Micropropagation is a method to produce large number of propagula from a mother plant under in vitro conditions. However, the method is still costly due to intensive hand manipulation of various culture phases and is not used commercially for all plant species. (Ziv, 1995). In addition, some plants have initial stage of establishment and response that is slow and survival of the plants in final stage ex vivo is often poor, which further reduces the micropropagation production potential (Ziv, 1995; Aitken-Christie et al., 1995). Efficient commercial micropropagation depends on rapid and extensive proliferation by using large-scale cultures in multiplication phases. (Preece and Stutter, 1991; Ziv, 1995). Many designs are considered, trials with different nutrient media and growth regulators as well as mechanization and automation are introduced. The challenges revolve around new technologies, scale-up and automation. Not only that, there is also problem with contamination. Thus far, further understanding and knowledge correlates to improving technologies. Therefore, in adopting new biotechnology innovations, we proposed agitated, thin-films liquid system for efficient micropropagation adapted from Jeffrey Adelberg of Clemson University, USA. (Figure 1) This advanced micropropagation was demonstrated to show greater increase in multiplication rate, cost-effective facility and space utility, nutrient availability, contamination control as well as worker efficiency compared to conventional constrained agar-based system. This new system conform to human environment which to be as simple, economic and robust (Adelberg, 2006). The ergonomic and biological benefits hope to reveal the significance to implement this new innovative system.
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Figure 1: Jeffery Adelberg with his large, rectangular custom designed vessel for agitated, thin-film liquid system micropropagation
2.0 Agitated thin-film liquid system
This system is a hybrid micropropagation method that is adapted from semi-solid gel, bioreactor technology and temporary immersion system. It uses large, rigid rectangular vessels in a slow pitching motion to intermittently wet and aerate plantlets (Adelberg and Simpson, 2004). It has two-dimensional growth surface area optimization for nutrient exchanges and adequate aerial environment as well as three-dimensional volumetric optimization of vessel. Larger vessels are used to increase productivity and liquid system to improve distribution of solutes in medium. The rocker system produced 1-rpm pitch motion every 15 minutes. (Figure 2) The immersion time contributes to efficiency of the system and eliminate hyperhydricity problem. Another advantage is that separation of nutrient during temporary immersion enables rapid assay variations in nutrient and hormonal changes over a large number of vessels. Conventional propagation in small flask vessels is time consuming to do any manipulation. Additionally, the plantlets are aerated through oxygenation using wave machines Eibl and Eibl. It is similar to temporary immersion system in term of accumulation of residual solutes from media to produce high shoot quality. Besides that, the culture room is different than conventional micropropagation in correlation to the designed vessel.
Figure 2: Agitated, thin-films are created by slowly pitching large rectangular vessels. (The angle is 5-30?)Reproduced from Adelberg,J. (2004) with permission from Society for In Vitro Biology.
Agar or other organic gelling agents face problems such as mineral impurities, limited hydraulic conductance, and limited availability of solutes to the tissue and binding of toxic exudates near the tissue interface (Smith et al., 1995; Williams R.R., 1995; Leifert C., 1995). Indeed, agar media moves primarily by diffusion and may proceed against concentration gradient at latter stages. The high humidity in sealed vessels limits transpiration and thus restricts mass flow of dissolved solutes through spaces xylem and intercellular space.
Fortunately, liquid system has been reported to show better growth. The system has lack of impurities from agar, better water availability, better nutrient availability and larger vessels (Smith and Spomer, 1995; Berthouly and Etienne, 2002).
Hence, selecting optimal plant density is important in terms of productivity and quality. For high-density agar gelled cultures, highest plant densities had lowest multiplication rates and lowest rate of nutrient uptake per plant. This resulted from the diffusion one-dimensional concentration gradient in sugar concentration with time. Ficks law equation coefficient, boundary layer resistance at interface surface of plant and medium as well as resistance corresponding to biochemical sink and transport causes this slow diffusion.
In fact, the boundary resistance at the plant/medium interface was approximately 6000 times greater in agar than liquid media per unit surface area. That is, plantlets on agar gel have smaller surface area for exchange compared to plantlet wet with nutrient across its entire surface. On the contrary, turbulent media does not develop gradient and thus less resistance in solute transfer.
