Breeding “Super Brassica” cultivars at hexaploid level

Guijun Yan1 and Shyama R. Weerakoon2 1School of Plant Biology (MO84), Faculty of Natural and Agricultural Sciences, The University of Western Australia Crawley, Perth, W.A. 6009 Australia, gyan@plants.uwa.edu.au 2Department of Botany, Faculty of Natural Sciences, The Open University of Sri Lanka, P.O. Box 21, Nawala, Sri Lanka, [email protected]

ABSTRACT Plants with higher ploidy levels tend to be more adaptive to harsh growing conditions than their lower ploidy counterparts as multiple copies of genomes can serve as buffers for the changing environments. All the natural Brassica plants are either at diploid or tetraploid levels. A new initiative has recently started to synthesis several hexaploid Brassica populations from different approaches. It is hoped that the inter-cross of the synthesised populations will result in the selection of “Super Brassica” cultivars for oilseed or vegetable production. There are many successful natural hexaploid crop plants including bread wheat, oat and kiwifruit as well as man- made hexaploids such as the hybrid (Triticale) of Triticum and Secale. Among crop species, common wheat (Triticum aestivum L.) is the best example of hexaploid plants having wider adaptation, better quality and higher yielding capacity than tetraploid durum wheat (Triticum durum Desf.). The three most important tetraploid Brassica species (B. napus, AACC; B. juncea, AABB; B. carinata, BBCC) are from the pair wise genome combination of three diploid species (B. rapa, AA; B. oleraceae, CC; B. nigra, BB). Each genome has its own specific genes controlling agronomic important traits. The creation of the allohexaploid will combine the three genomes and those useful genes, hopefully resulting in “Super Brassica” cultivars for agricultural or horticultural exploitation. There are at least three potential approaches to produce hexaploid Brassica using members in the U triangle i.e. using three tetraploids as parents by unilateral unreduced gametes, using one tetraploid and one diploid as parents followed by chromosome doubling (4X – 2X), and using three diploids as parents (2X – 2X – 2X). In addition to diploid and tetraploid Brassica species in the U triangle, there is a wide range of other Brassica species which may also provide useful genes/traits when hybridized with species in the U triangle, to produce hexaploid “Super Brassica” cultivars.

Key words: “Super Brassica”, Allohexaploids, Polyploidy, Interspecific hybridization

INTRODUCTION Brassica is a genus of plants in the mustard family (Brassicaceae). The genus Brassica is exceptionally important for containing more important agricultural and horticultural crops than any other genus. It also includes a number of weeds, both wild taxa and escapees from cultivation. It includes over 30 wild species and hybrids, and numerous additional cultivars and hybrids of cultivated origin. Due to their agricultural importance, Brassica plants have been the subject of much scientific interest (Wikipedia encyclopedia). The close relationship between six particularly important species (B. carinata, B. juncea, B. oleracea, B. napus, B. nigra and B. rapa) is described by the triangle of U theory (U, 1935).

After palm oil and soybean, Brassica oil seed species are the third important oilseed crop in the world. They are an important source of vegetable oil. Among many Brassica species the most common oilseed crops cultivated commercially are rapeseed (B. napus L. and B. rapa L.) and mustard (B. juncea [L.] Czern & Coss). Besides carbohydrates and proteins, fats or oils are an important part of human nutrition being a high-energy food and necessary for the absorption of fat-soluable vitamins. In today’s health conscious world, the fatty acid composition of food in terms of saturated and unsaturated fatty acid is important. Thus oils with low saturated fatty acids (<6%), high oleic acid (>50%), moderate linoleic acid (<40%) and low linolenic (<14%) are considered ideal for edible purposes (Potts et al., 1999).

