13 Breeding: An overview

Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 9 1999 Elsevier Science B.V. All rights reserved.

413

13 B R E E D I N G : AN O V E R V I E W Heiko C. Becker (1), H a r m L6ptien (2) a n d G e r h a r d R6bbelen (1)

(1) Institut ffir Pflanzenbau und Pflanzenzfichtung, Georg August Universitdt, G6ttingen (2) Marner GZG Saaten AG, K6nigstrasse 47, D-25709 Marne, Germany Plant b r e e d i n g is a technology b a s e d on n u m e r o u s empirical skills a n d e s t a b l i s h e d practices, b u t also on critical scientific knowledge s u c h as t h a t described in the p r e c e d i n g c h a p t e r s . The sole aim of p l a n t breeding is the d e v e l o p m e n t of p l a n t cultivars with high p e r f o r m a n c e in farm p r o d u c t i o n s a n d the i m p r o v e m e n t of those p r e s e n t l y grown. F r o m its s t a r t to this very end every p l a n t breeding p r o g r a m requires more t h a n a decade of intensive research. Therefore, the financial i n v e s t m e n t r e q u i r e d t o d a y for a single new cultivar of a m a j o r crop species m a y easily exceed a few million US dollars. B e c a u s e of this long period a n d high financial i n v e s t m e n t the economic success of a p l a n t breeding p r o g r a m is highly d e p e n d e n t on the effectiveness of the s e p a r a t e o p e r a t i o n s employed as well as on a n optimized efficient succession of these individual steps within the general outline (Schnell, 1982a). Convincing evidence h a s been p r e s e n t e d above on the i m p o r t a n t p r o g r e s s which h a s been m a d e due to an i n c r e a s e d the u n d e r s t a n d i n g of the p l a n t genome a n d the t e c h n i q u e s of its m a n i p u l a t i o n at a m o l e c u l a r level d u r i n g the last one or two decades. High e x p e c t a t i o n s have b e e n p u t forward regarding their benefits in p l a n t breeding applications. However, so far only a few of these h o p e s have been proven a n d m o s t of the new cultivars p r o d u c e d until now still r e s u l t from r a t h e r traditional p r o c e d u r e s . With this backg r o u n d in mind, p r e s e n t c h a p t e r a i m s to elaborate on the p r e s e n t s t a t u s a n d future potential of p l a n t breeding within the Brassica coenospecies. The g e n u s Brassica c o m p r i s e s a considerable n u m b e r of crops of greatly diverging biological characteristics. The extent of this diversity clearly relates to their u s e s as well as to the history of their d o m e s t i c a t i o n (see c h a p t e r 2 a n d also P r a k a s h a n d Hinata, 1980). The h i g h e s t variability in p l a n t morphology is exhibited w h e r e the p l a n t s are c o n s u m e d as vegetables with root, stems, leaves, a n d t e r m i n a l or axillary b u d s s o m e t i m e s drastically modified to a t t a i n high nutritive qualities. This is p a r t i c u l a r l y true for Brassica oleracea, which h a s a long tradition of c o n s u m p t i o n in Europe. As early as in the 4th c e n t u r y B.C., T h e o p h r a s t o s described the u s e of two different types of

414 cabbage in Greece. Plinius described the cultivation of even six distinct Brassica cultivars in the Roman empire including types which resembled varieties of cabbage, kohlrabi, curly kale, or broccoli of today (K6rber-Grohne, 1987). During the distribution of these novel cultivated forms, new genetic characters were generated also p r e s u m a b l y by genetic introgression through hybridization with other related wild B r a s s i c a species (Gustafsson, 1981). Most interestingly, a parallel evolution occurred in China with B. rapa and B. j u n c e a . In these species, too, ideotypes were developed by m e a n s of selective cultivation, which formed modified plant organs with particular qualities for vegetable uses. For example, as in the E u r o p e a n B. oleracea, varieties of B. rapa and B. j u n c e a evolved which exhibited head forming terminal buds, or thickened shoot stems, or c om pr e s s e d a nd enlarged inflorescences (Wen, 1980; Toxopeus et al., 1987). Much less diversity in phenotype is evident from species cultivated for foraging p u r p o s es , where the growers were mainly interested in the total m a s s of green m a t t e r of the above-ground stands. Plants in which seed is the only harvest, s u c h as the condiment m u s t a r d or in seedoil uses, plant phenotypes r emain e d even more uniform, and they differ from their ancestral weeds in little more t h a n seed retention and seed size. However, two oilseed forms have evolved in the temperate climate of Central Europe within B. nap u s and B. rapa, one for prewinter sowing and one for spring sowing. These do not only differ in their tolerance to cold b u t also in their photoperiodical adaptation, constituting a n o t h e r i m p o r t a n t agronomical distinction. Recently, the oilseed producing brassicas have attracted considerable attention in the world m a r k e t after their seedoil u n d e r w e n t f u n d a m e n t a l quality improvements by genetic selection, which primed rapeseed oil to become one of the world's m o s t valuable health food commodities. This change-over was started 25 years ago by the amphidiploid B. n a p u s , b u t dragged other "rapeseed" species along, s u c h as B. rapa, B. j u n c e a and B. carinata. Notwithstanding all this morphological diversity, the activity of the breeder is still more affected by differences in reproductive systems of the plants. Most of the diploid B r a s s i c a species represent self incompatible, outcrossing forms, while the amphidiploids are predominantly self compatible and inbreeding. The latter, of course, are the phylogenetically younger species (see c h a p t e r 3). Correspondingly, B. n a p u s is still subject to insufficient shattering resistance, while because of their longer history of cultivation B. j u n c e a and B. c a r i n a t a contain more s h a t t e r resistant types. However, the greatest diversity resides within the B. oleracea and B. rapa group, where the diploid n a t u r e and the n a t u r a l cross fertilization coincide with the most ancient cultivation history allowing for wide ecological dispersal and variable crop characteristics. Again, 'Yellow Sarson', an Indian oilseed subspecies of the diploid B. rapa, not only attained large seeds and s h a t t e r resistant siliques, b u t also self compatibility. In this way, crop development is contin u o u s l y s u p e r i m p o s e d by the effects of (unconscious or conscious) selection a n d corresponding genetic changes.