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In the proposed system, greater plant densities is used prior to larger vessel and showed increase macro-nutrient uptake and thus leading to an increase in yield new plants per vessel. This also leads to increase in dry weight gain. The advantage is when sugar is depleted at high densities, plant continue to grow on more water relative to solutes. Therefore, increase in sugar concentration allows higher density cultures to maintain high relative dry matter content.
With Hosta plantlets in liquid system, initial levels of sucrose from 1-7% w/v relates to endogenous sucrose level, glucose and fructose. The increase in sucrose, increases shoot and root dry weights, lowered mortality and less leaf chlorosis, following rooting, cold-translocation, storage and re-growth in greenhouse (Gollagunta et al., 2005). Modelling this cycle can be developed to maximize value of young plants for shipping and commercialization. Futhermore, Hosta multiplied faster and develops into larger plants than on agar. Greater dry weights of Alocasia, Colocasia and Hosta in liquid are due to greater availability of sugar compared to agar (Adelberg, 2004).
2.2 Design Consideration
The main design aim for this system is increase surface area for plant growth within the vessel and larger contact surface of plants and media allowing greater sugar availability.
Compare to "baby-food" jars used for agar micropropagation, liquid system design rectangular vessel for thin-film culture. 180ml cylindrical baby food jar has 18cm2 bottom surface and eight jars (4x2 arrangement) for one "footprint" on culture room shelf takes up 144cm2 that is less than half of the 297cm2 of rectangular vessel. Large rectangular vessels create less void space between vessels. The liquid allowed plants to multiply more vigorously and grow more densely in the same volumes of medium compared to agar. This also allows better space utilization on the shelves.
Physically, the vessel has a base with one longer dimension allowing a slight pitch with angle of 5-30? (Figure 2 and 4) to create a wave capable of immersion of entire plantlet. It enables liquid nutrient renewal and still uses minimal liquid to be cost effective. The rectangular vessels facilitated a slow moving wave of media to migrate the length of the vessel with motion of the shelf. (Figure 3)
Figure 3: Rectangular vessels arrange on horizontal platform moving together with the shelf.
It is rectangular because length to width any less or greater than 3 are often considered awkward for handling. Not only that, the 10cm base creates large growth surface and taper to a 6cm upper surface made for easy grip. For this, the vessels were large enough for approximately 75-150 plants to be harvested per cycle for labor efficiency. The height is common for the trade. The side-mounted closure allows greater surface accessible to forceps with advantages in aseptic hood process. There is also a route for steam penetration. Stacking of vessels during storage is facilitated by internally nested, tapered vessels or collapsed flexible film bags (Adelberg, 2004). (Figure 9)
Figure 4: Side elevation view of a pivoting platform showing additional details of the pivoting motion brought about by linkage of the platform to a variable speed motor.
In addition, the opening is large to allow cut buds and larger plantlets in and out of the vessel. Circular closures using threaded screw-caps apply uniform pressure on seal. Thread patterns trap condensed water and potentially provide refuge for contaminants that can be drawn out. However, the seal should not have broad horizontal surface that allow condensation to collect. Other than that, membrane filters laminated to openings can be constructed to ventilate and exclude microbe but adhesive ventilation patches (Figure 5) are more cost effective for larger surfaces for growing more plants in larger vessels. Repeated aseptic sampling of liquid media is possible using silicone rubber septa and syringe needles.
Figure 5: Liquid Lab Vessel? for agitated, thin-film micropropagation with adhesive ventilation patches
Nonetheless, the vessel is inert, inexpensive and easy to handle. It can withstand 121?C at 1.2 kg cm2 pressure generated from convenient stream sterilization. There are few parts therefore less part to clean, re-use and assembly. The critical parts are all accessible. Rigid multiple-use and flexible single-use components allow innovation vessel construction using glass and polycarbonate materials.
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About the facility, the white fluorescent light used provides photosynthetic energy and signals for plant growth. Long tubes enable even distribution of light flux density on the culture shelf (Fujiwura K., 1995). They are mounted at underside of the shelf. The vessels are translucent and provide unobstructed view for visual inspection. Moreover, the shelves are reduced to open support frames. Reflectors and canisters are removed so light are transmitted radially. Despite that, the open frames do not trap heat, even when packed with vessels. The rocking motion also removes any "hot pockets".
