Title of Invention

AN IMPROVED DEVICE FOR ACCLIMATIZATION

Abstract

An improved device for acclimatization characterized by chamber (1) having an airtight lid (2) for permitting incident light, an adjustable perforated platform (3), a light source (4) being optionally provided above the said lid (2) of the said chamber (1), an inlet (5) alongwith monitor (6) being provided at the side wall of the said chamber (1) for supply of CO2, the said chamber is optionally provided with an inlet (7) connected to reservoir (8) through a pump (9) and a valve (V1) for inter-connecting units, conventional sensors are also provided inside the said chamber for monitoring pH, relative humidity, temperature and light intensity, an outlet (10) being provided at the bottom of the said chamber (1) for draining the liquids

Full Text

The present invention relates to an improved device for acclimatization. Particularly this invention relates to a device useful for hardening of tissue culture raised plants, the efficient acclimatization of microshoots before their transfer to the fields. More particularly, the unit provides simultaneous hardening and rooting of the microshoots. Micro-propagation of plants has many obvious advantages like disease-free planting material, true-to type plants, rapid propagation, induction and screening of variability. The success of micropropagated plants necessarily requires proper acclimatization of microshoots before their transfer to ambient conditions. The basic drawbacks with in vitro raised plants are: a) lack of a well defined protective covering (cuticle) on the leaf surfaces, b) presence of non-functional stomata for regulating gaseous exchanges, c) under developed leaf mesophyll and d) inferior vascularization. This happens because the plants grow under very high humidity on a nutrient medium under heterotrophic conditions. Acclimatization is a method whereby such heterotrophs are tuned to autotrophic mode of growth when there is cuticle development as well as the stomata become functional thus preparing the plantlets for the harsh conditions outside the culture vessels. This assumes greater importance as all plants can not be easily acclimatized because of their specific physico-chemical requirements and therefore, many micro-shoots are lost during the process of their transfer out of the culture vessels. For any successful tissue culture venture, it is more important to find out optimum conditions for hardening in order to increase productivity as well as profits. Hardening of In vitro' raised plants has been variously attempted and mainly two different strategies adopted are in vitro hardening which includes In vitro1 rooting of shoots and 'ex situ' hardening and root induction. Traditionally, the tissue culture produced micro-shoots are excised and put in the rooting media containing specific root promoting substances and then transferred to low PGRs (plant growth regulators), low carbohydrate concentrations, reduced relative humidity and gradually increased light intensities. This takes about 4 weeks before the plants could be transferred to the potting mix and shifted in green houses. But, this method suffers from an additional step of In vitro' root induction which takes about 4-6 weeks and also the roots are brittle and tend to break quite easily. It has been proven that 'in vitro' raised roots perform poorly because of lack of functional vascular under tissue with poor connection between shoot and the root systems often restricts water uptake (Grout, B.W.W. and Aston, H. 1977. Hort. Res. 17: 1-7). According to Smith, M.A.L and McClelland, M.T. 1991. In Vitro Cell, Deveh. Biol. 27 pp: 52-66; the choice of rhizogenesis method, 'in vitro' or 'ex vitro', determines the longterm structure and function of the root system. An auxin pre-initiation treatment for rooting woody plant micro-cuttings (Zimmerman, R.H. and Fordham, I. 1985. J. Amer. Soc. Hort. 110:34-38) induced root primordia which developed into roots once the microshoots were transferred 'ex vitro'. In vitro' acclimatization has been recently reviewed by Ziv, M. 1995. In: J. Artken-Christie, T. Kozai and L. Smith (Eds.), Automation and Environmental Control in Plant Tissue Culture, pp. 493-516. Kluwer Academic Publishers, The Netherlands. According to her, the unfavourable consequences of the 'in vitro1 environment on plant development can be effectively circumvented by modifying the 'in vitro1 microenvironrnent which entail fluctuations in temperature, high relative humidity, diurnal changes in C02, the presence of sugars, growth factors and regulators, to more closely parallel "ex vitro1 conditions. In vitro plantlets are known to have limited photosynthetic capacity, require sugars as an energy source and specific levels of nutrients and growth regulators. During hardening, a defined physical environment with controlled irradiance, gas (C02 and O2) exchange and relative humidity are pre-requisite for plant acclimatization (Kozai T, 1991. In: Y.P.S. Bajaj (ed.) Biotechnology in Agriculture and Forestry. Vol. 17. High Tech Micropropagation I. pp. 127-129. Springier Verlag, Berlin; Preece, J.E. and Sutter E. 1991. In: P.C. Debergh and R.H. Zimmermann (eds.). Micropropagation: Technology and Application pp. 71-91. Kluwer Academic Publishers, Dordrecht; Sutter E. G., Shackel, K. and Diaz, J.C. 1992. Acta Hort. 314: 