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RESYNTHESIS OF B.NAPUS FROM B.OLERACEA AND B.CAMPESTRIS TO PROVIDE USEFUL TRAITS FOR CANOLA BREEDING.

Ramnik Kahlon 1 Eddie Pang 2, Phil Salisbury 1, Gururaj Kadkol 3, Paul Taylor 1

1 Molecular Plant Genetics and Germplasm Development Group, The University of Melbourne, Parkville, Victoria 3052, Australia.
2
Department of Applied Biology and Biotechnology, RMIT University of Melbourne, Victoria 3000, Australia.
3
Ag-Seed Research, Pty Ltd., P.O. Box 836, Horsham 3042, Victoria, Australia.

ABSTRACT

The lack of genetic information in canola for economically important traits such as resistance to siliqua shatter and tolerance to acid soils hampers the breeding of elite cultivars. Ten lines of B.rapa and B.oleracea were screened for their tolerance to manganese toxicity using a sand culture bioassay with the nutrient solutions at pH 4.1, 4.6 and 5.7. The tolerant genotypes selected were further screened for their tolerance to aluminium. The symptoms of manganese toxicity were most pronounced at pH 4.6 but the plants remained healthy. At pH 4.1 the symptoms of manganese toxicity were not obvious due to plant growth inhibition. Three B.rapa lines - Chitose, Span and Duro were symptomless after first screening at six weeks. In B.oleracea ATC94708 was found to be most tolerant whereas Cauliflower Snowball and Boreal were fairly tolerant to manganese toxicity. F3 progeny of a cross between DS-17-D (shatter resistant) and Torch (shatter susceptible), eight B.oleracea, five B.napus, three B.rapa lines were assessed for the presence of the sh2 shatter resistant gene using SCX-7 primers. The sh2 gene was not present in B.oleracea but was present in resistant B.rapa accessions and some B.napus cultivars.

KEYWORDS: Canola, siliqua shatter, acid soils.

INTRODUCTION

Canola is an important commercial crop in Australia, on an average 1,000,000 ha grown in different regions. As with many crop species, the amount of genetic variation in canola is quite limited when compared to its progenitors. The diploid parents of canola, B.rapa and B.oleracea are well known for their diversity in form, environmental adaptation and end uses. Certain accessions of these species possess many economically important traits, such as shatter resistance and tolerance to acid soils, which are lacking in B.napus. The transfer of these traits to B. napus has not been possible due to post-fertilisation barriers, but with recent advances in tissue culture techniques, it is now possible to resynthesise B. napus from its putative parents. The synthetic B. napus may then be used as a “vehicle” to transfer useful traits into canola.

At present the seed loss from ruptured siliquae can cause yield losses upto 60% after hot dry winds. Windrowing prior to ripening reduces seed loss, but adds to production costs and is not completely effective. Genetic improvement through selection and hybridisation, backcrossing or resynthesis can provide a better alternative. Shatter resistant cultivars can have the potential to save $10 per ha through no windrowing.

Acid soils is another important factor, limiting the production of canola in eastern parts of Australia. Brassica crops grown in the wetter, eastern edge of the wheat belt, (southern NSW and north eastern Victoria), are likely to be affected due to toxic levels of manganese and aluminium. Accessions of B.oleracea and B.rapa will be screened for tolerance to manganese, aluminium toxicity and resistance to siliqua shatter with the aim of reconstructing B.napus genotypes with improved traits. A range of B.oleracea genotypes will be screened for siliqua shatter resistance using a pendulum test that assesses the energy required to rupture siliqua; and PCR based molecular markers will be used to assess the presence of specific shatter resistant genes. Genotypes of B.oleracea and B.rapa exhibiting increased shatter resistance and/or soil acidity tolerance will be used to create synthetic B.napus.

METHODS AND MATERIALS

Source of germplasm : A range of accessions of B.oleracea and B.rapa were obtained from the

Australian Temperate Field Crops Collection (ATFCC) based at the Victorian Institute for Dryland Agriculture, Horsham, Victoria; National Institute for Agrobiological Resource, Ibaraki, Japan; .Department of Vegetable Crops,UC Davis, California and FAL Institute, Federal Republic of Germany.

