The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis. Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail. Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.

Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh

Plant stress responses are a key factor in steering the development of cells, tissues, and organs. However, the stress-induced signal transduction cascades that control localized growth and cell size/differentiation are not well understood. It is reported here that oxidative stress, exerted by paraquat or alloxan, induced localized cell proliferation in intact seedlings, in isolated root segments, and at the single cell level. Analysis of the stress-induced mitotic activity revealed that oxidative stress enhances auxin-dependent growth cycle reactivation. Based on the similarities between responses at plant, tissue, or single cell level, it is hypothesized that a common mechanism of reactive oxygen species enhanced auxin-responsiveness underlies the stress-induced re-orientation of growth, and that stress-induced effects on the protoplast growth cycle are directly relevant in terms of understanding whole plant behaviour.

Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ, and cellular level

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7-10 months.

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High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

Somatic embryogenesis (SE) has been studied as a model system for understanding of molecular events in the physiology, biochemistry, and biology areas occurring during plant embryo development. Stresses are also the factors that have been increasingly recognized as having important role in the induction of SE. Plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D), ABA, ethylene, and high concentrations of 2,4-D are known as stress-related substances for acquisition of embryogenic competence by plant cells. Gene expression analysis in both the proteome and transcriptome levels have led to the identification and characterization of some stress-related genes and proteins associated with SE. This review focuses on the molecular basis for stress-induced acquisition of SE.

The molecular basis for stress-induced acquisition of somatic embryogenesis.

Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops. Over the last decade, significant progress has been made in the development of new and efficient transformation methods in plants. Despite a variety of available DNA delivery methods, Agrobacterium- and biolistic-mediated transformation remain the two predominantly employed approaches. In particular, progress in Agrobacterium-mediated transformation of cereals and other recalcitrant dicot species has been quite remarkable. In the meantime, other transgenic-enabling technologies have emerged, including generation of marker-free transgenics, gene targeting, and chromosomal engineering. Although transformation of some plant species or elite germplasm remains a challenge, further advancement in transformation technology is expected because the mechanisms of governing the regeneration and transformation processes are now better understood and are being creatively applied to designing improved transformation methods or to developing new enabling technologies.

Recent advances in plant transformation.

A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton (Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20–24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.

High-frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress.

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

A simple and rapid Agrobacterium -mediated transformation protocol for cotton ( Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

S. Leelavathi

Papers

High-frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress.

A simple and rapid Agrobacterium -mediated transformation protocol for cotton ( Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants