In 1980s, genetically modified plants were developed to improve their quality, such as resistance to certain pests, diseases or environmental conditions, or for the production of certain nutrients or pharmaceutical agents. It became possible due to the cloning vectors developed for higher plants.

Three types of cloning system have been used with varying degrees of success with higher plants:

  1. Vectors based on naturally occurring plasmids of Agrobacterium.
  2. Direct gene transfer using various types of plasmid DNA.
  3. Vectors based on plant viruses.

Vectors based on Agrobacterium

Agrobacterium tumefaciens, the cause of economically important disease, crown gall, has also been studied for years because of its remarkable biology. The mechanism this bacterium uses to parasitize plant tissue involves integration of some of its own DNA into the host genome resulting in unsightly tumors that also leads to changes in the plant metabolism. A. tumefaciens prompted the first successful development of a biological control agent and is now used as a tool for engineering desired genes into plants. This delivery of viral or viroidal sequences to plants using this bacterium is called agroinfection or agroinoculation. Although harmful to plants it is useful to scientists as it transfers DNA into plant genomes. Found in soil worldwide, A. tumefaciens causes disease in plants by transferring its own DNA into plant cells. But in the laboratory, the ability to move all sorts of genes into plants has made this microbe standard tool for investigating plant genetics and modifying crops.

A. tumefaciens genome has a very unusual structure. Around 5,400 genes reside on four DNA elements, a circular chromosome, a linear chromosome, and two smaller circular structures called plasmids. Agrobacteria are the only species known to have both circular and linear chromosome structures together. A. tumefaciens can be effectively isolated for identification from gall tissue, soil or water.

Crown gall disease caused by A. tumefaciens
Figure 1: Crown gall disease caused by A. tumefaciens

Between the 1970s and 1980s, some striking aspects were discovered about the biology, biochemistry, and molecular biology of Agrobacterium. Tumorous plant cells were found to contain DNA of bacterial origin integrated in their genome. Furthermore, the transferred DNA (named T-DNA) was originally part of a small molecule of DNA located outside the chromosome of the bacterium. This DNA molecule was called Ti (tumor-inducing) plasmid.

The Ti plasmid contains most of the genes required for tumor formation. Wounded plants exude phenolic compounds that stimulate the expression of the virulence genes (vir genes), which are also located on the Ti plasmid. The vir genes encode a set of proteins responsible for the excision, transfer and integration of T-DNA into the plant genome. Genes in T-DNA region are responsible for the tumorigenic process. Some of them direct the production of plant growth hormones that cause proliferation of the transformed plant cells. T-DNA region is flanked at both ends by 25 base pairs (bp) of nucleotides called T-DNA borders. T-DNA left border is not essential, but the right border is indispensable for T-DNA transfer.

Basic elements of the vectors designed for Agrobacterium-mediated transformation that were taken from the native Ti-plasmid are as following.

  • T-DNA sequences, which initiates integration of T-DNA region into the plant genome
  • vir genes, which are required for the transfer of T-DNA region to the plant, and
  • A modified T-DNA region of Ti plasmid, in which genes responsible for tumor formation are removed by genetic engineering and replaced by foreign genes of diverse origin, e.g., from plants, bacteria, virus.
Ti plasmid of A. tumefaciens
Figure 2: Ti plasmid of A. tumefaciens

Agrobacterium was initially believed to be restricted to the transformation of certain dicotyledonous plants, but later it was discovered that it can also be used for monocotyledonous plants.

T-DNA transfer efficiency via Agrobacterium to a plant varies, not only among the plant species, but also among various tissues. Tissues like leaves, shoot apices, roots, hypocotyls, cotyledons, seeds and calli are derived from various parts of a plant are used in Agrobacterium mediated transformation. The bacterium used for callus, immature embryo, pollen, shoot apex, floral, and seed transformation is Agrobacterium or specifically A. tumefaciens. Transformation of pollen with Agrobacterium is broadly protected in the United States and Australia.

