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The following points highlight the four main techniques adopted for in vitro regeneration of plants. The techniques are: 1. Micro-Propagation Technique 2. Embryo Culture Technique 3. Gene Transfer Techniques 4. In vitro Techniques.
1. Micro-Propagation Technique:
Micro propagation has been defined as ‘in vitro regeneration of plants from organs, tissues, cells or protoplasts’ and ‘the true-to-type propagation of a selected genotype using in vitro culture techniques’. True-to-type propagation has important benefits for plants that have no named varieties, such as Australian dioecious papaw genotypes where traditional plant breeding has failed to produce stable lines.
Micro propagation has also been useful for the rapid initial release of new varieties prior to multiplication by conventional methods, e.g.. pineapple and strawberry, and germplasm storage for maintenance of disease-free stock both in controlled environment conditions and long term via cryopreservation.
However, the ability to propagate plants in vitro free of genetic off- types is dependant on the technique used for micro propagation. Failure to understand this principle has resulted in disastrous consequences with some species, for example the commercial production of banana clones containing 90% dwarfs.
Protocols which have been developed for in vitro propagation of plant species can be divided into three systems. System 1 is based on callus culture followed by organogenesis or embryogenesis. System 2 comprises proliferation of axillary buds and/or adventitious buds, resulting from repeated subculture on multiplication media containing cytokinin.
In system 3, micro-cuttings obtained from axillary buds of apically dominant shoots are grown on hormone-free or low cytokinin media. System 3 has been used in the author’s laboratory for clonal propagation of papaw, neem, coffee and passion-fruit and is not prone to production of genetic off-types.
Exploitation of the variation that exists in populations has led to the development of many commercial varieties and hybrids. With the rapid expansion of tissue culture technologies came the observation that genetic variation was occurring in plants regenerated from somatic cells and this was seen as a novel source of variation.
The frequency of off-types varies with species, culture type and number of sub-cultures and has been attributed to a number of variations within cultured cells. However, the cause of somaclonal variation is not fully understood; therefore, it cannot be controlled and changes can be epigenetic and unstable.
Variation can be promoted by the use of radiation or chemical mutagens and the use of colchicine to change ploidy. The potential of somaclonal variation involves the ability to change one or a few characters without altering the remaining part of a genotype.
Initial notes of potentially useful variation included increases in cane and sugar yield, and resistance to eye- spot disease in sugar cane; improved tuber shape, colour, and late blight resistance in potato; and, increased solids and Fusarium resistance in tomato.
In vitro selection involves the screening of cell cultures that are exhibiting genetic variation, for tolerance or resistance to pathogens, herbicides, low or high temperatures, metals and salt. It necessitates a reliable method of regeneration from callus.
For successful application of the technique, tolerance at the cellular level must also occur in the regenerated plant in the field and be transferable to other plants via conventional plant breeding.
The use of in vitro screening to select for disease resistance is most effective for diseases which produce toxins. This was initially demonstrated by Carlson who regenerated tobacco plants resistant to Pseudomonas tabaci, from haploid cells resistant to methionine sulfoxamine, an analogue of the disease toxin.
Similar successes have been noted with a range of diseases including Phoma lingam in canola, Helminthosporium oryzae in rice, Pseudomonas and Alternaria in tobacco and Pseudomonas solanacearum in tomato. Addition of herbicides to culture is an ideal system for in vitro screening as defined concentrations can be used.
However, effectiveness is dependent on the mode of action of a herbicide on the whole plant being similar at the cellular level, and is therefore better suited to herbicides that interfere with basic metabolic functions. Resistance or tolerance has been developed in vitro to a range of herbicides including paraquat in tomato, picloram amitrole and triazine in tobacco and 2, 4-D in Lotus corniculatus.
Carrot suspension cultures which were resistant to 35mM glyphosate in solution were selected after 8 subcultures on low concentrations of glyphosate (0.3 to 0.6 mM). Carrot plants regenerated from these cultures were resistant to glyphosate sprays in field plantings. A similar stepwise selection procedure for gene amplification produced tobacco cell lines with high tolerance to glufosinate.
Cell cultures that are resistant to increased salt (NaCl) in vitro do not necessarily produce regenerants which tolerate high salinity levels in soil. For example, the use of somatic cell cultures of rice did not lead to heritable salinity tolerance in the field; however, this was eventually achieved after in vitro selection of callus cultures initiated from immature ovaries and anther, culture-derived lines.
Examples of genotypes which had been released or were in field trials in the USA and Canada. Many of his references were patent applications or personal communications.
These included chlorsulfuron and imidazilinone resistance in canola, imidazilinone resistance in corn, high solids and Fusarium race 2 resistance in tomato, a white flowered form of lucerne, potato virus Y resistance in tobacco and fall armyworm resistance in sorghum.
