1 Objective. 06. June 2013

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1 Polar regeneration of auxin induced adventitious roots in hypocotyls of beans Regeneration of Linum usitatissimum seedlings Comparison of internodal growth of dwarf and normal peas Comparison of development in light and darkness (D, E, F, G) Authors: Niels Sievertsen, Stefan Zeyen, Connie Müller, Melanie Weis, Assistant: Yvonne Steinbach, 06. June Objective The aims of the four experiments were to take a look at plant hormones and their effect on an individual plant. In the following experiments, we studied the polar 1

2 2 INTRODUCTION Figure 1: Strucure of Indol-3-yl-acetic acid used in the experiment. 2 Introduction 2.1 D - Polar regeneration of auxin induced adventitious roots in hypocotyls of beans The purpose of this experiment is to study the effect of auxin on the polar regeneration of bean hypocotyls. Auxins are a class of plant hormones. They mainly contribute to cell growth and plant behavior. They are essential for the correct development of the plant. Indole Acetic Acid (IAA) is one of the most common natural auxins, its structure is shown in figure 1. It is produced in the tip of the plant and establishes a concentration gradient decreasing from the tip of the shoot all the way down to the root. IAA is transported by two different ways - passive diffusion and active transport. The active transport assures the polarity in a cell. This transport system is described by the chemioosmotic model (T aiz&zeiger, pp ). The IIA anions are transported in a symport system with 2H + ions in a cell. The ATP dependent efflux carriers are situated only on the other side of the cell. Normal concentrations of IAA mediate cell growth, whereas too high concentrations of auxin lead to reduced growth but augmented side and adventitious root growth. If a plant is cut in two halves, this type of basipetal active transport leads to an accumulation of auxin at the lower end of the upper half. This will build the new tip. 2.2 E - Regeneration of Linum usitatissimum seedlings In this experiment, the regeneration of Linum usitatissimum seedlings cut at various shoot lengths was analyzed. 2

3 2.3 F - Comparison of internodal growth of dwarf and 2 normal INTRODUCTION peas Figure 2: Structure of Gibberellic acid, the phytohormone used in the experiment. 2.3 F - Comparison of internodal growth of dwarf and normal peas Giberellin or Gibbelleric Acid (GA3) are phytohormones. GA3 is a pentacyclic diterpene acid and its structure is shown in figure 2. The direct effects of GA3 treatment are augmented growth of the shoot axis. For example, in dwarf mutants by cell division, elongation of cells, promotion of germination of shoots. GA3 also affects decomposition of plants. Used in small amounts, it stimulates growth, but cells can become tolerant to it. Whereas too much GA3 will have the opposite effect. In our experiment, we wanted to confirm the effect of GA3 as a plant hormone on a plant. In particular, we measured internodal growth of dwarf and normal Konservenkönigin pea plants in order to make statements about the diterpene. 2.4 G - Comparison of development in light and darkness The purpose of this experiment was to analyze the development of hypogeic, dicotyledonous pea seedlings, epigeic, dicotyledonous mustard seedlings and monocotyledonous barley seedlings in light and darkness alike. The analysis consisted of measuring the seed lengths and describing the photomorphogenesis of the different seedlings. 3

4 3 METHODS Figure 3: Method for this experiment as shown in the course material. 3 Methods 3.1 D - Polar regeneration of auxin induced adventitious roots in hypocotyls of beans To analyze the effect of auxin on polar regeneration of hypocotyls of beans, ten plants of seven days old hypocotyls of beans were cut in half. Five tips and five roots were put with the cut ends in a IAA solution (0.1 mm, 10 ml). The other five tips and five roots were put in the same way in 10 ml deionized water as control samples. The samples were incubated in these solutions for 2 hours. Then they were transferred into Petri dishes. The hypocotyls were put on five layers of wet filter paper. The five water treated tips and the five water treated roots were put in control petri dishes. The auxin treated tips and roots were put into other petri dishes, separately. The dishes were sealed using Parafilm and put in a grey box and kept in darkness. The methodology of the course material was followed and a sketch describing the method is given in figure 3. 4

