|Title of Invention||
AUTOMATED HYDROPONIC SYSTEM FOR POTATO MICROTUBER PRODUCTION IN VITRO
|Abstract||An Automated Hydroponic System (AHS) for large-scale micropropagation and subsequent induction of disease-free microtubers in vitro from single node cuttings/ axillary shoot cuttings in potato. This system comprises a specially designed sterilisable polycarbonate hydroponic culture chamber, its stainless steel (SS) cover lid with various retrofitting port extensions (for culture inoculation, medium dispensing, sterile aeration and sterile venting), a 4 litre or higher volume polypropylene media reservoir (with a sterilisable polypropylene filling/ venting cap with thermoplastic elastomer gasket, a medium dispensing port and a sterile venting port), 0.2 j,i PTFE vent microfilters, autoclavable silicone tubings, a computer-controlled reversible and variable speed programmable peristaltic pump, a single-head air pump, a programmable timer and a computer with support software for system operation and data interface. The inlets of the medium dispensing ports of the hydroponic chamber and medium reservoir are fitted with 20"-long silicone tubes with polypropylene barbed retrofit at their ends. The outlets of the medium dispensing ports of the hydroponic chamber and medium reservoir are connected by a 4'-long silicone tube, which, at its centre, is mounted on pump-head of the peristaltic pump. The peristaltic pump is connected to the computer through RS-232 interface. The sterile vent ports of the hydroponic chamber and medium reservoir is fitted with 0.2 µ PTFE vent microfilters at the outlets and with 8"-long silicone tubes at the inlets. The sterile aeration port of the hydroponic chamber is vertically fitted with a 0.2 µ PTFE vent microfilter, which, in turn, is connected to the single-head air pump through a silicone tube. The air pump is connected to the programmable timer. The computer and the programmable timer are connected to the external power source through a UPS.|
|Full Text||This invention pertains to designing and fabrication of an innovative cylindrical hydroponic culture chamber, and development of an integrated and automated hydroponic system for efficient and rapid production of microtubers (miniature sized potatoes induced in vitro) completely free from systemic viral diseases with multiple innovative components facilitating hassle-free, biologically safe and economically efficient seed potato production and multiplication system. This system is devised to allow production of disease-free microtubers from high-value potato cultivars infected by systemic diseases through multiplication of axillary shoot cultures followed by microtuber induction.
Hydroponic system for in vitro plant culture is a well-known technique. It is simply the science of cultivation of plants on artificial nutrient solution instead of soil. The science of hydroponics predates to the present plant cell and tissue culture techniques. In potato, nutrient film technique (NFT), a modified hydroponic approach, was developed in 1990s (Wheeler et al., 1990) to produce seed tubers in PVC trays under ex vitro conditions (in glasshouses) at the United States National Aeronautics and Space Administration (NASA) as an adjunct for long-term space travel. Subsequently, other variations of hydroponic systems such as deep flow technique (DFT), aeroponic etc. (Boersig and Wagner, 1988; Chang et al, 2000) were developed for ex vitro production of potato seed tubers in greenhouses. For in vitro production of potato microtubers, iermcnter or bioreactor technology with different modifications was also attempted for in vitro microtuber production (Hao et al., 1998; Watad et al., 1998). Fermenter or bioreactor technology for microtuber production is cumbersome, complicated and involves expensive and sophisticated facility at prototype and scale-up levels. As a result, fermenter or bioreactor technology for potato microtuber production is yet to become functional. Whenever fermenter or bioreactor technology may prove functional for large-scale potato microtuber production, it may be commercially not viable in developing countries for its high initial and operational cost.
