Tissue culture callus

Aside from chemical methods, several patents have appeared on the biochemical production of natural vitamin from callus tissue cultures (41). 

Feung, C. S., Hanrilton, R.H., and Mumma, R.O. Metabolism of 2,4-dichlorophenoxyacetic acid. V. Identification of metabolites in soybean callus tissue cultures. J. Agric. Food Chem., 21(4) 637-640, 1973. 

The work by Scott and Lee 165) on the isolation of a crude enzyme system from a callus tissue culture of C. roseus was followed by studies of Zenk et al. on an enzyme preparation from a cell suspension system which produced indole alkaloids 166). The cell-free preparation was incubated with tryptamine and secologanin (34) in the presence of NADPH to afford ajmalicine (39), 19-epiajmalicine (92), and tetrahydroalstonine (55) in the ratio 1 2 0.5. No geissoschizine (35) was detected. In the absence of NADPH, an intermediate accumulated which could be reduced with a crude homogenate of C. roseus cells in the presence of NADPH to ajmalicine (39). Thus, the reaction for the formation of ajmalicine is critically dependent on the availability of a reduced pyridine nucleotide. 

P. Aksenova. Study of histones of calluses with vegetative and generative morphogenesis in trapezoid tobacco. Dokl AkadNauk SSR 1974 216 226. Ackermann, L. Evidence of a glycoprotein, until now unknown, in tissue culture of hlicotiana tabacum. C R Seances Soc Biol Ses Fil 1974 168 344. 

In the process of developing an in vitro system to select phosphate starvation resistant cell lines we simultaneously selected for a line that was constitutively induced for APase excretion. Tissue cultured tomato cells were plated onto solid medium containing starvation levels of phosphate. While most cells died, we identified isolated clumps of callus capable of near-normal rates of growth. Starvation-resistant cells were used to start suspension cultures that were kept under phosphate starva-... 

Obaata-Sasamoto H, Komamine A (1982) Suppression mechanism of DOPA accumulation in Stizolobium callus. In Plant tissue culture 1982. Japanese Association for Plant Tissue Culture, Tokyo, p 345... 

Yasuda, T., Yajima, Y., and Yamada, Y., Induction of DNA synthesis and callus formation from tuber tissue of Jerusalem artichoke by 2,4-dichlorophenoxyacetic acid, Plant Cell Physiol., 15, 321-329, 1974. Yeoman, M.M., Tissue Culture and Plant Science, Street, H.E., Ed., Blackwell s, Oxford, 1974, pp. 1-17. Zubr, J. and Pedersen, H.S., Characteristics of growth and development of different Jerusalem artichoke cultivars, in Inulin and Inulin-containing Crops, Fuchs, A., Ed., Elsevier, Amsterdam, The Netherlands, 1993, pp. 11-19. 

In the appropriate culture medium, tissue explants give rise to callus tissue. Callus tissue is comprised of large, thin-walled parenchyma cells. It is similar to the undifferentiated tissue produced by plants as a repair mechanism when they are injured. In tissue culture, dedifferentiated callus can be induced to form plantlets that grow into normal plants. The induction of callus occurs when a sterile explant is brought into contact with a nutrient medium, which contains substances that initiate cell division and support growth. An explant may be a uniform piece of tissue or tissue derived from different cell types (Yeoman, 1973). Storage parenchyma tissue from Jerusalem... 

Electron microscopy (EM) has been used to study the development of Jerusalem artichoke explants in tissue culture. Tulett et al. (1969) described procedures for preparing tuber explants and callus tissue for EM to look at cell structure. Small pieces of callus were fixed in 6% glutaraldehyde in 0.1 M phosphate buffer at pH 6.9, at room temperature for 2 h, and then at 5°C overnight. After fixing, tissue was washed in several changes of phosphate buffer. Postfixation treatments involved immersion in a 1 to 2% buffered osmium solution for 1 h or a 2% aqueous solution of potassium permanganate for 1 to 2 h. 

An antimicrobial alkaloid that was isolated from roots of Ruta graveolens and from callus tissue cultures proved to be rutacridone epoxide (31).17 The structure was determined by 2H and 13C n.m.r. and by mass spectroscopy, although the configurations at C-2 and C-18 are not known. The epoxide, rather than rutacridone (37), is a major root alkaloid clearly, the plant is chemically different from that studied previously, but the reason for the variation is unknown. 

Figure 2. Tissue culture crop improvement. Sequence shows the integration of cell biology techniques into crop improvement. Hurdles to using the scheme include callus initiation, protoplast preparation, selection in culture, and plant regeneration.

Figure 9. Somatic cell selection for herbicide resistance. Bottom left, a flask of alfalfa cells in suspension. Top left, addition of herbicide to the cells. Center, cells plated onto solid medium containing herbicide a resistant callus growing on herbicide-containing medium. Top right, resistant plantlets regenerating. Bottom right, tolerant plants selected from tissue culture growing in the field after being sprayed with the herbicide.

The entire tissue culture sequence, used to obtain the transformed petunia plants, Is shown in Figure 18. Two types of callus are growing in the petri plate containing kanamycin media. 

Figure 18. Tissue culture sequence to obtain transformed petunia plants expressing a foreign gene, kanamycin resistance. The petri plate at the bottom contains two calli. The callus not forming shoots received the "long transfer", and the shoot-forming callus, the "short transfer". The "short transfer" shoots are removed from the callus and rooted in the container in the center. The rooted plant is transferred to the greenhouse. The leaves of the regenerated plant express the foreign gene.

Adventitious Developing from unusual points of origin, such as shoots or root tissues from callus or embryos from sources other than zygotes. This term can also be used to describe agents that contaminate cell cultures. 

There has been a demand for the development of cryopreservation methods for plant cells to avoid the troublesome maintenance of tissue cultures and the danger of microbial contamination. The most successful method for cryopreservation of plant cells reported so far has been the freezing of callus cultures or shoot tips [36, 37]. As the system here enables us to obtain sufficient initial shoot materials, its potential practical application to cryopreservation is in progress. In addition, the system of adventitious shoot formation might be a promising tool to investigate relationship between morphogenesis (shoot formation) and alkaloid biosynthesis. 

Many researchers have so far investigated tissue culture of P. somniferum [131, ref. cited therein], and most cultured P. somniferum cells, either as callus or cell suspensions, readily produced sanguinarine and its analogs [130-137], but rarely, if even, produce morphinan alkaloids [138]. Kamo et al. [139], Schuchmann and Wellmann [134], and Yoshikawa and Furuya [140] reported the production of morphinan alkaloids in redifferentiated organs, either shoots or somatic embryos, and their results emphasize the importance of the degree of cell differentiation for the biosynthesis of morphinan alkaloids. 

An additional insight into the importance of specific media components on production of secondary products can be gained by examining the case history of shikonin production. It had been shown that callus cultures of Lithospermum erythrorhizon could be induced to produce shikonin on Linsmaier-Skoog medium supplemented with lpM indole acetic acid (IAA) and lOpM kinetin (KIN) (52). The effects of specific nutritional components of the tissue culture medium on growth of the cell cultures and production of shikonin were also investigated i53). Fujita et al. (54,55) found that the levels of NO,, Cu+, and SO had profound effects of shikonin biosynthesis. Optimal concentrations were identified for each ion (I 8 ) as well as optimal levels of key organic components. The resultant medium supported production of shikonin at a rate approximately 13 times that obtained on previous media formulations. 

The five xanthone compounds, demethylpaxantonin, patulone, garcinone B, tripteroside, and 1,3,5,6-tetrahydroxyxanthone, purified from a callus tissue culture of H. patulum were evaluated for their anti-inflammatory activity. 

In the meantime, the formation of the main alkaloids in C. ipecacuanha under a variety of conditions has been extensively investigated emetine (1) in callus cultures (49) and under the effects of L-tyrosine supplementation (5t)) emetine (1) and cephaeline (2) in Panamanian ipecac (57), in Nicaraguan ipecac (52), in regenerates obtained by clonal propagation (53,54), in tissue cultures (55) and under the effects of exogenous feeding of shikimic acid and L-phenylalanine (55), in cell suspension and excised root cultures (57), in adventitious root cultures (58), and in callus cultures (56,59) and the effects of age and electrokinetic potential (60) ipecoside (7) in the roots (61) and the effect oi Azotobacter, leaf mold, and farmyard manure on alkaloid content (62). In addition, micropropagation systems for C. ipecacuanha have been developed (63-65). 

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