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Plant Tissue Culture: Scale up | Problems & possibilities
#1
This is post no. 1 under the main topic.
Plant Cell/Tissue Culture-the scientific art of growing plant cells/tissues in-vitro, has for long been considered a bright hope towards providing a sustainable source for producing plant derived therapeutically/commercially important chemicals. It not only lowers the burden on natural flora (whose reckless commercial exploitation might endanger the target plant species), but also provides a route for continuous production of plant biomass in laboratory controlled environment, independent of seasonal/climatic/geographical limitations! Following is a brief account of possibilities associated with successful cultivation of plant cells at large scale:

Possibilities in Large Scale Plant Cell Culture:

a. Fast Rate of Production
(Unlike the whole plant, cells grow very fast (doubling time of 1-2 days) under optimized culture conditions of Bioreactors)

b. Continuous Production
(Unlike whole plants, which often seed (seeds are often known to have high contents of metabolites) seasonally and according to climatic conditions, cells in bioreactor can be propagated regularly, leading to continuous production of metabolites/biomass.

c. High & Consistent Content of Target Product
(Whole plant parts are not rich in the target metabolite concentration (which is often a secondary metabolite) . Plant cells can be genetically modified and propagated in the large scale for unusually high productivities. Also, the quality of product obtained from different plants of different agro-climatic conditions is not consistent, which is not the case with the homogenous cultures of plant cells in reactors.)

d. No Pressure on Land Use
(Growing the plants on land (which is already very limited) for obtaining commercial products creates a huge pressure and imbalance. This can be completely avoided by concentrated cultivation of the plant cells in confinement of Bioreactors)

Considering the advantages of the in-vitro production of plant biomass, a lot of research has underwent in the last few decades to attempt the commercial utilization of in-vitro plant cell cultivation. Though the literature is brimmed with the laboratory scale production of numerous plant species viz Azadirachta indica (Neem; for Biopesticides), Lithospermum erythrorhizon (for Shikonin), Taxus cuspidata (for Taxol, a Cancer drug), Catharanthus roseus (for Ajmalicine) etc, very few attempts at the industrial scale have been made in the past. Some of the significant industrial attempts for large scale plant cell cultivation include Nicotiana tabacum (Tobacco, in 1500L Bubble Column Reactor, during 1970s), Lithospermum erythrorhizon (in 750L Stirred Tank Reactor, in 1985), Taxus cuspidata (in 500L Bubble Column, in year 2000) and Panax notoginseng (in 30L Stirred Tank Reactor, in year 2005). Very few of these could continue for long, and most of the attempts faced some major limitation and hurdles in sustaining the large scale cultivation of plant cells in Bioreactors. Following is a brief account of the major hurdles in scaling up the Plant cell cultivation:

Problems in Large Scale Plant Cell Cultivation:


A. Shear Stress Augmentation
The large size of the plant cells (10-100 times larger than microbial cells) and huge vacuoles possessed by them makes them extremely sensitive to shear stress and osmotic pressures. Maintaining the non-shear environment in huge vessels containing large impellers/sparging apparatus is a big challenge. Shear stresses tend to damage the cells, reducing their viability and ultimately death!

B. Aggregation
Secretion of Extra-Cellular Polysaccharides (ECP) induces aggregation and clumping of the plant cells, greatly affecting the nutrient and oxygen transfer. Clumping is an inherent characteristic of plant cells, but it’s not favorable for homogenous culture requirements. In small scale, anti-clumping agents can be economically used, but in huge bioreactors, the requirement of anti-clumping agents also shoots up! Which is unhealthy for cells as well as economy of production. Also, aggregates tend to sediment which asks for the need of increased agitation rates, which could rather lead to increased shear stress!

C. Slow Growth Rate
The slow growth rates and low biomass yields of the plant cells is another issue worth consideration during large scale cultivation of plant cells. The batch times are very large, so maintenance and monitoring needs are big. And, being one of the slowest growing living systems, the risk of contamination by fast growing bacteria/fungus also remains a factor of concern during large scale cultivation.