415

Breeding objectives Vegetable brassicas Since each Brassica vegetable is considered typical for its special characteristics, individual breeding p r o g r a m s have to observe r a t h e r different goals and priorities. Here, for r e a s ons of limited space the situation is mainly presented for the case of breeding white cabbage. This B. oleracea form can roughly be grouped according to the u s e s of its harvested heads. If cultivars are to be grown for industrial processing, highest possible yields, i.e. high head weights, d e m a n d first priority. However, the reverse is also followed in cabbage breeding, i.e. varieties with relatively small h e a d s and low n o n - w r a p p e r leaf size allow for dense planting. This cabbage is to be delivered fresh to the vegetable m a r k e t s and greengroceries, and in this trade low head weights are w ant ed convenient for the small sized family of today. This m a r k e t sector again requires two plant types, one of which is sold directly from the field, while the other needs to tolerate longer storage in a cold depot; the latter type is u s u a l l y distinguished by high dry m a t t e r content as well as by a tight waxy layer. This waxiness of the leaves additionally provides effective protection against biotic and abiotic stress not only during storage, b u t also in the field. For instance, a positive correlation was found to exist between the degree of leaf waxiness and resistance a g a i n s t infestation with Albugo candida and Alternaria ssp. (Subudhi and Raut, 1994). In designing the cabbage ideotype, the breeder h a s to consider t h a t m a r k e t d e m a n d s can be highly divergent in different countries. In Central Europe, a c o m p a c t head is requested for industrial processing, while in the former Yugoslavia or in Turkey more loose leafed h e a d s are preferred. Many m a r k e t s see special value in r o u n d heads, b u t others desire pronouncedl y fiat and even pointed he a de d cabbages. The situation is similar in Chinese cabbage, B. rapa subsp, pekinensis: more ovate and more cylindrical heads are favored in different geographical regions. With regards the main production characters, white cabbage is known to exhibit a wide variation, e.g., in harvest date. For cultivation in Central Europe varieties are available which are ready for harvest from 55 to 150 days after transplanting. A similar s p e c t r u m of cultivars is offered for the Chinese cabbage with m a t u r i t y dates between 55 and 110 days after sowing (Opena et al., 1988). For some forms, for instance curly kale or the late ripening savoy cabbages, winter h a r d i n e s s is of considerable relevance. Here, FI hybrid varieties between white and the m o s t h a r d y Savoy cabbages have been developed specifically for English productions. Based on their frost tolerance these intermediate types with attractive head qualities can be marketed in England all t h r o u g h the winter u p to March directly from the field. F u r t h e r e m i n e n t breeding aims consist in resistance against bolting and splitting of the heads. Good s t a n d i n g ability is a n o t h e r i m p o r t a n t condition for adequate plant health a nd easy mechanical harvesting. For r e a s o n s of

416 high p r o d u c t quality, p a r t i c u l a r a t t e n t i o n m u s t be paid to physiologically conditioned d i s e a s e s s u c h as t i p b u r n c a u s e d by local calcium deficiency in the leaf tissue. Finally, it goes w i t h o u t saying t h a t genetic p a t h o g e n resist a n c e (see c h a p t e r 12) is a n essential r e q u i r e m e n t for all types of vegetables, since c h e m i c a l m e a s u r e s of p l a n t protection are as u n d e s i r e d today as for example m y c o t o x i n s derived from fungal infestations. For obvious r e a s o n s , the b r e e d e r m u s t pay careful a t t e n t i o n to the chemical composition of the m a r k e t e d vegetable. For i n s t a n c e , a high c o n t e n t of vitamin C gives rise to a n improved n u t r i t i o n a l value a n d also prevents greyish discoloration of the " s a u e r k r a u t " , the pickled white cabbage (K~nsch et al., 1989) on i n d u s t r i a l processing. Another role is a s s i g n e d to the glucosinolates, which for long were considered to be noxious a l t h o u g h the positive value of some of t h e s e c o m p o u n d s as antimicrobial or a n t i c a n c e r o g e n i c factors is now well recognized. Glucosinolates are also e s s e n t i a l flavor c o m p o n e n t s a n d it is r e c o m m e n d e d to reduce the c o n t e n t s of sinigrin a n d progoitrin, which both g e n e r a t e b r e a k d o w n p r o d u c t s with a bitter taste in favor of other glucosinolate c o n s t i t u e n t s (Olsson, 1993). Oilseed brassicas

The r a p e s e e d h a r v e s t from the respective B r a s s i c a species is processed to p r o d u c e two m a i n products: oil a n d meal. The oil is u s e d for h u m a n cons u m p t i o n , as salad oil or in the form of m a r g a r i n e , as well as for technical p u r p o s e s , s u c h as l u b r i c a n t s a n d h y d r a u l i c oils, as base chemicals for the o l e o c h e m i s t r y or biodiesel fuel (Shahidi, 1990; Kimber a n d McGregor, 1995). The meal which r e m a i n s after the oil extraction is high in nutritionally firstclass proteins. However, today b e c a u s e of the c r u d e traditional "oil-milling"process, the defatted meal or oilcakes are largely only a s s i g n e d to a n i m a l feed m i x t u r e s or even to fertilizer uses. Recently, the valuable proteins of the r a p e s e e d received new attention, including for u s e in h u m a n c o n s u m p t i o n , a n d gentle oil e x t r a c t i o n t e c h n i q u e s b a s e d on "green chemistry" u s i n g enzyme m a c e r a t i o n a n d avoiding organic solvents are being developed (Jensen et al., 1990). These m a y finally allow f u r t h e r c o - p r o d u c t s , s u c h as lecithins, tocopherols (i.e. vitamin E), hulls a n d fibres to be o b t a i n e d as well as glucosinolates with a h i g h e r quality t h a n ever before (Bagger a n d Sorensen, 1996). T h u s , t a k i n g all potential p r o d u c t s into a c c o u n t , s u c h new concepts will promise economic feasibility a n d greatly add value to even the high-valued r a p e s e e d c o m m o d i t y of today.