Canola oil extracted from the seeds has a fatty acid composition of 5% saturated fats, 57% monounsaturated fat (oleic acid), 23% polyunsaturated fats (linolenic) and 10-15% linoleic

- 32 - acids. The high levels of monounsaturated fatty acids (oleic acid) and polyunsaturated fatty acids are beneficial to prevent cardiovascular diseases by reducing cholesterol levels and inhibiting platelet formation. In a review of types of dietery fat and risk of coronary heart diseases, Hu et al. (2001) showed that the type of fatty acid has a more important role than total amount of fat in the diet. They suggest that ω-3 fatty acids from fish or plant sources (α-linoleic acid) substantially lower the risk of cardiovascular mortality. Canola is an important source of α- linoleic acid. Extensive nutritional studies in India have shown that linolenic acid in mustard oil is highly beneficial for vegetarians and low income sections of the society (Ghafoorunissa, 1998). B. oleracea and B. rapa are species highly consumed as leaf vegetables in many countries. Brassica vegetables provide high amounts of vitamin C and soluble fiber and contain multiple nutrients with potent anti-cancer properties: diindolylmethane, sulforaphane and selenium.

Successful polyploids/hexaploids as food crops: Polyploidy is recognized as a major mechanism in plant evolution and adaptation (Ramsey and Schemske, 1998; Bennett, 2004). From a plant breeding point of view, induction of polyploidy may originate new genetic combinations, providing the breeder with more variability. It has been observed in nature that an increase in genome size (polyploids) of plant species is associated with an increase in cell size and dry matter production (Arnold, 1997).

Although, canola is an important crop as a main source of plant-based oil in the world, the highest natural ploidy in the genus Brassica is tetraploid (4x). There are many successful hexaploids (6x) such as bread wheat, kiwifruit, triticale and oat. However, no plant in the genus Brassica is naturally hexaploid. Common wheat (Triticum aestivum L.) is considered as a fine example of hexaploid plants having wider adaptation, better quality and higher yielding capacity than tetraploid durum wheat (Triticum durum Desf.). Durum wheat has limited growing areas and accounts only for 5 % of total wheat production in the world (Anon, 1992). The hexaploid wheat is by far the most important wheat in terms of utilization and geographical distribution (Gooding and Davies, 1997).

Possibilities of wide hybridization in Brassica: The cytology and relationships between six Brassica species are well understood. The six species possess three types of diploid genomes designated as A, B, and C either singly or in pairs (U, 1935). B. napus (AACC), B. juncea (AABB) and B. carinata (BBCC) are the tetraploids reported in nature.

Brassica species provide an opportunity to study rapid genome changes related with polyploidy. Some amphidiploid species, B. napus, B. juncea and B. carinata have been resynthesized by hybridizing diploid species followed by doubling the chromosomes (Song et al., 1993) resulting in completely homozygous polyploid lines. However, little work has been done on the synthesis of hexaploid Brassica species except one case. Hexaploids were synthesized from interspecific crosses between B. rapa (AA) and B. carinata (BBCC) (Meng et al., 1988). Interspecific hybridition between tetraploid species is used in the Brassica genus to broaden the genetic diversity and to transfer valuable traits from one species to another. Success in crosses between B. napus and B. juncea was reported in 1970s (Roy 1978). Meng et al. (1988) succeeded in crossing B. napus x B. carinata using traditional breeding and the hybrids were fertile. The UWA canola group has used double haploid technology to further advance this hybridization (B. napus x B. carinata) and rapidly develop homozygous populations for cultivar development (Nelson et al., 2006).

Interspecific hybridization between a tetraploid and a diploid species is difficult and failures occur at many stages starting from pollination incompatibility to pre/post-germination barriers. Most interspecific crosses do not produce mature seeds due to failure of endosperm development (Nishiyama et al., 1991). Variation in successfulness in F1 hybrids is observed at various stages (Brown and Brown, 1996). Hybridizations between allotetraploid species, B. napus, B. juncea, B. carinata and the diploids B. nigra, B. oleracea and B. rapa are naturally highly infertile. Fertilization may take place, but abortion occurs early in the development of the embryo. Embryo rescue techniques can produce viable hybrids. Ovule culture was used to overcome postzygotic interspecific incompatibility in reciprocal crosses between B. rapa and B.

