Abstract. Keywords: Controlled biochamber, PLC, Biochamber, Controlled Environment.

Transcription

1 A PLC (Programmable Logic Controller) Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants. Norlida Buniyamin 1, Zainuddin Mohamad 2, Rofina Yasmin Othman 3, Norzulaani Khalid 3. 1 Faculty of Electrical Engineering, 2 Faculty of Mechanical Engineering of Universiti Teknologi MARA, Malaysia, 3 Institute of Biological Sciences, Universiti Malaya, Malaysia Abstract The application of a PLC (Programmable Logic Controller) in the design of a biochamber for use in micropropagation of in-vitrio tissue cultured plants is presented. Several methods of plant propagation and their advantages and disadvantages are briefly discussed.the function and features of the chamber that addresses some of the identified problems in plant propagation is then highlighted. Further, the system hardware, modes of operations and operation flowchart are presented. The biochamber capabilities as a research tool for plant biologist is also discussed. Keywords: Controlled biochamber, PLC, Biochamber, Controlled Environment. 1.0 Introduction The technology for plant propagation in soilless culture has progressed from the bed method (since 1732) to hydroponics (since 1938), tissue culture and most recently to the aeroponics method (Carruther 1992). Tissue culture or in vitro micropropagation involves the mass culture of plants in a sterile environment such as jars, using soiless nutrients either in gel or liquid form. In tissue culture the use of liquid medium for mass propagation is considered ideal and simplifies subculturing procedures. However a major disadvantage of liquid medium is vitrification, a physiological disorder which causes plants to be abnormal. To overcome this problem, different procedures have been developed such as a raft to support plants over stationary liquid (Connor and Meredith, 1984; Hamilton et al, 1985), mist culture (Wheathers and Giles, 1988) and temporary immersion (Teisson et al, 1995). Hydroponics on the other hand is a planting technique which involves immersion, temporary or permanent, in liquid nutrient, in environments which are not sterile. Aeroponics is considered to be an improved version where hydroponics often causes water root formation. This method is usually adopted for the production of vegetables and in some cases for plant propagules. Aeroponics is a technique that involves intermittent spraying of required nutrients and water to plants suspended on a support raft, at the required intervals. As with hydroponics a non-sterile environment is used. Each of these methods has its own advantages and as well as problems. In tissue culture, because of the sterile conditions used, problems faced using standard aeroponics such as A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 1

2 contamination from diseased plants and also growth of fungi and bacteria due to the mist could be overcome. Hence a combination system of tissue culture and aerophonic technology could be seen as a further improvement to existing systems. Based on the idea of intermittent media supply to the tissue cultured plants, an apparatus called a biochamber is proposed. The chamber will be automatically controlled by a PLC thus reducing labour and handling. Efficiency in plantlet production should also improve due to the reduction of the plant growth period and the production of higher quality plantlets. The advantage of this biochamber for micropropagation is short production time, high survival rate for tissue culture plants and less dependency on labour. This chamber could also be used for other physiological experiments and tests for pathogens. Our initial model plant is banana and we hope to extend trials on other crops. 2.0 The Need For An Automated Biohamber The tissue culture method of propagation is time consuming and labour intensive due to numerous times of changing and sterilizing the containers; each time new media needs to be introduced and plantlets separated. Abnormal and non-functional roots also develop due to abundant food supply and container constraints. As a result, there might be problems when transplantating to the field. Plantlets grown in culture vessels are exposed to a different microclimate as compared to the field. The humidity is high and there is no air movement. As a result tissue culture plants are normally fragile and sensitive. The main problem with the aeroponic method is contamination since growth takes place in a non-sterile condition. A new technique that combines the above methods was proposed. The design is based on principles of tissue culture mainly bioreactors and basic mechanisms of aeroponics. The problems associated with each method are to be minimized while the advantage of each method retained. For example labour will be minimized, bacteria controlled (compared to aeroponic method) and food absorption by plant optimized. This new technique needs a biochamber that is automated. In the biochamber, the media is to be sprayed intermittently onto plantlets (as in the aeroponic method) suspended in a sterile condition (as in a tissue culture environment). A system capable of doing the following functions is needed: 1) sterilize the chamber, 2) pump media and water through nozzles at predetermined conditions to ensure the best growth environment for the plantlets. Since the required process control for this system is not complex, it was decided that a PLC could be used as the control needed is mostly repetitive, discrete and sequential. A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 2

3 3.0 The Automated Biochamber The biochamber is designed to grow plantlets in a controlled environment. The partly transparent chamber will allow for observations and light to penetrate. The design of the chamber allows for portability and easy connection/disconnection of the services (piping, cablings, etc.). A schematic diagram of the biochamber is shown in figure 1. Figure 1: Biochamber Schematic Diagram The chamber must first be sterilized before the plantlets are transferred into it. The suspended plantlets are sectioned into two parts since the lower and upper part of the chamber is separated to allow individual feeding (through spraying of the media) of the roots and adaxial surface of the plantlets (spraying water). There is also a provision for the release of ethylene gas produced by the plantlets via a filter. The main objective of the chamber is to promote plant growth in a controlled environment. The system will allow the user to simulate day and night condition (manual A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 3