Yield p e r f o r m a n c e In the b r e e d i n g of rapeseed, the first a n d ever i m p o r t a n t t a s k is to increase the seed h a r v e s t s per u n i t l a n d surface (see Table 13.1, a n d reviews by A n d e r s s o n a n d Olsson, 1961; Downey a n d R6bbelen, 1989; Downey a n d

417 Rimmer, 1993; Buzza, 1995). This trait, however, is n e i t h e r simple nor indep e n d e n t of e n v i r o n m e n t a l influences a n d agronomic practices. Primarily, the seed yield is c o m p o s e d of three d e t e r m i n i n g c o m p o n e n t s , i.e. the n u m b e r of siliques per u n i t area (determined as n u m b e r of siliques per p l a n t + the n u m b e r of p l a n t s per u n i t area), the n u m b e r of seeds per silique, a n d the individual seed weight. Since these traits are negatively correlated with each other, yield i n c r e a s e s of cultivars u s u a l l y r e s u l t from small i m p r o v e m e n t s r a t h e r t h a n from a m a r k e d step u p w a r d s in one of these comp o n e n t s . Principally all seed m a t t e r is derived from p h o t o s y n t h e s i s in the green leaves a n d s h o o t surfaces a n d the s u b s e q u e n t allocation of these prim a r y a s s i m i l a t e s into the developing seed. This is why the breeder aims for an o p t i m u m h a r v e s t index, i.e. a high seed weight h a r v e s t e d as p e r c e n t of the total p l a n t dry m a t t e r produced. This index averages a b o u t 35% for the best E u r o p e a n w i n t e r - r a p e s e e d cultivars, b u t m a y reach values above 40% by c o n t i n u e d selection. However, for e x p e r i m e n t a l r e a s o n s the breeder rarely if ever d e t e r m i n e s the dry m a t t e r p r o d u c t i o n directly, b u t i n s t e a d selects for a n imagined p l a n t ideotype, which he a s s u m e s to secure a sufficient productivity in the vegetative p h a s e a n d a high t r a n s l o c a t i o n efficiency after flowering (Thurling, 1991). This ideotype conception is b a s e d on his long p e r s o n a l experience with high yielding genotypes at his e x p e r i m e n t station. It not only includes morphological p l a n t c h a r a c t e r s , s u c h as total p l a n t height, internode p a t t e r n , b r a n c h i n g s t r u c t u r e , or leaf a r e a index, b u t also implies developm e n t a l traits, s u c h as seedling vigor, leaf rosette formation, flowering initiation, or length of the seed filling period. Some of the morphological c h a r a c t e r s , in p a r t i c u l a r p l a n t height or basal s h o o t diameter, also c o n t r i b u t e to lodging resistance, which is essential for the final low-loss m e c h a n i c a l h a r v e s t of the seed produced. In addition, early lodging of the green p l a n t greatly i m p a i r s assimilation a n d translocation p r o c e s s e s a n d t h e r e b y m a y reduce seed yield a n d quality considerably. In the same way, a n y other d a m a g e to the green p l a n t surface is d e t r i m e n t a l to yield, w h e t h e r it is yellowing or withering u n d e r salt, d r o u g h t , or h e a t s t r e s s or p l a n t decay from frost d a m a g e . Genetic differences in tolerance to these abiotic s t r e s s e s are well k n o w n between the different oilseed B r a s s i c a species. B. n a p u s , particularly its winter form, is best a d a p t e d to the coastal or at least cooler n o r t h e r n areas. In the c o n t i n e n t a l or dryer a n d w a r m e r regions, B. j u n c e a or some spring types of B. rapa, a n d in the tropical highl a n d s of Ethiopia, B. c a r i n a t a forms are the m o s t productive B r a s s i c a oilseed crops. In order to extend the growing a r e a of the oilseed crop "canola", extensive efforts have recently been directed in C a n a d a to the exploration a n d breeding of these "other" B r a s s i c a species (Rakow, 1995, 1997). In the future, it m a y be possible to transfer s t r e s s tolerance from these species to B. nap u s by interspecific hybridization. In all cases, however, screening for perform a n c e a n d a d a p t a t i o n h a s been of little success, if b a s e d on morphological or physiological traits. Breeders, therefore, still select for the best lines in the field u n d e r h a r s h n a t u r a l conditions. As w e a t h e r c h a n g e s from year to year,

418 T a b l e 13.1 Main target characters for yield improvement in oilseed brassicas.

Component traits:

Number of siliques per unit area Number of seeds per silique Seed weight

Securing traits:

Tolerance to late sowing Winter hardiness Effective regeneration for damage repair Fertiliser use efficiency Tolerance against heat, drought, or salt stress Lodging resistance Uniform seed ripening Early maturity Shattering resistance Disease and pest resistance

T a b l e 13.2 Main diseases of oilseed rape (Brassica napus) in Europe and the prospects of breeding for resistance. Scores range from 1 - low, to 9 = high (after R6bbelen, 1994, modified).

Pathogen

Occurrence

Damage potential

Resistance derived from

Breeding potential

Phoma lingam Sclerotinia sclerotiorum Plasmodiophora brassicae Verticillium dahliae Alternaria brassicae Cylindrosporium concentricum Botrytis cinerea Peronospora parasitica Erysiphe cruciferarum

7 7 4 6 7 5 6 5 2

8 7 7 6 4 4 3 3 1

7 (nap, jun, nig) 1 6 (ole, rap, nig) 5 (napus) 5 (S. alba) 7 (napus) 3 (napus) 2 (napus)

9 1 6 7 3 7 3 2 1

Virus (TuYV)

7

3

7 (rapa)

5

Heterodera spec.

6

2

5 (Sin, Raph)