- 33 - oleracea (Diederichsen and Sacristan, 1994). Similarly, the cross between B. napus and B. oleracea is normally unsuccessful, but the use of embryo culture techniques can produce hybrids (Gowers and Christey, 1999).

In nature, most if not all polyploids have arisen by sexual polyplodization through unreduced (2n) gametes (Ramanna, 1992; Ramsey and Schemske, 1998). The production of unreduced gametes is genetically controlled (Bretagonolle and Thompson, 1995) and has been reported in cultivated plants such as Alstromeria (Ramanna et al., 2003), potato (Peloquin et al., 1992; Ramanna and Jakobsen, 1992), sweet potato (Lopez-Lavalle and Orjeda, 2002), alfalfa (Vorsa and Bingham, 1979; McCoy and Smith, 1983; Mariani et al., 1992; Barcaccia et al., 2003), ryegrass (wagenvoort and Den Nijs, 1992), white clover (Hussain and Williams, 1997), red clover (Parrott and Smith, 1984, 1986; Mousset-Declas et al., 1992, Simioni et al., 2004), kiwifruit (Yan et al. 1997), Rosa hybrida (Crespel and Gudin, 2003) and others.

Although somatic polyploidization is still an important tool in plant breeding (Elliot, 1967; Taylor et al., 1976; Taylor and Quesenberry, 1996), sexual polyploids are gaining increasing importance (Ramanna and Jacobsen, 2003). Alfalfa sexual polyploids are more productive than somatic ones (McCoy and Rowe, 1986). In potato (Carputo and Barone, 2005), alfalfa (Sledge et al., 2002), unreduced gametes are used to transfer desirable characteristics from the wild into the cultivated species. In red clover sexual means can produce polyploid plants more consistantly than somatic tetraploids (Simoni et al., 2006). Identification of unreduced gametes could be achieved by examining sporads (Lyrene et al., 2003) and progeny analysis (Bing et al., 1995; Ramana and Jacobsen, 2003).

Possible approaches to produce hexaploid Brassica cultivars:

Fig. 1 Possible approaches of producing hexaploid Brassica genotypes

Development of hexaploid Brassica plants is a process of learning from nature and applying innovation to advance Brassica production for edible oils, bio-diesel and vegetables in a wide range of environments with higher yield. The synthesis of hexaploid plants with all the A, B and C genomes together will have very good potential to create new crops for domestication. The synthesized new hexaploids may provide traits for resistance to a number of biotic and abiotic stresses including drought, salt, pests and diseases, increased yield in terms of seed production, higher oil content, higher nutrition value, enhanced anti-carcinogenic, anti-oxidant and other medicinal properties as well as phytoremediative properties.

There are at least three possible approaches to produce hexaploid Brassica:

Approach 1: Use one tetraploid and one diploid as parents (4X – 2X) followed by the chromosome doubling of triploid hybrids (Fig. 1) j j j j o o (a) Cross between B. juncea (A A B B ) and B. oleracea (C C ) to obtain interspecific F1 hybrids (AjBjCo) and double the chromosome number to produce hexaploids (AjAjBjBjCoCo). n n n n ni ni (b) Cross between B. napus (A A C C ) and B. nigra (B B ) to obtain interspecific F1 hybrids (AnBniCn) and double the chromosome number to produce hexaploids (AnAnBniBniCnCn).

- 34 - ca ca ca ca r r (c) Cross between B. carinata (B B C C ) and B. rapa (A A ) to obtain interspecific F1 hybrids (ArBcaCca) and double the chromosome number to produce hexaploids (ArArBcaBcaCcaCca).