5 3.2 E - Regeneration of Linum usitatissimum seedlings 3 METHODS Figure 4: Method for experiment E as shown in the course material. 3.2 E - Regeneration of Linum usitatissimum seedlings The seedlings were cut using a scalpel on a milimeter paper. The four different shoot lengths (0, 5, 10, 15 and 20 mm) were measured from the cotyledon downwards. Ten upper parts of the seeds for each length were put on three layer of wet filter paper into Petri dishes. The hypocotyls were covered in wet cotton in order to keep the seedlings from drying out. The dishes were then closed, sealed with Parafilm and exposed to sunlight, so that they could grow. After 2 days of growth, the number and root lengths for the different lengths were measured and discussed. The procedure can be seen in figure F - Comparison of internodal growth of dwarf and normal peas To analyze the effect of GA3 on plants, quantitatively, two 11 days old plants of two different pea varieties, Konservenkönigin (normal size) and früher Zwerg (dwarfed peas) were treated. One of each kind was treated using the control solution (deionized water and 1% Tween 20) and the other plants 5

6 3.4 G - Comparison of development in light and darkness 3 METHODS Figure 5: Method for experiment F as shown in the course material. were treated with gibberellin solution (0.1 mm GA3 in 1% Tween 20). Before treating the plants, the lengths of the internodes from five 11 days old seedlings of the variety Konservenkönigin and five 11 days old seedlings from the variety früher Zwerg were measured for reference in the results. The methodology of the course material was followed and a sketch describing the method is given in figure G - Comparison of development in light and darkness In two plastic boxes filled with Vermiculite, three rows of fifteen seedlings in each row were planted. In one row, we planted hypogeic (pea) seedlings, in another row epigeic (mustard) seedlings and in the third row the monocotyledonous (barley) seedlings. After planting, the seeds were covered in a new layer of Vermiculite. In one box, the seedlings were grown under sunlight whereas in the second box, the seedlings grew in darkness. The procedure can be seen in figure 6. 6

7 4 RESULTS & DISCUSSION Figure 6: Method for experiment G as shown in the course material. 4 Results & Discussion 4.1 D - Polar regeneration of auxin induced adventitious roots in hypocotyls of beans As the auxin is produced in the tip of a seedling and transported downwards (basipetal transport), we would assume that the largest accumulation of this phytohormone would be found in the low part of the upper half. Therefore the expectation was to see the most developed roots in the upper half of the untreated peahypocotyles (control group) and roots all over the hypocotyl in the auxin treated plants, because there, the phytohormone is available everywhere and the effect of basipetal transport is diminished. The adventitious roots of the control group plants have been visible as assumed. However, they were not present in the upper parts of the auxin treated seedlings. The reason for this failure could not be clearly established, but it is to guess, that an error happened in the procedure, for example too much or too little auxin could have been added. An overdose of auxin makes the growing of primary roots stop. The rhizogenic activity of auxin could still be demonstrated with the end part of the auxin treated seedling. There, the adventitious roots grew as expected all along the hypocotyl. Logically, we did not see any 7

8 4.2 E - Regeneration of Linum usitatissimum seedlings 4 RESULTS & DISCUSSION growth in the end part of the control group. Because of the missing tip, this lower hypocotyl part has no access to any auxin at all and cannot develop adventitious roots. 4.2 E - Regeneration of Linum usitatissimum seedlings In the Praktikum, we were introduced to two phytohormones which work as antagonists regarding the growth and development of adventitious roots. First, there is auxin, which is responsible for root growth, and secondly, we have cytokinin, which inhibits this procedure. Both hormones are produced in the tip of the plant, but while auxin travels basipetally through the hypocotyl, cytokinin stays concentrated at the tip. That is why the effects of auxin get more visible at farther distances away from the tip (near the tip: higher concentration of cytokinin inhibition of root growing, far from the tip: higher concentration of auxin stimulation of root growing). Consequently, we expected the shorter hypocotyles to have smaller or even no roots, as the taller ones should demonstrate many, larger roots in the low parts. Unfortunately, we were not able to show this effect very well, because of the overall incubation time was to short for the Linum seedlings. At least two tiny roots showed up in plants with a hypocotyl length of 15 mm and 20 mm, respectively. Although it is not very significant, we can hint that the roots were observed in the expected seedlings. 4.3 F - Comparison of internodal growth of dwarf and normal peas The different pea plants, Konservenkönigin (normal size) and früher Zwerg (dwarfed peas), showed different results in this experiment. In order to make assumptions of the effect of GA3 in the growing plants, we compared the present internal lenthgs with the measurements we took right before incubation of the trays. Table 1 shows the beginning lengths of each internode and average length of internodes in normal and dwarf sized pea plants. The internodes were labelled from the bottom, so that an internode labelled 1 reflects the length of the first internode above the soil. The results are listed in table 2 with the average growth of each plant and each intenode per plant. 8

