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Barley mutants with short roots
Malgorzata Nawrot, Iwona Szarejko and Miroslaw Maluszynski
Department of Genetics, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
e-mail: HYPERLINK "mailto:mnawrot@us.edu.pl" mnawrot@us.edu.pl
Abstract
Nine mutants showing significant shortening of seminal roots have been identified in the collection of dwarf and semi-dwarf forms obtained after mutagenic treatments of spring barley varieties with N-nitroso-N-methylurea and sodium azide. Genetic analysis, performed at the seedling and the spike-emergence stage of plant development, indicated that a single recessive gene was responsible for root shortening in each of analyzed mutants. One short-root mutant developed also very short root hairs. Short roots and short root hairs were controlled by separate genes. The reciprocal crosses of four mutants revealed that they were non allelic.
Introduction
Roots play a decisive role in plant growth and development. The mutational analysis of root system provides a useful tool for revealing the genetic basis of root characters, such as root apical organization, root cell proliferation, secondary root formation, root hair development, and root elongation. The most advanced results on genetics of root system development and morphology were achieved in a model plant species Arabidopsis thaliana, where several root mutants have been described (Baskin et al., 1992; Benfey et al., 1993; Aeschbacher et al, 1994; Baskin et al, 1995; Hauser et al., 1995; Di Laurenzo et al., 1996). In agronomically important crop plants, only a few root system mutants are known (Yao et al., 2003; 2002; Inukai et al., 2001; Tsyganov, 2000; Tsuchiya, 1974). In the presented paper we report on barley mutants characterized by seminal roots significantly shorter than roots of parent varieties during whole vegetation period.
Material and methods
The studies included nine dwarf and semi-dwarf (sd) spring barley mutants developing shorter seminal roots than parent varieties. The mutants, obtained after mutagenic treatment with N-nitroso-N-methylurea (MNH=MNU), were identified in the collection of dwarf and semi-dwarf forms of the Department of Genetics, University of Silesia, Poland. Additionally, one genotype (225DV from cv. Diva) developed also very short root hairs.
Root length at the early developmental stage (8-day old seedling) was evaluated using a paper roller method. The method based on growing seedlings in rollers made of filter paper wrapped tightly around a glass tube (( 2.5 cm). The plastic-coated wires were enclosed between each layer of paper, and the surface of rollers was covered with black foil. Sterilized and pre-germinated seeds (with coleorhiza emerged by 1-2 mm) were placed embryo down, one beside each of two sides of a wire. The rollers were placed in containers with equal level of distilled water and kept in a growth chamber under controlled conditions: illumination 180 Em-2s-1, 16/8h photoperiod and temperature 24/22oC day and night, respectively. At the stage of 8-day old seedling, the length of the longest seminal root and the length of the first leaf were measured. Three replications of each mutant and its parent variety (10 seedlings per replication) were included in each analysis.
The second analysis of root growth was performed at 6-week stage. Plants were grown in the PVC tubes, 125 cm long and 7.5 cm in diameter, filled with sand. To facilitate the extraction of intact roots, plastic foil was used to line the inside of each tube. Pre-germinated seeds were sown into the tubes, and covered with 2-3 cm layer of soil. The experiments were conducted in a glasshouse, under semi-controlled conditions: illumination 200 Em-2s-1, 16/8h photoperiod and temperature 25/15oC day and night, respectively. Every two days, plants were nourished with 100 ml of MS mineral medium (Murashige and Skoog, 1962). The excess of solution flew out freely from the tubes. Plants were gently sprinkled with tap water throughout to prevent desiccation. The experiments were performed in three replications with 3-5 plants per replication. Plants were harvested and washed after 6 weeks and the length of the longest seminal root, and the number of roots was measured.
The analysis of F1 and F2 generation of the crosses between mutants and their parent varieties as well as among selected mutants was performed at the seedling stage. Ten to 20 F1 plants and 100-200 F2 plants were examined for the longest root and the first leaf length.
Results
The selected sd mutants developed significantly shorter seminal roots at both analyzed stages of plant development (Table 1). Mutant 225DV from variety 'Diva' presented the highest level of root and shoot reduction, with root length reaching only about 40% of parent variety at the seedling and 50% at the spike-emergence stage. The root length reduction observed in mutants from variety 'Aramir' ranged from 25% - 48% at both analyzed stages. The results obtained for four mutants from variety 'Delisa' indicated similar level of root length reduction. In most mutants, the shoot length was slightly more reduced at the seedling stage than at maturity.
Table 1. Analysis of root and shoot length in mutants and their parent varieties at the seedling and spike-emergence stage.
