The following points highlight the eight experiments on translocation of plants. Some of the experiments are: 1. Demonstration of Upward Translocation from Germinating Seeds 2. To Show the Downward Translocation of Food in a Woody Stem (Or Effect of Ringing Upon Food Movement) 3. Demonstration of Translocation from Leaves 4. Demonstration of Upward Translocation of Food in Woody Stem and Others.
Contents
Experiments # 1
Demonstration of Upward Translocation from Germinating Seeds:
Experiment:
About 100 seeds of pea or gram or Vicia seeds are soaked in distilled water. The seeds are divided into two lots. One lot of seeds is taken out at the stage when seed coats can be removed. The fresh weight, dry weight and ash weight of this lot are determined.
The other lot is allowed to grow in dark.
As soon as the primary leaves appear in this lot, the cotyledons are separated from a few seedlings:
(i) The average dry weight of the cotyledons and the shoot,
(ii) The average ash weight and
(iii) The average loss or gain of the cotyledons and the shoots are separately determined.
Results:
(i) Percentage loss of dry weight and ash weight of the cotyledons and
(ii) The corresponding percentage increase of dry and ash weights of the shoot are calculated from the data.
Discussion:
The cotyledons are the storehouse of growing embryo. As the embryo grows to a seedling, reserved food material is translocated to the seedling from cotyledons. So long the leaves are not formed, photosynthesis cannot take place and seedlings remain entirely dependent on the cotyledons for their nourishment and growth.
In this experiment the seedlings are grown in dark in order to preclude the possibility of getting nourishment of the seedlings from the photosynthate.
Decrease of the food materials from the cotyledons and concomitant increase in the shoot indicates that food materials are translocated upward to the shoot at the cost of cotyledons. Some amount of food materials is lost by way of respiration or other catabolic processes in both cotyledons and shoots which may be taken here as insignificant.
Experiments # 2
To Show the Downward Translocation of Food in a Woody Stem (Or Effect of Ringing Upon Food Movement):
Experiment:
The experiment is to be performed with a woody plant in which the apical growth has ceased. It is best performed in growing season. One or more stems or branches are selected which have no side branch for 50 cm or more and which are several centimeters in diameter.
Ring is made by removing the bark and phloem tissue approximately in the middle of the clear portion. The removed rings of bark should be about 1 cm wide and ringing should be done carefully so that the xylem is not damaged.
The exposed surface of the xylem should be carefully scrapped with a sharp knife so that all traces of cambium are removed. The exposed surface is covered with paraffin wax. After three weeks, final growth measurements are taken.
Sections are cut 25 to 50 cm from the regions immediately above and below the ring and following data are taken:
(i) Sections from just above and just below the ring are tested for starch with iodine solution and compared.
(ii) The volumes of 100gm of tissue from below and above the ring; is determined by displacement of water and compared.
(iii) The percentage of dry matter in each tissue is determined and compared.
Results:
Results are tabulated or plotted to show the effect of ringing upon translocation of food material based on the above three indices.
Discussion:
Food materials are synthesized in leaves and translocated downwards through the phloem. Removal of phloem tissue hampers this downward translocation and accumulation of food materials above the ring occurs. This experiment indicates that organic solutes flow downward through the phloem into root and other organs, when the above the indices below and above the ring are compared.
Experiments # 3
Demonstration of Translocation from Leaves:
Experiment:
Several seedlings of kidney bean (Phaseolus vulgaris) are chosen on which the first pair of primary leaves is well developed. Three sets of plants are exposed to bright sunlight until the leaves show heavy starch accumulation on test with 1% iodine.
One petiole of each pair of leaves is then treated by the following methods, leaving the second petiole and leaf intact as control:
(i) One petiole is cut off and kept in water for comparison with control leaf;
(ii) Killing a portion of one petiole by a hot forceps (heating any other portion of the plant is avoided; the leaf may be kept in position with the help of a thread); and
(iii) A portion of one petiole is anesthetised with chloroform or ether soaked in cotton. All the plants are kept in dark in a moist chamber at 20°C for 24 hour’s. Chlorophyll is removed from the leaves with alcohol and a few drops of lactic acid and tested for starch with 1 % iodine solution.
