Micropropagation is a method to bring forth big figure of propagula from a female parent works under in vitro conditions. However, the method is still dearly-won due to intensive manus use of assorted civilization stages and is non used commercially for all works species. ( Ziv, 1995 ) . In add-on, some workss have initial phase of constitution and response that is slow and endurance of the workss in concluding phase ex vivo is frequently hapless, which farther reduces the micropropagation production potency ( Ziv, 1995 ; Aitken-Christie et al. , 1995 ) . Efficient commercial micropropagation depends on rapid and extended proliferation by utilizing large-scale civilizations in generation stages. ( Preece and Stutter, 1991 ; Ziv, 1995 ) . Many designs are considered, tests with different alimentary media and growing regulators every bit good as mechanisation and mechanization are introduced. The challenges revolve around new engineerings, scale-up and mechanization. Not merely that, there is besides job with taint. Therefore far, farther apprehension and cognition correlatives to bettering engineerings. Therefore, in following new biotechnology inventions, 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 demo greater addition in generation rate, cost-efficient installation and infinite public-service corporation, alimentary handiness, taint control every bit good as worker efficiency compared to conventional constrained agar-based system. This new system conform to human environment which to be every bit simple, economic and robust ( Adelberg, 2006 ) . The ergonomic and biological benefits hope to uncover the significance to implement this new advanced system.
Figure 1: Jeffery Adelberg with his big, rectangular usage designed vas for agitated, thin-film liquid system micropropagation
2.0 Agitated thin-film liquid system
This system is a intercrossed micropropagation method that is adapted from semi-solid gel, bioreactor engineering and impermanent submergence system. It uses big, stiff rectangular vass in a slow pitching gesture to intermittently wet and air out plantlets ( Adelberg and Simpson, 2004 ) . It has planar growing surface country optimisation for alimentary exchanges and equal aerial environment every bit good as 3-dimensional volumetric optimisation of vas. Larger vass are used to increase productiveness and liquid system to better distribution of solutes in medium. The rocker system produced 1-rpm pitch gesture every 15 proceedingss. ( Figure 2 ) The submergence clip contributes to efficiency of the system and extinguish hyperhydricity job. Another advantage is that separation of food during impermanent submergence enables rapid assay fluctuations in food and hormonal alterations over a big figure of vass. Conventional extension in little flask vass is clip devouring to make any use. Additionally, the plantlets are aerated through oxygenation utilizing wave machines Eibl and Eibl. It is similar to impermanent submergence system in term of accretion of residuary solutes from media to bring forth high shoot quality. Besides that, the civilization room is different than conventional micropropagation in correlativity to the designed vas.
Figure 2: Agitated, thin-films are created by easy fliping big rectangular vass. ( 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 jobs such as mineral drosss, limited hydraulic conductance, and limited handiness of solutes to the tissue and binding of toxic exudations near the tissue interface ( Smith et al. , 1995 ; Williams R.R. , 1995 ; Leifert C. , 1995 ) . Indeed, agar media moves chiefly by diffusion and may continue against concentration gradient at latter phases. The high humidness in certain vass bounds transpiration and therefore restricts mass flow of dissolved solutes through infinites xylem and intercellular infinite.
Fortunately, liquid system has been reported to demo better growing. The system has deficiency of drosss from agar, better H2O handiness, better alimentary handiness and larger vass ( Smith and Spomer, 1995 ; Berthouly and Etienne, 2002 ) .
Hence, choosing optimum works denseness is of import in footings of productiveness and quality. For high-density agar gelled civilizations, highest works densenesss had lowest generation rates and lowest rate of alimentary uptake per works. This resulted from the diffusion unidimensional concentration gradient in sugar concentration with clip. Ficks jurisprudence equation coefficient, boundary bed opposition at interface surface of works and medium every bit good as opposition matching to biochemical sink and conveyance causes this slow diffusion.
In fact, the boundary opposition at the plant/medium interface was about 6000 times greater in agar than liquid media per unit surface country. That is, plantlets on agar gel have smaller surface country for exchange compared to plantlet moisture with alimentary across its full surface. On the contrary, disruptive media does non develop gradient and therefore less opposition in solute transportation.
In the proposed system, greater works densenesss is used prior to larger vas and showed increase macro-nutrient uptake and therefore taking to an addition in yield new workss per vas. This besides leads to increase in dry weight addition. The advantage is when sugar is depleted at high densenesss, works continue to turn on more H2O relation to solutes. Therefore, addition in sugar concentration allows higher denseness civilizations to keep high comparative dry affair content.
