Title of Invention

A BIOREACTOR FOR PLANT SHOOT CULTURE

Abstract

A bioreactor for plant shoot culture comprising a main bioreactor portion and a perfusion vessel portion, the former being placed in a plexiglass enclosure with lights to provide the required illumination; a programmable timer for regulating the illumination; an inoculation port for introduction of micro-propagules into the bioreactor portion for placement on a rotatable perforated plate; a medium inlet for introduction of a growth medium from the perfusion vessel portion into the bioreactor portion; misting means employing compressed air for converting the growth medium introduced into the bioreactor portion into a spray or fine droplets; a humidifier for humidifying the compressed air, a medium outlet for returning the growth medium to the perfusion vessel.

Full Text

This invention relates to a bioreactor for plant shoot culture. Clonal propagation of plants by tissue culture is now internationally established as the best method of obtaining planting material for vegetatively propagated plants. Tissue culture ensures production of large numbers of identical, disease free propagules. Many commercial tissue culture factories have been set up in India to cater to the ever increasing international and local demand for tissue cultured plants of fruit plants such as banana and strawberry, besides ornamentals. A problem with the tissue culture system is the high cost of the plants due to the specialized inputs. Micropropagation of plants is largely a manual operation, and is restricted by the limitations of ease of handling by skilled operators and stringent sterility conditions. Bioreactor systems are an alternative method to increase the scale of operations and rate of production of the plants. Considering the increased output efficiency at lower costs, if large-scale micropropagation of plants is changed to the bioreactor method, the horticultural industry will better realize the advantages of using tissue cultured plants for cultivation. Although bioreactors are very common, and are extensively used in the bio-industry, bioreactors for plant shoot culture are rare. According to the known art, a bioreactor for plant shoot culture consists of a sterile plastic bag with a facility for sterile gas exchange. The plastic bag is filled with the liquid growth medium, and the plantlets are grown at the surface of that liquid. Under these conditions, the shoots of the majority of plant species get hyper-hydrated or 'vitrified' leading to abnormalities in morphogertesiraiid problems in hardening. Further, the bioreactor does not allow easy, continuous monitoring of the conditions in the bioreactor such as dissolved oxygen (DO), pH, temperature, shoot mass, and others that are essential for optimization of the process. Also, it is not possible to manipulate the atmosphere in the bioreactor, which is essential to modify conditions during cultivation toward desirable results. In addition, the sterile plastic bag cannot be re-used. The advantages of this invention are: The proposed bioreactor does not require plantlets to grow on the liquid surface. Thus, the main drawback of hyper-hydration or vitrification is completely avoided when the proposed bioreactor is used. Further, the proposed bioreactor is re-usable, and allows easy, continuous monitoring of many important bioreactor variables such as dissolved oxygen (DO), pH, temperature, shoot mass, and others that are essential for optimization of the process. Also, in the proposed bioreactor it is possible to manipulate the atmosphere toward desirable results such as high growth rates, desirable contents of plant parts such as the leaf, and others. The proposed bioreactor is re-usable and can be steam sterilized for re-use as easily as sterilizing any regular lab-scale apparatus, FtAxJfd* ftj. caiAXA^r f^^^ • Modular design - main bioreactor and a perfusion vessel - allows for easy modifications as recmired at a later Hatfi • The perfusion vessel has been designed as a complete bioreactor so that it can also double as a separate bioreactor for other cultivations, when it is not used for shoot culture. • The proposed bioreactor allows for continuous monitoring of the medium parameters (temperature, DO, pH, air flow rate, and others). Also, the periods of lighting for appropriate ligh^/dark cycles can be programmed. • The design allows for manipulating the medium variables easily to find out the optimum medium conditions for the particular shoot culture. • The proposed bioreacttfallows for manipulating the atmosphere in the main shoot bioreactor; any gaseous nutrient/elicitor. be added. • The proposed bioreactor also allows for continuous monitoring of the gaseous atmosphere in the main bioreactor by hooking up to the appropriate existing measuring instrument such as a gas chromatograph. • Provision has been made for continuous monitoring of shoot mass. The main bioreactor has been provided with a misting system. Normally misting systems need a certain distance over which the misting can occur. We needed the misting to occur inside the bioreactor over a very short distance of 7 cm. The required misting over a distance of 7 cm was ultimately achieved by using a specially designed misting head. A schematic diagram of the bioreactor is given in Figure 1. The key to the legend in Figure 1 is as follows: A Air inlet through sparger Ac Acid container Al Alkali container AOF Air outlet through filter or to online measurement systems B Balance CA = Compressed air DAS Data acquisition system DO w Dissolved Oxygen probe EN -• Enclosure for the main bioreactor H Humidifier IP Inoculation port L Lights (many) M Motor Ma - Manipulator for shoots MB Main Bioreactor. The shoots are grown in the main bioreactor. Ml Medium inlet MO Medium outlet pH pH probe PF Perfusion vessel (jacketed, stirred vessel that is temperature controlled, and can be used as a stand-alone