115-119). Current in vitro' procedures for acclimatization are still unsatisfactory in providing quality micropropagated transplants for the greenhouse or the field. The alternative solution appears to be in providing culture conditions 'in vitro, prior to transplanting, which resemble photoautotrophic conditions ex vitro and provide an optimal water balance for plant development (Kozai, T. 1991. In: IK. Vasil (ed.) Cell culture and Somatic Cell Genetics of Plants, pp. 313-343. Springer Verlag, Berlin). The in vitro raised plants suffer from certain structural deficiencies such as poorly developed protective coating (cuticle) on the leaves, generally fewer and open stomata with large, thin walled guard cells, thin pallisade layers with large spongy tissue in the leaves, involve an additional step of In vitro' rooting and the roots remaining adventitious with poorly developed vascular bundles and slightly brittle with tendencies to break easily (Ziv, M. and Ariel, T. 1992. Acta. Hort. 314: 121-129. These traits lead to poor survival of the micropropagated plantlets 'ex vitro'. Among the strategies adopted to acclimatize the plants 'in vitro' include selection of the vessel including the type of lid allowing gaseous exchange and avoiding hyperhydricity, enhanced illumination and temperature, reduction in minerals, carbohydrates relative humidity, growth regulators and agar in a phased manner and enrichment of the environment with C02 to enhance photosynthesis (Kozai, T. Fujiwara, K, Hayashi, M. and Aitken-Christie, J. 1992. In K. Kurata and T. Kozai (eds.) Transplant Production Systems, pp. 247-282, Kluwer Academic Publishers, Dordrecht; Ziv, M. 1991. In: P.C. Debergh and R.H. Zimmerman (eds.). Micropropagation Technology and Application, pp. 45-69, Kluwer Academic Publishers, Dordrecht). Another recent method in vogue for 'in vitro' propagation and hardening include scaled-up cultures in bioreactors controlled environment chambers, continuous flow of medium using floating rafts, cultures with double layer liquid phase, use of growth retardants, a production of corms, tubers and bulbs as storage organsin vitro'. A culture vessel incorporating a permeable membrane allowing gaseous exchange and reduced RH was described by Roberts A.V., Walker, S. Horan, I, Smith, E.F. and Mottley, J. 1992. Acta Hort. 319: 153-158 enabling support to mixotrophic or autotrophic mode of plant growth under greenhouse. But, this suffers from high maintenance costs. Alternatively, the cut ends of microshoots are treated with high concentrations of root promoting substances and directly transferred to soil and kept under high humidity in polytunnels or other covers (Donnelly, D.J., Vidaver, W.E. and Lee, K.Y. 1985. Plant Cell Tissue Organ Cult. 4: 43-50; Rogers, R.B. and Smith, M.A.L. 1992. J. Horticult. Sci. 67: 535-540). This method also suffers from defects that close monitoring of light, humidity and temperature is not possible. Further sophistication was seen in micro-computer controlled acclimatization chambers developed by Fujiwara, L, Kozai, T. and Watanaba, I. 1988. Ada Hort. 230:153-158 and Hayashi, M., Nakayama, T. and Kozai T. 1988. Acta Hort. 230: 189-194. The unit provided controlled CO2 levels, irradiance, RH, air temperature and air flow to plants in the culture vessels which showed better survival "ex vitro'. But normally cost of such a construction is quite prohibitive and medium sized growers can not afford these. Secondly, production, maintenance and other costs also increase, leading to lower profits. Plant acclimatization in liquid media usually on some kind of a support system (even otherwise) can provide a suitable microenvironment for growth of root and shoot systems, eliminate the need for agar removal and decrease handling costs. Although an improvement in growth was seen in many plant species, in others hyperhydration was observed. Ziv, M. 1992. In: YPS Bajaj (ed.) Biotechnology in Agriculture and Forestry, 19, pp 72-90, Springer Verlag, Berlin reported use of plugs 'in vitro' in cucumber plants which when transferred ex vitro alongwith the plants, showed better survival. Yet in those plants which produce storage organs, acclimatization can be obviated by 'in vitro1 formation of tubers, corms or bulbs because the latter show easy storage, better survival after planting directly in soil. Bioreactors were used for mass propagation of lilium by Takayama, S., Swedlind, B. and Miwa, Y. 1990 In: I.K. Vasil (ed.) Scale up and Automation in Plant Propagation Cell Culture and Somatic Cell Genetics of Plants vol. 8, pp. 112-131, Academic Press, New York, which subsequently developed into plantlets without a need for cold treatment. In recent years, Kozai and his co-workers have shown the importance of CO2 enrichment, increased light intensity and high humidity for hardening of tissue culture raised plants. They have designed a few hardening chambers controlling these parameters through microprocessors (Hayashi, M. and Kozai, T. 1987. Symp. Florizel on Plant Micropropagation in Horticultural Industries, Arlon, Belgium pp. 123-134) but again the cost is beyond the means of many nurserymen particularly in developing countries. It, therefore, becomes quite obvious that all the hardening units explained above suffer from one or the other defects and the methods require dextrous handling of the plantlets/shoots