In order to tackle the problems associated with scale-up of plant cell culture, different groups have tried designing various kinds of bioreactors which might offer less shear stresses, ensure good mixing and keep the aggregates well dispersed in the reactor. Following is a brief account of favorable bioreactors for Large Scale Cultivation of Plant Cells:

A. Stirred Tank Reactor With Low Shearing Impellers:
Use of novel design of impellers like Paddle and Centrifuge impellers has proved successful in some cases of Large Scale Plant Cell Cultivation. They create an axial flow regime in the reactor, leading to low shear forces on the cells. Following is a visualization of the impellers:
[Image: F8287_01_wl.jpg][Image: centrifugal_pump_impeller.jpg]
Paddle Impeller and Centrifuge Impeller

Source:http://2.bp.blogspot.com/-NeOyeb5zaYU/TZ1byItYEOI/AAAAAAAADmg/x4aIdBS9v4U/s1600/centrifugal+pump+impeller.jpg


B. Air-Lift Reactor
An air lift reactor is characterized by the absence of any impeller. A special design inside the vessel (consisting of a draft tube, through which air circulates between the vessel and tube) leads to a circulation of medium through out the reactor, by the force of air. This leads to low shear stresses and high mixing.
[Image: bab0450001f03.gif]
Air Lift Reactor Design(s)

Source:http://www.babonline.org/bab/045/0001/bab0450001f03.gif

C. Bubble Column Reactor
A bubble column reactor belongs to a family of impeller less reactors. Unlike Air-lift, it lacks any draft tube and mixing is induced by the force of rising bubbles from the sparger. The rate of air sparging needs to be closely controlled, otherwise fast flowing air may lead to "bullet" action of the bubbles, creating huge shear forces on the cells.
[Image: 200px-Bubble_column.svg.png]
Bubble Column Reactor


D. Rotary Drum Reactor
It has not been used for very large scale -productions yet. But it's use in lab scale-productions has been reported in few cases. It consists of a drum (containing the biomass) partially submerged in the medium. The rotatory action of the drum ensures periods of exposure of the cells to the medium, rather than complete submergence. This arrangement provides extremely less shear force, though proper nutrient transfer is a limitation.
[Image: T0831_E12.gif]
Scheme of Rotary Drum Reactor


So, these were some of the highlights of challenges in Scale-up of plant cell cultivation. The possibilities are undoubtedly vast, and rather many companies across the globe are fast developing the technology for the exploitation of these possibilities. Recently  (2010) Protalix Ltd. filed a patent for it's novel bioreactor design for cultivation of Carrot Cells in 400L bioreactor to produce the Gaucher Drug-Glucocerebrosidase! It can be used for many other plant species too!  (Link to Patent Info). With such developments, it is hoped that the industrial production of plant cell biomass will soon catch the race with the well developed microbial systems.

Thanks
Sunil Nagpal
MS(Research) Scholar, IIT Delhi (Alumnus)
Advisor for the Biotech Students portal (BiotechStudents.com)
Computational Researcher in BioSciences at a leading MNC


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#2
This is post no. 2 under the main topic.
Plant Tissue Culture: A field of possibilities

I would like to share my knowledge of Plant Tissue Culture here in a way that might help a newbie/layman to understand what Plant Tissue Culture is; and what are the possibilities associated with this field of extra-ordinary biotechnical talent.

Let's start with: What is Plant Tissue Culture ?
Plant Tissue Culture refers to the art of growing "plant cells or tissues" inside the confinements of a laboratory in glass tubes/petridishes/bioreactors using synthetic growth media and plant hormones (growth regulators). Whenever an experiment is performed in glass apparatus (typical of a laboratory set-up), we call it an "in-vitro" approach. Thus, the art of growing plant cells/tissues in-vitro, may be referred to as "Plant Tissue Culture".