bioreactor for other cultivations).. The medium is circulated from the perfusion vessel to the Main Bioreactor and back. Also, the liquid parameters, pH, DO, etc., are measured in the PF. PP Perforated plate S System for Misting T Temperature pi-o.be The bioreactor for plant shoot culture, according to his invention, comprises a main bioreactor portion and a peifusion vessel portion, the former being placed in a plexiglass enclosure with lights to provide the required illumination", a programmable timer for regulating*the illumination; an innoculation port for introduction of micro-propagules into the bioreactor portion for placement on a rotatable perforated plate; a medium inlet for introduction of a growth medium from the perfusion vessel portion into the bioreactor portion; misting means employing compressed air for converting the growth medium introduced into the bioreactor portion into a spray or fine droplets; a humidifier for humidifying the compressed air; a medium outlet for returning the growth medium to the perfusion vessel This invention will now be descriWd"with reference to the accompanying drawings, the single Figure in which illustrates, by way of example, and not by way of limitation, one of possible embodiments of the bioreactor proposed herein: The embodiment consists of two pails, the main hioreactor MB and the perfusion vessel PF. The main bioreactor MB is placed in a plexiglass enclosure with lights to provide the rquird illumination. The lights are on a programmable timer. Micro-propagules or small plant parts that can give rise to a whole plant are rawn in the main bioreactor linger controlled conditions until they become plantlets that can be transferred to the SS«for further growth. The micro-propagules are placed in the mam bioreactor under sterile conditions through the inoculation port IP, and are manipulated for proper placement on the perforated plate PP using the manipulator for shoots Ma and by appropriately rotating the PP. The growth medium e.g. Murashige Skoog medium or any other is added to the perfusion vessel in a sterile fashion, and is fed to the main bioreactor through the medium inlet MI using a suitable pump. Any changes /.. to the medium are done by additions/removal to/from the PF liquid. The gas atmosphere in the main bioreactor can be changed by suitable additions/removals to/from the gas space in the main bioreactor. The medium is added to the main bioreactor using the system for misting S; where the medium is converted into a spray of micron-sized fine droplets using compressed air CA. The CA is humidified using a humidifier H to prevent loss of water from the medium during operations extending over many days at a stretch. The medium is returned to the perfusion vessel PF through the medium outlet port and a suitable pump. The mass of shoots in the main bioreactor can be monitored using the balance B placed inside the enclosure, by switching off the inlet and outlet flow rates appropriately to ensure no liquid flow inside the main bioreactor. Other gas phase parameters of interest such as concentrations or levels can be measured by attaching suitable instruments to the air exiting the filter AOF from the main bioreactor. The perfusion vessel PF is a stand-alone bioreactor for microbial culture when not used for perfusing medium during plant shoot culture. In the perfusion vessel, PF, the dissolved oxygen level^ temperature, aeration rate, agitation rate are continuously monitored using the DO probe, DO, pH probe, temperature probe, such as, a thermocouple T, I'otameter and rpm meter attached to the motor M, respectively. Tfre rpm vai' e and temperature in the PF are automatically controlled through appropriate electronics. The pH value is controlled through appropriate acid Ac and alkali At addition, automatically. Oxygen is supplied to the medium by bubbling air through a sparger A. All the data measured are electronically fed to the data acquisition/ control system DAS. We Claim: 1. A bioreactor for plant shoot culture comprising a main bioreactor portion and a perfusion vessel portion, the former being placed in a plexiglass enclosure with lights to provide the required illumination; a programmable timer for regulating the illumination; an inoculation port for introduction of micro-propagules into the bioreactor portion for placement on a rotatable perforated plate; a medium inlet for introduction of a growth medium from the perfusion vessel portion into the bioreactor portion; misting means employing compressed air for converting the growth medium introduced into the bioreactor portion into a spray or fine droplets; a humidifier for humidifying the compressed air; a medium outlet for returning the growth medium to the perfusion vessel 2. A bioreactor as claimed in Claim 1 wherein a pump is provided for pumping the growth medium into the bioreactor portion through the medium inlet. 3. A bioreactor as claimed in Claim 1 or Claim 2 wherein a pump is provided for returning the growth medium into the perfusion vessel portion through the medium outlet. 4. A bioreactor as claimed in any one of the preceding Claims wherein a balance is placed inside the said enclosure. 5. A bioreactor as claimed in any one of the preceding Claims wherein a sparger is provided for bubbling air therethrough for supply of oxygen to the growth medium. 6. A bioreactor as claimed in any one of the preceding Claims wherein means are provided for continuously monitoring the dissolved oxygen level foH, temperature, aeration rate, agitation rate. 7. A bioreactor for plant shoot culture substantially as herein- described and illustrated.

Documents:

0565-che-2005-claims.pdf

0565-che-2005-correspondnece-others.pdf

0565-che-2005-description(complete).pdf

0565-che-2005-description(provisional).pdf

0565-che-2005-drawings.pdf

0565-che-2005-form 1.pdf

0565-che-2005-form 26.pdf

0565-che-2005-form 5.pdf

565-che-2005-abstract.jpg