PROSPECTS AND LIMITATIONS FOR APPLICATION OF BIOTECHNOLOGY IN CROP IMPROVEMENT
ANCHA SRINIVASAN
* School of Agriculture, Department of Applied Biology, University of Cambridge, Pembroke Street, Cambridge, U.K.
An invited article written in 1990 primarily for senior high school students undergraduates in science.
In the recent past, there has been a considerable optimism in application of biotechnological tools in industry, medicine, animal husbandry and agriculture. Insofar as agriculture is concerned, biotechnology is indeed considered a harbinger of a second green revolution. Much of the appeal lies in its promise of creating entirely new opportunities for crop improvement and of modifying the biological constraints to a greater productivity. This is particularly so in the arid and semi-arid tropics where crop plants are exposed to a wide range of biotic and abiotic stresses and are generally grown in marginal environments.
Plant biotechnology is a term coined to encompass all laboratory-based manipulations aimed at altering the genetic make-up of higher plants while agricultural biotechnology is a term having much wider scope, which includes biotechnology related to food processing. There are several laboratory-based techniques which can be grouped under plant biotechnology. While some of them (e.g., cell and-tissue culture ) have been in use for a considerable time, the others (recombinant DNA-technology) are more recent. Indeed, the development of techniques which enabled gene transfer and expression in higher plants has generated a considerable interest and excitement amongst agricultural scientists. This article attempts to describe the various techniques available and their potential application in crop improvement and utilization.
Most methods of biotechnology presently used or envisaged require the culture of cells and subsequent regeneration of plants. Therefore tissue culture may be considered a gateway through which all forms of genetic engineering must pass. Besides this, tissue culture techniques are employed in many ways, as listed below:
(i) The most important area in the field of tissue culture with proven practical application is the meristem culture technique. This has been used for a considerable time in rapid clonal propagation and in the production of virus-free plants, particularly when used in combination with heat treatment. Indeed, it is as long ago as 1957, that Kassanis first demonstrated that paracrinkle virus could be removed from potato using meristem culture. The technique is relatively simple and is adaptable to a wide range of plants. Since the constituent cells of shoot apical meristems are less differentiated than the cells of other organs, in vitro culture of meristems would also result in the recovery of genetically identical progeny and this unique attribute makes plant meristems ideal candidates for the long term storage of germplasm and their international exchange.
(ii) Tissue culture techniques are also used in massive rapid screening for susceptibility/tolerance to various diseases (e.g., in corn for Helminthosporium maydis). However, such screening would be valid only when individual tissue or organ responses are similar to the whole plant responses.
(iii) Precocious flowering: It is desirable to induce precocious flowering to save time in breeding of plants where flowering is delayed until after juvenile phase. For example, in Ginseng breeding, induction of precocious flowering by embryoids derived from root callus could save about 3 years.
(iv) Reversing the developmental growth phase : In long lived woody plants such as forest trees, selection of elite individuals is based on old specimens. Very often the cuttings of shoots from these individuals show a tendency to mimic the growth habit of mature branches and this results in non-uniform growth, thereby making evaluation very difficult. However, juvenility can be restored by in vitro culture methods.
(v) Somatic embryogenesis: Plant cells are considered totipotent as they have ability to regenerate a complete plant. The ideal pathway to produce plants from single cells is through production of somatic embryos, in which cells undergo structural and organizational changes similar to those which occur during zygotic embryogenesis. Somatic embryos are produced in several crops but the problem of ensuring the genetic stability and phenotypic uniformity of the plants produced from somatic embryos limits its wide application in crop improvement.
(vi) Haploidization: The initial discovery of Guha and Maheshwari that anthers of Datura could be cultured and haploid plants regenerated was made in 1964. The technique aims at redifferentiating gametophytic cells to give divisions leading to embryogenesis or callus growth. Although embryo formation is to be preferred as the route to stable haploids for genetics and breeding, callus formation sometimes occurs, particularly in cereals. After haploid plants have been regenerated, dihaploids can be produced by treatment with colchicine. The main advantage with this is that homozygous lines can be created quickly.
(vii) Somaclonal variation : Tissue culture systems induce variability in cultures and the genetic variation introduced in this manner has been termed by Larkin and Scowcroft as somaclonal variation. Such variability is valuable in crop improvement as it serves as an important source of germplasm.
(viii) Wide crosses: Widening of genetic base for effective crop improvement sometimes involves crosses between related genera. For example species of Atylosia, a genus taxonomically close to Cajanus, possess desirable characters such as drought and salt tolerance. disease and insect resistance etc. Certain Atylosia species hybridize successfully with Cajanus while others fail to cross. Even after successful hybridization, wide crosses are often hampered by post-fertilization barriers such as failure of endosperm development and subsequent degeneration of hybrid embryos. In such cases, rescue and culture of embryos would facilitate production of viable hybrids.
