Internode sections

The First Internode Bioassay (15) is based on the curvature of the first internode section after insertion of a paper disc containing auxin to one side of the morphologically basal part of the internode. When it is used for the testing of BRst, the paper discs containing tested BRst are applied to the internode 1 h prior to auxin application. BRst stimulate the lag-phase of auxin action and its activity is expressed as a difference in internode curvature between BRst + IAA tested sections and controls treated with auxin alone. 

Our improvement of this test is based on the incubation of internode sections in the inversed position (9). Under these conditions the substances undergoing test are applied to young auxin-sensitive apical internode tissues and auxin is translocated basipetally toward the zone of curvature (Figure 1). Using this simple improvement the sensitivity of the test to IAA was increased 1,000 times as little as 10 fmoles of IAA can be detected. 

High sensitivity to auxin was closely related to the elevated sensitivity to 24-epiBR (I). As shown in Figure 2, the dependence of curvature of internode sections on the amount of 24-epiBR applied is expressed by a two peak curve. When evaluated on the first peak basis, inversion of sections caused an increase in the sensitivity of bioassay by factor 100. The unusual two-peak-response is probably a result of interactions of the two growth regulators applied successively to the internode sections, that is, 24-epiBR and IAA. The first peak was always recorded when equimolar quantities of the two regulators were used. 

Figure 1. The effect of inversion of bean first internode sections on distribution of 14C-IAA and its accumulation in auxin-sensitive zone. (Reproduced with permission from reference 9. Copyright 1985.)...

Figure 3. Effect of sequential treatments on bending response of bean first internode sections. Sections were treated with disks containing BR (947-B) (1.0 pg/segment), lAA (0.1 nmol/segment). Order of treatment is as indicated (947-B—> lAA) indicates that sections were first treated for 10 minutes with BR followed by lAA, (lAA—> 947-B) indicates the reverse. Sections treated with disks containing only solvent did not bend.

The rate of change of horizontal displacement of the apical portion of bean first internode sections treated unilaterially in the manner described above (bioassay) with brassinolide and/or lAA was monitored by using an angular transducer (in accordance with the technique described by Meudt and Bennett (37). 

Figure 4. Rate of horizontal displacement ot apical portion of bean internode sections treated with lAA (A) and BR (B) and lAA plus BR (C).

Figure 5. Effect of alternating BR and lAA treatments on curvature of bean first internode sections. Sections were treated alternately either with 100 pmol of BR followed by 100 pmol of lAA (solid line) or first with lAA followed by BR (dotted line). Treatments were exchanged at hourly intervals and measurements were taken at the end of each treatment.

Figure 6. Prevention of lAA induced autogenous growth inhibition (bending of bean intemode sections) (upper data) by BR. Bean internode section were pretreated for 1 hour with either lAA (dotted line) or lAA plus 200 pmol of BR (solid line). Data in lower graph show longitudinal growth of the internodes.

Analysis of tissues for lAA as determined by reverse isotope dilution assays reveals however, that brassinolide does not affect auxin uptake or auxin movement within the tissues (41). In this same study we also observed that bean internode sections treated with brassinolide contained significantly less lAA after 2 hours than tissues treated with lAA alone, even though BR potentiated lAA-induced growth by more than 400%. 

Figure 7. Effect of BR treatment of curvature (left) and elongation (right) of bean first internode sections treated with disks containing either 1 nmol of lAA or NAA (naphthalene-acetic-acid) or 2,4,-D (2,4-dichloro-phenoxy acetic acid) or GA3 (gibberellic acid) plus and minus 42 nmoles of BR.

Figure 9. Correlation between ethylene production (left) and growth (right) of bean first internode sections from 7- and 9-day old seedlings. Treatments consisted of 1 nmol lAA and 1 g BR.

Figure 10. Effect of 0.01 ppm BR on geotropic induction of Curvature of isolated bean internode sections.

To determine internodal distance, dissect a 1-2 cm segment of peripheral nerve such as the femoral or sciatic and fix as above (see Section 3.1.2). 

At the first harvest in 1999, stem internode pieces from the middle of two stems from each plot were immediately preserved in 50% ethanol. Thin sections were examined by light microscopy with and without staining for pectin to determine whether changes in the pattern of pectin accumulation occurred. Ruthenium red was used to visualize the presence of pectin in stem thin sections (12). 

Fig. 7. Cross sections of stems at internodes of a low THC fiber variant (left) and of a high THC variant (right). Fiber varieties have hollower stems at the internodes, allowing more energy to be directed into the production of fibers in the phloem. (Reprinted from Small, Plant Science Bulletin, vol. 35, 1975.)...

Bean First Internode Bioassay. This assay was originally designed for the detection of auxin activity (37). It takes advantage of the fact that unilaterally applied auxin causes bending of the treated bean internode, which reaches a maximum rate about 20 minutes after auxin application. The sections will respond to 10 pmol of lAA and BR increase the sensitivity of the sections to auxin by one order of magnitude. 

The test material is first dissolved in ethanol and lOpl are transferred to a small (5mm diameter) filter paper disk. The disks are dried and sandwiched between the lower part of the internode and the moistened sponge close to the vial. The position of the apical portion of the section is recorded and the vial is placed in a high humidity chamber. Subsequent measurements are taken at hourly intervals, over a period of 4 hours. 

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