1. Introduction. 2. Laboratory Organization and Equipment. 3. Media Preparation. 4. Collection, Sterilization, Excision and Culture. 5. Clonal Propagation. 6. Special Cultures. 7. Secondary Metabolite Production. 8. Pathology. 9. Nitrogen Fixation. 10. Storage of Cultures. 11. Genetic Modification. 12. Epilogue. 13. References. 14. Indices.

In Vitro Culture of Trees

As a general, long-lived perennial plants probably present the most challenging obstecles to the researcher, breeder or propagator utilizing microculture as a tool. These challenges appear during all stages of the microculture process, but are probably most resplendent during the stabilization phase. What may be particularly frustrating is that much of this ‘recalcitrance’ is genetically driven and is thus difficult to control by environmental and nutritional manipulations in microculture. Perennials have complex seasonal cycles and life cycles, which complicate control of their growth in microculture. As shoot cultures have provided useful tools for overcoming these limitations, the inability to establish stabilized shoot cultures is a major form of recalcitrance. Plants having seasonal growth dynamics dominated by strong episodic or determinant shoot growth are some of the most recalcitrant species because stabilized shoot cultures cannot be readily generated. In some cases, episodic growth may be tied closely to phase state and can thus be controlled by manipulating phase; nevertheless, adequate controls have not been identified for many problematic plants. Another trait contributing to recalcitrance of perennials is the relatively slow growth rate in microculture. Slow growth complicates such procedures as selection of transformed tissues. The high phenolic content of many perennial tissues can interfere with the efficacy of transgenic traits such as β-glucuronidase. Developmentally determined growth characteristics such as plagiotropism may persist through all stages of microculture and complicate the recovery of commercially useful micropropagules. Although some technical approaches can occasionally circumvent immediate microculture limitations, general solutions await the development of a deeper understanding of physiological bases of such genetically predetermined phenomena.

Special symposium: In vitro plant recalcitrance recalcitrance of woody and herbaceous perennial plants: Dealing with genetic predeterminism

Vitrified shoots were produced in multiplication cultures of Castanea sativa Mill. when Murashige and Skoog mineral medium was used during successive subcultures, whereas normal shoots were obtained when Heller's macronutrient formula was used with or without addition of 1 mM (NH4)2SO4. Vitreous and normal plantlets were compared both anatomically and chemically. Xylem and sclerenchyma tissues are less differentiated and lignified in vitrified than in normal shoots. On the other hand, there is a hypertrophy in the cortical and pith parenchyma. Leaves of vitrified shoots are characterized by the lack of clear differentiation between the palisade and spongy mesophyll, where a system of large intercellular spaces develops. The cytohistological zonation of vitrified apices is characterized by the comparatively small meristem tissue in the apical dome. Phenolic content of explants, assayed by high pressure liquid chromatography, was lower in vitreous than in normal cultures. Gallic acid was identified in both types of cultures.

Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro

Among the prevalent tissue culture techniques of agricultural and horticultural promise, meristem and shoot tip culture has been exploited at a much wider scale primarily due to its application in diverse areas such as rapid clonal multiplication of vegetatively propagated crop plants, virus elimination and germplasm preservation of both vegetatively and seed propagated crops (Kartha, 1981; 1986; George and Sherrington, 1984; Hussey, 1986), and more recently in the development of genetic transformation protocols for crop improvement through gene transfer (Ulian, 1988; Gould et al., 1991).

Meristem and Shoot Tip Culture: Requirements and Applications

Under the common name Walnut are included species belonging to the genus Juglans, order Juglandales, family Juglandaceae. There are three main groups in the genus Juglans: (1) Common walnuts: J. regia L. (Fig. 1); (2) Black walnuts: J. nigra L.; J. hindsii Jeps; J. californica Watson; J. major Heller and J. rupestris Engelm; (3) Grey walnuts and White walnuts: J. cinerea L., J. sieboldiana Maxim, J. catayensis Dode and J. manchurica Maxim (Germain et al. 1975; Luna Llorente 1979). Trees of the Caraya group are also called American walnuts.

A.M. Vieitez

Papers

Castanea sativa plantlets proliferated from axillary buds cultivated in vitro