Developing Genetically Modified Rice Variety Using Recombinant DNA Technologies

 

Plant breeding utilizes conventional crossing and selection procedures- aims to improve morpho-physiological traits and yield of a crop species. The basis of breeding is to select plants with desirable traits (agronomic traits, disease and stress tolerance, and grain quality). Selection involves - evaluating breeding populations to combine desirable genes in selected varieties. In pedigree breeding method, large population size are required - selection of superior homozygous plants involves visual assessment for improving useful traits. The entire process involves considerable time.

Biotechnology utilizes techniques other than crossing to modify a living organism - carrying genes from other species, that couldn’t be possible by conventional breeding. Biotechnology utilizes several techniques - resulting in a genetically modified organism (GMO), where one or more foreign gene has been inserted artificially. Biotechnology is used for developing GMO crops- disease-resistant plants, tolerance to stresses and improved nutrition. Call modification is called transgenic. From transgenic cells - a transgenic plant could be obtained using recombinant DNA technology.

The most useful area of biotechnology is modification of crop through genetic engineering. Genetic engineering tools are used to modify an organism  using recombinant DNA technology- emerged from microbial genetics. Genetic engineering based on recombination- cut DNA into fragments, rejoin different fragments and insertion of recombinant genes into  bacteria for reproduction. Gene cloning means-  modification of any kind of biological material such as a piece of DNA or an individual cell. American biochemists S. N. Cohen and H. W. Boyer (1973) was pioneer of this technology.

Pro-vitamin-A (bita-carotene) gene is not present in traditional rice varieties. Through transgenic technologies - two daffodil genes and one bacterial gene were inserted in rice genome, called transgenic golden rice. These three genes produce pro-vitamin-A in rice endosperm. Molecular markers linked with bita-carotene gene were used by IRRI to transfer bita-carotene gene into BRRI dhan29. Now, BRRI dhan29 (golden rice) carry bita-carotene gene are in the field trial in Bangladesh. Golden rice brings the hope that vitamin-A deficiencies in the diets of poor could be alleviated.

BRRI emphasized to develop high-yielding rice varieties through conventional breeding and bio-technological techniques for favourable and unfavourable areas. Thus, both conventional breeding and biotechnology could achieve genetic gains in yield, grain quality, earliness and tolerance to abiotic and abiotic stresses. These techniques could be  applied for improving rice  yield, aroma, cooked rice elongation, grain amylose and nutritional quality- high iron, Zn and fiber. These techniques could also be applied for improving color, shape, shelf life and smile of fruits.   

Knowledge on gene located on different chromosomes is important for a breeding program.  From rice genetic map- position of markers/genes on each chromosome are known. A character is carried in a fragment of DNA or in a gene. Different combination of a linked chain of 4 bases: A, G, C and T (or U) constitute different DNA molecule; called a gene. Of the two stands - each DNA strand acts as template for synthesis of a new strand and transfers genetic information to offspring.

 In rice, 715 primer pairs were synthesized having distinct sequence. Markers (short segment of DNA/ a gene/ a qualitative trait) are used for identifying variation at DNA level. Identification of a major QTL (quantitative trail loci)/genes linked with useful traits is possible from a F2 population. If the association between a marker and a trait is tight- that helps for selection of   target traits based on marker data. Then, gene specific marker for a trait could be designed using high resolution fine mapping.

Basic research on gene isolation, marker design, map-based sequencing, single nucleotide polymorphisms, gene cloning, cDNA synthesis and gene expression have become important for understanding gene function and recombinant DNA technology. Knowledge on gene or DNA (deoxyribonucleic acids) and gene expression is important. DNA sequencing determines the order of nucleotides within a strand of DNA molecule. A linked chain of 4 bases (adenine, guanine, cytosine, and thymine) constitute DNA molecule; called a gene located on chromosome - stores their genetic information.

A gene or DNA transcribed to mRNA that encodes specific protein to produce a specific trait/phenotype. Any small change (single nucleotide polymorphisms) in a gene sequence could alter the phenotype- that small change could be detected through gene expression analysis. Recombinant DNA technology is used to modify the DNA sequence of a crop and to observe the change on the phenotype. Modification of the DNA sequence of target organism- could be achieved by Recombinant DNA technology.

Recombinant DNA technology synthesizes a new copy of donor DNA and transfers it into host plant to develop a genetically modified crop. Gene cloning is isolation of a DNA sequence/a gene from any species and its insertion into a vector for propagation to generate a genetically identical copy of a cell.  For this purposes, map-based gene cloning utilizes genetic relationship between a marker and a gene. Steps of gene cloning are: Isolating source DNA, preparing cloning vector, formation of recombinant DNA molecule and introducing into the host recipient, and finally transformation.

