Tipping Shelf Temporary Immersion Micropropagation Apparatus

Temporary Immersion Bioreactors are used in micropropagation in contrast to gel culture to enable the more fluid movement of the medium around the plantlets that are being multiplied. This lifts diffusive constraints to nutrient supply to the plantlets and can increase their rate of growth and multiplication. Some temporary immersion systems (TIS) are produced commercially, but are relatively expensive and often reserved for well-funded research programs. Hobbyist tissue culturists are left with the need for inexpensive equipment that can accomplish the same task as commercial TIS. This project outlines the construction of a tipping shelf system and bioreactor for the successful multiplication of hazelnut.

Tools:

Drill and phillips head driver bits

Circular saw

Computer with Arduino IDE for programming Arduino

Materials:

Wide Mouth Mason Jars

Pebbles - for holding explants in place

Stainless Steel Lids - normal lids will rust

1/8" Luer bulkhead adapter - filter attachment

Nuts for bulkhead adapter

.22 micron Syringe Filter

1/4" O-ring for sealing bulkhead adapter

Heavy Duty Wire Shelf

32" x 14" x 1/2" plywood - for tipping shelf

1" x 4" x 8' pine board - for tipping shelf

3" screws

arduino uno

arduino stepper shield

Flange coupling connector

Stepper Motor Mounting Bracket

Geared Stepper Motor

2" x 1/8" aluminum bar - for mounting motor

4 M3 x 3mm bolts

4 M8 x 10mm bolts

Traxxas ball joints - lift arm joint

M4 x 20mm set screws - lift arm

carbon fiber tube - lift arm

4" to 5" hole saw - cutting lift/drive wheel (a jigsaw would also work here)

Timer - for lighting control

24" LED shop light 

M4 tap set

zip ties

The tipping action of the shelf causes the nutrient fluid within the bioreactor to flow intermittently to submerge the base of the plantlets and then drain the fluid from around the plantlets. The tipping shelf must accommodate numerous bioreactors and tip on an automated schedule.

Begin by assembling the wire shelf, placing the bottom shelf 16 inches from the ground. Place the subsequent shelves 12 inches apart. The last shelf should be nearly at the top. This will allow you to store supplies or static cultures below the bottom shelf and have 3 tipping shelves.

LED lights require being wired to a cord and plug and then can be zip-tied in a central position under each of the top 3 shelves. Cords can be made different lengths to accommodate varying heights of shelves so you don't have to deal with extra cord.

Assemble the wooden tipping shelves by cutting 1/2" plywood into 32" by 14" rectangles. Also cut the 4" x 1" board into 32" lengths. Align the long edges of the 4" by 1" board with the plywood and affix with a screw or nail.

Scrap pieces of wood can be nailed to either end of the shelf to prevent jars from rolling off. A screw is inserted near the vertex at either end of the boards to act as an anchor point for the shelf to pivot. Zip ties anchor the tipping shelf to the wire shelf.

Anchor the wooden shelf to each side of the wire shelf using several zip ties at each pivot point.

Cut a 4" section off of the 2" by 1/8" aluminum bar and mark the center, at 2 inches. Place the stepper motor mounting bracket over the aluminum bar and mark the position of 4 holes centered on the 4" aluminum bar. Drill the 4 holes through the aluminum bar large enough to accommodate an M8 bolt.

Sandwich the wire shelf between the stepper motor mount bracket and the aluminum bar at the middle and side of the wire shelf. Place the M8 bolts through the holes in the aluminum bar and the stepper motor mount bracket to affix the assembly to the wire shelf.

Use the 4 M3 bolts to mount the stepper motor to the mounting bracket.

Cut a 5" circle out of the 1/2" plywood and thoroughly sand the edge to smooth the wood. Use 4 short wood screws to mount the flange coupling connector to one side of the wood circle so it will be turned eccentrically. This is the lifting mechanism for the shelf. Mount this on the shaft of the stepper motor ensuring that one set screw seats on the flat side of the shaft.

