Protocol for Mass Propagation of Plants Using a Low-Cost Bioreactor

Abstract

Conventional in vitro mass propagation methods are labour-intensive, costly and have a low degree of automation. Bioreactor or automated growth vessel systems using liquid media were developed to overcome these problems. The use of liquid instead of solid culture medium for plant micropropagation offers better access to medium components and scalability through automation. However, the cost of setting up a bioreactor system is one of its disadvantages as such systems are expensive with limited number of manufacturers. A low-cost bioreactor system was set up using recycled, low biodegradable plastic bottles. This low-cost bioreactor, based on temporary immersion principle, has proven to be effective as a vessel for rapid plant propagation. It is designed to reduce the production cost of plant micropropagation. This chapter explains the step-by-step methods for setting up a low-cost bioreactor for banana seedling production. This low-cost bioreactor system has the potential to be adapted for large scale in vitro cultivation of the plant seedlings.

1 Introduction

The conventional micropropagation technique requires regular sub-culturing, manual handling at various stages of the process (labour-intensive) and more shelf space that contributes to high running and labour cost. Scaled-up and automated systems are therefore desirable to reduce the amount of handling, increase multiplication rates, hence overcome and/or minimize production costs of the conventional micropropagation technique as initially reported by Aitken-Christie et al. (1995). This can be achieved by using a bioreactor to scale up propagation. Bioreactors are usually described in a biochemical context as a self-contained, sterile environment which incorporates liquid nutrient or liquid/air inflow and outflow systems, designed for intensive culture and affording maximal opportunity for monitoring and control over micro environmental conditions (agitation, aeration, temperature, dissolved oxygen, pH etc). The use of bioreactors in controlled condition increases the multiplication rate and plant quality and has been proven as an efficient tool for rapid production of plant cells, tissue or organ culture and metabolites. The first reported use of bioreactor for micropropagation was in 1981 for Begonia propagation (Takayama and Misawa 1981). Since then, it has been widely used and proved applicable to many plant species including cassava (Golle et al. 2019), carnation (Marzieh et al. 2017), gerbera (Frómeta et al. 2017).

Various types of bioreactor systems, with different types and different sizes of vessels and agitation mechanisms (non-agitated, mechanical or pneumatically agitated) have been developed and used as described by Paek et al. (2005), Eibl et al. (2018) and Alireza et al. (2019). Among them, temporary immersion system (TIS) bioreactor is highly suitable for use in semi-automated micropropagation. This principle of temporary immersion was first tested by Harris et al. (1983) through alternate exposure and submergence of explants by tilting a flat-bottomed vessel to opposite direction using semi-automatic system. TIS bioreactor allows immersion of explants in medium for a specific duration at specified intervals, control of contamination, adequate nutrient and oxygen supply and mixing, relatively infrequent subculturing, ease of medium changes and limited shear damage. The temporary immersion of the plant with the media is a good technique to avoid damage, since long exposure can lead to physiological malformation which causes poor regeneration. In comparison with both, solid and liquid culture systems, TIS has technological and quantitative advantages such as higher multiplication rate and reduction of production cost (Etienne and Berthouly 2002). The use of TIS for large scale micropropagation produces better plant quality and higher multiplication rate (Ziv 2005). Examples of TIS bioreactor available today include BIT® twin-flasks system (Escolana et al. 1999), Reactor with Automatized Temporary Immersion (RITA®) (Alvard et al. 1993) and Bioreactor of Immersion by Bubbles (BIB®) (Soccol et al. 2008).

1.1 Low-Cost Bioreactor System

Many established TIS bioreactor systems were patented and are quite costly, hence less preferable for large scale mass propagation. Option for a simpler and cheaper TIS bioreactor system was explored through the development of a TIS bioreactor prototype called BIO-TIS (Ibrahim 2017). BIO-TIS consists of two glass vessels, one for the in vitro shoots and the other for liquid culture media which is connected by silicone tubing that permits the flow of the liquid medium from one vessel to the other. It has been tested for mass propagation of horticultural crops such as: fruit trees (pineapple, banana), ornamental plants (orchids, chrysanthemums) and herbal plants (Eurycoma longifolia Jack, Labisia pumila and Stevia rebaudiana). In a study on pineapple propagation, the multiplication rate with BIO-TIS was found to be much higher in comparison to the established RITA® bioreactor (Ibrahim 2017).