Apart from being a novel source of variability, wide hybridization in barley proved to be a potential means of haploid production via selective chromosome elimination. This technique, called "bulbosum technique", involves use of Hordeum vulgare as female parent and H. bulbosum as male. Following cross pollination and fertilization the resulting embryo would be haploid as the chromosomes of H. bulbosum are selectively eliminated.
Notwithstanding the above diverse applications, there are several constraints to application of tissue culture in crop improvement.
This is a form of genetic engineering in which protoplasts are isolated and fused and involves both nuclear and cytoplasmic interactions. The successful outcome of somatic hybridization, whether it is to produce nuclear hybrids, cytoplasmic hybrids (cybrids) or limited gene transfer will depend upon a basic knowledge of the many steps in this procedure. These steps include isolation and controlled fusion of protoplasts, identification and selection of heterokaryons, hybrid cells and the regeneration of plants. The fusion of protoplasts from different sources requires an inducing agent such as polyethylene glycol (PEG). Electrically induced protoplast fusion (electrofusion) was also found to enhance fusion frequency and reproducibility.
Somatic cell fusion also offers an opportunity for the geneticist to manipulate extra-chromosomally inherited characters such as cytoplasmic male sterility as the procedure results in merging of cytoplasm of the two parents while in sexual crosses only the female contributes cytoplasm. Thus protoplast fusion makes it possible to produce hybrids containing different types of cytoplasm which otherwise would never occur through sexual crosses.
Example : Red clover is the most promising sward component to be grown together with timothy or meadow fescue, but it is not persistent beyond 2 seasons. Aslike clover, a wild relative of red clover, is however known for good persistence. Because their hybridization by conventional means is difficult, hybrid production is attempted by fusion of mesophyll protoplasts using PEG and electrofusion.
Somatic hybridization may also be used to overcome some of the limitations inherent in wide crosses between divergent species or even genera. However, a major impediment to hybrid production via somatic fusion arises from genome imbalance, loss of unpaired chromosomes or simply lack of nuclear fusion. Even if fusion occurred, recent reports suggest that there was a selective loss of chromosomes and resulting nuclear imbalances could reduce the viability and regenerative potential. Such inability to regenerate plants at will from fused cells is the major constraint in application of somatic hybridization in crop improvement. Indeed, the first somatic hybrid was produced as long ago as in 1972 by Carlson, Smith and Dearing but the impact of somatic hybrids on crop production has been meager so far.
Central to the new methods is the development of recombinant DNA technology. A great transition took place in genetics since the discovery of structure of DNA by Watson and Crick in 1953. Subsequent to the discovery of enzymes which cleave and join DNA or RNA, attempts were made to clone foreign DNA into a bacterium, Escherichia coli. In 1983, it was shown that foreign genes will express in host plants also.
Although the use of recombinant DNA technology in crop improvement has been recent, the technology itself is not something that has been discovered in the last 10 years. Recombination occurs naturally in vivo but there are some limitations. Firstly, it occurs only between molecules that are strongly homologous to one another. Secondly, it occurs only when two DNA molecules get inside the same cell. However. the new recombinant DNA technology allows scientists, at least in theory, to recombine DNA from completely different and diverse sources.
There are 3 essential components in a recombinant DNA experiment: (l) the vector DNA molecule (the molecule that will act as the vehicle or the carrier for the genes that are to be cloned, (2) foreign DNA (that is the DNA to be cloned and manipulated), and (3) the host cell. In brief, the foreign DNA is cut into pieces using a restriction endonuclease (an enzyme that cleaves DNA molecules at specific short nucleotide sequences). Likewise, the vector is cleaved with a restriction endonuclease that specifically opens up the DNA molecule at a non-essential site. These two DNA molecules are mixed together and treated with an enzyme, DNA ligase, which has the capability of joining the DNA molecules at their terminal points. DNA ligase does not recognize the sequence of DNA molecules, but only their ends, and therefore it has no problem with joining together molecules that are from diverse species or sources. Once the recombinant molecules are constructed in vitro, they must be introduced back into living cells (bacteria) in order to be propagated.
The efficacy of a plant vector is determined on the basis of its ability to transfer DNA into the host cell in such a way that it is maintained through cell division and plant propagation and that it is expressed as a new genetic factor. Although some DNA viruses (e.g., Cauliflower mosaic virus ) were demonstrated to be useful vectors, the only well developed and widely used gene transfer system for higher plants is the Ti (Tumor inducing) plasmid of Agrobacterium tumefaciens which works well with most dicotyledons.
A. tumefaciens is a soil inhabiting bacterium which causes crown gall disease in dicotyledons. It invades wounded plant tissues and causes hypertrophy which appears macroscopically as an undifferentiated gall at the wound site. This bacterium contains a large Ti plasmid of which a portion called T-DNA is transferred into the plant chromosome thereby resulting in a genetic change. The Ti plasmid has a virulence region (vir) which is necessary for the transfer of the TDNA to the plant. Foreign genes to be transferred must be inserted between the borders of the T-DNA region.