Measure the distance between two adjacent shelves, subtracting the length of two of the Traxxas joints (2 x 5/8"). This should be about 10-3/4". Cut two carbon tubes to this length.

Wrap 1.5 cm of each end of the carbon tube with thread to reinforce the end. Soak the tread with cyanoacrylate (super glue). Use a M4 tap to cut threads into the ends of the carbon tubes to accept the M4 set screws.

Screw the set screw into the Traxxas ball joint. Slip two 1" long pieces of heat-shrink tubing over the carbon tube and then screw the Traxxas ball joints into each end to the carbon tube. Use a hair dryer, heat gun, or flame to shrink the heat-shrink tubing over both the Traxxas ball joint and carbon tubing. This finished rod is called the "lift rod".

Screw two lift rods into the far ends of two adjacent shelves. Repeat this process with each pair of shelves. Your lift shelves will finally all be linked to raise and lower in unison, driven by the single drive wheel.

Mount the stepper shield onto the Arduino and mount the wires in the 5 contact strip on the side of the stepper shield next to the USB port. The black and green wire go in adjacent connectors and blue and red wires go in adjacent connectors, leaving the middle connector open. Note these colors are for the wires coming out of the motor, my extender wires don't match the colors coming out of the motor.

Arduino Sketch:

//139:1 Stepper Motor Drive Sketch for Temporary Immersion Tilt Table 

#include <AFMotor.h>

// to motor port #1 (M1 and M1)
AF_Stepper motor(100, 1);

void setup() { 
  motor.setSpeed(50);  // 50 rpm    
}
  
void loop() {
  
  motor.step(3500, BACKWARD, MICROSTEP); 	//Amount of rotation backwards. 3500 steps for 51:1 motor
  motor.step(3500, BACKWARD, MICROSTEP);        //10500 total steps for 139:1 motor
  motor.step(3500, BACKWARD, MICROSTEP);        //Steps must be broken into increments for large step numbers
  delay (60000);				//Soak time
  motor.step(3500, FORWARD, MICROSTEP); 	//Amount of rotation forwards
  motor.step(3500, FORWARD, MICROSTEP);
  motor.step(3500, FORWARD, MICROSTEP);
  motor.release();				//shuts off power to motor
  delay (3500000);  // 30min ~ 1500000		//Tilt time
  
  //This code down below is is for other step modes if you wish to experiment

  //Serial.println("Double coil steps");	
  //motor.step(100, FORWARD, DOUBLE); 
  //motor.step(100, BACKWARD, DOUBLE);

  //Serial.println("Interleave coil steps");
  //motor.step(100, FORWARD, INTERLEAVE); 
  //motor.step(100, BACKWARD, INTERLEAVE); 

  //Serial.println("Micrsostep steps");
  //motor.step(100, FORWARD, MICROSTEP); 
  //motor.step(100, BACKWARD, MICROSTEP); 
}

Connect your computer to the Arduino board with a USB cable, select the proper COM port and Board under the "Tools" tab. Select "Verify" and then select "Upload" to upload the sketch to your Arduino board. The motor should now begin to turn. Unplugging the USB chord will reset the sketch, so you can allow the wheel to rotate and unplug when the wheel and shelf are at their highest position. Adjust the "Soak time" and "Tilt time" to achieve your desired results and then re-upload the sketch.

Jar lids must be drilled with a 1/4" drill bit to allow the bulkhead adapter and 0.22 micron filter to be attached. Use a silicon o-ring to seal the bulkead fitting.

A commercial bioreactor is available from We Vitro that would work well with this tipping shelf setup and would lift some constraints that the mason jars pose. It is difficult to place many plantlets through the open mouth of the mason jar as it lies on its side. However, the mason jar remains as a budget option.

This assembly tutorial is also available on my website at: https://www.democratek.org/temporaryimmersion.html