Modification was done by replacing the glass bottles in BIO-TIS with recycled plastic bottles as an alternative for a cheaper setting up cost. A silicone cap with stainless steel tubing is fabricated for liquid nutrient or liquid/air inflow and outflow. This low-cost bioreactor is capable of supplying planting materials in large quantities for various plants, able to increase the multiplication rate of in vitro plantlets up to ten-fold (Ibrahim 2017; Mustapha et al. 2017), improve the quality of tissue culture plantlets by reducing vitrification and is environmentally friendly. This system can be used by the plant biotechnology industry and agro-industry to save on the production cost. Furthermore, recycling of plastic bottles helps to reduce issue of the disposal of unused material in landfills thus reduce environmental pollution from disposal of used plastic bottles.

2 Materials

2.1 Liquid Media

• 1. Murashige and Skoog medium including vitamins (Murashige and Skoog 1962) (Cat Nr. M0222, Duchefa, Haarlem, The Netherlands).

• 2. 6-Benzylaminopurine (BAP) stock solution (Concentration: 1 g/1 l) (Cat Nr. B0904, Duchefa, Haarlem, The Netherlands) (see Note 1).

• 3. α-Naphthalene acetic acid (NAA) stock solution (Concentration: 1 g/1 l) (Cat Nr. N0903, Duchefa, Haarlem, The Netherlands) (see Note 2).

• 4. Sucrose (table sugar).

• 5. Sodium hydroxide (NaOH).

• 6. Hydrochloric Acid (HCl).

• 7. Distilled water.

• 8. Beaker (Volume: 1 l).

• 9. Bottle (Scott, Volume: 1 l).

• 10. Spatula.

• 11. Pipette.

• 12. Pipette tips.

• 13. pH meter.

• 14. Electronic balance.

• 15. Autoclave.

2.2 Bioreactor System (Fig. 11.1)

• 1. Recycled plastic bottle (Capacity: 5-10 L).

• 2. Silicone cap.

• 3. Silicone tube (Diameter: 6 mm).

• 4. Air filter (Sartorius, Midisart® 2000 PTFE or equivalent).

• 5. Air compressor pump (2 unit) (Rocker 320, Taiwan or equivalent model).

• 6. Timer (2 unit).

• 7. PVC Pipe (Length: 1 m, Diameter: 15 mm).

• 8. Cling film (3 cm width).

• 9. Scissors.

Fig. 11.1

(a) Recycled plastic bottles. (b) Modified silicone cap. (c) Silicone tube. (d) Air filter. (e) Air compressor pump. (f) Timer. (g) PVC pipe

2.3 Culture Initiation

• 1. 30–40 day old in vitro shoots of banana measuring 1.5 cm with 2–4 leaves.

• 2. Forceps.

• 3. Scalpels.

• 4. Blades.

• 5. Ethanol (70% v/v).

• 6. Tissue paper.

• 7. Hot bead sterilizer or bunsen burner.

• 8. Laminar air flow cabinet.

3 Methods

3.1 Preparation of MS Medium

• 1. Fill 800 ml of distilled water into a beaker.

• 2. Add MS medium, 30 g of sucrose, 2.5 ml of BAP stock solution and 0.1 ml NAA stock solution.

• 3. Stir the solution until dissolved.

• 4. Adjust the pH to 5.6–5.8 by adding NaOH/HCl.

• 5. When the desired pH is achieved, bring the volume to 1 l with distilled water.

• 6. Transfer the media into a bottle and sterilize for 15 minutes at 15 p.s.i and 121 °C.

• 7. Allow medium to cool down to room temperature prior to use.

3.2 Preparation of Bioreactor Set and Culture Initiation (See Note 3)

• 1. Sterilize plastic bottle using 5.25% sodium hypochlorite solution (see Note 4).

• 2. Prepare the modified silicone cap by connecting tube, cap and air filter as shown in Fig. 11.2 and sterilize by autoclaving at 15 minutes/15 p.s.i/121 °C.