Manganese assessment: Accessions of B.oleracea and B.rapa were screened for tolerance to manganese. This bioassay involved growing seedlings in pots filled with sand and irrigating with a modified Hoagland No.2 nutrient solutions at final pH levels of 5.7, 4.6 and 4.1. Seedlings were first established in sand then transferred to the culture tanks immediately prior to the secondary root formation. Manganese was added as MnSO4 and solutions were changed once a week. At the first screening, after six weeks, the tolerant genotypes were transferred to potting mix and selfed to produce second generation. The same assay was repeated on second generation selfed plants to identify true to type genotypes. The design of the experiment was a randomised block design RBD, consisting of 3 treatments and 5 replications with three plants of each line grown in each replication.

Aluminium assessment: A bioassay, similiar to the bioassay for manganese tolerance was performed for aluminium tolerance. Culture solutions were supplied with 50 ppm aluminium as Al2( SO4)3, in the first two and the last two weeks, and the pH of the culture solutions was maintained at pH 4.6, 5.7 for the total duration of the of six weeks trial.

Siliqua shattering assessment: Thirty B.oleracea accessions were planted in pots in the glasshouse to study siliqua shatter resistance under controlled conditions. Another experiment has been set up at Horsham, Victoria, for screening accessions for shatter resistance in the field. Shatter resistance will be assessed upon ripening. The procedure for determining shatter resistance is based on the rupture strength of individual siliquae using a pendulum test (Kadkol 1986; Mongkolporn 1998).

Screening of B. oleracea lines with SCX-7 primers: Eight B.oleracea lines used in the trials have been screened with specific oligonucleotide primers (SCX-7) that are linked to one of the known shatter resistant genes (sh2) in Brassica rapa (Mongkolporn 1999). Five B.napus lines, B.rapa accessions IB5 and B46 and a F3 progeny from a cross between DS17-D and Torch.lines were also tested. Young leaf material was taken from the B.oleracea accessions and DNA isolated using a modified CTAB method (Taylor et al 1995). PCR products were separated using gel electrophoresis and the presence of a marker, approximately 1000 bp indicated the presence of the sh2 gene.

RESULTS AND DISCUSSION

Screening for manganese and aluminium tolerance.

The manganese tolerance bioassay showed that B.rapa was very sensitive to manganese toxicity. The symptoms, first appeared in the older leaves at the margins, included necrotic lesions, rolling of edges followed by the appearance of a bumpy leaf surface.(Fig. 1). Later the leaves turned light green and the lesions changed from yellow to brown and the deposition of callose occurred in the leaf tissue (Fig. 2) B.oleracea genotypes developed dark brown spots, another symptom of manganese toxicity not seen in B.rapa.

Fig.1 Symptoms due to manganese toxicity

Fig.2 Callose deposition in the leaf tissue due to excess of manganese

The plants were scored for manganese and aluminium tolerance as described by Carter et al. (1975). The performance of the B.rapa lines in the manganese bioassay is shown in Table1. Cultivars Chitose, MexicoJ, Span and Candle were found to be the most tolerant. The plants of these genotypes were symptomless. Accessions PI 324507 and Duro were assigned symptom rating 3, plants showed slight symptoms. The Dichotoma lines and Hakusu Zairi plants showed the least tolerance with most plants showing distinct symptoms.

Table 1. Symptom rating of 10 lines of B.rapa at pH 4.6

Cultivar

Symptoms

Chitose

 

MexicoJ

plants symptomless

Span Candle

 

PI 324507

plants showed slight
symptoms

Duro

3

Dichotoma lines

 

Hakusu Zairi

plants with distinct symptoms
1,2,3

   

Symptom rating 1-leaf wrinkling; 2-marginal chlorosis; 3-necrosis

Amongst the B. oleracea genotypes, accession ATC94708 (capitata group) was the most tolerant with most plants showing no symptoms (Table 2). Genotypes Snowball, White Rock and Boreal from non- capitata showed slight symptoms whereas, the least tolerant genotypes were Yoshin and Fuji (capitata); Alboglabra, Neckperle and Hammer (non capitata). The manganese tolerant genotypes of

B. oleracea and B.rapa were also assessed for aluminium toxicity. All the genotypes tolerant to manganese toxicity were also tolerant to aluminium in the solution.