4 control), control the amount of media fed to the plantlets, the feeding interval and the feeding time. Upon starting up, the user will select a mode of operation. The PLC will then send signals to open or close the appropriate valves to pump media (food) and water to the plantlets via nozzles in the chamber according to the selected mode. The PLC will send or receive signals to/from the following components: 1) Pump: For pumping of media and water into the biochamber through pipes and nozzles. 2) Control valves: one-way valves and three way valves. To control the amount of media or water sprayed to the planlets. 3) Level sensors: To monitor the media and water level in the vessels. 4) LEDs: System condition indicators (Red LED: alarm, Green LED: Growth in progress/system on, Blue LED: Sterilization in progress). 4.0 Biochamber Operations The user can select the following modes: Mode A: Plant growth In this mode, the plants are intermittently sprayed from the top with water and the roots with media. The user is only allowed to choose the total growing period (with the maximum of 3 weeks). Media and water volume and spraying period have been predetermined for the best growth conditions for plantlets. Mode B: Hardening Process This mode is almost similar to Mode A where the user is only allowed to choose the total number of days for the plantlets to undergo the hardening process. The media volume and humidity conditions are selected so as to prepare the plants for a slightly more hostile conditions so as to enable the plants to adapt easier when transplanted into the fields. Mode C: Research Mode C is specifically designed to enable researchers to conduct experiments. Here the user is allowed to select the following parameters. The media and water volume, spraying intervals, and spraying period. Once a mode is designed, the user can then select the number of days for this new mode to be implemented. A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 4

5 Mode D: Sterilization The sterilization process is to prepare the chamber before new plantlets can be introduced. After the chamber is manually cleaned with alcohol, it is then sprayed with hot water (100 O C) for 20minutes. This process will remove/control almost all of the bacteria or virus harmful to the plantlets. The flow of the modes of operation is as shown in Figure 2 below. Figure 2: Biochamber Operation Flowchart The biochamber is designed for use in a laboratory hence internal temperature control is not provided. (The chamber temperature is assumed to be at 25 0 C). Since this is a first prototype, the design will be of a smaller dimension and the economics of production is not yet a major design criterion. A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 5

6 6.0 Research Capabilities It is important that plants are assessed for susceptibility to pathogens. These experiments require a closed environment to avoid other contamination and careful disposal of the pathogens. The chamber (with ideal growth conditions) would be useful for this purpose. Experiments involving effects of various physiological conditions such as diurnal regulation, volatile hormone requirements, different phytochrome conditions on plant growth could also be conducted in this chamber. To facilitate the research further, the biochamber will be equipped with various sensors, e.g. ph, humidity and thermometer probes. In the case where complex calculations and data manipulations is required, the PLC can be replaced with a computer so that the display of data, averaging, charting etc will be easier. The system will also allow for the collection of used media for the purpose of investigating its composition. Examples of investigation that could be carried out are: 1) Root morphology and adaptation period. 2) Determining the right media composition for the plantlets. 3) Determining whether to feed only through roots, leaves or combination of both. 4) Finding the appropriate feeding requirements such as feeding frequency, interval and volume. 5) Determining a suitable photoperiod for plants and explants growth. 6) Manipulate cultural practices to obtain the shortest production time. 7) Determining the spacing between plantlets for optimum growth. Conclusions This paper presented the design of a PLC controlled biochamber for the micropropagation of plantlets. It is expected that the chamber will optimize plant growth and development process while reducing the growing period of the plantlets thus improving production rate. Upon successful implementation of the method, the plantlets would be grown commercially. It is hoped that the chamber will also be useful for laboratories and research centres. The improvement of the production rate would contribute towards the drive towards the use of technology in agriculture, which is of strategic interest. References 1 Astrom, K.J, Wittenmark, B. Computer Controlled Systems, Prentice-Hall International, A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 6

7 2 Carruther,S. Aeroponics-system Review. Practical Hydroponics July/August 1992, pp Hamilton R, Pederson H and Chin CK. Plant Tissue Culture On Membrane Rafts. Biotechniques March p Johnson, C.D. Process Control Instrumentation Technology, Prentice-Hall International, Kevers,C and Gaspar T. Vitrification Of Carnation In Vitro Changes In Water Content, Extracellular Space, Air Volume And Ion Levels. Physiol Veg 24(6) Mohamad, Z, Buniyamin, N., Othman, R.Y.,Khalid, N. Development Of An Innovative Biochamber For Micropropagation Of Banana Plantlets. Proceedings of the 11 th National Biotechnology Seminar, Ministry of Science, Technology and the Environment, Malaysia. 22 nd 24 th Nov Sani, W, N.Khalid, R.Y.Othman. Competent Cell For Mass Propagation Of Local Banana, Proceedings of abstract for Malaysian Society of Molecular Biology and Biotechnology, Pasir Salak 2-4 September Soloman, S. Sensors And Control Systems In Manufacturing. Mc Graw Hill, Wheathers PJ and Giles KL Regeneration Of Plants Using Nutrient Mist Culture. In Vitro Cellular And Development Biology 24 (7): , Precision Farming Technology. Jurutera, Institution of Engineers Malaysia, ISSN , January 99, pp A PLC Controlled Biochamber For Micropropagation Of In-vitrio Tissue Cultured Plants 7