With Hosta plantlets in liquid system, initial degrees of saccharose from 1-7 % w/v relates to endogenous sucrose degree, glucose and fructose. The addition in sucrose, additions shoot and root dry weights, lowered mortality and less leaf greensickness, following rooting, cold-translocation, storage and re-growth in nursery ( Gollagunta et al. , 2005 ) . Modeling this rhythm can be developed to maximise value of immature workss for transporting and commercialisation. Futhermore, Hosta multiplied faster and develops into larger workss than on agar. Greater dry weights of Alocasia, Colocasia and Hosta in liquid are due to greater handiness of sugar compared to agar ( Adelberg, 2004 ) .
2.2 Design Consideration
The chief design purpose for this system is increase surface country for works growing within the vas and larger contact surface of workss and media leting greater sugar handiness.
Compare to “ baby-food ” jars used for agar micropropagation, liquid system design rectangular vas for thin-film civilization. 180ml cylindrical babe nutrient jar has 18cm2 underside surface and eight jars ( 4×2 agreement ) for one “ footmark ” on civilization room shelf takes up 144cm2 that is less than half of the 297cm2 of rectangular vas. Large rectangular vass create less null infinite between vass. The liquid allowed workss to multiply more smartly and turn more dumbly in the same volumes of medium compared to agar. This besides allows better infinite use on the shelves.
Physically, the vas has a base with one longer dimension leting a little pitch with angle of 5-30? ( Figure 2 and 4 ) to make a moving ridge capable of submergence of full plantlet. It enables liquid food reclamation and still uses minimum liquid to be cost effectual. The rectangular vass facilitated a slow traveling moving ridge of media to migrate the length of the vas with gesture of the shelf. ( Figure 3 )
Figure 3: Rectangular vass arrange on horizontal platform traveling together with the shelf.
It is rectangular because length to width any less or greater than 3 are frequently considered awkward for managing. Not merely that, the 10cm base creates big growing surface and taper to a 6cm upper surface made for easy clasp. For this, the vass were big plenty for about 75-150 workss to be harvested per rhythm for labour efficiency. The tallness is common for the trade. The side-mounted closing allows greater surface accessible to forceps with advantages in sterile goon procedure. There is besides a path for steam incursion. Stacking of vass during storage is facilitated by internally nested, tapering vass or collapsed flexible movie bags ( Adelberg, 2004 ) . ( Figure 9 )
Figure 4: Side lift position of a swiveling platform demoing extra inside informations of the pivoting gesture brought approximately by linkage of the platform to a variable velocity motor.
In add-on, the gap is big to let cut buds and larger plantlets in and out of the vas. Round closings utilizing threaded screw-caps apply unvarying force per unit area on seal. Thread patterns trap condensed H2O and potentially supply safety for contaminations that can be drawn out. However, the seal should non hold wide horizontal surface that allow condensation to roll up. Other than that, membrane filters laminated to gaps can be constructed to air out and except bug but adhesive airing spots ( Figure 5 ) are more cost effectual for larger surfaces for turning more workss in larger vass. Repeated sterile sampling of liquid media is possible utilizing silicone gum elastic septa and syringe acerate leafs.
Figure 5: Liquid Lab Vessel? for agitated, thin-film micropropagation with adhesive airing spots
However, the vas is inert, cheap and easy to manage. It can defy 121? C at 1.2 kilograms cm2 force per unit area generated from convenient watercourse sterilisation. There are few parts hence less portion to clean, re-use and assembly. The critical parts are all accessible. Rigid multiple-use and flexible single-use constituents allow invention vas building utilizing glass and polycarbonate stuffs.
About the installation, the white fluorescent visible radiation used provides photosynthetic energy and signals for works growing. Long tubes enable even distribution of light flux denseness on the civilization shelf ( Fujiwura K. , 1995 ) . They are mounted at bottom of the shelf. The vass are semitransparent and supply unobstructed position for ocular review. Furthermore, the shelves are reduced to open support frames. Reflectors and case shots are removed so light are transmitted radially. Despite that, the unfastened frames do non pin down heat, even when packed with vass. The swaying gesture besides removes any “ hot pockets ” .