Approach 2: Use three tetraploids as parents (a) Cross between B. napus (AnAnCnCn) and B. carinata (BcaBcaCcaCca) to produce unbalanced allotetraploids (AnBcaCnCca – unreduced gametes: gametes with the somatic chromosome number, Fig. 2). and cross with B. juncea (AjAjBjBj) to obtain allohexaploids (AnAjBcaBjCnCca ) (Fig. 3). (b) Cross between B. napus (AnAnCnCn) and B. juncea (AjAjBjBj) to produce unbalanced allotetraploids (AnAjBjCn – unreduced gametes) and cross with B. carinata (BcaBcaCcaCca) to obtain allohexaploids (AnAjBcaBjCnCca). (c) Cross between B. carinata (BcaBcaCcaCca) and B. juncea (AjAjBjBj) to produce unbalanced allotetraploids (AjBcaBjCca - – unreduced gametes) and cross with B. napus (AnAnCnCn) to obtain allohexaploids (AnAjBcaBjCnCca).

Fig. 3 A cross of B. napus, B. carinata & B. Fig. 2 Production of unreduced gametes juncea

Approach 3: Use three diploids as parents (2X – 2X – 2X) Cross between B. rapa (ArAr), B. nigra (BniBni) and B. oleracea (CoCo) sequentially to obtain hexaploid hybrids (ArArBniBniCoCo).

Beyond U Triangle: In addition to the commercially important Brassica species included in the U triangle, there is a wide range of important Brassica species such as; B. elongata (Elongated Mustard), B. fruticulosa (Mediterranean Cabbage), B. narinosa (Broadbeaked Mustard), B. perviridis (Tender Green, Mustard Spinach), B. rupestris (Brown Mustard), B. septiceps (Seventop Turnip), B. tournefortii (Asian Mustard) etc. A screening of the above species for resistance to the important fungal pathogens has been carried out (Plummer, 1995; Scholze and Hammer, 1998; Siemens, 2002) and obtained very interesting results. As such, these species may provide genes/traits for resistance to a number of biotic and abiotic stresses as well as genes/traits for yield, oil content, nutrition value and medicinal properties, when hybridized with six Brassica species in the U triangle to produce hexaploid Brassica cultivars.

- 35 - CONCLUSION Polyploidy is accepted as a key mechanism in plant evolution and adaptation (Ramsey and Schemske, 1998; Bennett, 2004). Thus, increase in genome size (polyploids) of plant species is linked with an enhanced cell size and dry matter production (Arnold, 1997). Many successful natural hexaploid crop plants are available including bread wheat, oat and kiwifruit as well as man-made hexaploids such as the hybrid (Triticale) of Triticum and Secale. No natural hexaploid Brassica exists and the production of hexaploid Brassica is still at its initial stages. Meng et al. (1988) has succeeded in crossing B. napus x B. carinata using traditional breeding and able to obtain fertile hybrids. The UWA canola group has gone further and used double haploid technology to advance this hybridization (B. napus x B. carinata) to develop homozygous populations for cultivar development (Nelson et al., 2006). We report here the potential diverse approaches which may possibly be used to produce hexaploid Brassica cultivars. The use of three tetraploids as parents by unreduced gametes, use of one tetraploid and one diploid as parents (4X – 2X) and use of three diploids as parents (2X – 2X – 2X) are the three most possible approaches to produce hexaploid “Super Brassica” cultivars. In addition to the economically important Brassica species included in the U triangle, a wide range of important Brassica species available may provide genes/traits for resistance to a number of biotic and abiotic stresses as well as genes/traits for yield, oil, nutrition value and enhanced medicinal properties when hybridized with six Brassica species in the U triangle.

ACKNOWLEDGEMENTS We thank Associate Professor Julie Plummer, Associate Professor Wallace Cowling, Drs Ping Si, Matthew Nelson, Zakaria Solmain and Sheng Chen, Ms Linda Thompson, Ms Aneeta Pradhan and Ms Annaliese Manson for useful discussions and suggestions.

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