In some cases, electrical treatment of protoplasts (electroporation) would also allow the direct uptake of DNA. For example, Walbot and her colleagues electroporated foreign DNA into maize cells. Once the transfer of desirable genes for chosen traits takes place, the transformed lines are selected using marker genes. Tests are also done to see whether the genes are expressed through different generations.
There are several areas where conventional breeding has not met with an anticipated measure of success and the prospects for application of recombinant DNA are bright in such cases. For example, breeding programs for high protein rice and wheat, and for higher levels of sulfur containing amino acids (Cysteine, Cystine and Methionine) in peas, soybean and faba beans, have so far been unsuccessful. The manipulation of seed storage protein primary gene sequence through the use of recombinant DNA technology might alter the quantity and quality of protein. Grain characteristics may also be altered to improve the utilization and processing of grains.
Active genes can be constructed using DNA from various sources and the expression of a foreign gene can be directed in a plant by having a promoter operating at the desired place and time. For example, Flavell and others at the Plant Breeding institute, Cambridge succeeded in putting the promoter for the storage protein glutenin of wheat in front of a gene determining chloramphenical acetyl transferase. When this plasmid was vectored into tobacco, the enzyme is expressed only in the tobacco seed. This means that the promoter gets activated only in the seed but not in the rest of the plant.
Recombinant DNA technology may also be used in improvement of the quality of food products. The presence of condensed tannins is a major nutritional problem with utilization of some cultivars of sorghum, as tannins combine with protein to make it non-digestible. Tannins also decrease the activity of digestive enzymes. As the presence of tannins is under genetic control, biotechnology may be used to remove them from the new product.
Genetic engineering may also be used to introduce disease resistance into crop plants. For example, the cauliflower mosaic virus can have, independent of its genome, a satellite sequence of DNA of about 335 bases in length. When the satellite sequence is present, the expression of viral symptoms is greatly reduced. When DNA for the cauliflower mosaic satellite sequence was transferred into tobacco, there was essentially absence of viral symptoms on infection.
Limitations: As mentioned above, there are several potential benefits of recombinant DNA technology but there is a great deal of work to be done before realizing its fruits. Agrobacterium has a very limited host range and therefore gene transfer systems are largely tested in model species such as plants of solanaceae (petunia, tobacco, tomato, potato) which are highly susceptible to infection by Agrobacterim. Other reasons for choosing these species include (1) their conventional genetics and cytology are well studied, (2) they are easy to manipulate and regenerate in vitro, (3) they have short regeneration time (seed to flower). Many agriculturally more important cereal crops are less amenable to transformation with Agrobacterium and plant regeneration from single cells is often poor for these species.
Another limitation is that very few genes are available at present for successful genetic engineering. These are primarily antibiotic (kanamycin or hygromycin) or chemical (e.g., herbicide) resistance genes whose functions can easily be detected. These genes provide useful markers but for practical crop improvement, many additional genes which control physiologically important functions will need to be cloned and characterized. For example. in many disease systems, the gene products which are directly involved in conferring resistance are not known and therefore critical DNA sequences cannot be isolated. Similarly, it is difficult to visualize major alterations in the patterns of photosynthetic efficiency or biological nitrogen fixation as long as we do not have a clear understanding of the genetic and biochemical complexities of these processes. The lack of knowledge on the genes that regulate the amounts of (un)desirable products in different foods precludes the application of biotechnology to improve their utilization.
Besides the introduction of new methods, the sensitivity of other techniques such as HPLC (High Performance Liquid Chromatography - a tool for separating complex mixtures of molecules), PAGE ( Polyacrylamide Gel Electrophoresis - an extremely powerful tool for the analysis of complex mixtures of proteins), protein blotting (Southern blotting or capillary blotting, contact-diffusion blotting and electroblotting which enable further analysis of separated proteins - identified on the basis of their ability to bind a particular probe) and protein sequencing (chemical or enzymatic breakdown of a protein chain into smaller polypeptides and determination of amino acid sequences after isolation of peptides) is being greatly improved upon.
Conclusion
Plant biotechnology is a rapidly developing subject which offers many new opportunities for crop improvement. Besides providing novel approaches to create genetic diversity in plants, the technology can be used especially where the conventional breeding methods have failed to bring about a desirable level of improvement. However, as is generally accepted, any new and developing technology leaves more questions unanswered than answered and plant biotechnology is no exception to this rule. Considering the complexities involved in determining the number of genes that regulate a trait, and in gene transfer and expression, lit is unlikely that biotechnology alone will significantly shorten the time span required to develop a superior variety. Therefore, we should first of all identify the need for particular improvement, and assess the potential use of biotechnology. Finally, plants which have been derived from biotechnology will need to be subjected to comprehensive testing and evaluation for yield, quality or stress tolerance just as is needed for evaluating the progenies of crosses made as part of the conventional breeding procedures. Considering all these points, we should hope that progress in the nineties would reveal the extent of usefulness of this new technology in crop improvement.
Acknowledgment
The information for this article has been collected from several sources including text books, class notes and personal discussions with various research workers at the University of Cambridge. The author wishes to acknowledge all these sources.
January 15, 1999