Following is a photographic summary of what plant tissue culture looks like:
[Image: ptc.gif]

Now, let's shift to: How Plant Tissue Culture is carried out?
(i) Explant: Plant Tissue Culture often starts with the 'choice of Explant'. Now, "What is an Explant?" Explant refers to the part/tissue of the plant which is used to start tissue culture. For example, if you take plant leaves as the part for initiating the tissue culture, then explant in that case is Leaf tissue. Whereas in most plants, the tissue culture can be initiated from explant obtained from any part of leaf/stem/root/shoot/flowers, in some plants, explants obtained from different parts exhibit different growth rates. It is thus advisable to choose explant carefully. Not only this, your choice of explant can greatly decide the haploid/diploid nature of the tissue grown (anther used as explant will lead to haploid culture).

(ii) Sterilization of Explant: The next step towards plant tissue culture involves sterilization of the explant. Sterilization is very important for following reasons:
a) Explant exposed to natural conditions is expected to be contaminated with plethora of microbes and fungi.
b) The contaminants (microbes/fungi) have very high growth rate as compared to plant cells, thus they can easily overgrow on the media on which plant cells are expected to grow.


But, sterilization doesn't mean heating/autoclaving the explant (which would lead to death of the sensitive cells). Careful surface sterilization of the explant is carried out in a well defined protocol. Following is the protocol for Sterilization of Explant (assuming a leaf):

1. Wash your hands with soap and hot water. Excise several healthy leaves, both young and old, and discard the petioles.
2. Wash the leaf blades briefly in cool soapy water. Rinse them in running tap water, and prepare for aseptic procedures.
Aseptic procedure means that your entire working area inside the laminar hood should be aseptic (sterilized). Aseptic conditions can be maintained as follows:
  • Ensure that you have washed your hands well with an anti-septic soap/solution before handling any cultures or reagents.
  • Switch on the laminar flow hood (this should start the air flow from HEPA filters). DONOT switch on the UV light right now, while you are creating aseptic environment!
  • Wipe down the sides, top and bottom with a clean paper towel and
  • Wipe the surface of all materials you will need with 70% ethanol, before placing them into the hood.

  • Bottles, pipettes, forceps, etc. should be placedtogether in a rear corner of the hood to avoid any obstructeions in the workspace (Be sure not to block the airflow!).

  • To maintain sterility always work toward the back of the hood and try not to put your hands behind the cultures (this could blow spores from your hands into the culture).

  • All apparatus should be well autoclaved (cutting blades, forceps, pipettes, scissors, empty flasks etc)

  • Media transfers and preparations should be performed inside the hood in front of a flame.

  • Culture bottles shouldn't be left open inside the hood.

  • Tools like scoops, blades should be sterilized by flaming before reusing them.

  • When finished, remove all materials from the hood then ethanol wipe all surfaces in the hood.
3. Dip the leaf blades in ethanol (70% v/v) for 5 seconds and rinse in sterile DDH2O  (Double Distillled Autoclaved water) in a 250-cm3 beaker.

4. Complete the surface sterilization by immersing the blades in the 10% hypochlorite solution for 10 min.

5. Make sure to handle the leaves with only sterile, flamed forceps (but not with hot blades/forceps!).

6. Each blade is transferred to a sterile Petri dish containing filter paper, and explants are prepared with forceps and scalpel. The filter paper will remove the excess moisture from the final DDH2O rinse.

7. The leaf tissue most effective in organogenesis is located in the central part, and the outer margins and leaf tip are relatively unproductive. Slice the blade into rectangles approximately 10-12 mm of a side, ensuring that each explant contains a portion of the mid-vein of the leaf.


Keep watching this space for further information on this topic.



References:

http://passel.unl.edu/
wikimedia
Bhojwani and Razdan's PTC

 
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