Pollen bioassay

As pointed out above, bioassay choice is extremely important since different sensitivities and different responses are observed that are dependent on the bioassay specimen. For example, comparison of four bioassay methods for detection of destruxin B, a host-specific toxin produced by Alternaria brassicae, indicated a lack of rapid electrolyte leakage and insensitivity of host protoplasts. However, leaf and pollen bioassays were sensitive to low toxin levels. Pollen germination was the most rapid, sensitive and quantitative bioassay for the toxin. Significant inhibition of Brassica campestris pollen germination and pollen tube growth occurred only 30 min after incubation with 2.5 pg mL 1, and 7.5 pg mL 1 caused total inhibition.3... 

Ma, T-H. Tradescantia micronucleus bioassay and pollen tube chromatid aberration test for in situ monitoring and mutagen screening. Environ. Health Perspect. 37 85-90, 1981. 

The partially purified biologically active extract from rape pollen will be referred to as "brassins" in this Chapter. The extract was partially purified via a thin layer silica gel chromatographic procedure. Biologically active fractions were detected and monitored via the "bean 2nd internode bioassay" (6). Fractions from the... 

A second column chromatography clean-up step similar to the first was then conducted on the active material from the first column. Sixty g batches of the active residue were applied and the column successively eluted with increasing concentrations of methanol in chloroform. Bioassay monitoring showed that essentially all the activity was eluted from the column when the methanol concentration reached 10-20%. The second chromatography step reduced the 450 g of active material to ca. 200 g of active brassins per 181 kg pollen (ca. 252 g active brassins per 227 kg pollen, equivalent) ... 

In 1970, Mitchell and colleagues reported that a lipoidal extract obtained from rape (Brassica napus L. ) pollen elicited strong elongation activity in the bean second-intemode bioassay (1). The activity was distinguished from other known plant hormones in that the lipoidal extract promoted not only cell elongation, but also cell division. The activity was termed brassins activity and extracts showing the same type of activity were also obtained from pollens of other plants (see ref. 2). Thus they proposed that new types of lipoidal plant hormone coined the brassins were contained in pollen. 

The bean second-intemode bioassay was used for the isolation of BL from the pollen of rape (Brassica napus L.) (7). The procedure of the bioassay is described in... 

Recently, by the ligand-accelerated osmium catalyzed asymmetric hydroxylation using a cinchona alkaloid derivative, the 22/ ,23/ -epimer was obtained as the major product of the oxidation (77). Thus, epibrassinolide may be ideal for practical use, and, therefore, is one of the most desirable brassinosteroids because of the ease with which it can be synthesized. This compound showed about one tenth the activity of brassinolide in Raphanus and tomato bioassay (5). In field trials, the activity of epibrassinolide was about the same as of brassinolide. Epibrassinolide is a natural sterol. This was confirmed by GC-MS analysis, which identified epibrassinolide, along with co-existing brassinolide, brassinone, and castasterone, from the bee pollen of the broad bean Vida faba obtained in China (72). 

The early USDA efforts to isolate hormones from corn pollen were not very fruitful. The USDA finally terminated the pollen research project in 1944 due to lack of (a) suitable plant bioassay systems to detect the hormones present in pollen and (b) proper analytical methods to isolate and identify the pollen hormones. 

Figure 1. Bean second internode bioassay after 4 days controls (left) and bean plants treated with gibberellic acid at 10 /ig (center) and with pollen extracts at 10 fig (right). (Reproduced from reference 1. Courtesy of Marcel Dekker, Inc.)...

Aprotinin (2.5 mg/g in pollen-based food) has also been fed for 7 days to newly-emerged adult bees which were tagged and then returned to outdoor hives and monitored. These bees began to fly and also died about 3 days sooner than control bees fed with plain pollen-food, in accordance with the survival effects noted in some of the laboratory bioassays reported above. The period during which flights took place was not altered by the PI treatment [47]. 

There are as yet few published measurements of PI expression levels in pollen. It is therefore difficult to extrapolate from the results of experiments with purified transgene products to making predictions about the effects of GM plants on bees. However, if we assume that the bees in the bioassays described above received a diet which was 25 percent protein, then the doses of Pis administered ranged from 0.004 to 4 percent of total protein received. GM Pl-plants that are effectively protected from pest attack typically have leaf expression levels ranging from... 

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]. 

The development of bioassays for isolating bioactive compounds from natural sources has played an important role in recent smdies of namral BR phytochemistry. The development of highly sensitive and specific bioassays was essential for the isolation and purification of BRs from plant tissues because of the very low physiological concentrations of these hormones. The bean second intemode assay was used to isolate BL from rape pollen [1], and the rice-lamina inclination test was used to isolate CS from chestnut insect galls [14]. Following the publication of these results, the rice-lamina inclination test has been widely used to isolate many BRs from various plant sources because of its simplicity, high sensitivity, and specificity for BRs [25]. 

Malik CP, Mehan M (1975 b) Interaction between cycocel and gibberellic-acid in pollen-tube elongation of Calotropis procera. Curr Sci 44 785-786 Manos GE (1961) The effects of growth substances on attached and detached root tips of Pisum sativum L. Physiol Plant 14 697-711 Manos PJ, Goldthwaite J (1976) An improved cytokinin bioassay using cultured soybean hypocotyl sections. Plant Physiol 57 894-897 Marinos NG (1960) Some responses of Avena coleoptiles to ethylene. J Exp Bot 11 227-235... 

We performed a series of replicated bioassays using bodi purified toxins and pollen samples from a number of com hybrids created widi different transformation events. In these, we measured the effect of increasing doses of Bt pollen on feeding behavior and weight gain of first-instar monarch larvae. We established a no-observable-effect level (NOEL) from these results and compared that dose with the density of pollen that we estimated to occur on milkweed leaves in and around com fields to determine the likelihood of encounter with an effective dose. To validate our laboratory observations and estimates of intact, we performed a series of field experiments over two years in tiuee different locations within the Com Belt. Monarch larvae were eitiier caged on milkweed plants or fed milkweed leaves from plants in and around com fields... 

Five field bioassays were undertaken to determine the outcome of exposure of larvae under field conditions on milkweed plants growing or placed in die field. In Iowa, reduced weight gain was noted for larvae exposed to event 176 pollen on milkweeds within cornfields at densities of 20-25 pollen grains/cm. Bodi survival and weight gain were affected in Maryland, where a series of assays using milkweed leaves collected firom plants in an event 176 cornfield were carried out over the pollen-shed period (6). 

Mattila, H.R., M.K. Sears, and J.J. Duan. 2005. Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay. Entomol. Exp. Appl. 116 31 1. 

Sears, M. and H. Mattila. 2001. Determination of the Toxicity of Corn Pollen Expressing a CrySBbl Variant Protein to First Instar Monarch Butterfly Larvae Danaus plexippus) via Laboratory Bioassay Lab Project Number MSL-17235 01-01-39-26. Unpublished study prepared by University of Guelph. 33 p. 

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