This method can help answer key questions about dormancy in woody plant, such as why do the flower buds need to accumulate cold during winter to flower properly. The main advantage of this technique is it allows the quantification of the starch in very small samples of flower primordium. This approach is very useful to detect the physiological activity of the flower primordium during dormancy and can be also applied to other tree species like apricot or plum.
Though this method can provide insight into the dormant flower bud, it can also be applied to other processes such as the reproductive phase from pollination to the onset of fruiting. To begin, collect five shoots from the field. Then weigh and fix 10 flower buds in a 10-milliliter glass tube with fixative solution for at least 24 hours at four degrees Celsius.
After this, discard the fixative and add enough 75%ethanol to cover the samples. Obtain three shoots that are 15 to 30 centimeters in length and five millimeters in diameter with 10 flower buds each. Then place the shoots on water-soaked florist's foam in a growth chamber.
After 10 days in the growth chamber, remove the flower buds from the shoots and weigh them. Every week from the beginning of autumn until budburst in spring, evaluate the growth of the flower buds. Remove the external bud scales from each sample, then place each bud in a watchmaker's glass containing 75%ethanol.
Use precision forceps and an ophthalmologic scalpel to dissect the buds and obtain at least one flower primordium from each bud. Embed the samples individually in paraffin wax to form a block and section them on a rotary microtome. After sectioning the samples, apply a drop of fresh Lugol's iodine over a section.
After five minutes, remove excess stain with blotting paper. Next apply a small drop of synthetic mounting media, place a small cover glass over the sample, and press hard. Once the mounting media is dry, observe the stained section under a brightfield microscope.
First adjust the aperture diaphragm and the brightness control according to the text protocol. Then make sure there are no filters in the filter holder and select a brightfield condition in the microscope. After this, adjust the camera settings for the stained preparation without tissue.
Fix the brightness at 50%Next activate the overexposure/underexposure function and adjust the exposure time at the limit of overexposure. Apply the white balance function and the shading correction. Measure the gray level of the resulting black-and-white image of the stained preparation without tissue.
Then apply a 4N filter, acquire another black-and-white image of the preparation and measure the gray level. Next acquire an image of the same preparation without light and measure the gray level of the resulting image and calibrate. After this, acquire a color image of a sample section in TIFF format with a resolution of at least 300 dpi.
Create a binary image corresponding to the stained area, then set the three color thresholds until the binary image exactly reflects the stained starch granules. Repeat this process with other preparations and tissues to fine-tune the final detection levels. Using the image analysis system, measure the sum of the optical density of every pixel under the mask to quantify the starch content in the field.
In this protocol, flower bud growth and dormancy status were evaluated by measuring the increase in bud weight after 10 days in suitable conditions. During dormancy, no changes were observed after 10 days in suitable conditions. Once dormancy was overcome, the buds swelled and burst in the growth chamber.
Starch content in the ovary primordium was quantified via image analysis in each microtomed section. Consistently, the amount of starch in early winter presented an optical density value less than 40, 000 and the maximum amount reached a value between 120, 000 and 140, 000 during both years of the study. Taking dormancy into account, the maximum amount of starch occurred concomitantly with the chilling fulfillment during both years.
It's important to remember that the detection levels used by the image analyzer are directly dependent on the calibration of the system. This includes light condition, stain intensity, and magnification of the microscope. This condition must be fixed for all the preparations.
The combination of histochemical techniques with image analysis has resulted very useful to examine the carbohydrate reserve of the flower primordium during the different phases of dormancy. It can be also applied to other species and tissues. The relationship between dormancy release and starch accumulation in the ovary primordia unveiled by the use of this method provide a sound basis to understand the biological basis of dormancy and chilling requirements.
Future effort have to be focused on studying reliable biological indicators that could easily indicate the dormancy status of the tree. Meanwhile, the pattern of starch variations along dormancy can be used to frame further physiological and genetic studies.