Large-scale microtuber production in potato currently followed in India and elsewhere consists of three basic steps: 1. maintenance and multiplication of disease-free clones through axillary shoot cuttings in culture tubes on semisolid (agar-gelled) propagation medium; 2. initiation of axillary shoot cultures in 250 ml capacity Erlenmeyer flasks or Magenta boxes or other appropriate culture vessels on liquid
propagation medium; and 3. induction of microtubers in aforesaid diverse culture vessels under tuber-inducing conditions in vitro (Sarkar et al., 1997; Sarkar and Naik, 1998). Each step of the above protocol has its own specific requirements in terms of nutrient medium, culture conditions, culture space, inoculation schedules and incubation intervals. Consequently, the production efficiency of microtubers is dependent upon the propagation efficiencies of individual culture stages. Therefore, this method starting from initial multiplication through axillary shoot cuttings to final microtuber induction is not only highly time-consuming, but also labour-intensive. It is estimated that about more than 60 % of microtuber production cost accounts for labour input on time-consuming medium preparation, medium transfer to culture vessels and culture inoculation. Besides, the present schedule of microtuber production involving conventional culture tubes and 250 ml Erlenmeyer flasks/Magenta boxes/Melli jars are enormously space-intensive. For example, according to a modest estimate, production of 10,000 potato microtubers involves initial multiplication in 1000 culture tubes, each containing three microplants, followed by liquid propagation in 500 Erlenmeyer flasks/Magenta boxes/Melli jars with subsequent microtuber induction in these 500 culture vessels by replacing the liquid propagation medium from each vessel with fresh microtuber induction medium under aseptic conditions. It can, therefore, be well appreciated the extent of culture space and rigid culture sequence and/or schedule required for large-scale potato micropropagation and microtuber production on a commercial scale. Moreover, because of multiple containers being used and multiple transfer of media made during this culture, the frequency of contamination and loss of plants at different stages are higher. This cumbersome procedure and space-cost disadvantage are preventing the existing microtuber production technology from commercialisation for seed potato production.
It was this drawback of the existing disease-free seed potato production technique and the high commercial need for an alternate economically viable and feasible technology that compelled us to undertake this research for innovating and developing an integrated automated system for potato microplant culture and microtuber production in vitro. The system we have developed deploys only liquid nutrient media both for growing of potato microplants as well as for their subsequent induction to produce microtubers. Therefore, our method is designated as "Automated Hydroponic System for Potato Microtuber Production in vitro".
Boersing, M. R. and S. A. Wagner. 1988. Hydroponic systems for production of seed
tubers. Amer. Potato J. 65: 741. Chang, D-C., S-Y. Kim, Y-L. Hahm and K-Y. Shin. 2000. Hydroponic culture system
for the production of seed tubers without soil. Amer. J. Potato Res. 77: 394. Hao, Z. L, O. Y. Fan, Y. X. Geng, X. D. Deng, Z. M. Hu and Z. G. Chen. 1998.
Propagation of potato tubers in a nutrient mist bioreactor. Biotechnol. Technique
12: 641-644. Sarkar, D. and P. S. Naik. 1998. Effect of inorganic nitrogen nutrition on cytokinin-
induced potato microtuber production in vitro. Potato Res. 41:211-217. Sarkar, D., P. S. Naik and R. Chandra. 1997. Effect of inoculation density on potato
micropropagation. Plant Cell Tissue Org. Cult. 48: 63-66. Watad, A. A., Y. Alper, R. Slav, R. Levin, A. Altman, M. Ziv and S. Izhar.
Mechanization of micropropagation. Curr. Plant Sci. Biotech. Agril. 36: 663-666. Wheeler, R. M., C. L. Mackowiak, J. C. Sager, W. M. Knott and C. R. Hinkle. 1990.
Potato growth and yield using nutrient film technique (NET). Amer. Potato J. 67:
Automated Hydroponic System for Potato Microtuber Production in vitro This system allows large-scale multiplication of potato microplantlets free from all systemic viral diseases, and subsequent induction (on these microplantlets) of abundant microtubers in vitro with the help of a precisely scheduled delivery of nutrient media into the specially designed and fabricated cylindrical-shaped hydroponic culture chamber through a specially developed computerized operating system. The merit of this integrated automated system lies in its simplicity and high production efficiency with optimisation of time and space, and minimisation of contamination during culture, all leading to significant cut in cost of production. The technique developed and claimed herein is unique and involves low-cost technology, which could be affordable in most of the countries. Moreover, the method under this application is amenable for scaling-up to suit to the large-scale commercial microtuber production for integration into disease-free seed potato production system.