Observation:
The untreated leaves show little or no response with iodine test compared to treated ones.
Inference:
Marked loss of starch from a leaf is taken as an evidence of downward translocation. Considerable retention of starch in a treated leaf indicates that starch has not been translocated due to interference in the translocatory path. The experiment thus shows that food material is translocated from leaves.
Experiments # 4
Demonstration of Upward Translocation of Food in Woody Stem:
Experiment:
The experiment can be best performed in growing season. Four approximately uniform twigs on a woody plant are selected. Potted woody plants or even cut branches with their basal ends immersed in water can be used in this experiment. When tested with iodine they show considerable quantity of starch.
Smooth-barked species with rather stout stems and with true terminal buds are most suitable If possible twigs should be selected from plants which bear no side branches for a distance of about 40 cm back from the terminal bud.
The selected branches are numbered. Twig number 1 is ringed about 5 cm, twig number 2 about 20 cm, and twig number 3 about 40 cm below the respective terminal buds. Twig number 4 is kept as control.
During ringing, a strip of bark is removed 0.5 cm wide and the exposed wood is carefully scrapped to remove all traces of cambium avoiding any damage to the xylem.
The exposed wood is coated with paraffin wax. Leaves and lateral buds are removed from the portions of the twigs above the rings as fast as they emerge from the stem.
On the control stem all the leaves and lateral buds which start to develop about 40 cm below the original location of the terminal bud are removed. The twigs are observed from time to time as growth proceeds from the terminal bud, noting especially differences in the rate of longitudinal growth.
Results:
After three weeks the stem elongation which has occurred from the terminal bud of each stem is measured and recorded in millimeters. Cross sections are cut from above and below the ring of each stem and tested for starch with iodine solution.
Discussion:
The rate of growth above the ring in case of number 1, 2 and 3 twigs is very much checked as compared with control. The growth that has occurred in the ringed stems is only due to upward translocation of food materials through xylem.
In case of control twig the upward translocation has taken place both through xylem and phloem. It is also evident from the experiment that the more is the distance of the ring from the terminal bud, the less is the rate of growth above the ring. The accumulation of starch is always maximum just below the ring.
N.B. From the above experiment a correlation with translocation and growth may be made. The influence of phloem upon translocation and growth may also be studied by removing different amounts of phloem tissue from a particular region on the stem.
Experiments # 5
To Demonstrate the Exudation from Phloem Tissue:
Experiment:
Cucurbita seedlings are grown under favourable conditions until they have attained a length of 30 cm. The stem of one of the plants is cut off with a sharp scalpel from 5 to 10 cm above the soil surface. The cut end of the excised portion of the stem is held in an inverted position and observed under a powerful hand lens or binocular microscope for exudation.
Exudation may also be studied by puncturing a sharp needle through the bark to a sufficient depth to just reach the inner layer of phloem.
Observation:
It is observed from what tissue (xylem or phloem?) the exudate comes. If exudate cannot be discerned clearly in the excised by stem the first drop of exudate is blotted with a filter paper and the cut end is re-examined.
Inference:
It is clear from the study that the sap comes out mainly through the phloem tissue in the form of droplets.
Experiments # 6
Demonstration of Translocation of Food into Developing Fruits:
Experiment:
Suitable species of fruit trees on which fruits of considerable size usually develop are selected. The experiment should be started when the fruits on the tree are half-matured.
At least ten fruits are tagged and numbered and their circumferences are carefully measured. The fruits are isolated from the main phloem system of the plant by means of proper ringing. Rings may be made at the base of fruiting branch or both above or below its point of attachment on the stem.
All precautions are followed in ringing the stem. The circumferences of an equal number of fruits from un-ringed branches are also measured to serve as controls. From time to time both sets of fruits are measured and rates of growth in diameter of the fruits from the ringed and un-ringed branches are compared.