5

419 stress resistance is a difficult trait to breed for. The same holds true for nutrient use efficiency, which is a m ode r n d e m a n d advanced for r e a s o n s of lowering agricultural i n p u t s and minimizing loads to the environment. In particular, ground water pollution by mineral nitrogen fertilizer is u n d e r public debate, since rapeseed with its high nitrogen r e q u i r e m e n t for o p t i m u m yields and low nitrogen export by the harvested seed, m ay leave unfavorably high a m o u n t s of nitrogen in the harvested fields (Yau and Thurling, 1987 a,b). This draws attention to the last, b u t not the least i m p o r t a n t character for o p t i m u m yields, i.e. the agronomic suitability of the crop within the agricultural production system and its ecological a d a p t a t i o n to the given climatic conditions. In Central Europe, for example, winter forms of rapeseed are essential to m a t c h with the winter cereals in the same rotation. However, not the winter h a r d i n e s s of the rapeseed cultivars, b u t r a t h e r the missing rainfalls in August at sowing are the main obstacle to a satisfactory prewinter e s t a b l i s h m e n t of the stands. In these not infrequent situations, the p r o n o u n ced intolerance of B. n a p u s to late sowing (or late field emergence) may cause severe growth r e t a r d a t i o n s u n d e r the diminishing daylengths and temperatures. This photo- or thermoperiodic sensitivity is expressed to a m u c h greater extent in winter forms of B. n a p u s t h a n of B. rapa. Improved genetic neutrality in this respect might also help to escape late frosts by retarding precocious growth in early spring. But vice versa, every favorable day not utilized for dry m a t t e r production does m e a n a loss to yield potential (Diepenbrock and Grosse, 1995). Because of these complex relations, breeding of high performance cultivars will remain a regional business. Disease an d pest resistance In B r a s s i c a breeding, a great effort is always devoted to improve plant resistance against diseases and pests (see Table 13.2, and reviews by Davies, 1986; Paul an d Rawlinson, 1992; Rimmer and Buchwaldt, 1995). As in every other crop, several diseases cause problems in m o s t growing areas, others are of more local importance, while also individual species m ay suffer from the attack of specialized pathogens. Separate breeding p r o g r a m s towards disease or pest resistance will be rewarding only for the major cultivated species and against the m o s t h a z a r d o u s pathogens, irrespective of possible local calamities. When promising, the breeder will first try to establish effective ways of screening for r e s i s t a n t genotypes and to find sources of disease resistance, which t hen have to be transferred into a useful cultivar. Some principles of this "resistance breeding" are described here for three of the p a th o g en s which affect brassicas. In any rape growing area, particularly at coastal, h u m i d and wind-protected sites, epidemic infections by Sclerotinia s c l e r o t i o r u m can cause premature ripening, deficient seed filling, and extensive shat t eri ng in oilseed stands. Since the f u n g u s covers a wide host range, it is r a t h e r unlikely t h a t durable resistance can be established in cultivars, a l t h o u g h moderate tole-

420 r a n c e h a s b e e n reported to o c c u r in some B r a s s i c a lines (Kolte, 1985; S e d u n et al., 1989). Therefore, a p e t a l o u s m u t a n t s have been p r o p o s e d to provide a n alternative m e a n s of r e d u c i n g Sclerotina a t t a c k s . Usually the s e n e s c e n t petals, w h i c h fall to the leaves or are lodged in the leaf axils after flowering, serve as a n ideal m e d i u m for the a s c o s p o r e s to g e r m i n a t e a n d the mycelium to p e n e t r a t e into the stems. This route is blocked in a p e t a l o u s forms; b u t productive cultivars of this type are still to be developed (Buzza, 1983; Fu et al., 1990; Thurling, 1991). O t h e r m e a n s of protection m a y be provided by gene technology. For example, w h e n the f u n g u s invades the plant, p r o d u c e s oxalic acid as a phytotoxin, which c a u s e s necrosis in the h o s t tissue. After i n t r o d u c t i o n of a gene isolated from w h e a t , which codes for oxalate oxidase, the extent of disease, i.e. the surface of leaf necrosis, was significantly smaller t h a n in n o n - t r a n s g e n i c isolines (Thompson et al., 1995; F r e y s s i n e t et al., 1995). Nowadays, m a n y similar strategies interfering with the p a t h w a y of pat h o g e n e s i s are being extensively studied by biotechnologists (see c h a p t e r 12). Worldwide of u t m o s t i m p o r t a n c e (except in China) is the 'phoma' disease, n a m e d after the a s e x u a l stage of the f u n g u s , Phoma lingam, belonging to the a s c o m y c e t e L e p t o s p h a e r i a maculans. This "blackleg" infection can proceed from b o t h p y c n o s p o r e s a n d a s c o s p o r e s , a n d the r e s u l t i n g mycelia after p e n e t r a t i o n c a n completely girdle the s t e m b a s e ("stem canker") and kill the plant. C h e m i c a l control is not economical p a r t i c u l a r l y in r a p e s e e d winter forms b e c a u s e of the long e x p o s u r e period to infection. B u t breeding h a s been p r o m i s i n g since genetic r e s i s t a n c e w a s first identified a n d widely u s e d in the F r e n c h cultivar 'Jet Neuf (Anonymous, 1982). Selections have been p u r s u e d in field n u r s e r i e s after n a t u r a l or artificially enforced (using diseased stubbles) infections. Meanwhile, g r e e n h o u s e tests by inoculation at the s t e m base have also been successfully applied. New s o u r c e s of resistance are now available in B. napus, being derived from species with the C-genome of B. nigra (Rimmer a n d van den Berg, 1992; Chevre et al. 1996) or from wild forms, e . g . B , rapa s u b s p , sylvestris (Crouch et al., 1994). It is, however, imp o r t a n t to observe t h a t for s u c h genetic testing isolates of L. m a c u l a n s from geographically d i s t a n t s o u r c e s are not t r a n s p o r t e d negligently from region to region on s e e d s or p l a n t residues, b e c a u s e genetic r e c o m b i n a t i o n between t h e m m a y r e s u l t in new i n c r e a s e d virulence (Petrie a n d Lewis, 1985). White r u s t from Albugo c a n d i d a infection m a y c a u s e s u b s t a n t i a l losses of seed yield w h e n it a t t a c k s a n d deforms the floral parts. However, serious d a m a g e is only k n o w n for B. rapa (with race 7 of the fungus) a n d B. j u n c e a (with race 2), while m o s t cultivars of B. n a p u s a p p e a r to be r e s i s t a n t to the p r e v a l e n t r a c e s (Fan et al., 1983). B u t for B. rapa, a high proportion of plants r e s i s t a n t to race 7 were found in the C a n a d i a n cultivar 'Tobin', a n d m a n y yellow seeded oriental m u s t a r d a c c e s s i o n s of B. j u n c e a were r e s i s t a n t to race 2 (Tiwari et al., 1988). M a n y i n s e c t s feed on the l u s h B r a s s i c a p l a n t s a n d those c a u s i n g serious d a m a g e vary with the region a n d p l a n t d e v e l o p m e n t (for review see Lamb, 1989; E k b o m , 1995). Most insect p e s t s of the oilseed b r a s s i c a s are

421 crucifer specialists, particularly a d a p t e d to the glucosinolates which are u b i q u i t o u s in this family. The same s e c o n d a r y c o m p o u n d s which act as feeding d e t e r r e n t s or toxins to livestock a n i m a l s , are a t t r a c t a n t s , feeding stimuli, or oviposition stimuli for the major insect p e s t s of rapeseed. So far, all these can be controlled by insecticides w i t h o u t risk of r e s i d u e s in the processed products; b u t the risk is always p e n d i n g t h a t insect p o p u l a t i o n s m a y develop pesticide resistance. So far, breeding for genetic h o s t p l a n t r e s i s t a n ce to insects h a s yielded very little success. Only selection for rapid seedling development or s h o r t e r flowering period h a s c o n t r i b u t e d to reduce insect damage. In c o m p a r a t i v e trials differences in insect a t t r a c t i o n have been demostrated: e.g., pod midges d a m a g e B. j u n c e a less t h a n B. n a p u s , or seedlings of Sinapis alba are less preferred by flea beetles. Transfer of these characters into B. n a p u s a n d B. rapa m a y be possible; b u t all screening, u s i n g mobile insect agents, is highly laborious indoors a n d unreliable outdoors.