3.0 Efficiency in procedure
3.1 Generation rate
BRIX is a unit of representative of the sugar content of an aqueous solution. 1 grade of BRIX correlatives to 1g of saccharose in 100g of solution and therefore represents strength of solution as per centum by weight ( % w/w ) . The refractometer step BRIX is used as it is rapid, cheap and provides real-time feedback. It does non utilize any reagent and the real-time feedback is readily adaptable to practical state of affairss. The reading includes sucrose and its monomers, fructose and glucose.
To the heterotrophic works civilization, fresh weight addition is chiefly due to uptake of H2O and the dry weight addition is due chiefly to uptake of sugar and inorganic ions. Plants grown in this liquid system is in high denseness but had lower residuary sugar concentrations compared to agar medium. In the instance of Hosta, a liquid medium with 50g l-1 saccharose was concentrated plenty so that sugar depletion did non restrict growing even at the highest densenesss. The comparative dry affair of workss ( dry weight/ fresh weight ) was reciprocally correlated to concentration of sugar in residuary media. ( Figure 6 )
Figure 6: Correlation between residuary sugar in media and comparative dry weight of Venus flytrap, Drosera muscipula. ( Vessels initiated for agar and liquid medium with 3 and 5 % w/v saccharose over a scope of explants densenesss. Each information point represents tissue sampled from one vas )
Liquid medium with high sugar concentration ( 5-12 % w/v ) consequences in higher dry weight and stored saccharide in better quality seting stock. For case, workss with specialised storage variety meats and modified shoot systems. Examples are such as lily ( Lian et al. , 2002 ) , garlic ( Kim et al. , 2006 ) , murphy ( Ziv and Shemesh, 1996 ) , turmeric ( Salvi et al.,2002 ) and cocoyam ( Zhou et al.,1999 ) . In contrast, those with leafy shoots has superior shoot quality ( Etienne and Berthouly, 2002 ) in liquid system excessively because altered impermanent submergence system with right timed rhythms to avoid hyperhydricity, limit shear force, supply equal O and sufficient mix of medium ( Figure 7 ) . The shoots were larger with greater foliage surface country with more dry weight in spite addition in about 10- crease in sugar and nitrate assimilation on fresh weight footing ( Escalona et al. , 2002 ) . Besides, sugar usage in Alocasia, Colocasia, Hosta and Hemerocallis is better correlated to biomass than generation rate.
Figure 7: Hosta plantlets of agitated, thin-film liquid system produce larger leafty and more plantlets compared to agar-cultured.
On agar, works denseness did non affect generation rate. As a affair of fact, the generation rates with denseness of 200 workss have an norm of 1.6 for agar and 2.3 for liquid media. In one-year harvest rhythm and six-week subcultures, liquid system with 40 per liter would bring forth 14, 858 workss while agar would merely bring forth 256 workss. Obviously, there is a rapid addition and efficient usage of liquid system. The output is higher in liquid than on agar, per volume media. ( Table 1 )
Initial denseness ( plants/L )
Table 1: Generation rate and figure of new workss per sample square metre of bench infinite per hebdomad generated in agar incorporating babe nutrient jars and big rectangular vass in agitated, thin-film liquid system at varied initial works densenesss for Hosta spp. ( informations pooled for three assortments over two, 6-week civilization rhythms on 100M benzyladenine and calculated from Adelberg, 2004 )
Furthermore, Hosta workss grew more rapidly in liquid system than agar in the nursery and baby’s room ( Adelberg et al. , 2000 ) . This is because rocker system avoids changeless impregnation of non-pivoting platform hence green goods stronger, unvarying, physically robust workss which can be hardened and acclimatized outside vas.
2.2 Hormone and chemical
This liquid system adapted utilizations of assorted concentrations of endocrines and chemicals particularly in phase II of civilization in shoot bud division to retard leaf organ proliferation while optimized the sugar consumption. With Hosta plantlets in shake-glasks, initial degrees of saccharose ( 1-7 % w/v ) were straight related to endogenous degrees of saccharose, glucose and fructose following 5-weeks of civilization ( Gollagunta et. al. , 2004 ) . At 5 % media saccharose, shoot bud generation is optimized. The addition in sucrose degree, linearly addition the dry weight as in medium incorporating benzyladenine. Alternatively, 5 % sucrose degree corsets even with dry weight addition in agar medium.