Potentially useful somaclonal variants have been identified in a number of fruit crops. These include Phytophthora resistance in papaya and apple, Xanthomonas resistance in peach, Erwinia resistance in pear and salt tolerance and 2, 4-D tolerance in orange.
2. Embryo Culture Technique:
Embryo culture was probably the first tissue culture technique to be applied to plant improvement. Applications of embryo culture are rescuing embryos after interspecific hybridisation, clonal propagation of families such as Gramineae and conifers which contain recalcitrant species, and overcoming seed dormancy and seed sterility.
Interspecific crosses are often attempted to transfer desired traits such as disease resistance, stress tolerance or high yield from wild species into important crop species, e.g.. cotton, soybean and papaya.
Incompatibility after these crosses normally results in embryo abortion and this is often caused by breakdown of the endosperm or embryo- endosperm incompatibility. In vitro culture of hybrid embryos often successfully bypasses post zygotic incompatibilities.
There are numerous references of embryo rescue of incompatible hybrids. Another approach to wide hybridisation is in vitro fertilisation of cultured ovules and ovaries. This was achieved initially with opium poppy. The major application of this technology in monocotyledonous crop species is in maize.
In an extensive research program funded by the Australian Centre for International Agricultural Research (ACIAR) techniques have been developed for interspecific hybridisation and embryo rescue of papaya and wild relatives.
Hybrids have been produced between C. papaya and C. cauliflora, C. quercifolia, C. pubescens, C. parviflora and C. goudotiana. Useful characteristics of the wild species include papaya ring spot virus resistance, Phytophthora resistance, high sugar content, and ornamental characteristics.
3. Gene Transfer Techniques:
Many years of research have culminated in an increasingly detailed understanding of biology and genetics at the molecular level. Combined with tissue culture technology, this knowledge is being applied to the modification of plant genomes. Because of the universality of the genetic code, potentially useful genes can be transferred to plants from any organism.
This has lead to the development of new and improved genotypes by the addition of single genes that code for traits such as insect or disease resistance, or by the inactivation of single gene faults. Future potential includes the possibility of transferring larger DNA constructs that code for multiple genes conferring more complex traits.
Applications of gene transfer are increasing rapidly, driven by an extensive research effort worldwide. Initial successes were in the development of disease, insect and herbicide resistant crops.
More recently, gene transfer is resulting in control of plant development in individual species. Inhibition of expression of polygalacturonase in a tomato genotype lead to the first commercial release of a genetically engineered plant – the FLAVR-SAVR tomato. Extensive research is being directed towards gene inactivation techniques for control of ethylene production, and thus ripening, in climacteric fruits.
Gene transfer techniques have also lead to the development of carnations with increased vase life and new colour forms. Isolation of a flower-meristem-identity gene from Arabidopsis has the potential to dramatically reduce time to flowering and hence length of the juvenile phase in plant species.
Developing applications include transformed plants which will produce more or increased levels of valuable oils, proteins and starches, and plants that produce vaccines and can be used for oral immunisation against a range of serious human diseases.
Useful agricultural applications of gene transfer necessitates incorporation of a foreign DNA construct into a plant genome, the regeneration of transformed plants, the stable expression of the introduced gene and inheritance in subsequent generations.
The successful application of plant transformation techniques is dependent on plant tissue culture protocols to regenerate transformed plants. Since the first reports of gene transfer with species that are relatively easy to tissue culture, petunia and tobacco, transformation procedures have been published for a wide range of species including tree crops, such as apples and papaws.
Transgenic plants of a number of fruit species have been produced, including kiwi-fruit, citrus, strawberry, grape, cranberry, peach and plum. Numerous DNA delivery systems have been noted.
Direct gene transfer systems such as microinjection, electroporation and polyethylene glycol have been used to transform protoplasts. Protoplasts are easily damaged and regeneration of plants from them is very difficult with many species.
Consequently, such systems are not widely used. The popular method of direct gene transfer is particle bombardment. DNA coated micro particles are bombarded into tissues, and major crop species have been transformed using this technique, for example monocots such as wheat, barley, rice and recalcitrant dicot species such as cotton, and soybean.
The advantages of the particle gun in terms of tissue culture regeneration is that it can be used on callus and suspension cultures and on organised tissue of both monocots and dicots.
Increasing the number of options for tissue culture technology increases the probability of successful regeneration. The preferred method of gene transfer for dicots is Agrobacterium mediated transfer.
Co-cultivation of callus, suspension cultures or leaf discs with Agrobacterium has been used to successfully transform many species, for example efficient systems have been described for major crop species such as potato, tomato and sugar beet.
Virus, insect and herbicide resistance in a range of species including tobacco, potato, tomato, lucerne and soybean have been successfully trialed in field conditions and are in the early stages of commercialisation. Transformation of cereals, which are the world’s most important food, has been by direct DNA uptake systems, particularly biolistics.