Agrobacterium rhizogenes

Agrobacterium rhizogenes (Rhizobium rhizogenes), soil-borne bacterium, is the causative agent of hairy root disease in dicotyledonous plants. This disease is characterized by extensive proliferation of roots from an infected plant wound. It results from the transfer and integration of transferred-DNA (T-DNA) of root-inducing (Ri) plasmid to the plant genome. Transfer of T-DNA is mediated by virulence genes, which form the vir region of the Ri-plasmid and the bacterial chv genes.

Hairy root disease caused by A. rhizogenes
Figure 3: Hairy root disease caused by A. rhizogenes

As transformed roots can be excised and grown in vitro as hairy root cultures, they are very useful in biotechnology research, genetic manipulation of plants for the production of phytochemicals, large-scale secondary metabolite production, phytoremediation, and monoclonal antibody production. Fast growth, genetic as well as biosynthetic stability, low doubling time, ease of maintenance, and the ability to synthesize a range of chemical compounds makes hairy root system suitable for in vitro production of the secondary metabolites.

There are advantages and disadvantages of Agrobacterium mediated transformation need to be considered

Advantages are as follows:

  1. It is a natural means of a transfer so more acceptable than any other vector.
  2. There is no limitation of tissue culture as Agrobacterium is capable of infecting intact plant cells, tissues, and organ.
  3. Agrobacterium has high efficiency in transfer of large DNA fragments without major rearrangements.
  4. Highly stable gene transfer occurs via Agrobacterium.

Various shortcomings or disadvantages associated with Agrobacterium mediated transformation are as follows:

  1. Most important limitation in Agrobacterium mediated transformation is associated with its capability to infect few hosts.  Some important food crops cannot be modified by this technique.
  2. Few cells present in tissues are deep. So, Agrobacterium is unable to reach them.
  3. Some cells are not suitable targets for T-DNA transfer.

Cloning genes in plants by direct gene transfer

Direct gene transfer plays key role in the study of gene regulation and function in bacteria, fungi, animal, and plant cells. In plants, the direct transfer method involves delivery of foreign gene of interest into the host plant cell without help of a vector. Transfer can be done in nucleus as well as chloroplast of the plant cell. Although there are several methods of direct gene transfer in bacteria and animal cell (These methods will be described in detail later in this book), but the methods mentioned below are commonly used for the direct gene transfer in plants.

  1. Chemical mediated gene transfer: Chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts. Calcium phosphate is also used to transfer DNA into cultured cells.
  1. Microinjection: In this method DNA of interest is directly injected into plant protoplasts or cells (specifically into nucleus or cytoplasm) using fine tipped (0.5-1.0μm diameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
  1. Electroporation: It involves application of high voltage pulse to protoplasts/cells/ tissues to make temporary pores in the plasma membrane. This facilitates the uptake of foreign DNA. Cells are placed in a solution containing foreign DNA and subjected to electrical shocks to form holes in the membranes. Foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
  1. Particle gun/Particle bombardment: Primary explants and the proliferating embryonic tissues are commonly used for particle bombardment. In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 μmeters) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).
  1. Liposome mediated gene transfer or Lipofection: It is an efficient technique used to transfer genes in bacterial, animal as well as plant cells. Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus.

Binary vector systems include most commonly used vectors devised for Agrobacterium gene transfer to plants. In these systems, the T-DNA region containing a gene of interest is contained in one vector and the vir region is located in a separate Ti plasmid (without tumor-genes). The plasmids co-reside in Agrobacterium and remain independent.

Co-integrated vectors are constructed by recombining an Agrobacterium Ti plasmid lacking tumor-causing genes (also known as “disarmed” Ti plasmid) and a small vector plasmid, which is engineered to carry a gene of interest between a right and a left T-DNA border of the T-DNA region (engineered or modified T-DNA region). Recombination takes place by a single crossover between a homologous region present in both plasmids.