Benzyladenine is effectual in bring oning 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 growing surcease. As a effect, there is an addition in consumption of nitrate, Ca and K related to greater fresh weight. Plant cells have approximately 50 % transition efficiency of organic C provender to concluding cell dry weight ( W. Curtis, 1999 ) . That is to state the addition consumption of foods will lend to dry weight addition.
Obviously, Alocasia and Colocasia from agitated, thin-film liquid system had 2.5 times greater dry weight per works than from agar ( Table 2 ) . Benzyladenine concentration raised and added ancymidol in equimolar cut down works size and jobs. This resulted in 45 % decrease in dry weight per works but greater average dry weights in liquid than agar at all densenesss.
Table 2: Average dry weight per works of Alocasia macrorrhiza after 4 hebdomads of civilization in agar and agitated, thin-film liquid system at different initial works densenesss. Equivalent ratios of explants per volume media was used for both agar and liquid media. ( Average dry weight per works was calculated as the merchandise of biomass per works and comparative dry weight per vas from informations of Adelberg and Toler, 2004 ) .
Larger workss are besides expected from this liquid system and made sterile transportation more hard. Therefore far, smaller workss are used to better efficiency. Shorter, thicker microcuttings with decreased leaf country are more immune to H2O loss during acclimatisation ( Ziv, 1994 ) . Growth retardents such as ancymidol or paclobutrazol are used to suppress gibberellins synthesis. Shoot size is significantly reduced in Cucumis sativus ( Ziv, 1992 ) , philodendron ( Ziv and Ariel, 1991 ) and poplar ( Vincour et al.,2000 ) .
In phase II of shoot bud division, old foliages and roots are removed before replanting. This causes nitrogen depletion hence inordinate root elongation in peculiar to birch ( Adelberg et al. , 1997 ) and orchid plantlets ( A.J.S. Mcdonald, 1994 ) . Root giantism is boring and increase cutting clip in crop. For this, ancymidol is used to cut down leaf size. Ancymidol ( 0.3200M ) in liquid civilization of Hemerocallis “ Todd Monroe ” decreased works size about 50 % , increasing the Numberss or workss per vas from 60-120 ( Maki et al. , 2005 ) .
Not merely that, Acnymidol in liquid media besides increased sugar consumption and endogenous saccharide concentrations in shoot bud bunchs. Obviously, ancymidol addition both amylum and soluble sugar concentrations in foliages of liquid cultured Narcissus ( Chen and Ziv, 2001, 2003 ) . The bud bunchs are little and dense which are easier to reassign manually ( Adelberg and Toler, 2004 ) and besides conformable to mechanical film editing ( Ziv et al. , 1994 ) . Besides that, ancymidol and paclobutrazol besides better dessication opposition in portion of in vitro indurating procedure in acclimization ( Ziv, 1995 ) .
3.3 Technician Labor and other cost
Labor is the largest cost constituent in micropropagation ( Chu, 1995 ) . Liquid system allows advanced methods in workstation mechanisation particularly in transportation and film editing of workss. As explained in Section 3.2, bud bunchs are easier to reassign or cut. This predict economy of cost up to 50 % ( Gross and Levin, 1999 ) . The downstream processing affecting manual, single film editing and replanting are necessarily required in micropropagation and estimated to be 60 % of labour cost ( Chu, 1995 ) .
In conventional agar media, the civilized works required 7 % of the clip to take workss, 48 % to cut and 45 % to replant into a new vas ( Alper et al. , 1994 ) . Replanting gelled media is insistent and needs careful orientation and spacing for single plantlet. However, in liquid system, majority transportation allows inactive spacing and orientation due to the larger vas. Therefore, technician clip is reduced. The “ sorting and placing ” buds in fresh media are replaced with “ cut and shit ” .
The decreased clip in transportation, therefore allows exclusive focal point on cutting procedure. From subdivision 3.2 besides, the decreased size of Hemerocallis “ Todd Monroe ” shows addition figure of workss cut per hr from 110 to 230 ( Maki et al. , 2005 ) . The cutting procedure returns at a changeless gait and was non interrupted often by off-task maps unlike transportation procedure.