3.0 Efficiency in process
3.1 Multiplication rate
BRIX is a unit of representative of the sugar content of an aqueous solution. 1 degree of BRIX correlates to 1g of sucrose in 100g of solution and thus represents strength of solution as percentage by weight (%w/w). The refractometer measure BRIX is used as it is rapid, inexpensive and provides real-time feedback. It does not use any reagent and the real-time feedback is readily adaptable to practical situations. The reading includes sucrose and its monomers, fructose and glucose.
To the heterotrophic plant culture, fresh weight gain is primarily due to uptake of water and the dry weight gain is due mainly to uptake of sugar and inorganic ions. Plants grown in this liquid system is in high density but had lower residual sugar concentrations compared to agar medium. In the case of Hosta, a liquid medium with 50g l-1 sucrose was concentrated enough so that sugar depletion did not limit growth even at the highest densities. The relative dry matter of plants (dry weight/ fresh weight) was inversely correlated to concentration of sugar in residual media. (Figure 6)
Figure 6: Correlation between residual sugar in media and relative dry weight of Venus flytrap, Drosera muscipula. (Vessels initiated for agar and liquid medium with 3 and 5% w/v sucrose over a range of explants densities. Each data point represents tissue sampled from one vessel)
Liquid medium with high sugar concentration (5-12% w/v) results in higher dry weight and stored carbohydrate in better quality planting stock. For instance, plants with specialized storage organs and modified shoot systems. Examples are such as lily (Lian et al., 2002), garlic (Kim et al., 2006), potato (Ziv and Shemesh, 1996) , turmeric (Salvi et al.,2002) and taro (Zhou et al.,1999). In contrast, those with leafy shoots has superior shoot quality (Etienne and Berthouly, 2002) in liquid system too because adapted temporary immersion system with correctly timed cycles to avoid hyperhydricity, limit shear force, provide adequate oxygen and sufficient mix of medium (Figure 7). The shoots were larger with greater leaf surface area with more dry weight in spite increase in approximately 10- fold in sugar and nitrate assimilation on fresh weight basis (Escalona et al., 2002). Also, sugar use in Alocasia, Colocasia, Hosta and Hemerocallis is better correlated to biomass than multiplication rate.
Figure 7: Hosta plantlets of agitated, thin-film liquid system produce larger leafty and more plantlets compared to agar-cultured.
On agar, plant density did not affect multiplication rate. As a matter of fact, the multiplication rates with density of 200 plants have an average of 1.6 for agar and 2.3 for liquid media. In annual crop cycle and six-week subcultures, liquid system with 40 per litre would produce 14, 858 plants while agar would only produce 256 plants. Evidently, there is a speedy increase and efficient use of liquid system. The yield is higher in liquid than on agar, per volume media. (Table 1)
Initial density (plants/L)
Table 1: Multiplication rate and number of new plants per sample square meter of bench space per week generated in agar containing baby food jars and large rectangular vessels in agitated, thin-film liquid system at varied initial plant densities for Hosta spp. (data pooled for three varieties over two, 6-week culture cycles on 100M benzyladenine and calculated from Adelberg, 2004)
Furthermore, Hosta plants grew more quickly in liquid system than agar in the greenhouse and nursery (Adelberg et al., 2000). This is because rocker system avoids constant saturation of non-pivoting platform hence produce stronger, uniform, physically robust plants which can be hardened and acclimatized outside vessel.
2.2 Hormone and chemical
This liquid system adapted uses of various concentrations of hormones and chemicals especially in stage II of culture in shoot bud division to retard leaf organ proliferation while optimized the sugar uptake. With Hosta plantlets in shake-glasks, initial levels of sucrose (1-7% w/v) were directly related to endogenous levels of sucrose, glucose and fructose following 5-weeks of culture (Gollagunta et. al., 2004). At 5% media sucrose, shoot bud multiplication is optimized. The increase in sucrose level, linearly increase the dry weight as in medium containing benzyladenine. Alternatively, 5% sucrose level stays even with dry weight gain in agar medium.
