Plant Genetic Manipulation LECTURE 8 Introduction To Plant Genetic Manipulation Dr. Aparna Islam

What do you understand by plant genetic manipulation? How can you do that? What is plant genetic transformation? What is transgenic plant? Is GM crop and transgenic plants same? What can we achieve through plant transformation? What are the types of plant transformation techniques?

What do you understand by plant genetic manipulation? Inclusion or deletion of genetic material. How can you do that? Conventional breeding and transformation. What is plant genetic transformation? Transformation of gene of interest from any plant or non-plant sources into any plant species. What is transgenic plant? Plant regenerated following transformation. Is GM crop and transgenic plants same? Yes. In the beginning GM crop meant Genetically Manipulated. But as ‘manipulation’ has a negative notation, it was then changed to genetically modified crop. What can we achieve through plant transformation? Stress resistance, quality improvement, yield improvement, etc What are the types of plant transformation techniques? Two types: direct and indirect methods

Conventional Breeding Wheat Rye X Triticale New species, but NOT biotechnology products

Scientific Milestones 1907 Discovery of the causative agent of crown gall disease (A. tumefaciens) 1930 Discovery of the causative agent of hairy root disease (A. rhizogenes) 1975 Identification of Ti plasmids responsible for tumor induction in plants 1977 First isolation of a plant gene and regeneration of tobacco from A. rhizogenes-transformed roots 1983 Plant genetic engineering with a modified T-DNA 1985 Transfer of herbicide tolerance in transgenic tobacco though Electroporation 1987 Popular transformation technique: Gene gun 1994 First commercialization of genetically engineered crops

Major Methods of Transgene(s) Delivery Direct methods Protoplast transformation -polyethyleneglycol (PEG) method -electroporation -microinjection Plant cell transformation -particle bombardment 2. Indirect method -Agrobacterium-mediated transformation can’t be applied to organelle

Direct Methods of Plant Transformation Advantages: Suitable for all species and all plant materials Expression vectors usually easy to prepare Disadvantages: Expensive equipment required Transient gene expression Fragmentation and rearrangement of introduced sequence

Direct Methods of Plant Transformation 1. PEG Method Principle: Use chemicals to permeabilize the protoplasts membrane to stimulate uptake of DNA into the plant cell. Method: DNA is added to a suspension of protoplasts in the presence of PEG and the mixture shocked with an electrical field of 200-600 V/cm to facilitate DNA uptake. Drawbacks: Requires lengthy and skillful tissue culture regeneration from the protoplast. Uses and Advantages: For use with species not susceptible towards Agrobacterium infection.

2. Electroporation The electric field causes holes in the plasma membrane allowing DNA to be taken up by the cell. Advantages: Alone, produces fair results. When coupled with PEG, transformation success can be dramatically increased. 2. Has been applied on a variety of species and tissue types. Disadvantages: A high mortality rate (25-50% survival). Requires protoplast regeneration which is difficult.

3. Microinjection Principle: Advantages Disadvantages Direct and precise delivery of DNA into the plant cell or its nucleus using a micro-syringe. Dyes in combination with fluorescence microscopy are used to visualize the nucleus. Advantages High transformation efficiencies (14-66%) Precise delivery Disadvantages Tedious procedure Only a limited number of cells can be microinjected in one exp. 3. Expensive equipment required

4. Particle gun bombardment Particle gun bombardment is injection of DNA directly into whole cells with intact cell walls Drawback: Feasible but with limited success Modification: Use of minute metal beads (tungsten, gold, etc.) coated DNA to shoot directly into cells (1987) THAT IS PARTICLE GUN BOMBSRMENT

4. Particle Gun Bombardment Method: The DNA is precipitated onto the surface of the beads, which are fired from the “gun” with velocities ~ 430 m/s Intact leaves, callus and suspension cultures plated on filters. Cells in the direct line of fire are killed, but there is a concentric zone. GENE GUN

Gene Gun

Hand Held Gene Gun

Drawbacks: Advantages: Transient expression Tissue damage Low efficiency (1-5%) Size of the plasmid Advantages: Natural transgene flow avoided: maternally inherited Applicable to most crops High copy number/cell Molecular farming (harvestable proteins)- high level expression Limit toxicity - recombinant proteins in chloroplast No gene silencing Choose integration site

Indirect Method of Plant Transformation Agrobacterium-mediated transformation

Agrobacterium-mediated Plant Transformation "Nature's Genetic Engineer"

Comparing Agrobacterium with bombardment Agrobacterium is effective mostly to dicots; bombardment is effective on all plant cells, monocots and dicots. Agrobacterium takes less time (e.g In tobacco ~3-5 months to complete; bombardment takes longer, about 5-7 months. Agrobacterium places DNA only into the nucleus of a plant cell; bombardment can place DNA into any of the three plant cell compartments that contain DNA: the nucleus, mitochondria, and chloroplasts. Agrobacterium insert transgenes anywhere within the genome while bombarded plasmid has spefic target site within the organelle DNA.

How A. tumefaciens cause plant transformation Three important issues in plant transformation Naturally Agrobacterium cause tumor formation upon transformation of plant cells resulted from transfer and integration of T-DNA and the subsequent expression of T-DNA genes Any foreign DNA placed within the T-DNA borders can be transferred to the plant cells, no matter where it comes from. T-DNA genes are transcribed only in the plant cells and do not play any role during the transfer process.

