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More recently, Gagliano et al., investigated short-term and long term habituation along with effects of light intensity on the leaflet closure response of habituation in Mimosa pudica. The results of the experiment confirmed the findings of Holmes and Grunberg.
The apparatus consists of a marked vertical steel railing fixed to a foam base. A clear acrylic vessel is mounted onto the steel railing using variable hangers in which potted plants will be placed during the experiment. The foam base is cut out and hollowed in the center making a shallow depression for the acrylic vessel to drop into from a height by sliding along the steel railing.
Set up a growth room with the size of 5.30 m2 for the experiments. Make three compartments in the room using black plastic sheets. The training trials will be conducted in the middle compartment and the test trials will be conducted in the side compartments. In each of the side compartments fit fluorescent lights, one compartment with low intensity lights (LL) while the other compartment with high intensity lights (HL). Measure light intensity just above the plants in each compartment before starting the experiment. Grow each individual Mimosa pudica plant with similar heights (6-8 cm) in a 10 cm round acrylic pot with a standard mixture of loamy soil and organic compost with a 1:1 of volume. Fertilize and water the plants as per requirement. Maintain a 12-h light: 12-h dark cycle, 60%-70% relative humidity and 21-24oC temperature.
Randomly assign potted individual plants to either HL or LL conditions. Leave them undisturbed for 5 days until training day.
Place a potted plant in the acrylic vessel and drop it from a set height of 15 cm in the morning and then repeat the drop from same height 8 hours later. Repeat this for all the plants from each of the side compartment.
Place a potted plant in the acrylic vessel and drop it from a set height of 15 cm. Repeat this procedure 60 times with an interval of 5 to 10 seconds between each drop. Repeat another set of 60 drops, for a total of seven times consecutively one after the other in a single day with increasing intervals between each train of 60 drops. Repeat this for all the plants from each of the side compartment.
Place individual plant in a close fitted foam container that is attached to a shaker plate. Turn on the shaker plate and set it to 250 revolutions per minute for 5 seconds. Observe the leaf folding behavior.
Test for Short term memory of the habituated leaf‑folding response
Test each of the trained plant from both side compartments 6 days after 1 day training using 60 consecutive drops from set height of 15 cm.
Test for Long‑term memory in Mimosa exposed to a new environment
Transfer plants from the side compartment with LL to the compartment with HL and vice versa. Test the plants after 28 days using complete training protocol mentioned above under training 2.
After each drop, for each individual plant select three random leaves. Measure the breadth of the leaf (mm) from tip-to-tip using digital calipers. Take an average of the three measurements. The response is then represented as the maximum leaf breadth immediately after the drop relative to the maximum leaf breadth before the drop. The observations should be taken in quick succession from the three leaves both before and after the drops.
Mimosa pudica is an ideal plant for habituation experiments as they are easy to maintain, a lot of information is available about its natural history and they have a visible leaflet closing response to external stimuli (Abramson et al., 2016). Experiments related to habituation are generally easy to perform as they require little equipment.
Mimosa pudica has a long recovery time of up to 15 minutes which can be problematic as several training variables like inter-stimulus time interval and the time period between a response and its outcome must be very short for any association to be formed. This can also produce a hindrance when comparing animal and plant behavioral studies and techniques as animal responses are much quicker than responses in plants. Researchers need to develop an automatic way of presentation of stimuli as well as recording of the response in order to further develop the field of research related to plant learning. It is crucial to know the rate, length and temporal pattern of the target response before studying habituation. A control group must be used that will be subject to training situation but no habituation training will be given (Abramson et al., 2016).
Some researchers do argue that Mimosa has a neural capacity for learning behavior. If this is agreed upon then further studies will broaden understanding of nervous system and clarify mechanisms involved in Mimosa’s response. One advantage for using Mimosa for studying hormonal secretion induced by nervous system is that the cells are relatively large, accessible and excitable as compared to the small size and inaccessibility of the cells involved. This can provide new opportunities for studying interactions between electrical and chemical mechanisms of control.
Studies related to plant learning that involve habituation are an excellent way of collecting data to compare them with animals as habituation is universally observed in the animal kingdom. Habituation although considered simplest form of learning, shares a lot of properties of complex learning such as ability to recover from a response over time, forming new behavior patterns and improved performance over repeated sessions. These properties and sensitivity to training variables like intensity, frequency and pattern of stimulation can be further investigated in plants.
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