during in vitro or ex vitro' conditions or sophistication in instrumentation leading to an ultimate escalation in the costs. Besides, such methods can not be adopted by small growers especially in developing countries like India. Thus, none of these units can bring about rooting and acclimatization as a one step procedure. On the other hand, the present invention overcomes the aforesaid limitation and provides a comparatively cheaper and versatile module for use for different plant species. Moreover, this facilitates not only hardening but rooting also takes place simultaneously. The intensity of light, CO2 concentration and medium flow could be regulated with comparative ease and thereby increasing the achievable success in acclimatization and preparing micro shoots for transplantation in the fields and saving in terms of time and costs also. Further, the unit can be used independently or a series of units can be interconnected to increase area of operation. Fig.l of the drawings accompanying this specification shows one such device and Fig.2 shows 5 units interconnected with each other. The main object of the present invention is to provide an improve device . Another object of the present invention is to provide a quicker method of bringing about rooting in microshoots. Yet another object of the present invention is to develop a module for raising hydroponics under controlled physical conditions. Accordingly, the present investigation provides an improved device for acclimatization characterized by chamber (1) having an airtight lid (2) for permitting incident light, an adjustable perforated platform (3), a light source (4) being optionally provided above the said lid (2) of the said chamber (1), an inlet (5) alongwith monitor (6) being provided at the side wall of the said chamber (1) for supply of CO2, the said chamber is optionally provided with an inlet (7) connected to reservoir (8) through a pump (9) and a valve (Vi) for inter-connecting units, conventional sensors are also provided inside the said chamber for monitoring pH, relative humidity, temperature and light intensity, an outlet (10) being provided at the bottom of the said chamber (1) for draining the liquids. In an embodiment of the present invention micro-shoots were planted in the holes of the platform in moss plugs tightly. In another embodiment of the present invention micro-shoots were planted in pro-trays. In yet another embodiment of the present invention micro-shoots were planted in root trainers (Hikkotrays). In yet another embodiment of the present invention juvenile plantlets were planted directly in the potting mix spread over the flat platform. The outer chamber ' 1' may be made of biocompatible hard material like metals, alloys, plastic, fiber glass or wood. The outer chamber be protected with anti-corrosion paint like red-oxide, aluminium paint, enamel paint or epoxy coating, rubberised paint. The openable panel '2' may be joined with hinges or pivots with the body of outer chamber '1' and provided with handles and locking facilities and a rubber/polyurathene lining for making the chambers air tight. The transparent sheet may be made of glass, polycarbonate, polythene or any material permitting passage of incident light to the inside of Chamber '1'. The removable perforated platform '3' may be made of metal, plastic, aluminium sheet with holes or of wire mesh resting on metallic or suitable solid frame fitted inside Chamber '1'. The removable perforated platform '3' may be coated with any bio-compatible anti-corrosion paint or coating. The light source may be selected out of incandescent, fluorescent or optic fiber illuminator or the acclimatization units may be kept under direct/diffused sunlight. The sub-units may be interlinked or independent by using suitable valves. The microshoots after excision are separated and cut ends are treated with high concentrations of auxins before transplantation in the potting mix in either of the containers (moss plugs pro-trays, Hikko trays or pots) depending upon the plant species. The platform (3) height can also be adjusted accordingly and the chamber (1) may be filled up with water or suitable nutrient solutions/liquids through valve V1. The trays/pots are kept over the platform and in case of microshoots in moss plugs, the plugs can be fitted directly in the holes of the perforated platform (3) with their lower ends touching the liquid/water in chamber (1). The C02 is supplied through an inlet(5) in the sidewall and light source (4) is switched on to facilitate the acclimatization. Spraying of liquids is optional and the fogging system can be made operational by switching on the pump (9). Various physical parameters like pH, C02 concentration, temperature and relative humidity inside the chamber can be monitored by suitable sensors. The water/liquid in the chamber can be easily replaced by using the drain (10). The following examples are given by way of illustration of the use of the unit of the present invention and should not be construed to limit the scope of present invention. Example-1 Microshoots of tea were obtained from 'in vitro1 proliferating cultures raised from axillary buds. Only those shoots which were 3.0-4.0 cm with short inter nodes (0.5-1.0 cm) and broad leaves (1.0 cm in the middle) were excised and the cut ends were treated with an auxin solution (lndole-3-butyric acid; IBA 500 mg/l) for 30 min and the treated shoots were transferred to a soil mix consisting of Sand: Garden soil: Farm yard manure 4:5:1 and having a pH of 5.4 taken in Hikko trays or root trainers (supplied by M/s Wimco, India) and the trays were placed inside the acclimatization unit over the adjustable platform. Carbon dioxide was supplied through a side port with the help of a rubber tube and its level was maintained at 800 ppm. After putting the plants inside the chamber, the lids were closed and light was provided by the overhung electrical bulbs (1600 lux). The lights were kept on for 9 hours every day. Root initials were noticed after 40 days and it took another 20 days for rooting and establishment. Upto 91.0% shoots showed successful acclimatization and establishment. The above example shows the usefulness of this unit for hardening of a woody plant like tea under high C02 concentration, high humidity and light. The hardened plants could then be transferred to the polysleeves till their field transplantation. Example-2 Microshoots of another economically important perennial plant Damask rose (Rosa damascene) were obtained from proliferating cultures raised from axillary buds in liquid static cultures. Two-three cm long shoots were excised and fixed in the holes of the metallic platform in moss plugs in such a way that the cut ends were immersed in the solution containing an auxin IBA (5mM-7 days or 10 mM-5 days) and then replacing the auxin supplemented medium with basal medium (Murashige and Skoog, 1962). The CO2 levels were again maintained at 800 ppm and light (1,600 lux) for 9 h was provided as given under Example-1. The root induction takes about 2 weeks and well rooted hardened plants ready for transplantation in soil mix are obtained in 40-45 days. Example-3 Young plantlets of philodendrons/fems raised 'in vitro were taken out and their roots washed in lukewarm water to remove any traces of agar. These were then transplanted directly in a soil mix spread over the platform over a layer of sphagnum moss. Rest of the conditions remaining the same as in Example-1. The plantlets were established in soil in 4-6 weeks and then transferred to the pots in polytunnels. In these examples, the versatility of the hardening (acclimatization) unit is established whereby, rooting as well hardening could be accomplished as a single step procedure. Moreover, the same unit is suitable for different plant varieties. Hardening of the grafted plants under high C02 concentration, high humidity and light proved beneficial for preparing the plants for field transfer. The main advantages of the present invention are: 1. A low cost versatile portable, time effective acclimatization unit for hardening. 2. A unit where both acclimatization and rooting of micro shoots can be brought about simultaneously. 3. Hardening of rooted plants under high C02 concentration and high light intensity, gets facilitated at faster rate. 4. Monitoring of physical parameters like CO2, relative humidity, pH, light and nutritional aspects becomes easier. 5. The unit can be used independently or many units can be inter-connected through suitable stop cocks/gate valves. 6. The shoots/plantlets can be grown while in touch with the auxin supplemented medium in moss plugs or directly planted in soil mix or any potting mix spread over the flat platform. We claim: 1. An improved device for acclimatization characterized by chamber (1) having an airtight lid (2) for permitting incident light, an adjustable perforated platform (3), a light source (4) being optionally provided above the said lid (2) of the said chamber (1), an inlet (5) alongwith monitor (6) being provided at the side wall of the said chamber (1) for supply of CO2, the said chamber is optionally provided with an inlet (7) connected to reservoir (8) through a pump (9) and a valve (Vj) for inter-connecting units, conventional sensors are also provided inside the said chamber for monitoring pH, relative humidity, temperature and light intensity, an outlet (10) being provided at the bottom of the said chamber (1) for draining the liquids. 2. An improved device as claimed in claim 1 wherein the outer chamber (1) platform (3) are made of bio-compatible hard materials selected from metals, alloys, plastic, fiber glass or wood. 3. An improved device as claimed in claim 1 to 2, wherein the outer chamber (1) and platform (3) are coated with anti-corrosion agent selected from red oxide, aluminium paint, enamel paint, epoxy coating or rubberised paint. 4. An improved device as claimed in claim 1 to 3, wherein the lid (2) is joined with hinges or pivots with the body of outer chamber (1) and provided with handles and locking facilities and a rubber or polyurathene lining for making the unit air tight. 5. An improved device as claimed in claim 1 to 4, wherein the lid (2) is made of glass, polythene, polypropylene, polycarbonate or any material. 9, An improved device for acclimatization substantially as herein described with reference to the examples and drawing accompanying this specification.

Documents:

279-del-1999-abstract.pdf

279-del-1999-claims.pdf

279-del-1999-correspondence-others.pdf

279-del-1999-correspondence-po.pdf

279-del-1999-description (complete).pdf

279-del-1999-drawings.pdf

279-del-1999-form-1.pdf

279-del-1999-form-19.pdf

279-del-1999-form-2.pdf