Embryo genic cultures in cereals have generally been developed from immature embryos and inflorescences and subsequently transformed in the form of protoplast, suspension or callus cultures. The most recalcitrant of these species is wheat, where difficulty in developing reliable regeneration systems is exacerbated by low frequency of transformation.
Other problems associated with tissue culture of cereals are loss of regenerative capacity with increasing number of subcultures and occurrence of abnormalities such as sterility in regenerated plants. Agrobacterium was thought incapable of infecting monocots, however recent notes describe Agrobacterium mediated transformation of rice, maize and barley which offer considerable potential.
Systems have been noted for transformation of haploids via particle bombardment of pollen and via microinjection of DNA into microspore derived embryoids of canola. More recently, maize has been transformed by pollen which was mixed with Agrobacterium containing binary plasmids.
Such systems may provide a method that eliminates the need for in vitro regeneration, which is a major limiting step for some species and is prone to somaclonal variation.
Although in theory tissue of any species can be transformed, it is currently not possible to regenerate transformed plants at high frequencies for many species. Current methods of gene transfer and selection can be disruptive to growth of in vitro cultures, thus it can be difficult to develop efficient transformation procedures for species that are recalcitrant in vitro.
In addition, there is a need to be able to deliver gene constructs to tissue that is competent both for transformation and regeneration.
Limitations of current technology include low frequency of transformation, high frequency of undesired genetic aberrant, unpredictability of transgene expression, the need for techniques to transfer large DNA sequences coding for multiple genes and the absence of efficient repeatable regeneration protocols for many species in vitro.
4. In vitro Techniques:
In vitro Collecting:
In vitro collecting involves initial disinfestation and placement of plant explants in sterile culture medium, before transport to a tissue culture laboratory for further in vitro procedures. In vitro collection is particularly useful for species that are vegetatively propagated and for those with recalcitrant seeds or embryos which deteriorate rapidly.
The technique has much potential to facilitate the collection of germplasm of tropical and subtropical fruit species, as has already been demonstrated with Cassava and coconut. Recently, 300 Musa accessions were collected in Papua New Guinea using this technique, before being transported to a collection in Australia.
An added advantage of this exercise is that it complied with quarantine regulations that are in place to stop the spread of Fusarium and other diseases.
In vitro Culture:
In vitro culture offers some major advantages for conservation and use of plant genetic resources. In vitro cultures can be established from disease-free parent material and maintained in a disease-free state. Alternatively, meristem culture, in combination with treatments such as chemo- or thermotherapy, is a proven technique for elimination of specific viral and other diseases.
Consequently, disease-free germplasm can be safely and rapidly transferred between countries. However, it is not safe to assume in vitro cultures are free of specific viruses, unless the initial parent material has been cleared by appropriate ELISA or equally stringent testing. Caution should always be exercised when dealing with export/ import of plant tissue cultures.
In vitro culture has been used to facilitate international collaboration in a research project between Australia and the Philippines on development of PRSV-P resistance in papaya. Sterile cultures of Carica species, interspecific hybrids and embryo genic cultures were regularly transferred between the two countries and contained in quarantine approved laboratories.
For example, cultures from the Philippines have been transported to Australia, subjected to in vitro procedures in the author’s laboratory and returned to the Philippines. Australian quarantine restrictions that prevent early field release of this material are not contravened.
In vitro Germplasm Storage:
Ninety percent of all accessions held in germplasm collections are stored as seeds. There are three limitations of seed storage. Firstly, it is not applicable to species with no seeds eg. Musa species and Artocarpus altilis (breadfruit). Secondly, for highly heterozygous species, e.g.. dioecious papaya, it is preferable to conserve vegetative or clonal material.
Thirdly, seeds of recalcitrant species are sensitive to desiccation and/or chilling, and tropical recalcitrant species often lack a natural dormancy mechanism, e.g.. Mangifera indica (mango), Nephelium lappaceum (rambutan) and Cocus nucifera (coconut).
In vitro storage also represents a viable alternative to field collections for species included in the three groups for rare and endangered species. Field collections require regular maintenance and are prone to damage from disease and insect attacks, extremes of weather and natural disasters.
By comparison in vitro collections are safe from these problems, although failures in environmental control can result in loss of cultures. Thus duplicate collections are advisable. Another advantage of in vitro storage over field collections is that large numbers of cultures can be stored in a small volume in a growth room.
In vitro collections are generally maintained in either slow growth storage or by cryopreservation. Slow growth storage employs various methods to reduce sub-culture frequency, and thus labour and media costs. Cryopreservation involves long term growth suspension in liquid nitrogen. Cryopreservation was achieved initially using cryoprotectants and controlled cooling.
This classic technique has been successfully applied to cell suspension and callus cultures. Newer methods involve dehydration of cultures before rapid freezing by immersion in liquid nitrogen. This method shows potential with organised tissue, including embryos and meristems.
There exist a number of different dehydration/freezing procedures, including vitrification, encapsulation dehydration, desiccation, pre-growth and droplet freezing.