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Genetic evaluation and improvement of acid stress tolerance in lucerne breeding

Xianguang Zhang1, Trevor Garnett1, Kathi Davies2, David Peck1, Alan Humphries1 and Geoff Auricht1

1 South Australian Research and Development Institute (SARDI), Waite Campus, GPO Box 397, Adelaide 5001.
www.sardi.sa.gov.au
Email zhang.xianguang@saugov.sa.gov.au
2
Great Southern Agricultural Research Institute, Department of Agriculture, Western Australia, 10 Dore Street, Katanning, WA
6317. www.agric.wa.gov.au Email kdavies@agric.wa.gov.au

Abstract

Screening systems were developed for testing and selecting lucerne plants tolerant to acidic soils containing aluminium (Al) at low pH. A hydroponic technique was used to screen for Al-tolerant germplasm at the Waite Campus, SARDI. The technique allows examination of effects of Al stress on individual plants by taking into account individual seedling vigour. Results indicated the existence of considerable within-population variation in Al tolerance in the plants tested; these variations in Al response provide the opportunity for genetic improvement of acid stress tolerance through selective breeding of individual plants with relatively long root growth. A glasshouse experiment using pot culture was initiated at Katanning, WA to investigate differential cultivar responses to 3 different acid soils, and the results indicated significant differences of early plant growth characters among the soil types and cultivars tested. The 2 systems are complementary and can be used both alternatively and consecutively in the development of new acid-tolerant lucerne lines.

Media summary

Screening systems were developed for testing and selecting lucerne tolerant to acidic soils containing toxic Al. This will facilitate the genetic improvement in performance of lucerne on acid soils.

Keywords

Selective breeding, nutrient solution, pot culture, field selection, progeny test, farming systems

Introduction

Across Southern Australia dryland salinity or groundwater recharge is a major concern in agricultural sustainability (Cocks 2001; Humphries and Auricht 2001). Reduction of this recharge or deep drainage is an important goal that can be achieved by using perennial crops with year-round high water usage. Lucerne (alfalfa) has been found to be the best choice since it is better suited to many farming systems, compared with many other perennial and annual pasture legumes and grasses. However, a large proportion of the soils in this agricultural region are acidic and/or Al-toxic (Zhang and Jessop 1998), making them inhospitable for many economically important crops including lucerne, a high quality and high value but acid-intolerant pasture crop. Soil acidity and/or aluminium (Al) toxicity seriously limit the production and wider adoption of lucerne. Therefore, it is imperative to develop lucerne cultivars with acid stress tolerance. Improved performance of lucerne on acid soils will ultimately lead to a significant increase in the acreage under lucerne in dryland farming systems and thus boost regional economies. To achieve this objective, research was undertaken to establish a screening system for acid/Al stress tolerance using nutrient solution culture (Garnett et al. 2003) and pot culture. These techniques are being used to develop elite lucerne germplasm with Al tolerance, at low pH, together with other desirable characters suited to the current farming systems in Southern Australia.

Methods

Nutrient solution screening system

A solution culture system was developed under controlled glasshouse conditions to test the relative response to solution Al toxicity in lucerne cultivars and breeding lines, and to select Al-tolerant germplasm at low solution pH (pH 4.5). Preliminary experiments determined the most appropriate Al concentration (ca. 3.0 M) imposed for effective screening, with relative magnitude of root growth as an indicator of apparent Al response. A concentration of 1.5 M Al was found to substantially reduce root elongation of some lucerne cultivars whilst at 10 M Al, few lucerne cultivars and lines showed any root elongation (Garnett et al. 2003).

Experiments were conducted at the Waite Campus, SARDI, South Australia, using a large volume (120 L) solution culture system. The solution used was 1 mM CaC12 (pH 4.5) with AlCl3 as the Al stress. The technique incorporated measuring individual root elongation rates both before and after the imposition of Al stress. Root lengths were measured from digital root images, and growth rates before and after the addition of Al were checked and calculated. In this way the seedling vigour of each genotype within a lucerne population was discriminated. Approximately 200 seedlings of each lucerne population were tested. The experiment used a randomised complete block design replicated 4 times, with cultivar SARDI 10 as a check.