Oilseed quality _

One of the m o s t s p e c t a c u l a r a d v a n c e s m a d e in recent p l a n t breeding h a s been the i m p r o v e m e n t in n u t r i t i o n a l quality of the traditional r a p e s e e d cultivars. The c h a n g e s from high to low erucic acid c o n t e n t of the oil a n d from high to low c o n t e n t of glucosinolates in the meal to p r o d u c e -the so-called "canola"- or 0 0 - r a p e s e e d cultivars have o p e n e d a l m o s t u n l i m i t e d a v e n u e s into the food a n d feed m a r k e t s . This s u c c e s s achieved with traditional breeding m e t h o d s h a s been frequently reviewed (e.g. Downey et at., 1975; Kramer et al., 1983; Downey a n d Rimmer, 1993; Scarth, 1995; Rakow, 1995; Uppstr6m, 1995). Today, r a p e s e e d quality is in the top class c o m p a r e d to other major oilseeds, so at this point the focus m a y be directed on the c h a r a c t e r s still left for f u r t h e r i m p r o v e m e n t (Figure 13.1). Oil: The oil is the m o s t valuable fraction of the seed. Within e s t a b l i s h e d cultivars grown on a large scale, the oil c o n t e n t in the air dry B r a s s i c a seed varies between 40 a n d 47 % for B. n a p u s , 36 a n d 44% for B. rapa, a n d 28 a n d 34% for B. j u n c e a . Within the first two species, winter forms definitely s u r p a s s the seedoil c o n t e n t of the c o r r e s p o n d i n g spring sown forms. Seed of 'Yellow Sarson' (see above), however, p r o d u c e s oil c o n c e n t r a t i o n s m u c h higher t h a n the u s u a l I n d i a n forms of B. rapa, reflecting the s u c c e s s of the long traditional selection of local farmers t o w a r d s large seeded a n d yellow coated, high-oil t u r n i p rape. Altogether, sufficient genetic variability is at h a n d for the breeder, to still secure a slow b u t s t e a d y increase of the oil c o n t e n t in new cultivars, in p a r t i c u l a r b e c a u s e of a d v a n t a g e s from a relatively high heritability (as c o m p a r e d to yield) a n d the i n t r o d u c t i o n of new efficient screening m e t h o d s , i.e. n u c l e a r magnetic r e s o n a n c e (NMR, Madson, 1976) a n d n e a r infrared reflectance s p e c t r o s c o p y (NIRS; T k a c h u k , 1981) for its analytical d e t e r m i n a t i o n . Breeding for oil quality s t a r t e d with the identification of p l a n t s with essentially no erucic acid in their seedoil in B. n a p u s (Stefansson et al., 1961)

422

22.5

Jlllllllllllllllllll Crude protein

IIIIIIIIIIIIIIIIIIII Vitamines, Cholin ~

Minerals Glucose Saccharose

Starch-

(1) o0 tl)

(1) .Q

38.3

m

~

1.1 0.7

E

n

5.0 7.7 ~2.5 4.9

c-.

t-

Hemicellulose 13.0

(D J=

Oligosaccharidea-

Phe~olicsothers-

Phytates Sinapovl esters

4.7

tim

L _ ,

Lignin

m

>

2.5

2.23.2

,

m

t~ (o ~

100 %

C

. . m

43.5

Cr Jde fat !

i

100 % Figure 13.1 Chemical composition of the seed of a 00-cultivar of oilseed rape, Brassica napus (after Thies, 1994).

423 and B. rapa (Downey, 1964), followed by "zero erucic" B. j u n c e a (Kirk and Oram, 1981) and B. carinata (Alonso et al., 1991). This change in erucic acid content led to c o n c o m i t a n t shifts of all other fatty acids c o m p o n e n t s for purely calculatory r e a s o n s (see Table 13.3). While the relative increase in linoleic acid (vitamin F) was welcomed by h u m a n nutritionists, the technological disadvantage of the relatively high linolenic acid cont ent induced extensive breeding efforts (R6bbelen and Thies, 1980a; Scarth et al., 1995). These yielded promising lines with no more t h a n 3% linolenic acid (Rficker a n d R6bbelen, 1996), b u t none of these h a s yet been established at larger scales in agricultural productions. F u r t h e r d e m a n d s of the food i n d u s t r y were met by the breeders after m ut a ge ni c seed t r e a t m e n t a n d extensive screening, with B. n a p u s forms containing high oleic acid a n d others (B. rapa) with elevated palmitic acid c o n t e n t s (Table 13.3). For oleochemical purposes, where rapeseed oils with high erucic acid contents are desired, B. n a p u s lines with up to 60% erucic acid have been selected from interspecific crosses with preselected parents, i . e . B , oleracea cony. botrytis x B. rapa subsp, trilocularis 'Yellow Sarson' (Lfihs a nd Friedt, 1994). But m u c h beyond any n a t u r a l variability, molecular t r a ns f or m a t i on today offers the potential to produce transgenic varieties with u n u s u a l fatty acid profiles or even principally new c o n s t i t u e n t s not synthesized in the conventional B r a s s i c a seed till now (Table 13.4; for review see Murphy, 1994; Voelker, 1997). Evidently, the composition of storage c o m p o u n d s in the B r a s s i c a oilseeds is r e m a r k a b l y open to change, a n d breeding is highly promising provided efforts are sufficiently c o n t i n u o u s an d intense. Meal: The reduction of glucosinolates in the traditional rapeseed to less t h a n 10 % of their original contents (see reviews cited above) was a n o t h e r dramatic d e m o n s t r a t i o n of the potential of conventional breeding (R6bbelen and Thies, 1980 b). Oilseed meal from the new 00-rapeseed cultivars now allows full exploitation of the valuable protein in animal feed mixtures. However, the net protein utilization of rapeseed meal is still m u c h below soybean meal and the total of u n d e s i r e d c o m p o n e n t s still a m o u n t s to more t h a n 25% of the dry m a t t e r (Figure 13.1; for review see Bell, 1993; Uppstr6m, 1995). Hemicellulose with a b o u t 13 % of the dry m a t t e r const i t ut es a major share of the detrimental meal constituents. Youngs (1967; see also Downey et al., 1975) was the first to draw attention to the thin seed coat of yellowseeded B r a s s i c a forms. Since t hen breeders have wished to reduce the total fiber co n ten t of the meal by introducing this easy-to-screen c h a r a c t e r into their cultivars. However, the a p p r o a c h was more complicated t h a n expected and the first phenotypically stable lines of B. n a p u s were obtained only recently (Stringham et al., 1974; Chen and Heneen, 1992; Rashid et al., 1994; Tang et al., 1997). In the dehulled meal fraction, on the other h a n d , soluble oligosaccharides are the major u n d e s i r e d carbohydrates, i.e. raffinose (0,3 %, Gal-Glu-Fru) an d stachyose (2,5 %, Gal-Gal-Glu-Fru), which are undigestible for swine (and h u m a n s ; see also Slominski et al., 1995); they a c c u m u -