• 3. Inside a laminar air flow, take two sterile plastic bottles of the same size (see Note 5).

• 4. Fill 2 l of MS media into the first bottle and close the bottle with the sterile modified cap set.

• 5. In another bottle, transfer approximately 20 gram of in vitro shoots and close the bottle with the sterile silicone cap set (Fig. 11.3).

• 6. Wrap around the cover using cling film to ensure that no leakage occurs during operation.

• 7. Connect both bottles with silicon tubing as shown in Fig. 11.4.

Fig. 11.2

A close view of the modified silicone cap that consists of a fabricated silicone cap, stainless steel tubes to allow media/air outlet and inlet, and silicone tube

Fig. 11.3

Explants are transferred into sterile plastic bottle under aseptic condition in the laminar air flow cabinet (left). A complete low-cost bioreactor ready for incubation (right)

Fig. 11.4

Two bottles of the same size are used. Bottle 1 is filled with media and Bottle 2 is for explants

3.3 Setting up Low-Cost Bioreactor

• 1. Connect the low-cost bioreactor set to the air compressor pump (Fig. 11.4). A number of low-cost bioreactors can be combined and run simultaneously as shown in Fig. 11.5 and Fig. 11.6. For this protocol, the system was tested for a max of 4 sets merged together. However, the efficiency of the system must be revaluated if there is additional set added.

• 2. Program the timer of Air Compressor Pump 1 for 15 minutes to ensure all the media is transferred into the explants-containing bottles.

• 3. Immerse the in vitro culture for 30 minutes.

• 4. Program the timer of Air Compressor Pump 2 for 15 minutes in order to transfer the media back into the first bottle after the immersion is completed.

• 5. The immersion cycle for this system is every 6 hours. The immersion cycle is further explained in Fig. 11.7.

• 6. Place the bioreactor system in incubation room with 25 ± 2 °C and 16 hours photoperiod for a period of two months (see Note 6).

Fig. 11.5

Sets of low-cost bioreactor can be combined and run simultaneously

Fig. 11.6

Large-scale production of banana plantlets through low-cost bioreactor

Fig. 11.7

Operational principle of low-cost bioreactor system: (A) The liquid medium is located in Bottle 1 and explants in Bottle 2 (B) The air compressor pump 1 is run for 15 min and all medium flow from Bottle 1 to Bottle 2 (C) The explants are immersed into the liquid medium for 30 minutes (D) After immersion is complete, the air compressor pump 2 is run for 15 min to allow all medium flow back into Bottle 1

3.4 Harvesting

• 1. Detach all silicone tube from the cap and take out the cap from the culture bottle.

• 2. Carefully tilt the bottle and take out the plantlets using forcep.

• 3. Rinse the plantlets under running tap water before hardening.

4 Notes

• 1. To prepare 100 ml of BAP stock solution (1 g/l), dissolve 100 mg of BAP with 2–5 ml of 1 N NaOH. Then bring the volume to 100 ml with distilled water and mix well. The stock solution can be stored at 4 °C for several months.

• 2. To prepare 10 ml of NAA stock solution (1 g/l), dissolve 10 mg of NAA with 1–2 ml 1 N NaOH. Bring the volume to 10 ml using distilled water and mix well. The stock solution can be stored at 4 °C up to several months.

• 3. This protocol has been optimised for propagation of banana. However, the same protocol can be applied for propagation of other plants using a suitable media and optimisation should be carried out to make sure that the system is suitable.

• 4. Sterilisation of plastic bottles is performed using 5.25% sodium hypochlorite solution. Pour 200 ml sodium hypochlorite solution into bottle and shake for 5 minutes. Repeat this step for another 2 times. Lastly, rinse with sterile distilled water twice to ensure no trace of sodium hypochlorite left. This sterilisation work is done in the air laminar flow cabinet. Alternatively, gamma irradiation can be used to sterilise the plastic bottle.

• 5. Preparation of plastic bottle and in vitro initiation work must be performed using aseptic techniques in the laminar air flow cabinet to avoid contamination.

• 6. Due to the efficient gaseous exchange between plant tissue and gas phase inside the vessel, the yield obtained from this procedure can increase up to 10–15 old compared to solid media.