The role of activated charcoal in plant tissue culture.

The recent increase in organoid research has been met with great enthusiasm, as well as expectation, from the scientific community and the public alike. There is no doubt that this technology opens up a world of possibilities for scientific discovery in developmental biology as well as in translational research, but whether organoids can truly live up to this challenge is, for some, still an open question. In this Spotlight article, Meritxell Huch and Juergen Knoblich begin by discussing the exciting promise of organoid technology and give concrete examples of how this promise is starting to be realised. In the second part, Matthias Lutolf and Alfonso Martinez-Arias offer a careful and considered view of the state of the organoid field and its current limitations, and lay out the approach they feel is necessary to maximise the potential of organoid technology.

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The hope and the hype of organoid research.

Introduction. 1. Male Gametogenesis Development and Structure of Sperm D. Southworth, S.C. Russell. 2. Sperm and Generative Cell Isolation and Manipulation D. Southworth. 3. Pollen Germination and Pollen Tube Growth Tip Growth Mechanism in Sexual Plant Reproduction A. Moscatelli, M. Cresti. 4. Female Gametogenesis Ontogenesis of the Embryo Sac and Female Gametes S.D. Russell. 5. Embryo Sac Isolation and Manipulation D.D. Cass, J.D. Laurie. 6. In Vivo Fertilization T.B. Batygina, V.E. Vasilyeva. 7. In Vitro Fertilization E. Kranz. 8. Sexual Incompatibility F. Cruz-Garcia, B.A. MacClure. 9. Zygotic Embryogenesis Structural Aspects R. Czapik, R. Izmailow. 10. Zygotic Embryogenesis Hormonal Control of Embryo Development B. Fischer-Iglesias, G. Neuhaus. 11. Zygotic Embryogenesis: Developmental Genetics The Formation of an Embryo from a Fertilized Egg K. Schrick, T. Laux. 12. Somatic Embryogenesis T.A. Thorpe, C. Stasolla. 13. Synthetic Seeds of Asparagus officinalis L. K. Mamiya, et al. 14. Endosperm Development P.W. Becraft, et al. 15. Seed Maturation, Germination, and Dormancy A.B. Downie. 16. Gametophytic Apomixis A Successful Mutation of the Female Gametogenesis Y. Savidan. 17. Parthenocarpy State of the Art A. Spena, G.L. Rotino. 18. Androgenesis in Brassica A Model System to Study the Initiation of Plant Embryogenesis J.B.M. Custers, et al. 19. Androgenesis in Cereals S.K. Datta. 20. In Vitro Gynogenesis S.S. Bhojwani, T.D. Thomas. 21. Inheritance of Cytoplasmic Traits -- Embryological Perspectives T. Kuroiwa, et al. Subject Index.

Current trends in the embryology of angiosperms.

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny

Gene technology for grain legumes: can it contribute to the food challenge in developing countries?

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months.

In vitro organogenesis and plant formation in Acacia sinuata

Embryogenic callus was induced from primary leaves of Vigna unguiculata (L.) Walp. in MS medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D). Greenish-white, friable embryogenic calluses were used to establish suspension cultures. A shaking speed of 90 rpm and 0.4 ml packed cell volume per 25 ml medium were found to be optimal for maintaining suspension cultures. Globular, heart-shaped and torpedo-shaped embryos were developed in suspension culture containing 4.52 μM 2,4-D. Maturation of cotyledonary-stage somatic embryos was achieved on 0.05 μM 2,4-D, 5 μM abscisic acid and 3% mannitol. Twenty-two percent of the embryos were converted into plants and survived; survival in the field was 8–10%.

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA3). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Induction of somatic embryogenesis from nucellus-derived callus of Anacardium occidentale L

Suspension cultures initiated from calluses derived from seedling leaf explants of Acacia sinuata (Lour.) Merr. produced somatic embryos. Embryogenic callus was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.22 μM 6-benzylaminopurine. A high frequency of somatic embryos was induced in MS liquid medium supplemented with 4.52 μM 2,4-D and 10% coconut water. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent development led to the formation of globular, heart, torpedo-shaped and cotyledonary-stage embryos. The conversion of somatic embryos occurred on auxin-free MS medium. Effects of various auxins, cytokinins, carbohydrates and amino acids in enhancing productin, of somatic embryos were studied. MS medium supplemented with 87.64 mM sucrose and 342.46 μM glutamine promoted higher somatic embryo production whereas cytokinin had no effect and led to recallusing of embryos. About 8–10% of embryos converted into plants.

R. Prem Anand

Papers

In vitro organogenesis and plant formation in Acacia sinuata

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Induction of somatic embryogenesis from nucellus-derived callus of Anacardium occidentale L

Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr.