Shooting formats

Results presented in Table VII show that sufficient light is needed for new shoot formation on explants and that the herbicide fluridone causes chlorosis in new growth just as in whole plants (12,13). These data also confirm that the apical explant, which contains the terminal meristem, is a poor system for assaying inhibitors of new shoot production. 

Once these calli are formed, the hormone levels can be manipulated to induce shoot formation. This is followed by the rooting, hardening and transfer-to-greenhouse processes. During this sequence the selected lines can be screened at the cellular level, at the regenerated plantlet level, at the whole plant level in the greenhouse, and finally in the field which is the "acid test". 

Efficient micropropagation of ipecac by adventitious shoot formation on internode (Method II)... 

Table 8. Influence of basal media on shoot formation on intemodal segments...

Fig. (17). Effects of cytokinins and influence of light on shoot formation on intemodal segments The segments were cultured on WP solid medium containing cytokinin at 25 °C for 8 weeks whether in 16 hr/day light (80 gEm S" ) or in the dark. Bars represent standard deviation of the means, n=6.

Fig, (18). Comparison of shoot formation on intemodal (A) and nodal (B) segments. 

The effect of BA on shoot formation in liquid medium under 16 hr/day light on a rotary shaker (30 rpm) is shown in Fig. (19). In liquid media, 0.1 mg/1 BA resulted in 100% shoot formation and the best growth of shoots, though as stated above 0.01 mg/1 BA was the most effective for shoot formation on a solid medium. 

Since the optimal concentration of BA in liquid medium was 0.1 mg/1, scaled-up shoot proliferation in a rotating dmm fermenter (RDF) was undertaken in WP liquid medium containing 0.1 mg/1 BA to obtain the shoots more efficiently. Fig. (21). The frequency of shoot formation was 83.7% and approximately 10 shoots were formed on each segment. 

A two-litre airlift type fermenter was also applied to the scaled-up culturing in the same conditions as the RDF. However, the shoot formation frequency was below 30%. Tanaka [28] reported that Catharanthus roseus cells were damaged by hydrodynamic stress when cultured in a jar fermenter but not when cultured in a RDF. The low... 

Fig. (19). Effect of various concentrations of BA on shoot formation in liquid medium.

Theoretical number of ipecac to be micropropagated through adventitious shoot formation on internode and its application... 

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. 

Improved micropropagation of ipecac through adventitious shoot formation on cultured root segments (Method III)... 

Micropropagation systems mentioned previously (Method I and Method II) require two different cultural steps, shoot multiplication and rooting. In the course of tissue culture studies of ipecac, adventitious shoot formation on cultured root had been incidentally found. Therefore, one step micropropagation system of ipecac was established through adventitious shoot formation on the cultured root [14]. 

Adventitious shoot formation on cultured root segments... 

Table 10. Influence of basal medium on shoot formation from cultured root segments of C. ipecacuanha...

Medium Condition % of shoot formation No. of shoots / segment No. of lateral roots / segment % of establishment to soil... 

Fig. (24). Shoot formation of cultured root segment a Shoot formation in the dark. Segment was cultured on HF B5 solid medium in the dark for 1 month, b Development of shoots cultured in the dark for 2 months, c Plantlet was further cultured under 16 hr/day light for 1 month, d 17-month old regenerated plant cultivated in a greenhouse. Bars represent I cm (a, b, c) or 5 cm (d).

Adventitious shoot formation occurred when the roots were cultured onto auxin-free medium as mentioned above. Since the decrease of auxin levels seemed to be important for the adventitious shoot formation, the influences of auxin inhibitors on shoot formation on the cultured root segments were investigated. The auxin polar transport inhibitor, 2,3,... 

Table 12. Influences of auxin inhibitors on shoot formation on the cultured root segments...

The cultured root segments were divided into proximal, distal and newly developed lateral root parts and their endogenous lAA levels were periodically analyzed using enzyme-linked immunosorbent assay (ELISA) kits for lAA (PHYTODETEK iAA, Idetek Inc., USA) according to the procedure of Weiler et al. [39]. The lAA levels in the root segments are indicated in Fig. (26). The lAA levels in the roots cultured on HF B5 medium that initiated shoot formation increased once at day 9 and consequently decreased in both proximal and distal parts. On the other hand, lAA levels in proximal part of the roots cultured in the presence of 0.5 mg/1 TIBA increased along with the culture period. The roots cultured on the MS medium containing 0.5 mg/1 NAA (source for the experiments) accumulated low amounts of lAA and the lateral roots did not accumulate detectable amounts of lAA in all the cases. 

It is known that cytokinins induce shoot bud formation and the roots are the principal sites of cytokinin biosynthesis [18, 40]. Therefore, the adverse effects of TIBA on shoot formation might be explained by the secondary effects, such as the down regulation of cytokinin biosynthesis, caused by auxin accumulation, because auxin competitive inhibitor alone did not affect the shoot formation. 

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