Parts of Ti-plasmid: T-DNA the part of DNA that is exported into the plant cell and integrated into the plant genome vir region: encode proteins involved in this transfer, but stays within the bacteria. This region is 30 kb long and organized in six operons eg. virA, virB, virD and virG (involved in T-DNA transfer) and virC and virE (increase transfer efficiency) Gene involved in Opine catabolism Genes involved in bacterium-bacterium conjugation

How Agrobacterium tumefaciens can be used as a tool for genetic engineering Problem: tumor How can we engineer the Ti plasmid to make it useful? • Delete auxin and cytokinin genes • Retain vir genes, LB & RB, ori • Ti plasmid is huge (~120 kb) – need to make it smaller

Engineered Ti-plasmid T-DNA vir ori

Further modification and make binary vector Through further modification super virulent Agrobacterium strains are made.

Steps of Agrobacterium-mediated transformation Grow Engineered Agrobacterium Preparation of Agrobacterium solution for infection Infection of plant tissue/explant Co-cultivation Selection Effect of Antibiotic (selection and bacteria control: bactostatic and bactericidal) Duration, conditions: dark/ light or both Agro. medium, Agro. conc., inducers: sugar, acetosyringone Agro.-plant interaction, Agro. strain, plant species, variety or cultivars, tissue/ explant, reg. protocol, transformation technique (duration, wounding), addition of chemical to promote infected tissue survival rate (eg. antinecrotic), low pH Vector: super/ binary, reporter genes, promoter, mol. DNA manipulation of gene seq.

So, Agrobacterium transformation is not just the above picture So, Agrobacterium transformation is not just the above picture. It is more than this.

Following gene transfer • Consequences of the insertion: - Foreign DNA inserted - Insertional mutagenesis (does not kill the cell – the organism is diploid!) • Each cell is hemizygous for the insertion – only one of the homologous chromosomes gets the insertion Transformation involves one cell which then regenerates an entire organism under selection pressure

Optimal conditions for establishment of an Agrobacterium-mediated genetic transformation protocol in local Bangladeshi varieties of tomato (Lycopersicon esculentum Miller) Aparna Islam, Jebunnesa Chowdhury and Zeba I. Seraj Journal of Bangladesh Academy of Sciences 31

Tomato, which is considered as a genetic model for improving dicotyledonous plants is still regarded as a recalcitrant crop for transformation. The average transformation frequency ranged from 6.2-10.4% depending on regeneration protocol, transformation procedure The absence of highly efficient transformation methodology is the major difficulty in obtaining transgenic tomato. Reports on transformation of Bangladeshi tomato varieties are limited. Begum and Mia (1993) reported tomato regeneration in Bari varieties, while Sarker et al. (2009) reported transformation in Bari tomato-3 and Pusa Ruby where they observed chimarism of the marker nptII gene. 32

In the present study, using marker gene, attempts have been made to develop a transformation protocol for locally grown popular winter and summer tomato varieties. To achieve this various factors influencing transformation efficiency will be optimized to establish a simple, efficient and reliable transformation methodology. Four varieties of tomato (Lycopersicon esculentum Mill.), namely, Bahar (BR), Bina tomato-3 (B-3), Bina tomato-5 (B-5) and Pusa Ruby (PR) and Genetically engineered Agrobacterium tumefaciens strain LBA4404 were used in this study. 33

Determination of strain-cultivar compatibility: A. tumefactions strain LBA4404 with the cotyledonary leaf explants of Bina tomato-3 (B-3), Bina tomato-5 (B-5), Bahar (BR) and Pusa Ruby (PR) varieties, all the explants found susceptible towards infection, thus positive interaction between the bacteria and tomato varieties. 34

Influence of optical density of Agrobacterium suspension Three viz. 0.79, 0.64 and 0.42 optical density (OD600) was tested. Transformation efficiency examined through transient GUS expression revealed significant differences (F (2, 6) = 16.09, P = 5.14) in the three ODs. Transformation frequency was found to be increased with the increase of optical density of the Agrobacterium suspension. Maximum transformation (~95%) was observed at OD600 of 0.79 while minimum (≤50%) was at OD600 of 0.42 in all varieties. 35

Influence of incubation period on transformation The transformation efficiency showed significant differences (F (2, 6) = 206.7, P = 5.14) with the 3 different incubation periods. At OD600 0.79, transient assays showed cent percent transformation (GUS +ve) in BR and B-3 when explants were incubated for 15 mins. In the same condition, B-5 and PR also showed highest (92% and 90%, respectively) GUS positive result. But the GUS positive response was much lower at 20 min. of incubation period. Thus, increase in incubation period beyond a critical time length resulted in decrease in transformation efficiency in all the varieties. 36

Influence of pre-culture 20-30 15-18 90 PR 23-27 15-20 100 B-3 22-25 16-19 92 B-5 20-25 99 BR Non pre-cultured Pre-cultured Days required for regeneration initiation % of GUS +ve explants Varieties Transformation efficiency was not found to be influenced by pre-culture of explants in any of the four varieties. Pre-cultured explants were found to regenerate faster and performed better than the non-pre-cultured explants 37

Influence of co-cultivation period: co-cultivation found to influence the transformation efficiency and subsequent regeneration capacity. It was found that, percentage of transformation could be increased just by increasing the co-cultivation period. But, prolonged co-cultivation period (more than 3 days) was found to promote overgrowth of bacteria on the infected explants and also explants found to suffer from poor health showing browning at the cut surfaces and failed to regenerate. Correspondingly the transformation percentage was found to decrease with the decrease (less than 3 days) of co-cultivation period. Therefore, three days of co-cultivation was determined to be the best for all four varieties tested. 38 These studies needs to be done in a case by case manner.