GenotypeSeedling stageSpike emergence stageMaturityRoot length
(cm)
_
x SDReduction (%)First leaf length (cm)
(x SDReduction (%)Root length
(cm)
(
x SDReduction (%)Shoot length (cm)
(x SDReduction (%)Aramir32.00.7A*15.10.3A132.63.2A80.44.5A014AR19.80.2B38.112.90.4B14.598.95.3B25.456.60.4BC29.6035AR23.91.4B25.310.10.4B33.192.90.4B29.945.33.1D43.6037AR16.71.6B47.88.70.4C42.480.84.2C39.146.80.6D41.8090AR20.20.8B36.912.90.5B14.699.84.8B24.761.41.4B23.6Diva31.93.7A14.71.0A136.03.3A85.51.3A225DV11.80.5B63.05.60.4B61.966.62.2B51.041.01.1B52.0Delisa27.02.2A13.30.2A128.22.3A86.35.4A522DK15.50.1B42.67.90.4B59.480.65.2B37.158.92.0C31.8538DK16.90.6B37.47.90.3B59.488.62.7B30.966.32.1B23.1587DK16.11.3B40.47.00.9B47.487.12.5B32.065.53.4B24.1588DK15.91.3B41.17.00.4B47.495.62.3B25.466.60.7B22.8* - the same letter for the group of the mutant and parent variety indicates not significant difference for P=0.05.
Analysis of seminal root length in the F1 generation of the crosses mutant x parent variety revealed the recessive character of root phenotypes in all examined mutants. The segregation of F2 progeny indicated that short seminal roots were recessive and monogenically inherited (Tab. 2). The allelism test performed up to now for 4 mutants crossed to each other revealed four different loci responsible for the seminal root shortening. The allelism analysis of other mutants is in progress. Short roots and short root hairs of mutant 225DV from variety Diva were controlled by two separate but linked genes (Tab. 3).
Table 2. Analysis of F1 and F2 generation of the crosses mutant x parent variety and mutant x mutant.GenotypeNo. of analyzed plantsSeminal root length (cm)
(x ( SDNo. of F2 plants with the phenotype of(23:1parent varietymutantAramir3024.5(2.0A*014AR3018.7(1.8BF1014AR x Aramir1723.0(0.7AF2014AR x Aramir9769280.76Aramir3028.90.1A035AR3018.90.5BF1035AR x Aramir2827.71.3AF2035AR x Aramir109109360.002Aramir3024.5(2.0A037AR3012.9(0.5BF1037AR x Aramir1123.3(1.7AF2037AR x Aramir9065250.36Aramir3024.5(2.0A090AR3013.9(0.7BF1090AR x Aramir1324.9(1.2AF2090AR x Aramir8563220.01Diva3027.90.7A225DV307.00.8BF1225DV x Diva2726.50.9AF2225DV x Diva187134531.11Delisa3019.6(0.7A522DK3011.8(0.2BF1522DK x Delisa1121.8(1.1AF2522DK x Delisa12285371.84Delisa3019.6(0.7A538DK3014.2(1.0BF1538DK x Delisa1419.6(0.5AF2538DK x Delisa150113370.01Delisa3019.6(0.7A587DK3013.1(1.0BF1587DK x Delisa1221.9(1.1AF2587DK x Delisa10371322.03Delisa3019.6(0.7a588DK3012.1(1.2bF1588DK x Delisa921.8(2.0aF2588DK x Delisa6848200.18Diva3028.1(1.8A225DV306.4(0.9CAramir3030.41.4A014AR3015.51.5BF1225DV x 014AR828.9(1.7A
Table 2 (continued) . Analysis of F1 and F2 generation of the crosses mutant x parent variety and mutant x mutant.GenotypeNo. of analyzed plantsSeminal root length (cm)
(x ( SDNo. of F2 plants with the phenotype of(23:1parent varietymutantDiva3028.1(1.8A225DV306.4(0.9CAramir3030.41.4A035AR3017.61.2BF1225DV x 035AR929.4(2.7ADiva3028.1(1.8A225DV306.4(0.9CAramir3030.41.4A090AR3014.21.3BF1225DV x 090AR1028.6(1.0AAramir3030.41.4A014AR3015.51.5B035AR3017.61.2BF1014AR x 035AR929.8(1.3AAramir3030.41.4A035AR3017.61.2B090AR3014.21.3BF1035AR x 090AR1028.7(2.1AAramir3030.41.4A014AR3015.51.5B090AR3014.21.3BF1014AR x 090AR828.5(1.0A
* - the same letter in each individual cross indicates non significant difference for P=0.05
Table 3. Analysis of seminal root length and root hair morphology in the F2 generation of the cross mutant 225DV x Diva
Number of seedlings with a phenotype(23:1A,a(23:1B,b(2LA.B.A.bbaaB.aabb12957461.110.52152.72**(2L>3.84, P=0.05
A.B. parental phenotype (long seminal root, normal root hair), A.bb recombinant phenotype (long seminal root, short root hairs), aaB. recombinant phenotype (short seminal root, long root hairs), aabb mutant phenotype (short seminal root, short root hairs).
References
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Tsuchiya, T. (1974). Root characters of curly mutants in barley. BGN 4, 88-91.
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Yao, S.G., Tageta, S. Ichii, M. (2002). A novel short-root gene affects specifically early root development in rice (Oryza sativa L.). Plant Science 163, 207-215.
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