Benzyladenine is effective in inducing crown divisions both in vitro and in field production (Hartmann et al., 1997; Garner et al.,1998). Likewise, high concentration of benzyladenine causes ammonium depletion followed by growth cessation. As a consequence, there is an increase in uptake of nitrate, calcium and potassium related to greater fresh weight. Plant cells have roughly 50% conversion efficiency of organic carbon feed to final cell dry weight (W. Curtis, 1999). That is to say the increase uptake of nutrients will contribute to dry weight gain.
Evidently, Alocasia and Colocasia from agitated, thin-film liquid system had 2.5 times greater dry weight per plant than from agar (Table 2). Benzyladenine concentration raised and added ancymidol in equimolar reduce plant size and problems. This resulted in 45% reduction in dry weight per plant but greater mean dry weights in liquid than agar at all densities.
Table 2: Mean dry weight per plant of Alocasia macrorrhiza after 4 weeks of culture in agar and agitated, thin-film liquid system at different initial plant densities. Equivalent ratios of explants per volume media was used for both agar and liquid media. (Mean dry weight per plant was calculated as the product of biomass per plant and relative dry weight per vessel from data of Adelberg and Toler, 2004).
Larger plants are also expected from this liquid system and made aseptic transfer more difficult. Thus far, smaller plants are used to improve efficiency. Shorter, thicker microcuttings with reduced leaf area are more resistant to water loss during acclimatization (Ziv, 1994). Growth retardants such as ancymidol or paclobutrazol are used to inhibit gibberellins synthesis. Shoot size is significantly reduced in cucumber (Ziv, 1992), philodendron (Ziv and Ariel, 1991) and poplar (Vincour et al.,2000).
In stage II of shoot bud division, old leaves and roots are removed before replanting. This causes nitrogen depletion hence excessive root elongation in particular to birch (Adelberg et al., 1997) and orchid plantlets (A.J.S. Mcdonald, 1994). Root overgrowth is tedious and increase cutting time in harvest. For this, ancymidol is used to reduce leaf size. Ancymidol (0.3200M) in liquid culture of Hemerocallis "Todd Monroe" decreased plant size approximately 50%, increasing the numbers or plants per vessel from 60-120 (Maki et al., 2005).
Not only that, Acnymidol in liquid media also increased sugar uptake and endogenous carbohydrate concentrations in shoot bud clusters. Evidently, ancymidol increase both starch and soluble sugar concentrations in leaves of liquid cultured Narcissus (Chen and Ziv, 2001, 2003). The bud clusters are small and dense which are easier to transfer manually (Adelberg and Toler, 2004) and also amenable to mechanical cutting (Ziv et al., 1994). Besides that, ancymidol and paclobutrazol also improve dessication resistance in part of in vitro hardening process in acclimization (Ziv, 1995).
3.3 Technician Labor and other cost
Labor is the largest cost component in micropropagation (Chu, 1995). Liquid system allows innovative methods in workstation mechanization especially in transfer and cutting of plants. As explained in Section 3.2, bud clusters are easier to transfer or cut. This predict saving of cost up to 50% (Gross and Levin, 1999). The downstream processing involving manual, individual cutting and replanting are inevitably required in micropropagation and estimated to be 60% of labor cost (Chu, 1995).
In conventional agar media, the cultured plant required 7% of the time to remove plants, 48% to cut and 45% to replant into a new vessel (Alper et al., 1994). Replanting gelled media is repetitive and needs careful orientation and spacing for individual plantlet. However, in liquid system, bulk transfer allows passive spacing and orientation due to the larger vessel. Thus, technician time is reduced. The "sorting and placing" buds in fresh media are replaced with "cut and dump".
The reduced time in transfer, thus allows sole focus on cutting process. From section 3.2 also, the reduced size of Hemerocallis "Todd Monroe" shows increase number of plants cut per hour from 110 to 230 (Maki et al., 2005). The cutting process proceeds at a constant pace and was not interrupted frequently by off-task functions unlike transfer process.
Other factors that affect transfer rate are such as individual technician, plant variety, media formulation, time of day, day of week and number of plants in and harvested per vessel (Alderberg, 2002). The number of plants harvested has the biggest impact on technician efficiency. The efficiency at transfer station increased as number of plants harvested reach to about 100 per vessel. The optimal range is where a minimum of 40 buds were dumped into 200ml of liquid media and the yield was 77-103 per vessel. This variance has 95% confidence. (Figure 8) The constant off-task time does not impact the overall efficiency of transfer, such as assembling tools, opening and sealing the vessel and data recording.