424 T a b l e 13.3 Fatty acid c o m p o s i t i o n o f seed oils from B r a s s i c a species (abridged after U p p s t r 6 m , 1995); ~A u l d e t a l . , 1992; 2 including 4% 16" 1.

Cultivar/type (a)

Fatty acid content (%) (b) 16:0

18:0

18:1

18:2

18:3

20:1

22:1

Traditional types

n

Victor (winter)

3.0

0.8

9.9

13.5

9.8

6.3

52.3

n

Target (spring)

3.0

1.5

20.9

13.9

9.1

12.2

38.6

r

Duro (winter)

2.0

1.0

12.9

13.4

9.1

8.9

49.0

r

Echo (spring)

4.5

1.3

33.3

20.4

7.6

9.4

23.0

r

Yellow Sarson

1.8

0.9

13.1

12.0

8.2

6.2

55.5

j

Indian origin

2.5

1.2

8.0

16.4

11.4

6.4

46.2

c

Ethiopian mustard

3.2

0.9

9.8

16.2

13.9

7.5

41.6

Zero erucic types

n

Low 22:1

4

2

62

20

9

2

<1

n

Low 18:3

4

1

59

29

3

1

<1

n

High 18:1 1

4

2

80

4

5

2

<1

r

High 16:0

14 2

1

51

19

13

1

<1

a: n = B r a s s i c a n a p u s ; r = B. rapa; j = B. j u n c e a ; c = B. c a r i n a t a b: Fatty acids represented by carbon chain length and number of double bonds.

late in s e e d d e v e l o p m e n t a n d their c o n t e n t is highly v a r i a b l e with climatic c o n d i t i o n s , w h i c h m a k e s t h e m difficult to select for. Utilization of the seed a n d m e a l is f u r t h e r limited by phenolic acids not only with r e g a r d to a n i m a l feed, b u t also to u s e s of food-grade protein. Their c o n t e n t s in B r a s s i c a oilseeds e x c e e d s t h a t of cotton s e e d a n d p e a n u t by a factor of 10 a n d t h a t of s o y b e a n by 30. Similarly, p h y t a t e s exert a n t i n u t r i t i o nal p r o p e r t i e s in swine feeding, while s i n a p i c acid e s t e r s c a u s e the fishy taste of eggs laid by c e r t a i n h e n races. The l a t t e r two g r o u p s t o g e t h e r m a y r e a c h 10 % of the total seed d r y m a t t e r . B r e e d i n g for r e d u c e d c o n c e n t r a t i o n s h a s n o t yet b e e n s u c c e s s f u l . B u t sufficient genetic v a r i a t i o n is a p p a r e n t at l e a s t from o t h e r c r u c i f e r o u s s p e c i e s a n d q u i c k p h o t o m e t r i c m e t h o d s have b e e n e l a b o r a t e d r e c e n t l y for efficient a n a l y t i c a l s c r e e n i n g (Thies, 1991, 1994).

425 T a b l e 13.4 Varieties of oilseed rape with novel chemical compositions of their seed (Carruthers, 1995).

Non-transgenic varieties (1)

Transgenic varieties (2)

Available now

Zero-erucic acid ('double-low', 50-60% oleic acid) Medium-erucic acid (15-30%) High-erucic acid (40-55%)(HEAR)

Laurie acid (30-40%) Stearic acid (30-40%)

Anticipated in 5-15 years

Very-high-oleic acid (85%) Palmitic acid (10-20%) Very-low
Very-high-erucic acid (60-80%) Ricinoleic acid Petroselinic acid ~,-linolenic acid Epoxy fatty acids Wax esters Polyhydroxybutyrate Pharmac. peptides and proteins

1 Using conventional plant breeding and non-transformation based techniques 2 Using transformation based techniques

Genetical

resources

Because of the m a n y centuries of cultivation and their worldwide distribution, B r a s s i c a crops, - in fact vegetables more t h a n oilseed forms, - passed through an extensive differentiation into an a b u n d a n c e of morpho-, physioand chemotypes. Although regional centers originated during cultivation with specifically adapted landraces in popular use. For example, in Germany close to the city of Stuttgart a landrace of white cabbage, called 'Filderkraut', was grown in the fields until recently, which was conspicuous by its strong growth and its peculiar pointed head. Overall, such high diversity is the genetic base of all our present cultivars, and although no longer grown p e r se, these materials are still of great importance for every modern plant breeding programme; they are the primary resource for essential genes required for the development of new cultivars. Therefore, collections such as those in gene banks, but also individual genotypes existing in remote regions or in the h a n d s of hobby-gardeners will hopefully be conserved and publicly held available for future breeding exercises.