9 4.3 F - Comparison of internodal growth of dwarf and normal peas 4 RESULTS & DISCUSSION The red values show the relative average growth per internodium for each of the four setups. It is observed, that the normal pea plants grow between 2.16 and 2.49 cm. The difference between GA3 treated and non treated plants is only 0.33 cm. The dwarf pea plants, however, hint a strong response to GA3. The starting average internodal growth was 0.87 cm, whereas the growth in treated plants went all the way up to 1.79 cm. This means, that GA3 treated dwarf pea plants grew more than twice as much as control non treated dwarf pea plants and grew an average of 0.92 cm per internode. Therefore, it is shown, that GA3 can have a high impact on internodal growth. However, this impact depends on the plant you are looking at. The normal ( Konservenkörigin ) had the higher overall growth in the time period, but the GA3 treated dwarf pea plant ( Früher Zwerg ) presented a larger relative growth compared to the non treated control. The observations may be due to a higher GA3 production within normal pea plants, in general. They cannot utilise the GA3 as much as the dwarf plants can. 9

10 4.3 F - Comparison of internodal growth of dwarf and normal peas 4 RESULTS & DISCUSSION Plant # Control (Dwarf) Control (Normal) Average Avg. internodal length Plant # GA3 (Dwarf) GA3 (Normal) Average Avg. internodal length Table 1: Internodal lengths of the control and GA3 treated plants right before incubation, measured in cm. 10

11 4.3 F - Comparison of internodal growth of dwarf and normal peas 4 RESULTS & DISCUSSION Plant # Control (Dwarf) Control (Normal) Average from start Avg. internodal length from start Plant # GA3 (Dwarf) GA3 (Normal) Average from start Avg. internodal length from start Table 2: Internodal lengths of the control and GA3 treated plants after incubation, measured in cm. values represent growth from starting values in cm given in table 1. 1 The third internode of the fourth GA3 dwarf after incubation was ripped off, so no value was available. Calculations were done with the starting value of 1.1 cm (cell labelled... ). 11

12 4.4 G - Comparison of development in light and darkness 4 RESULTS & DISCUSSION 4.4 G - Comparison of development in light and darkness Plants need a specific amount of light to undergo healthy growth and development. However, there are other factors such as nutrients, water supply and temperature, which have a significant influence. Dependent on the state of development and the species itself, the extent of these external growing factors varies. The results of the experiment lets us assume, that the seedlings are very dependent on light during the state of beginning development. It seems like the plants without light supply are desperately sprouting straight upward to still reach a bit of light, they develop as if they were still inside the soil. All seedlings demonstrated the phenomenon of longer hypocotyles in darkgrown plants, but anyway it is possible to observe differences in the intensity. Whereas the length of dark-grown barley hypocotyls are almost doubled in length, the dark-grown pea plant is only slightly taller on average. Mustard acts as something in between. A similar effect can be seen in mustard seedlings under the same conditions. Unlike growth, which is, according to our measurements, depending on different factors, the abundance of green colour is specifically related to the factor light. The reason for the green colour is the absorbance of red and blue light by the chlorophyll a and b of chloroplasts during photosynthesis. It is explanatory that photosynthesis can only be accomplished with the help of light. 12