The Automated Hydroponic System (AHSJ
Following are the specification details of the 'Automated Hydroponic System for
Potato Microtuber Production in vitro' claimed herein:
1. Steam sterilisable polycarbonate hydroponic culture chamber with Stainless Steel
(SS) cover lid designed and fabricated to accommodate four different ports: i)
culture inoculation port, ii) medium dispensing port, iii) sterile aeration port, and
iv) sterile venting port (Figures 1 and 2):
2. Steam sterilisable polypropylene culture bottle of 4 litre or higher volume as
medium reservoir with similar sterilisable polypropylene filling/venting cap with
thermoplastic elastomer (TPE) gasket and medium dispensing plus sterile venting
3. Computer-compatible, reversible, variable-speed programmable peristaltic pump.
4. Computer with peripherals like RS-232 data interface, monitor and UPS system.
5. Single-head air pump for aeration, which could also be modified as multi-head.
6. Programmable timer for controlling the air pump for aeration.
7. Silicone peroxide-cured steam sterilisable tubings for medium dispensing and
8. Steam sterilisable polypropylene retrofittings with acetal nuts, silicone gaskets and
silicone cap as port extensions.
9. Steam sterilisable hydrophobic 0.2 j.t PTFE vent microfilters for sterile aeration
and venting the hydroponic culture chamber and/or medium reservoir.
The total system assembly is diagrammatically shown in Figure 3.
Design and fabrication of hydroponic culture chamber
The cylindrical hydroponic culture chamber is made of steam sterilisable (121 °C) transparent polycarbonate material of 0.25 cm thickness. The prototype chamber height is 30.5 cm with an internal diameter of 20.5 cm. The top Stainless Steel (SS) cover lid has a diameter of 22.0 cm. At the centre of the SS cover lid is placed the culture inoculation port (with a SS screw cap) of 7.4 cm diameter with the top SS screw cap having a diameter of 7.9 cm. The SS screw cap has a height of 1.8 cm. The culture inoculation port (with the SS screw cap) is located on the SS cover lid at a distance of 7.3 cm from the periphery. Medium dispensing, sterile aeration and sterile venting ports on the SS cover lid are located at a distance of 2.5 cm from the periphery. Each port has a diameter of 1.0 cm. The distance between the periphery of
the central culture inoculation port and other ports (medium dispensing, sterile aeration and sterile venting) is 3.5 cm. Each port excluding the inoculation port has a total height of 5.6 cm with internal and external extensions (Figures 1 and 2).
Assembly and operation of the Automated Hydroponic System (AHS) The SS cover lid with its inoculation ports sealed with a SS screw cap is used to close the hydroponic culture chamber. The sterile aeration and sterile venting ports in the hydroponic culture chamber are fitted with 0.2 µ hydrophobic PTFE microfilters by silicone tubes in a way so that the microfilters stand vertically. A 20"-long silicone lube with a polypropylene barbed retrofit (at the end) is fitted to the inlet of the medium dispensing port extension so that it touches the bottom of the culture chamber. The outlet of the medium dispensing ports of the hydroponic culture chamber and the medium reservoir is connected by a 4'-long silicone tube. The inlet of the medium dispensing port of the medium reservoir is fitted with a 20"-long silicone tube with a polypropylene barbed retrofit at its end touching the bottom. The sterile vent port of the medium reservoir is fitted with a hydrophobic 0.2 µ PTFE vent micro filter at the outlet, and with a 8"-long silicone tube at the inlet. The total system (i. e. cylindrical hydroponic culture chamber plus medium reservoir containing liquid nutrient medium) is sterilised at 121 °C for 20 min.