Table 2. Symptom rating of 10 lines of B. oleracea at pH 4.6

Cultivar

Symptoms

   

ATC 94708

plants symptomless

   

Cauliflower Snowball

 

Boreal
Cauliflower White Rock

plants showed slight symptoms
2

Yoshin

 

Fuji

 

Alboglabra
Neckperle
Hammer
Flora-Blanca RZ

plants with distinct symptoms
1,2,3

Symptom rating 1-leaf wrinkling; 2-marginal chlorosis; 3-necrosis

Manganese is present in the soil in two forms, a bivalent form and an higher oxide. Both the forms exist in dynamic equilibrium. As the pH of the soil lowers, higher oxide forms are converted to the readily available bivalent forms. Three B.rapa lines - Chitose, Span and Duro were selected as most tolerant, which can now be used in crosses with tolerant lines of B.oleracea. Compared to B.rapa, B.oleracea lines were less tolerant to high levels of manganese. However some of the genotypes exhibited tolerance. Results showed that cauliflowers have good tolerance to manganese toxicity. In general plant growth showed the same pattern, in B.rapa and B.oleracea, healthy growth, severe symptoms and death at pH 5.7, 4.6 and 4.1 respectively. Grouse (B.napus) and Dunkeld (B.napus) were used as control in the trials. Both the B.napus genotypes were found to be highly sensitive to manganese toxicity. Young leaves showed chlorosis whereas, older leaves showed necrosis, leading to the death of the plant.

Attempts to develop a breeding program for tolerance to soil acidity is difficult due to a serious lack of genetic information for this trait. Results showed that B.napus is far more sensitive to acid soils as compared to B.rapa and B.oleracea. A more logical approach would be to search for tolerant genotypes in both the parents of B.napus. Differences between species in their tolerance to metal toxicity may be interpreted as preliminary evidence indicative of some possibility for genetic inheritance of this character. Aluminium is universally known as a non- essential element to plants and has no economic role in agriculture. On the other hand it has been found to be extremely toxic to plants and under certain conditions, such as low pH. The tolerant genotypes of both B.rapa and B.oleracea showed no symptoms of aluminium toxicity with no injury to either shoot or root systems.

Siliqua shatter resistance

The presence of a marker at 1000bp in some of the B.napus B.rapa accessions after PCR with primers SCX-7 indicated the presence of the shatter resistant gene sh2 However none of the B.oleracea lines had this marker and thus the sh2 gene was absent. The absence of the sh2 gene in B.oleracea may correlate with the lack of shatter resistance in this species (Kadkol pers.comm). However the lack of shatter resistance in these lines will be confirmed at maturity using a pendulum test. Shatter resistant genes may be present in B.oleracea lines. In order to enhance shatter resistance in B.napus, lines for shatter resistance need to be determined in B.oleracea and B.rapa before the resynthesis of B.napus.

Line/accession

Phenotype

sh 2 gene

B.oleracea

   

Watanbe Seikou

ND

Aichi D

ND

Snowball

ND

Boreal

ND

Kohlrabi

ND

Kuroba

ND

Yoshin

ND

Vorbote

ND

B.rapa

   

IB5

Resistant

+

B46

Resistant

+

* F3 DS17-D Torch

Resistant

+

B.napus

 

Excel

Susceptible

+

Oscar

Tolerant

+

Eureka

Tolerant

+

Dunkeld

Susceptible

Grouse

Susceptible

* 9 families 5 individuals

ACKNOWLEDGMENTS

I wish to thank all my supervisors for their scientific aid and advice and The Melbourne University for providing Melbourne International Research Scholarship. Special thanks to Dr. Paul Taylor for his cooperation and everwilling support. I also wish to thank Manjit Singh and Bert Collard for constructive review of this manuscript.

REFERENCES

1. Carter, O.G., Rose, I.A. and Reading, P.F. (1975). Variation in Susceptibility to Manganese toxicity in 30 Soybean Genotypes. Crop Science 15:730-732.

2. Kadkol, G.P. (1985). Ph.D Thesis, The University of Melbourne, Parkville, Australia.

3. Kadkol, G.P, Halloran, G.M and Mc Millan, R.H. (1986). Inheritance of siliqua strength in B.rapa L. I. Studies of F2 and backcross populations. Cana.J.Genet.Cytol. 28: 365-373.

4. Mongkolporn, O(1998). Ph.D Thesis,The University of Melbourne, Parkville, Australia.

5. Mongkolporn, O, Pang E C K, Kadkol G P and Taylor PWJ (1999). Evaluation of SCAR Markers for Shatter Resistance in Brassicas. 10th International Rapeseed Conference, Canberra, Australia.

6. Taylor, P.W.J., Fraser, T.A., Ko, H-L and Henry, R J (1995). RAPD analysis of sugarcane during tissue culture. In: M.Terzi, Cella, R. and Falavigan, A. (Eds), Current issues in the Plant Molecular and Cellular Biology, pp 241-246. Kluwer Academic Int., Dordrecht, Netherlands.

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