In this article we will discuss about:- 1. Selection of Disease Resistant Variants at the Plant Level 2. Selection in Vitro with Toxins and Culture Filtrates against Diseases 3. Selection for Resistance to Drought and Flooding 4. Selection for Salt Tolerance.
Selection of Disease Resistant Variants at the Plant Level:
One approach to utilise somaclonal variation is to screen regenerated plants for disease resistance. Dominant and homozygous recessive traits can directly be selected. If the regenerants are heterozygous for a desired characteristic, recessive traits can be selected in the progenies of the regenerants. In case of self-fertilizing crops, it is recommended to screen the progenies of the regenerants to avoid epigenetic variations.
Table 9.3 shows most publications reporting successful selection at the plant level for somaclonal variants with an increased disease resistance. About half the number of studies listed show that the resistance is inherited in a stable manner, although for vegetatively propagated crops, such as potato and sugarcane, transmission of resistance is shown through vegetative reproduction. The table also shows that success was obtained with fungal as well as bacterial and viral pathogens, demonstrating the feasibility of the approach.
Selection in Vitro with Toxins and Culture Filtrates against Diseases:
The efficiency of a selection procedure may be increased by the application of a selection pressure to cells or tissues in culture. In this approach large number of individuals can be screened in limited space. A list of disease resistant plants obtained by in vitro selection is given in Table 9.4.
Helgeson (1972) proved that tobacco calli resistant to Phytophthora could be obtained from resistant plants, and that susceptible plants yielded only susceptible calli. The disease expression in vitro appeared to be closely related to expression at the whole plant level. Buiatti el al., (1985) found a correlation between in vivo resistance and in vitro response of Dianthus, not only to Fusarium, but also to elicitors of the fungus.
A number of experiments were done with crude culture filtrates of the pathogens, e.g., in potato with culture filtrates of Phytophthora, and Sacristan (1985) reported on screening in the Brassica/Phoma system. Not all regenerants from selected rapeseed cultures showed increased resistance, but the proportion of resistant or tolerant plants was up to six times higher among regenerants from selected cultures than among plants from controls.
The first three progeny generations of rapeseed were tested for their level of resistance; unexpectedly, segregation was found in the progenies of plants classified as resistant, although in all cases the proportion of resistant plants was higher for progenies obtained from resistant material, indicating genetically defined resistance. It became evident, however, in several experiments with and without in vitro selection that variation in characters, besides the one, aimed for, occurred in vitro, mostly in an undesired direction.
The first experiment where a controlled selection for resistance to a pathogen was made with the responsible toxin alone was reported for maize callus. The reversion through somaclone formation from susceptibility to resistance to Helminthosporium maydis was, however, associated with genetic changes in the cytoplasm leading to reversion from male sterility to male fertility. Thus, this result was of no practical importance.
Work with single cells would increase the chance of detecting differences normally covered by cross-feeding. Because the reproducibility in potato protoplast regeneration has been increased, experiments were started using the crude exotoxin to screen potato protoplast suspensions for Fusarium coeruleum and F. sulphureum.
The relative concentration of toxins was directly correlated with the inhibition of the respiration activity. Due to the rapid passage through the callus level, after regeneration all clones looked rather phenotypically uniform and were fertile. Retesting of leaves or secondary callus revealed a significantly increased stability against the Fusarium toxins.
Three years of field tests with these potato clones expressed visible differences between the selected resistant plants and the unselected control one; the differences could not be made significant, however, due to very strong environmental influence.
The contrasting results, particularly in the experiment aimed at disease resistance, demonstrate that in vitro screening cannot become a general procedure for a huge number of characters. To what extent correlations or causal dependence exists between an in vitro reaction and the phenotype of the whole plant must be checked first.
The relationship between reactions of calli with reactions of the same genotypes as complete plants can be very close when the toxic compound responsible for the virulence is used to detect sensitive individuals at an early developmental stage. In all such in vitro selection systems, it is quite common to observe habituation.
This means that the resistance or tolerance is present only as long as the selective agent is present. The desired new trait is not fixed in the genome or plastome but only epigenetically expressed. Before a stable new clone can be considered, further generations must be checked.
Selection for Resistance to Drought and Flooding:
Drought and flooding are perennial problems of agriculture. Tolerance to drought is a polygenic trait and involves complex osmoregulatory functions. Simulated drought conditions have been achieved in vitro through incorporation of non- penetrating osmotic solutes such as PEG (polyethylene glycol) and dextran’s in the media.
Water stress tolerant lines have been successfully isolated in tomato using PEG as the selective agent. It has been shown that concentration of free proline in a plant is a good indicator of its stress tolerance. The biotechnology company Agrigenetics has isolated mutant plants by tissue culture, which shows depressed synthesis of proline. These mutants show tolerance to water stress.
Crop injury due to flooding results from anaerobic respiration in roots. Consequently, alcohol accumulates due to catalytic activity of the alcohol dehydrogenase (ADH) enzyme. Therefore, selection for ADH deficient mutants could help to develop cell lines and plants tolerant to flooding.
Selection for Salt Tolerance:
The problem of salt affected soils is serious in arid and semiarid regions. The solution to this problem lies in developing varieties resistant to salt stress. Cell lines tolerating high levels of sodium chloride have been reported in Nicotiana, Medicago, Lycopersicon, and Avena.
Transmission of this trait to the succeeding generations has been demonstrated for Avena and rice. Similarly, there are reports regarding effective selection for metal ion toxicity and herbicide resistance. The details on role of biotechnology for crop improvement are available by Chopra and Narasimhulu (1990).