Results:
The rates of growth in diameter of fruits in ringed and control sets are recorded and compared.
Discussion:
The developing fruits are the active centres of mobilisation of carbohydrate from other regions of the plant. Since phloem is the principal path of translocation of food materials to the developing fruits, removal of phloem tissue greatly hampers this transport of carbohydrate to the growing fruits. Hence the growth rate of the ringed fruits is much less compared to controls.
Experiments # 7
Demonstration of Upward Translocation of Mineral Salts In-Woody Stem:
Experiment:
This experiment can be performed with potted woody plant as in Expt. 4. Three sets of at least five comparable branches on the plant are selected. In one Set all of the branches are ringed 5 cm below the base of each terminal bud. Rings are made about 0.5 cm wide, the exposed wood is carefully scrapped to remove all traces of cambium and the exposed surface is covered with paraffin wax.
In the second set all the branches are removed from the plant by making a sharp cut at the point corresponding to that at which the branches of the first set were ringed, i.e., 5 cm below each terminal bud. This set is used as ‘starting’ control.
In the third set all the branches are tagged at a point 5 cm below the terminal bud. At the end of three weeks the branches of the girdled set and the tagged set which is to serve as ‘end’ control are removed by cutting them off at the point of girdle and at the point of tagging respectively.
The ash content of the branches of the ‘starting’ control is determined at the beginning of the experiment and that of the ‘end’ control and of ringed branches at the end of the experiment, as follows.
The sample of stems to constant weight is dried in an oven at about 80°C and its dry weight is determined. Each sample is ground and mixed thoroughly. The ground dry tissue from each set of stem is weighed and heated to constant weight in a muffle furnace at about 600°C.
Results:
The estimated ash contents are expressed as percentages of the dry weight and fresh weight of stems.
Discussion:
The mineral from the soil solution is carried through the xylem in transpiration stream, though some amount of mineral is translocated through phloem. As minerals are mainly translocated through the xylem, the removal of phloem tissue from the ring will not debar the stem above the ring from the supply of mineral nutrients.
When the ash content of the stems of three sets is compared, it becomes clear that percentage of ash content is maximum in case of the set where the branches were originally removed 5 cm below the terminal bud and minimum in case of the set where branches remained intact. This shows that translocation of mineral solutes takes place mainly through xylem.
Experiments # 8
Demonstration of the Effects of Inhibitors on the Uptake, Distribution and Translocation of 32P in Plants:
Experiment:
One-month-old bean seedlings grown in sand culture may be suitably employed in this study. The plants are removed from the culture taking sufficient care not to injure the roots.
Roots are then washed well to remove the adhering particles. These are then selected for treatment. Two such plants are taken and the roots are inserted in each of the six test tubes containing the following solutions (10 ml) and three test tubes may be bubbled for aeration.
Now from each leaf, discs are prepared with the help of a corkborer at different intervals, dried under infra-red lamp and its radio-activity is measured in a Geiger-Muller Counter. After 1 to 2 hr. the plants are taken out, roots are washed well with carrier phosphate (0.01 M Na2HPO4) and the distribution of radio-activity is determined by autoradiography as follows.
The plant is kept for sufficient time in contact with a suitable film in a dark room under some uniform pressure. [Half-life of 32P is to be taken into consideration (14.3 days)]. After exposure the film is developed and the spots of radio-activity are determined.
Exposure time = 107/ x min.
+3 days, where x is the count per minute (CPM) during experiment.
Observation:
The uptake of 32P by plants as affected by aeration, sodium arsenate and sodium azide is noted and data are tabulated.
Discussion:
The above experiment clearly demonstrates the translocation of radio-active phosphorus to different organs of the plant. The translocation of phosphorus is a function of time and distance from the roots as indicated by the radio-activity in different plant parts.
Again metabolic inhibitors like azide or arsenate inhibit the rate of translocation indicating that it is regulated by the metabolism of living cells. Increased translocation with aeration suggests that respiratory energy is also involved in the translocation of phosphorus.