Brassinosteroids rice-lamina inclination test

Table L Biological Activity of Selected Brassinosteroid Analogs in the Rice Lamina Inclination Test...

Examination on the production of brassinosteroid-like active substances. The cells were extracted with methanol, and the NE fractions of the extracts were subjected to bioassay using the rice lamina inclination test by the same... 

Figure 8. Effects of 2,4-D on production of brassinosteroids in the cultures of C. roseus crown gall cells. Brassinosteroid contents in the cells are expressed as the response in die rice lamina inclination test...

Therefore, we examined the effect of 2,4-D added to the medium on production of brassinosteroids using V208 and V277 cells. However, addition of 10 ppm of 2,4-D to the culture medium at the start of the culture process caused growth inhibition of the crown gall cells. Therefore, 2,4-D was added to the medium when cultures were 10-days old. After culturing another 4 days, the cells were harvested and extracted with methanol. Brassinosteroid activity of the NE fractions of the cell extracts was examined by the rice lamina inclination test. The results are shown in Figure 8. 

Since the discovery of brassinolide, ten years ago, research on brassinolide and related steroids has attained a remarkable degree of development. So far, twenty-three brassinosteroids [BS] have been isolated and identified from twenty-two plant species. Furthermore, many unknown brassinolide-like active substances have been detected, so that the number of identified ones is likely to increase in the future. The background to successful isolation of new BS from various plant sources is due to the use of a rice-lamina inclination test for screening that is sensitive and specific for brassinosteroids. 

The results of the rice lamina inclination test (RLIT) indicated an extraordinary high activity of 25-hydroxy-24-epibrassinolide (90). This compound is about ten times more active than 24-epibrassinolide (12), indicating that the hydroxylation at C-25 is an activating step in the brassinosteroid metabolism. Therefore, 25-hydroxy-24-epibrassinolide (90) is, next to brassinolide (1), one of the most active brassinosteroids known until now. In comparison with 25-hydroxy-24-epibrassinolide (90), the 26-hydroxylated metabolite (91) was clearly less active. As in other groups of steroidal hormones, for instance vitamine D metabolites, hydroxylation at C-25 seems to be essential for high activity. ... 

The physiological concentrations of BRs in plants are extremely low (ng.Kg Fw), and it can be veiy difficult to analyze their abundance in plant tissues. A wide range of methods are currently employed for the determination and quantification of brassinosteroids in plants, including bioassays, diverse chromatographic procedures, radioimmunoassays [9], and enzyme-linked immunosorbent assays [10-13]. The most widely used bioassays are the second bean intemode bioassay and rice-lamina inclination test. These bioassays have been used in the isolation of brassinolide from rape pollen [1] and castasterone from chestnut insect galls [14]. Gas chromatography-mass spectrometry (GC-MS) analysis is the current standard technique for instrumental analysis of BRs [15-17]. 

Maeda E (1965) Rate of lamina inclination in excised rice leaves. Physiol Plant 18 813-827 Takeno K, Pharis RP (1982) Brassinosteroid-induced bending of the leaf lamina of dwarf rice seedlings an auxin-mediated phenomenon. Plant Cell Physiol 23 1275-1281 54. Wada K, Marumo S, Ikekawa N, Morisaki M, Mori K (1981) Brassinolide and homobras-sinolide promotion of lamina inclination of rice seedlings. Plant Cell Physiol 22 323-325 Wada K, Marumo S, Abe H, Morishita T, Nakamura K, Mori K (1984) A rice lamina inclination test - a microquantitative bioassay for brassinosteroids. Agric Biol Chem 48 719-726... 

We developed several methods for construction of the side chain and the A/B ring of brassinosteroids, and synthesized more than fifty analogs (6). In order to obtain the structure-activity relationship, those analogs were subjected to bioassay in the rice-lamina inclination (7), Raphanus, and tomato tests (5). In general, die structural... 

---