Pot culture techniques

A pot culture experiment was conducted at Katanning, Western Australia (WA) to establish a method of investigating acid tolerance response of lucerne and to select promising plants that are most suited to the acid soil conditions of the region.

Table 1. Description of three soils used in the pot culture at Katanning, WA, 2003

Soil name

Soil description

pH (in CaCl2)

Al level (mg/kg)

Katanning

Sand

5.1

0.7

Eyres

Sandy duplex

4.7

2.0

Ladyman

Loamy duplex

4.5

4.6

Three soils with relatively low pH were collected from the local Katanning district in WA (Table 1). Five cultivars of lucerne with varying winter vigour (in parentheses) were used: Rippa (10), Sceptre (9), SARDI 7 (7), Hunterfield (6) and Cimmaron (4). Planting tubes were sealed and filled with soil. Twenty seeds from each cultivar were sown per tube. There were 4 replications of each cultivar. Tubes were watered to 100% field capacity (FC) when sown, with initial watering containing a complete fertiliser ‘Thrive’. Plants were then watered every 2 days to maintain 100% FC for the first week, and 80% FC for the remaining 3 weeks. Plants were harvested 4 weeks after sowing, washed out of the soil and the number of plants per tube counted. Shoot height and taproot length of individual plants were measured. Roots and shoots were dried and weighed. Data were analysed by ANOVA in GENSTAT.

Field selections of acid-tolerant plants

To speed up the process of developing acid-tolerant lucerne breeding lines with suitability and general adaptation, field selections of individual plants were made in late 2003 from several field trials with low pH and/or moderately high Al concentrations, sown in 1999 (at Katanning), 2001 (Dumbleyung) and 2002 (Buntine), WA. Individual lines with desirable agronomic characters were also short-listed, based on their level of winter activity and persistence through at least 1 dry season. Other criteria used in the selection were herbage production, quality and leaf colour of individual plants within the elite lines. Selected plants were grouped on a line basis, either separately or bulked over lines with similar backgrounds. This constituted a selection intensity of 6.5–18.3%. Selected plants are raised in small plots at the Waite Campus, with plants to be selected for inter-crossing between more promising lines following further field observation. The breeding effectiveness in acid tolerance will be evaluated by estimating genetic gains.

Results and Discussion

Nutrient solution screening system

Root elongation of lucerne was severely reduced by Al toxicity in low pH nutrient solutions. Although only small overall differences in Al tolerance were found among the lucerne populations tested, considerable within-population variation was detected. Individual plants identified as Al-tolerant or moderately Al-tolerant were selected, and are being raised under pot culture conditions. They will be selectively inter-crossed for use in a recurrent phenotypic selection scheme for further evaluation.

By following the nutrient solution system, we will commence mass screening of a range of lucerne cultivars for Al tolerance, along with other desirable characters, such as optimal winter dormancy. Materials to be used in the screening will include current cultivars in commercial production, locally adapted cultivars, breeding lines under further evaluation, newly formed synthetics, and recent overseas introductions.

Based on further screening results, the level of Al stress tolerance/susceptibility of each lucerne population will be determined and quantified as described by Zhang et al. (1999). Selections of individual plants will be made at the seedling stage, using varying selection pressures (2–10 %), depending on the relative magnitudes of tolerance of the lucerne population under investigation or the breeding population to be desired. After approximately 2 cycles of artificial selection, the effectiveness of breeding for acid stress tolerance will be systematically analysed in terms of genetic gains following the approach of Zhang et al. (2002). In addition, selected plants will be grown using pot cultures for continual observation of plant growth and development to test the likely association of seedling Al tolerance with adult plant tolerance, as indicated by the high herbage dry matter production and/or persistence under acid soil conditions.