Mobilisable plasmids are not capable of promoting their own transfer unless an appropriate conjugation system is provided by a helper plasmid. Mobilizable vectors contain a site for transfer initiation called origin of transfer, oriT, and have sequences encoding proteins (Mob) involved in the mobilization of DNA during the conjugative process. As Mob proteins alone are not sufficient to achieve the transfer of the genome, additional proteins for transfer (Tra) are involved in the formation of a pore or pilus through which genome passes to the recipient bacteria. Mobilizable plasmids do not encode Tra proteins and for this reason they require a helper plasmid providing the tra genes

Vectors based on plant viruses

Several researches have been done for the development and application of plant viral-based vectors to develop novel disease control strategies and products for plant and animal diseases. As many viruses or their isolated genomes are capable of infecting intact plant tissue, they are used as plant transformation vectors. Vectors based on viruses are desirable because of their high efficiency of gene transfer that can be obtained by infection and amplification of transferred genes (occurs via viral genome replication). Infection of plant cells with virus results in addition of new genetic material, which is expressed in host. Once the biological characteristics of a virus is selected and manipulated, new genes are added to the virus in a way that it expresses itself when it enters plant cell. Vectors useful in transferring genes in plants are based upon DNA or RNA molecule. RNA viruses are not useful as potential cloning vectors because manipulations with RNA are difficult to carry out. These vectors are non-integrative in contrast to A. tumifaciens that are integrative.

Non-integrative vectors are vectors that do not integrate into the host genomes; rather, they spread systematically within a plant and accumulate to high copy numbers in their respective target cells.

Generally viral genomes are modified to accommodate the foreign sequences and mimic their wild type counterparts, so that they can be transferred in a plant as recombinant virus genomes successfully. A viral vector possesses following characteristics:

  1. Broad host range, virulence, rate, and ease of transmission.
  2. It has potential to carry additional genetic information. Thus, virus with a filamentous, rod shaped capsid or a multipartite genome are used.
  3. Its suitability depends upon the fact that genetic material must be able to be manipulated and infectious.

Caulimoviruses

Caulimoviruses infect plants. This group contains 6-19 viruses. Virions are not enveloped and contain one molecule of, or two segments of open circular, or linear double stranded DNA, due to which it is easily available for the manipulations. Nucleocapsids are of two types, bacilliform and isometric. The commonest are the carnations etched viruses (CERV), cauliflower mosaic virus (CaMV), dahlia mosaic virus (DaMV), mirabilis mosaic virus, and strawberry vein-banding virus. Among several caulimovirus (CaMV) is the most potential virus for introducing foreign DNA to the plant cell to generate various transgenic crops. Many species of Cruciferae and Datura stramonium are affected this virus. Aphids transmit CaMV, but they can also be transmitted mechanically. CaMV powerful promoter drives replication of the retrovirus and is active in both angiosperms and gymnosperms making it very useful in biotechnology research. CaMV can even recombine with insect viruses and propagated in insect cells.

The genome of CaMV consists of relaxed circular molecule of 8kb. Genome sequence analysis has revealed that six major and two minor ORFs are present in one coding strands. It replicates via reverse transcription. ORF II (codes for the insect transmission factor) and ORF VII (unknown function) present in this virus can be replaced by the gene of interest. It is very suitable as an experimental tool as naked DNA of this virus is infective as it directly enters plant cell (even if it is rubbed with mild abrasive), the genome of this virus is packed in nucleosomes, and are transcribed by RNA polymerase II.

The perceived hazards of CaMV in crop plants include the following:

  • The consequences of recombination and pseudo recombination (gene components of one virus are exchanged with the protein coats of another).
  • Tightly packed genomes with coding regions, so insertion of foreign DNA sometimes becomes difficult.
  • CaMV derived vectors are restricted to members of Cruciferae. However, few mutant strains of CaMV derived vectors are used for members of Solanaceae recently.
  • Multiple cleavage sites in CaMV DNA hinders in the usefulness of wild isolates of CaMV.
  • As CaMV infects whole plant, the DNA must be encapsidated, which causes serious constraints on the size of foreign DNA that can be inserted into the viral genome.