426 Records on the evolution of B. n a p u s as winter oilseed rape are particularly well d o c u m e n t e d in Central E u r o p e (for review see B a u r , 1944), where breeding s t a r t e d from a n u m b e r of l a n d r a c e s locally cultivated in the various rape growing regions. One of these, from which the y o u n g H a n s Lembke selected his ' L e m b k e s W i n t e r r a p s ' at the b e g i n n i n g of this c e n t u r y showed a n e x t r a o r d i n a r y potential for high crop p e r f o r m a n c e , since this landrace h a d evolved on the productive soils a n d in the suitable coastal s e a s o n s at the Baltic Sea, b u t also u n d e r frequent frost s t r e s s in the n o r t h e r n winters a n d d r o u g h t in the early s u m m e r s . A similar local l a n d r a c e from Oberschlesien provided the b a s e for ' J a n e t z k i ' s S c h l e s i s c h e r Winterraps', while other landraces also u s e d in Central E u r o p e were less successful in further cultivar evolution. Different winter rape m a t e r i a l s were derived in the first few deca d e s of this c e n t u r y from E a s t - E u r o p e a n , e.g., Polish l a n d r a c e s which were typically lower in height a n d less vigorous b u t highly frost tolerant, while W e s t - E u r o p e a n , e.g., F r e n c h origins were r a t h e r l u x u r i o u s vegetatively, b u t lacked w i n t e r h a r d i n e s s . Similar l a n d r a c e s are k n o w n for the other Brassica oilseed species a n d their c h a r a c t e r i s t i c s c a n be d e d u c e d from the local e d a p h i c a n d climatic local conditions of their respective c e n t e r s of origin a n d traditional d i s t r i b u t i o n (see c h a p t e r 2). On the other h a n d , long distance seed t r a n s f e r of the Old World B r a s s i c a oilseeds h a p p e n e d early. 'Mansholt's H a m b u r g e r Raps' was i n t r o d u c e d to J a p a n at the t u r n of the 20th century. D u r i n g World War II B r a s s i c a oilseeds were i n t r o d u c e d to C a n a d a , where they are still called 'Argentine' type (B. napus), or 'Polish' type (B. rapa) (Downey et al., 1975). W h e n ongoing available breeding l a n d r a c e s no longer offered sufficient variability to profit from selection, j u s t before the middle of this c e n t u r y b r e e d e r s began to s y s t e m a t i c a l l y compose the desired genetic variations in their basic breeding stocks. Infraspecific crossing between selected p a r e n t s of different l a n d r a c e s or p r e s e n t cultivars was the first a p p r o a c h to be applied. Next c a m e the knowledge t h a t a l t h o u g h spring forms of B. n a p u s exhibit a lower genetic variability, they often c o n s i d e r a b l y i n c r e a s e d selection gain in c r o s s e s with winter forms (Baur, 1944). Obviously, the gene pool of the spring forms is sufficiently different from t h a t of the winter types to provide positive c o m b i n i n g ability (Diers a n d Osborn, 1994; Knaak, 1996). Altogether, the existing high performing cultivars are the m o s t valuable initial breeding m a t e r i a l s a n d so far c o n s t i t u t e a n e s t i m a t e d 80 - 90 % of all genetic r e s o u r c e s applied in r a p e s e e d breeding p r o g r a m s . If r e q u i r e d for specific c h a r a c t e r s not available in the p r e s e n t cultivars, other more or less closely related B r a s s i c a species provide a "secondary gene pool" (Harlan a n d de Wet, 1971) to tap. After U (1935) h a d first synthesized B. n a p u s by interspecific crossing of B. rapa a n d B. oleracea (see c h a p t e r 3), r e s y n t h e s i s of the amphidiploid r a p e s e e d h a s repeatedly been reported in scientific p u b l i c a t i o n s (Andersson a n d Olsson, 1961). B u t the s t a t e m e n t of the last a u t h o r s in 1961 (p. 20): "The possibility of i n c r e a s i n g crop variation is of wide application to p r e s e n t a g r i c u l t u r a l practice" was definitely not true. The t e c h n i c a l difficulties of p r o d u c i n g interspecific h y b r i d s even between more d i s t a n t l y related g e n e r a in the B r a s s i c e a e today are minimized by mo-

427 dern in vitro techniques (see chapter 4). Nevertheless, relatively little use is made of experimentally resynthesized rapeseed in regular cultivar development. This m a y be so because, e.g., G e r m a n breeders clearly r e m e m b e r t hat for breeding the first zero erucic rapeseed cultivar, the semisynthetic cultivar 'Rapol' d e m o n s t r a t e d promising agronomic potential in its cross progenies, b u t had carried t h r o u g h u n n o t i c e d a high P h o m a sensitivity from its B. olerac e a p ar en t producing a devastating epidemic in the first year of commercial 0-rape production in Germany. Of course, these different related B r a s s i c a species contain m a n y useful genes (see the example given above on P h o m a resistance derived from the B. nigra C-genome). But again, allocating these genes into a well performing rapeseed cultivar is a serious m a t t e r of concern, the more alien and extended the transferred genetic s e g m e n t s are. For this reason, B r a s s i c a breeders have widely u s e d ionizing irradiation and chemical seed t r e a t m e n t to induce m u t a t i o n s and screen for desired traits in large progenies (e.g. Rakow, 1973; Velasco et al., 1995; R~cker and R6bbelen, 1996, 1997). This procedure is based on the a s s u m p t i o n t h a t except at the m u t a t e d site the rest of the genotype of the m u t a n t s represents the original high performing cultivar, which is, however, far from being confirmed. This expectation of genetic identity in the b a c k g r o u n d genotype is certainly more valid if molecular gene transfer is applied (see c h a p t e r 9). On the other h a n d , a new transgenic line does not m e a n a comprehensive improvement on the recipient cultivar. Except a single gene t r a n s p o s e d , it still represents the earlier genotype, which in its general characteristics of performance m a y soon be s u r p a s s e d by new cultivars as these c o n t i n u o u s l y emerge from breeders' activities. In this way, m u t a n t s a n d transgenics will finally only contribute some new genes to the c o m m o n pool of the species.

Operational s t e p s for breeding In order to create cultivars better t h a n existing ones and in compliance with specific breeding objectives, every breeding program is divided into three p h a s e s according to Schnell (1982a): 1) procuring appropriate initial variation; 2) establishing c a ndi da t e s for potential cultivars, a n d 3) selecting final cultivars via performance tests. Each of the p h a s e s involve different operations a n d it is the efficiency of these steps p e r s e as well as their effective positioning in the overall breeding method, which determines the pace of progress in breeding.