Pot culture techniques

Results in Table 2 indicated considerable differences in the agronomic characters of the 3 soils used. However, there were significant differences only in shoot height among the 5 lucerne cultivars. There were no significant interactive effects of lucerne cultivar soil for any of the 5 plant characters examined. Another experiment using a range of lucerne cultivars and breeding lines will be conducted to explore the genetic variability in acid tolerance response of lucerne.

Table 2. Shoot height, root length, shoot and root weight per plant and root/shoot ratios (R/S) for 5 cultivars grown for 4 weeks in 3 acid soils (Katanning, WA, 2003).

Cultivar

Soil

Shoot
height
(cm)

Root
length
(cm)

Shoot
weight
(mg)

Root
weight
(mg)

R/S

 

Katanning

8.0

25.5

39.2

54.2

3.27

Cimmaron

Eyres

7.3

18.9

44.4

31.6

0.83

 

Ladyman

3.5

17.4

11.9

18.8

2.33

 

Katanning

9.0

24.9

49.2

65.6

1.51

Hunterfield

Eyres

10.4

25.6

54.6

92.0

1.68

 

Ladyman

2.4

20.1

6.4

12.0

1.88

 

Katanning

11.8

26.2

72.1

122.9

1.52

SARDI7

Eyres

11.9

26.2

62.9

121.9

1.87

 

Ladyman

3.2

21.9

10.8

17.2

1.60

 

Katanning

13.0

25.2

83.3

97.5

1.27

Sceptre

Eyres

11.4

24.5

70.2

56.8

0.85

 

Ladyman

2.8

17.2

7.8

6.1

0.68

 

Katanning

11.5

23.0

65.5

44.8

0.66

Rippa

Eyres

13.4

26.5

75.6

98.2

1.37

 

Ladyman

3.3

18.4

5.1

10.5

2.79

lsd for cultivar

2.3*

4.2ns

22.8ns

40.2ns

1.02ns

lsd for soil

1.8*

3.2*

17.7*

31.2*

0.79ns

lsd for interaction

ns

ns

ns

ns

ns

* main effect significant at P = 0.05; ns, not significant at P = 0.05.

Field selection of adapted, acid-tolerant germplasm

240 individual plants were selected from the field trials of a wide range of lucerne genotypes. These selections were established under spaced field mini-plots for random inter-mating with honey bees and for half-sib seed production. Progeny tests for disease and insect pest resistance or tolerance on maternal parents will be conducted under controlled glasshouse conditions, followed by testing for Al stress tolerance in nutrient solutions to verify the acid tolerance response.

Field selection of lucerne plants from acid sites combined with both the hydroponic test for Al stress tolerance and pot culture of acid soils will more effectively facilitate the selective breeding of acid and /or Al-tolerant lucerne cultivars.

References

Cocks PS (2001). Ecology of herbaceous perennial legumes: a review of characteristics that may provide management options for the control of salinity and waterlogging in dryland cropping systems. Australian Journal of Agricultural Research 52, 137-151.

Garnett T, Peck D, Humphries A, Zhang XG and Auricht G (2003). Screening lucerne germplasm for aluminium tolerance. Proceedings of the Symposium of Genetic Solutions for Hostile Soils. 26-27 Nov. 2003, (CSIRO Plant Industry, Canberra).

Humphries A and Auricht G (2001). Breeding lucerne for Australian’s southern dryland cropping environments. Australian Journal of Agricultural Research 52, 153-169.

Zhang XG, Jessop RS (1998). Analysis of genetic variability of aluminium tolerance response in triticale. Euphytica 102, 177-182.

Zhang XG, Jessop RS and Ellison F (1999). Inheritance of root regrowth as an indicator of apparent aluminum tolerance in triticale. Euphytica 108, 97-103.

Zhang XG, Jessop RS and Ellison F (2002). Differential responses to selection for aluminium stress tolerance in triticale. Australian Journal of Agricultural Research 53, 1295-1303.

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