The explants (shoot cuttings/axillary shoots) are transplanted into the hydroponic culture chamber through the culture inoculation port by removing the SS screw cap on the cover lid under a Laminar Flow Clean Air (LFCA) work station. After inoculation, the system is placed in the tissue culture room with the medium dispensing tube (4'-long silicone tube connecting the chamber with the medium reservoir) connected to a peristaltic pump. The peristaltic pump is interfaced with a computer through RS-232 data interface cable. The outlet of the sterile aeration port (pre-fitted with a 0.2 µ vent microfilter) of the hydroponic culture chamber is connected to a air pump through a 6'-long silicone tube. The air pump is connected to a programmable timer. The power supply to the computer and programmable timer is provided through an UPS system (Figure 3). A computer programme has been customized for precise unattended scheduling of the medium flow rate, flow direction, flow duration, dispensing interval and culture bathing duration (to and fro the hydroponic culture chamber and medium reservoir) in this automated hydroponic
system. It allows automatic dispensing of desired volume of liquid nutrient medium from the medium reservoir to the hydroponic culture chamber at a desired flow rate bathing the cultures for desired duration, and recycling back to the medium reservoir.
Automated hydroponic culture
The automated hydroponic culture system in potato consists of two stages: i) shoot
multiplication and ii) microtuber induction and development. The base cultures of
disease-free potato cultivars are maintained and multiplied through single node
cuttings (SNCs) on semisoild propagation medium in the culture tubes (Sarkar et al.,
1997). The stock cultures are used for initiating hydroponic culture in the automated
1) Shoot multiplication stage: Shoot cuttings with 6-8 nodes collected from twenty to
thirty 28-day-old microplants are transferred into the hydroponic culture chamber,
and cultured for 6 weeks under a 16-h photoperiod (approx. 70-80 jamol m-2 s-1
light intensity) at 24 °C (Figure 4A). The cultures are periodically submerged in
liquid propagation medium recycled in and out of the hydroponic chamber (from
the medium reservoir) for a predetermined duration at 100 ml min"1 flow rate. The
culture in hydroponic chamber is aerated with sterile air. After 6 weeks'
incubation, the proliferating shoots attain an average height of 200 mm, and are
ready for microtuber induction (Figure 4B).
2) Microtuber induction and development: After shoot multiplication stage, the
liquid propagation medium in the reservoir is replaced with microtuber induction
medium. The cultures are incubated under dark at 20 °C for microtuber induction
(Figure 4C). The shoot cultures are initially submerged in 1 lit of induction
medium for 2 weeks, and thereafter, the medium level is maximized periodically
up to 2 lit at predetermined rale and interval. The stoloniferous microshoot growth
continues up to 3 weeks reaching more than 300-400 mm in length (Figure 4D).
The induction medium in the reservoir is replaced with fresh stock after every five
weeks. The microtuber cultures are aerated with sterile air. Microtubers start
developing epigeally within two weeks of culture initiation. Microtubers are
harvested after 10 weeks of incubation. On an average, 300-500 microtubers
develop in the hydroponic culture chamber with an average fresh mass of 250 mg
(Figure 4E). Microtuber production efficiency per microplant in the automated hydroponic system has been 4-7 times higher than that in conventional production system using 250 ml capacity Erlenmeyer flasks or Magenta boxes or Melli jars.
This method, therefore, overcomes all limitations of existing microtuber production methods. Moreover, it offers high efficiency in microtuber production with least incidence of loss from contamination, optimised use of costly culture media and minimised labour cost. Automation of the process places its operation ai clock-work-precision with total functional and economic efficiency.