Some researchers have proved that CaMV promoter is hazardous, but there are still controversies associated with these findings, some of which are shown below:

  • CaMV 35S promoter may be structurally unstable and prone to horizontal gene transfer and recombination.
  • Other hazards include mutagenesis, carcinogenesis, reactivation of dormant viruses, and generation of new viruses.
  • Plants contain mobile genetic elements. Transgenic constructs containing the 35S promoter may mobilize these elements. The elements may in turn provide helper-functions to destabilize the transgenic DNA, and may also serve as substrates for recombination to generate more exotic invasive elements.

Gemini viruses

Gemini viruses (family Geminiviridae) are small single-stranded (ss) DNA viruses infecting plants. They are transmitted by insects and infect either monocots or dicots. Symptoms vary from mosaic, yellow leaf, curling, rolling of leaves, thickening of veins, reduction of fruit set, yellow mottling, crinkling, severe stunting, reduced yields and at times death of plants. On infection typical mosaic-like leaf patterns of light and dark green occur.

Their virion morphology is unique in the known viral world as the two incomplete icosahedra are joined together to form twinned particles. These have small capsid size (18-20nm × 30nm). The names of gemini viruses have been standardized and a set of rules to derive names for newly identified species were laid down several years ago. In 2003, the International Committee on Taxonomy of Viruses established guidelines for the nomenclature of Gemini virus. According to the genome organization, host range and the insect vector used, gemini viruses are divided into four genera: Mastrevirus, Curtovirus, Begomovirus and Topocuvirus. Generally large number of begomoviruses possess bipartite genomes, i.e. genes are distributed on two separate ssDNA molecules that are usually both required for productive infection, while mastre-, curto- and topocuviruses encode all their genes on a single chromosome.

Gemini viruses replicate their genomes in the nuclei of infected (usually phloem tissue) cells via the rolling-circle (RCR) mechanism initiated by virus-encoded replication initiation protein (Rep) that ranges in size from approximately 320 to 400 amino acid residues. The potential of Gemini viruses as gene cloning vectors for plants is recognized due to ability of this virus to infect variety of plants. Replacement of coat protein coding sequences with a reporter gene or gene of interest is done to create suitable vectors. Gemini virus expression vectors are used to deliver, amplify, and express foreign genes in several different systems of protoplast, cultured cells, leaf discs, and plants. These viruses are suitable as cloning vectors as,

  • These viruses contain ssDNA that replicated via a double stranded intermediate that makes it suitable for in vivo manipulation in bacterial plasmid.
  • One of the most important features of bipartite Gemini virus is that it contains a deletion or replacement of virus coat protein sequences by foreign genes without interfering with the replication of the virus genome.

Problems associated with these viruses are that they are not readily transferred and small size of the particle creates problem in packaging of modified DNA molecules, which may also affect an ability of the viruses to infect the susceptible plants.

RNA Viruses

Two basic single stranded RNA viruses are generally used for this purpose. These are, monopartite and multipartite viruses. The monopartite viruses (For example, tobacco mosaic virus (TMV)) have undivided genomes containing all genetic information and multipartite viruses, as the name suggests, have their genome divided among small RNAs, either in same or different particles. The RNA components of these viruses are small and self-replicating. The second group consists of sub-genomic RNAs (e.g. RNA IV), are not used as cloning vectors as they are unable to self-replicate in infected plants.

Most RNA viral vectors carrying large or small inserts replicate stably in protoplast and/or inoculated leaves. Disadvantages associated are choice of vector is limited for use in inoculated leaves of whole plants, small insert size, and the induction of symptoms in the host.