizomania-resistant varieties are being evaluated in field trials. Transgenic plants that convert sucrose into fructan, a polymer of fructose, were also developed. Initial attempts to increase sucrose yields produced promising results, but it still requires additional work. Despite marked progress in improving regeneration and transformation of sugar beet, genotype dependence and low regeneration and transformation frequencies are still serious restrictions for routine application of in vitro culture and, more importantly, transformation technologies. Selected food safety and environmental impact, as well as regulatory and public acceptance issues relating to transgenic sugar beet are also discussed.     

<Emphasis Type="Italic">Echinacea</Emphasis> biotechnology: Challenges and opportunities

Echinacea, better known as purple coneflower, has received a global attention because of its increasing medicinal value. There is enormous potential for the discovery of new medicinal compounds in this species and an immediate need for techniques to facilitate the production of high quality, chemically consistent plant material for drug development and clinical trials. In vitro tissue culture of Echinacea can play a vital role in the development of novel germplasm, rapid multiplication, and genetic modifications for an enhanced phytochemical production. Recent establishment of liquid culture techniques, large-scale bioreactors, and Agrobacterium-mediated transformation are changing the production parameters of the Echinacea species. This review provides an overview of the recent developments in in vitro technologies and challenges that remain in the Echinacea biotechnology.     

Plant cell and biotechnology studies in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.     

Recent advances in Pelargonium in vitro regeneration systems

Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation of regeneration by growth regulators, choice of explant and characterization of induction and expression phases of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium opened up the possibilities for developing genotypes with novel characters, including resistance to some of the major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional crop improvement programs, thereby providing a valuable resource to the horticultural industry.     

Genetic transformation of green-colored cotton

Summary This study reports an Agrobacterium-mediated transformation of green-colored cotton (Gossypium hirsutum L.). A tissue culture procedure was optimized to induce callus formation from hypocotyl explants and subsequent differentiation into the embryogenic type. Callus formation could be induced by growing explants on Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Among the four genotypes studied, embryogenic calli and plant regeneration were observed only in var. G9803. Agrobacterium-mediated transformation of G9803 with the fiber-specific expansin gene GhExpl was achieved based on the establishment of these tissue culture methods. A total of 32 individual regenerants resistant to kanamycin were generated within 7 mo., with a transformation frequency of 17.8%. Transformation was confirmed by Southern blot analysis and RT-PCR. These results represent the first step towards genetic manipulation of the colors and fiber quality of green-colored cottons by biotechnology. These authors contributed equally to this work     

Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation

Cultivated strawberry (Fragaria ×ananassa) is a valuable crop, yet the absence of a rapid, high-throughput transgenic system has precluded meaningful application of biotechnology and translation of information from plant models to this crop. A new octoploid strawberry genetic line Laboratory Festival #9 has been identified, selected solely for its rapid regeneration and efficient transformation. Direct organogenesis has been achieved from all tissues tested, with rapidly-growing shoot initials visible in as few as 13 days. The conditions for optimal shoot regeneration, transformant selection, root generation, and plant acclimatization are presented. The progression from explant to plant in soil can be achieved in about 60 days. The development of transformation protocols in this rapid-cycling genotype allows high-throughput studies of gene function in the octoploid strawberry genetic background.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.