Legend to Figure 4
Figure 4: Automated Hydroponic System (AHS) for micropropagation and microtuber production in potato cultivar Kufri Sindhuri. A, microplants growing in the hydroponic culture chamber under a 16-h photoperiod (approx. 70-80 µmol m"2 s"1 light intensity) at 24 °C. B, 6-week-old microplantlets before microtuber induction. C, microplantlets growing on microtuber induction medium in the hydroponic culture chamber under the dark at 20 °C. D, stoloniferous microshoots developing in microtuber induction culture within the hydroponic culture chamber. E, microtubers initiating epigeally after 2 weeks' incubation of induction cultures. F, developing microtubers in the hydroponic culture chamber
1. An Automated Hydroponic System (AHS) for large-scale micropropagation and subsequent induction of disease-free microtubers in vitro from single node cuttings/ axillary shoot cuttings in potato. This system comprises a specially designed sterilisable polycarbonate hydroponic culture chamber, its stainless steel (SS) cover lid with various retrofitting port extensions (for culture inoculation, medium dispensing, sterile aeration and sterile venting), a 4 litre or higher volume polypropylene media reservoir (with a sterilisable polypropylene filling/ venting cap with thermoplastic elastomer gasket, a medium dispensing port and a sterile venting port), 0.2 µ PTFE vent microfilters, autoclavable silicone tubings, a computer-controlled reversible and variable speed programmable peristaltic pump, a single-head air pump, a programmable timer and a computer with support software for system operation and data interface. The inlets of the medium dispensing ports of the hydroponic chamber and medium reservoir are fitted with 20"-long silicone tubes with polypropylene barbed retrofit at their ends. The outlets of the medium dispensing ports of the hydroponic chamber and medium reservoir are connected by a 4'-long silicone tube, which, at its centre, is mounted on pump-head of the peristaltic pump. The peristaltic pump is connected to the computer through RS-232 interface. The sterile vent ports of the hydroponic chamber and medium reservoir is fitted with 0.2 µ PTFE vent microfilters at the outlets and with 8"-long silicone tubes at the inlets. The sterile aeration port of the hydroponic chamber is vertically fitted with a 0.2 µ, PTFE vent microfilter, which, in turn, is connected to the single-head air pump through a silicone tube. The air pump is connected to the programmable timer. The computer and the programmable timer are connected to the external power source through a UPS.2. The automated hydroponic system as claimed in claim 1 wherein the hydroponic culture chamber and retrofitting port extensions are fabricated using polycarbonate and polypropylene or such other material. The autoclavable cylindrical hydroponic chamber is made of transparent polycarbonate material of 0.25 cm thickness with a height of 30.5 cm and an internal diameter of 20.5 cm. The stainless steel (SS) cover lid of this hydroponic chamber has a diameter of
22.0 cm and has been fabricated to accommodate four different ports: i) centrally located (at a distance of 7.3 cm from the periphery of the SS cover lid) culture inoculation port of 7.4 cm diameter with a top SS screw cap of 1.8 cm height and 7.9 cm diameter; ii) medium dispensing port of a diameter of 1.0 cm located at a distance of 2.5 cm from the periphery; iii) sterile aeration port of 1.0 cm diameter located at a distance of 2.5 cm from the periphery; and iv) sterile vent port of 1.0 cm diameter located at a distance of 2.5 cm from the periphery. Each of the medium dispensing port, sterile aeration port and sterile venting port has a total height of 5.6 cm with internal and external extensions..
3. The automated hydroponic system as claimed in claim 1 facilitates automatic unattended circulation vis-a-vis dispensing and/ or recycling of desired volume of culture media to and fro the hydroponic culture chamber and medium reservoir through precise scheduling of medium flow rate, flow direction, flow duration, dispensing interval and culture bathing duration through customized computer-based operation routines.
4. The automated hydroponic system as herein described substantially increased the multiplication of potato microplants and subsequent microtuber production in vitro within a limited time and space, thus avoiding lengthy, labour-intensive and time-consuming conventional culture schedules of potato microtuber production consisting of serial culturing of axillary shoot cuttings/ single node cuttings on semisolid and liquid media for multiplication in vitro followed by microtuber induction on liquid media in 250 mi-capacity Erlenmeyer flasks/ Magenta boxes/ Melli jars.
5. This cylindrical hydroponic culture chamber with stainless steel cover lid as claimed in claim 2 can be moulded/ fabricated using transparent polycarbonate material and stainless steel of specified dimensions as described in Figures 1 and 2 therein, and the design offers scope and flexibility for alterations to meet any required number of ports or other extensions on the cover lid in accordance with changing requirements for in vitro hydroponic culture in other crop species.
|Indian Patent Application Number||824/DEL/2001|
|PG Journal Number||30/2008|
|Date of Filing||02-Aug-2001|
|Name of Patentee||COUNCIL OF AGRICULTURAL RESEARCH|
|PCT International Classification Number||A01G 31/00|
|PCT International Application Number||N/A|
|PCT International Filing date|