Figure 8: Labor input for 22 technicians propagating 40 varities of Hosta from more than 6 months of observation.
In agar media system, there is cost in preparing the media concerning the labor and the material as well as dish washing and manual removing of sugar-containing agar from plants for planting out. Meanwhile, in liquid system, Southern Sun Bio Systems (Hodges, SC, USA) devised a less costly vessel with fewer custom parts and still maintain asepsis during multiple uses. The vessel includes a microporous patch for gas exchange and septa port for addition and testing of fluids. (Figure 9)
Figure 9: Liquid Lab Vessel? created by Southern Sun Bio
As regard in section 2.2, the fewer custom parts avoid unnecessary cost. Fewer custom parts contribute to fewer moldings of parts. The rigid vessels need mould and will cost more than the materials until thousand units have been cast. Thermoforming techniques such as injection mould, blow mould and vacuum mould impact cost and limit choices of size, shape and precision of critical surfaces. Custom fabrication and unnecessary modification is avoided. For example, Nalgene Biosafe modifications are expensive consisting of 11 parts and required modifications for ventilation and media sampling. It deforms during steam sterilization and therefore need to be sterilized using gamma irradiation or ethylene oxide of biomedical trade that is too expensive for micropropagation. It is also too large to be easily maneuvered in hood station. Certainly, it is more important in deciding the desired qualities of culture vessel, leading to construction of agitated, thin-film culture vessel.
Further reduction in costs is the activation of rocker motors that is less compared to rotary shaker platforms. The rocker system only uses small electric motor and low speeds to bring a high torque output. It is designed to induce movement of about 1-4 inches in a vertical motion as to create a wave.
One of the major problems faced in micropropagation is contamination. Bacteria and fungi generally grow faster in agitated liquid than agar medium. However, liquid culture allows a proactive approach in greater lead time for appropriate action.
Agitated, thin-film vessel is a custom made vessel, hence does not require improvisation in parts. Improvised parts are unreliable and cause unaware potential of contamination. The well-designed vessel (specifications in section 2.2) fit most common autoclaves and stacking is possible. Ill-conceived autoclave packing and ad hoc cooling procedures are avoided.
The problem concerning contamination is likely about the protocol. Owing to the available protocols, agitated, thin-film liquid system uses process developed from Liquid Lab Vessel?. Vessel is placed in hood so laminar is parallel to the long linear dimension. Forceps can easily access the opening. The operator's hand is also shielded from the growth surface because the vessels has a slanted, fifth side.
Large or entangled plants in vessel that poses problem can be remedied by shorter culture with use of retardants such as ancymidol. The transfer of cut buds can be minimized to one motion and only the sterile jar only need to cross over the entry port. The opening is as big as a Petri dish which gives familiarity. Thus, the time for the vessel to remain open is minimized.
The rocker system is adapted to rapid screening for contamination. The periodic mixing enables sample to be withdrawn using a syringe. The conventional agar system requires swabbing is time consuming and do not provide thorough assessment for culture conditions.
Effective commercial propagation refers to quantitative propagation where there is efficient use of facility, least cost and still produces the highest yield. However, scale-up factor requires more active monitoring. Automation is also achievable by minimizing human intervention. It is up to the technicians to mechanize the process involved in micropropagation. In agitated, thin-film micropropagation, the minimum solutes are temporarily immersed and the vessel design is similar to bioreactor. When the liquid-cultured plant is subjected to high density, the multiplication rate increases thus, producing higher yield. Hormones and chemicals affect growth and proliferation of the plants such as benzyladenine and ancymidol. Other than that, this system reduces technician labor, time, material cost and container cost to be productive. Contamination is easily countered by early proactive control. In validating the system, reasonably sized factorial experiments may determine optimization of genotype , PGR or nutrient-use scenarios (Adelberg, 2006). The system offered many improvements in plant growth and system efficiencies compared to conventional methods. The benefits expressed in this proposed system help to justify significance to implement this new system.