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The in Vitro Genetics of Barley

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The in Vitro Genetics of Barley Heredity 59 (1987) 293-299 The Genetical Society of Great Britain Received 5December1986 Thein vitro genetics of barley (Hordeum vulgare L.): detection and analysis of reciprocal differences for culture response to 2,4-dichlorophenoxyacetic acid W. Powell and Scottish Crop Research Institute, Pentlandfield, P. D. S. Caligari* Roslin, Midlothian EH25 9RF, Scotland. The procedures of biometrical genetics have been used to detect and analyse reciprocal differences for the response of immature barley embryos to 2,4-dichlorophenoxyacetic acid in culture. Two genotypes were examined (4082 and R23) which had exhibited large reciprocal differences in culture in previous experiments. The F1, F2 and backcross generations, including full reciprocal families, were produced from these, true-breeding genotypes and evaluated in culture. The experiments confirmed the presence of reciprocal differences. Such differences were subsequently shown to stem from cytoplasmic effects alongside effects traceable to the mother tissue. The implications of these findings for the manipulation of barley genotypes in vitro are discussed. INTRODUCTION A number of factors may complicate the analyses of genotypic differences and in this paper Genotypicdifferences exist in the response of attention is focused on a relatively neglected area cereals to in vitro culture and it has become of tissue culture response i.e., the possibility of apparent that a thorough understanding of the reciprocal differences. A diallel analysis of in vitro genetical control of growth and differentiation in response in barley indicated the presence of large such systems is necessary for the efficient use of reciprocal differences when immature embryos cell culture technologies in crop improvement. In were cultured on media containing 2, or more, vitro selection (Bright, 1985), exploitation of soma- mg I_I of 2,4-dichlorophenoxyacetic acid clonal variation (Larkin, 1985) and the possibility (Caligari, Powell and Goodall, 1987). The presence of interspecific gene introgression (Armstrong, of reciprocal differences for culture response has Nakamura and Keller, 1983) rely on the accessibil- also been reported in wheat (Lazar, Collins and ity of commercially desirable cultivars for culture Vian, 1983) and maize (Nesticky et a!., 1983; establishment and plant regeneration. A pre- Beckert and Qing, 1984; Tabata and Motoyoshi, liminary evaluation of culture response (Powell 1965). In a more recent study (Tomes and Smith, and Dunwell, 1987) may allow the identification 1985) on the initiation of embryogenic callus from of responsive genotypes and the transfer of desir- maize genotypes, the reciprocal effects observed able genes controlling culture response may be were attributed to segregating nuclear factors and achieved by conventional methods. Knowledge of explant source rather than cytoplasmic genetic fac- the genetical systems underlying culture response tors. A distinction between maternal and cytoplas- will complement this approach and may even be mic factors is important since the participation of used to predict the response to selection (Mather the cytoplasm would imply that the organellar, and Jinks, 1982) and the probability of obtaining cytoplasmic DNA is involved in the control of desirable genotypes from a given cross (Jinks and culture response. The objective of the present study Pooni, 1976; Caligari, Powell and Jinks, 1985; was to investigate the underlying control of Powell et a!., 1985). differences in culture response to the synthetic *Presentaddress: Department of Agricultural Botany, Plant auxin 2,4-dichiorophenoxyacetic acid (2,4-D). Sciences Laboratories, University of Reading, Whiteknights, This was achieved using biometrical genetical tech- Reading RG6 2AS, U.K. niques which allowed not only the nuclear genetic 294 W. POWELL AND P. D. S. CALIGARI basis to be quantified and analysed but also the family was represented by 5embryosper petri-dish cytoplasmic component. therefore giving a maximum of 30 embryos per family. Examination of the raw data revealed large MATERIALS AND METHODS positive correlations between the estimates of the means and variances. It was therefore considered Experimental material necessary to transform the data. In practice it was found that taking the fourth root (x"4) removed The parental material used in the present study this scalar effect and all subsequent analyses were consisted of the genetically multiply-marked performed on the data transformed in this way spring barley stocks R23 and 4082. R23 possesses a range of morphological markers distributed (see Caligari etal,, 1987). among the seven pairs of homologous chromo- somes with the alleles being in the dominant condi- PRELIMINARYANALYSES tion. 4082 is recessive at the corresponding loci. The parental, F1, F2 and backcross generations Analysesof variance were performed on the trans- including full reciprocal families were produced formed data to test for differences between the 16 in a