Other factors that affect transportation rate are such as single technician, works assortment, media preparation, clip of twenty-four hours, twenty-four hours of hebdomad and figure of workss in and harvested per vas ( Alderberg, 2002 ) . The figure of workss harvested has the biggest impact on technician efficiency. The efficiency at transportation station increased as figure of workss harvested reach to about 100 per vas. The optimum scope is where a lower limit of 40 buds were dumped into 200ml of liquid media and the output was 77-103 per vas. This discrepancy has 95 % assurance. ( Figure 8 ) The changeless off-task clip does non impact the overall efficiency of transportation, such as piecing tools, opening and sealing the vas and informations 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 fixing the media refering the labour and the stuff every bit good as dish lavation and manual removing of sugar-containing agar from workss for seting out. Meanwhile, in liquid system, Southern Sun Bio Systems ( Hodges, SC, USA ) devised a less dearly-won vas with fewer usage parts and still keep antisepsis during multiple utilizations. The vas includes a microporous spot for gas exchange and septa port for add-on and testing of fluids. ( Figure 9 )
Figure 9: Liquid Lab Vessel? created by Southern Sun Bio
As respect in subdivision 2.2, the fewer usage parts avoid unneeded cost. Fewer usage parts contribute to fewer castings of parts. The stiff vass need molds and will be more than the stuffs until thousand units have been cast. Thermoforming techniques such as injection mold, blow mold and vacuity mould impact cost and bound picks of size, form and preciseness of critical surfaces. Custom fiction and unneeded alteration is avoided. For illustration, Nalgene Biosafe alterations are expensive consisting of 11 parts and needed alterations for airing and media trying. It deforms during steam sterilisation and hence necessitate to be sterilized utilizing gamma irradiation or ethylene oxide of biomedical trade that is excessively expensive for micropropagation. It is besides excessively big to be easy maneuvered in goon station. Surely, it is more of import in make up one’s minding the coveted qualities of civilization vas, taking to building of agitated, thin-film civilization vas.
Further decrease in costs is the activation of rocker motors that is less compared to rotary shaker platforms. The rocker system lone uses little electric motor and low velocities to convey a high torsion end product. It is designed to bring on motion of about 1-4 inches in a perpendicular gesture as to make a moving ridge.
One of the major jobs faced in micropropagation is taint. Bacteria and fungi by and large grow faster in agitated liquid than agar medium. However, liquid civilization allows a proactive attack in greater lead clip for appropriate action.
Agitated, thin-film vas is a usage made vas, hence does non necessitate improvisation in parts. Improvised parts are undependable and cause incognizant potency of taint. The well-designed vas ( specifications in subdivision 2.2 ) tantrum most common sterilizers and stacking is possible. Ill-conceived sterilizer wadding and ad hoc chilling processs are avoided.
The job refering taint is likely about the protocol. Owing to the available protocols, agitated, thin-film liquid system uses procedure developed from Liquid Lab Vessel? . Vessel is placed in goon so laminal is parallel to the long additive dimension. Forcepss can easy entree the gap. The operator ‘s manus is besides shielded from the growing surface because the vass has a slanted, 5th side.
Large or embroiled workss in vas that poses job can be remedied by shorter civilization with usage of retardents such as ancymidol. The transportation of cut buds can be minimized to one gesture and merely the sterile jar merely necessitate to traverse over the entry port. The gap is every bit large as a Petri dish which gives acquaintance. Therefore, the clip for the vas to stay unfastened is minimized.
The rocker system is adapted to rapid testing for taint. The periodic commixture enables sample to be withdrawn utilizing a syringe. The conventional agar system requires swobing is clip devouring and make non supply thorough appraisal for civilization conditions.
Effective commercial extension refers to quantitative extension where there is efficient usage of installation, least cost and still produces the highest output. However, scale-up factor requires more active monitoring. Automation is besides accomplishable by minimising human intercession. It is up to the technicians to mechanise the procedure involved in micropropagation. In agitated, thin-film micropropagation, the lower limit solutes are temporarily immersed and the vas design is similar to bioreactor. When the liquid-cultured works is subjected to high denseness, the generation rate additions therefore, bring forthing higher output. Hormones and chemicals affect growing and proliferation of the workss such as benzyladenine and ancymidol. Other than that, this system reduces technician labour, clip, stuff cost and container cost to be productive. Contamination is easy countered by early proactive control. In formalizing the system, moderately sized factorial experiments may find optimisation of genotype, PGR or nutrient-use scenarios ( Adelberg, 2006 ) . The system offered many betterments in works growing and system efficiencies compared to conventional methods. The benefits expressed in this proposed system aid to warrant significance to implement this new system.