A donor organism could donate source DNA that will be cloned. General protocols for DNA isolation are available.  To obtain the DNA comprising a gene- DNA is first extracted from cells. Map-based position of markers/genes on each chromosome and DNA sequence data of a crop is now available. PCR (Polymerase Chain Reaction) is a technique for amplifying DNA (or RNA), using taq polymerase and two specific flanking primers. Gel electrophoresis is used to separate PCR products and significant amount of DNA of interest could be isolated.

Preparing cloning vector is needed to insert cut up DNA from a donor genome into a cloning vector. A cloning vector is a small piece of circular DNA, extracted from a virus/Bacteria- where foreign DNA fragment could be inserted; used as a vehicle to carry a particular DNA segment into a host cell as part of cloning. These cloning vectors contain a site that allows DNA fragments to be inserted, which also has several restriction sites for ligation of DNA fragments.

Recombinant DNA technology emerged with the discovery of restriction enzymes (REs) that could specifically cut and join separate double-stranded DNA molecules. Both vector DNA and donor DNA are digested with same RE that cut at particular sequences of both stands. Then, vector DNA join with foreign DNA through binding cut DNA by addition of DNA lygase - thus a novel recombinant DNA vector is constructed. A cloning vector can be self-replicated inside host cell. It has a selectable marker with an antibiotic resistance gene for screening of recombinant organism.

A plasmid is a biological tool that allows any segment of DNA to be inserted into a carrier cell (bacterial cell) and replicated to produce more segments. Recombinant vector with gene of interest is inserted in a bacterial host (Escherichia coli) and in living bacterial cell- a recombinant vector with foreign DNA could multiply autonomously. In a cultural media, bacteria divides into millions of cells (called a DNA clone) - resulted in large number of copies of gene of interest.  The inserted DNA could replicate in recombinant vector within that bacterial host.

A collection of DNA clones that represents entire genome is called a genome library. After cloning of DNA fragment- it could be used for gene sequencing and transformation of a host plant. The nucleus of plant cell is targeted for transferring cloned DNA through two methods- “Agrobacterium” and “Direct gene transfer”. The Agrobacterium method is used to transfer foreign DNA into host plants (to be transformed) very efficiently. Agrobacterium called natural genetic engineer -has ability to transfer recombinant DNA into the chromosome of host plant from the vector.

The Agrobacterium infect plant cell- during infection; the piece of DNA is integrated into a host plant chromosome through a tumor-inducing plasmid (Ti plasmid). For this propose, dipping of flowering plant parts is done into a solution of Agrobacterium carrying the gene of interest, followed by the transgenic seeds being collected directly. Only those cells containing the cloning vector will grow when grown in antibiotics media. Thus, transgenic plants could be produced through introducing foreign gene into the genome with the technique other than crossing.

 Genome editing is also a special type of genetic engineering in which insertion, deletion or replication of genome DNA of a plant is done using engineered "molecular scissors”. Gene editing could produce benefit in plant trait modification. New gene editing tool- known as CRISPR/Cas9 could modifying a single-celled embryo of a plant. Researchers have successfully used CRISPR-Cas9 gene drives to modify genes associated with sterility in A. gambiae, the vector for malaria.

There is a research proposition to transfer more productive C4 photosynthetic pathway/genes from C4 maize into modern C3 rice through bio-technological tools. Biotechnology will be useful for transferring C4 photosynthesis path-ways (more synthesis of carbohydrates) from maize to rice. A program is going on at BRRI to identify major QTLs for efficient translocation of sources in to grains to maximize yield, as well identifying QTLs for efficient conversion of stored glucose/starch to lignin and fiber for cell wall strengthening and lodging tolerance.

Genetic engineering tools could be applied to modify rice plant with useful traits from other species. However,  before releasing transgenic rice- a bio-safety regulation might be formulated to protect risks associated with new GMO, as well to protect gene transfer between GMOs and their wild-type. It is more important to develop manpower in respect with application of molecular tools of recombinant DNA technologies. A National Biotechnological research institute could be build-up under leadership of BRRI. These Recombinant DNA technologies could be progressed based on advanced knowledge for assuring better food security in Bangladesh.


Dr. A S M Masuduzzaman is
Chief Scientific Officer, BRRI,
Gazipur. Nivea Nur Masud is a final
year student of EEE, UTM, Malaysia.