glasshouse with a minimum 16-hour photo- generation means for the characters: fresh weight period at a temperature of 20°—30°C. Florets were (F.Wt.), dry weight (D.Wt.) and water content emasculated by clipping the lemma and the palea (Wat), and these are presented in table 1. It should and the anthers were removed using fine forceps be noted, however, that the three characters recor- (Pope, 1944). The sixteen families examined in the ded here are not completely independent of one study are given in table 2. In order to avoid prob- another since D.Wt. and Wat make up F.Wt. lems associated with the effects of clipping, all the However, hearing this is mind, the analyses indi- families sampled were clipped at anthesis. cated that there were highly significant differences between families for the three characters scored. Cultureof immature zygotic embryos Furthermore, the interactions between replicates Caryopsescontaining embryos 1-2 mm in length and families were also highly significant (P< were removed from spikes representing each 0.001). The means and standard deviations for the family. In the case of the six-row parent (4082) three characters are given in table 2. These statistics and segregants from crosses with it, only primary may be used to compare the family means as well florets were excised. Caryopses were sterilised for as to detect and test for the presence of reciprocal 15 minutes in 2 per cent sodium hypochlorite with differences. Student's "t-test" was applied and the Tween 20 as a wetting agent. They were then values for "z" from a number of comparisons of washed in sterile distilled water. Embryos were interest are given in table 3. There were significant excised and placed with the scutellum uppermost differences between the parents for the characters on 20 ml of solidified medium (agar 07 per cent) F.Wt. and Wat. The comparison between families at a frequency of five per petri-dish (90 x 15 mm). 3 and 4 was also highly significant for these two The medium employed was based on that of characters indicating that there were large Murashige and Skoog (1962) with the major salts reciprocal differences between the responses of the at half the recommended concentration, 3 per cent F1 hybrids in culture depending on the direction sucrose and 4 mg l_l of 2,4-D. Dishes were sealed in which the cross was made. This reciprocal with paraflim and incubated at 20°C2°Cwith a difference is presented visually in fig. 1; where 16-hour photoperiod produced by white fluores- immature embryo derived callus from the parental cent tubes (30 pEm2s'). genotypes (R23 and 4082) together with the After 28 days in culture, each embryo and reciprocal F1 hybrids are shown. It can be seen attached callus was harvested. After blotting dry that the expression of the F1 derived embryos in with paper tissues the fresh weight (F.Wt.) was culture is clearly dependent on which direction the recorded in mg. Following drying overnight at cross is made. Thus when 4082 is used as the female 35°C,thedry weight (D.Wt.) was recorded and parent the resulting F1 hybrid embryos are much subsequently the water content (Wat) calculated. more responsive in culture than those derived from the reciprocal. Apart from the comparison of family 3 with family 1 in table 3, the rest of the Experimentaldesign and data transformation comparisons measure reciprocal effects in the F2's Theexperimental design was randomised complete and backcrosses, and most proved to be significant. blocks with six replicates (petri-dishes). Each The fact that not all of them are significant mdi- IN VITRO GENETICS OF BARLEY: RECIPROCAL DIFFERENCES 295 Table 1Analyses of variance for the 16 families raised from the inbred lines R23 and 4082 F.Wt. D.Wt. Wat Item df MS (102) MS (102) MS (102) Reps 5 12.08* 1691*** J3.47*** Families 15 5065*** 3012*** 33.49*** Reps x Families 72 (3)t 801*** 607*** 6.36*** Error 350 (34)f 311 249 237 t The number of missing observations. *PS-005—001; ***p <0001. cates that the cause(s) of such effects may not be tions of cytoplasmic determinants from the male simple and warrant further examination. and female gamete to the zygote (Jinks, 1964). This The comparison between families 3 and I is, type of inheritance is through the female parent in fact, the comparison of the higher scoring F1 which contributes by far the major part of the with the better parent and thus tests for heterosis, zygotic cytoplasm. Maternal or mother tissue although it should be noted that it is not formally effects are transient in nature in that they, by independent of the comparisons between I and 2 definition, change with the genotype of the mother. as well as 3 and 4. There are no significant levels A distinction between these two types of inherit- of heterosis observed, although the comparison for ance is important since a cytoplasmic component Wat approaches significance. There is, therefore would suggest that organellar DNA residing in the no evidence, in this study, for superiority of the chloroplasts or mitochondria is influencing in vitro F1 hybrid over the better scoring parent, for the development.
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