BIOLISTIC

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Dr. Shaveta Chauhan

Asstt.Professor

Botany Dept.

HRMMV,Jalandhar

• Introduction
• Mechanism
• Equipment
• Parameters:
• Physical parameters
• Environment parameters
• Biological parameters
• Advantages
• Disadvantages
• Summary

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CONTENTS

INTRODUCTION

• Biolistic is also known as particle bombardment, microprojectile bombardment ,particle acceleration.
• Biolistic or particle bombardment first introduced in 1987 by Kein et.al and Sanford et.al
• A procedure in which high velocity microprojectile (tungsten or gold particle) were utilized to deliver nucleic acid into living cell.

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MECHANISM

Biolistic equipments

• Gene gun

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• PDS1000/He

����Biolistic equipment -PDS1000/He

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• DNA coated microcarriers are loaded on microcarrier.
• Micro carriers are shot towards target tissues during helium gas decompression.
• A stopping screen placed allowing the coated microprojectile to pass through and reach the target cells.
• The microcarriers accelerate for maximum penetration as they move through barrel, while the helium pulse diffuses outward.
• The spacer maintains the optimal target distance for in vivo applications and vents the helium gas away from the target to minimize cell surface impact.

Parameters

The use of particle bombardment require a careful consideration of number of parameters. These parameters are classified into three category:

• Physical parameter
• Environmental parameter
• Biological parameter

Physical Parameter

• Nature, chemical and physical properties of the metal particle utilized the foreign DNA. Particles should be in high mass in order to posses adequate momentum to penetrate into plant tissue. A variety of metal such as gold ,tungsten ,platinum,pladium, rhodium, iridium and possibly other transition metals can be used , size range of the particle is 1micrometer. Metals should be chemically inert to prevent the adverse reaction with DNA or cell component .
• Nature preparation and binding of DNA onto the particles. The nature of DNA as single vs. double stranded condition but this may not be a significant variable in specific cases. In the process of coating the metal particles with DNA certain additives like Spermdine and Calcium Chloride appear to be useful. Alternate polyamines including Caldine and Termine have been studied but Spermdine dramatically increases of both transient and stable trans location

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• Target Tissue: It is a very important to target appropriate cell that is capable or cell division and transformation i.e. the cell which can be enriched by selection with antibiotic or herbicide that correspond to transforming DNA and then regeneration. It is apparent that different tissue have different requirement thus, extensive study needs to be performed in order to uncertain origin of regenerating tissue in particular transformation study. Penetration of microprojectile (DNA coated to inert metal gold and tungsten) is one of the most important variable and the ability to tune a system to achieve particle delivery to specific layers may result in success and failure in recovering of transgenic plant from given tissue. It has been reported that cells near target are injured and thus cant proliferate. This injury is attributed to physical trauma to cell from gas blast and acoustic shock generated by device. Present day devices used mesh screen devices which reduces cell death and increase transformation frequency significantly.

Environmental Parameters

These include variables such temperature,photoperiod and humidity of donor plant explants and bombardment tissue. These parameters effect physiology of tissue influence the receptiveness of the target tissue to foreign DNA delivery and also affect its susceptibility to damage and injury that may adversely affect outcome of transformation process . Some explants may require a healing period after bombardment under special regime of light, temperature and humidity.

Biological Parameter

• Nature of explant as well as pre and post bombardment culture condition .
• Explants derived from plants that under stress will provide inferior material for bombardment experiments.
• Metal particles need to be directed to the nucleus .
• Transformation frequencies may also be influenced by cell cycle stage.
• Osmotic pretreatment of target tissues has also been shown to be of importance .
• Physical trauma and tungsten toxicity were found to reduce efficiency of transformation in experiment performed with tobacco cell suspension culture/

Advantages

• Many different tissue and cell types can be transformed.
• No binary vector is required.
• It is possible to deliver multiple plasmids with high frequencies of co transformation.
• The transformation protocol is simple
• The large DNA fragment can be delivered
• The system can be adapted to deliver mRNA or protein

Disadvantages

• Messy integration pattern
• Requirement of equipment
• Shallow penetration of particles
• Associated cell damage
• The tissue to incorporate the DNA must be able to regenerate
• Equipment itself is very expensive

Summary

• For Biolistic transformation tungsten or gold particles are coated with DNA and accelerated towards plant tissues. Most devices use compressed helium as force to accelerate the particles .
• The particles punch holes in the plant cell and usually penetrate only 1-2cell layer wall.Particle bombardment is a physical methods of a DNA introduction.
• The DNA coated particle can end up either near or in the nucleus , where the DNA comes off the particles and integrated into plant chromosomal DNA