Procuring variation The first phase of breeding refers to the "genetic resources", i.e. to procure the initial breeding materials, which m u s t include the genetic variation

428 required for the p l a n n e d cultivar. In the breeding history of every crop, the origin a n d n a t u r e of t h e s e base m a t e r i a l s follow a typical s u c c e s s i o n starting with n a t u r a l l y o c c u r r i n g l a n d r a c e s of the species in q u e s t i o n a n d ending with highly e x p e r i m e n t a l genotypic p o p u l a t i o n s p r o d u c e d by sophisticated breeding p r o c e d u r e s . B r a s s i c a b r e e d e r s are u s u a l l y offered a wealth of genetic variations as was a l r e a d y described above. Often, for various r e a s o n s the desired c h a r a c teristics are c o n t a i n e d in a practically u s e l e s s genetic environment. Methods to trim s u c h initial genetic variation for first inclusion in a breeding prog r a m r a n g e from simple to r a t h e r s o p h i s t i c a t e d ones. Cultivation u n d e r appropriate n a t u r a l conditions with, e.g., high disease or p e s t incidence, d u r i n g cold w i n t e r s or u n d e r d r o u g h t or salt s t r e s s m a y preselect in given populations at low cost for desired genetic c o m b i n a t i o n s . In the case of in-breeding m a t e r i a l s desired r e c o m b i n a t i o n s m a y be a u g m e n t e d by a n introduction of male sterility s y s t e m s favoring cross-pollination (Rticker a n d Kr/iling, 1996). In directed crosses, b a c k c r o s s principles will often be applied. If the gene donor p a r e n t is a low yielding "exotic" genotype, the a d v a n c e d recipient parent will a g a i n be u s e d for a s u b s e q u e n t cross in order to increase its genetic proportion in the r e s u l t i n g progeny. A similar a d v a n t a g e m a y come from a "three way cross", where the first h y b r i d is again c r o s s e d with a n o t h e r high performing cultivar with expectedly good combining ability. The more distantly related the cross p a r e n t s are, the more s u c h p r e p a r a t o r y operations are r e q u i r e d to provide for a n initial v a r i a n t useful in f u r t h e r cultivar development. For example, from p r i m a r y a m p h i d i p l o i d s between a B r a s s i c a crop a n d a n alien species, u s u a l l y several b a c k c r o s s e s with the crop genotype a n d s u b s e q u e n t selections are required to remove m o s t of the exotic genotype, to be left with little more t h a n the desired gene or c h r o m o s o m e segment in a r e c o m b i n a n t or t r a n s l o c a t e d fashion within the productive genome. The potentially m o s t p r o m i s i n g genetic material, which is not p r e - a d a p t e d in a n y of these ways to the genotypes prevalent in a practical breeding program, m a y be lost even d u r i n g the first selection cycle b e c a u s e of destructive weakness. B a c k c r o s s i n g c a n be significantly enforced in effect by m a r k e r assisted selection for the genetic b a c k g r o u n d of the r e c u r r e n t p a r e n t , a n d n e a r isogenic lines, which m a y be e s s e n t i a l for the introgression of t r a n s g e n i c traits, can be p r o d u c e d in a more direct way (see c h a p t e r 9).

A highly direct a p p r o a c h of e s t a b l i s h i n g cultivar c a n d i d a t e s is molecular t r a n s f o r m a t i o n (for review see Poulsen, 1996). However, it s h o u l d be well recognized t h a t not every t r a n s f o r m a n t is a l r e a d y a p r o m i s i n g candidate. Apart from the more or less s o p h i s t i c a t e d m o l e c u l a r t e c h n i q u e s to verify the successful t r a n s f o r m a t i o n , copy n u m b e r a n d site of DNA insertion further inform a t i o n is required to identify relevant c a n d i d a t e s . At least twenty or even more i n d e p e n d e n t t r a n s f o r m a t i o n s m i g h t be n e c e s s a r y to s e c u r e the establ i s h m e n t of one p a r t i c u l a r t r a n s g e n i c line for potential application in breeding.

429

Establishing candidates Different types of cultivars can be distinguished by the last reproductive process taking place in the propagation of a cultivar (Schnell, 1982a): Self pollination will lead to line cultivars, panmictic cross pollination to population cultivars, and artificial crossing between seed and pollen parents to hybrid cultivars. Hence the methods to establish candidates are highly dependent on the n a t u r a l reproductive system of a crop. The diploid B r a s s i c a species B. rapa, B. o t e r a c e a and B. nigra are normally strictly cross pollinated due to their self incompatibility system (see chapter 5). However, exceptions exist like the self fertile B. r a p a subsp, trilocularis ('Yellow Sarson'). The amphidiploid B r a s s i c a species generally shows a mixed mating system with predominantly self pollination. The outcrossing rate is about 30 %, but depends on genotype and environmental conditions and may vary in B. n a p u s from 5 % to 70% (Rudloff and Schweiger, 1984; Rakow and Woods, 1987; Becker et al., 1991). Breeding of partially allogamous crops is very difficult and, therefore, in B. n a p u s breeding artificial selfing is widely used in early generations. In this way, B. n a p u s is converted to a self pollinating crop, and s t a n d a r d methods

like pedigree selection or single seed descent can be applied. Without selfing, at least in the early generations, an efficient improvement of quality characters is hardly possible. Moreover, in most countries the high d e m a n d s for uniformity, distinctness and stability of cultivars strongly favor pure lines or F1 hybrids as the desired type of cultivar. An alternative to artificial selfing in s u b s e q u e n t generations is the development of doubled haploid lines. B r a s s i c a crops are among the pioneer ones to use this technique in plant breeding (Chen et al., 1994; Gland-Zwerger, 1995). Already in 1975, the cultivar 'Haplona' was released in the UK, which originated from a spontaneous haploid plant selected in 'Oro' (Thompson, 1979). Today, microspore culture techniques, which have been successfully promoted particularly in B. n a p u s to high effectiveness with almost every plant genotype (see chapter 8), produce the desired homozygosity at once, fully replacing earlier methods with similar effects, e.g. single seed descent. They also work with B. j u n c e a , and even B. rapa, although this self incompatible, fully cross breeding species usually suffers from inbreeding depression, and useful dihaploids are rare (Dewan et al., 1995; Ferrie and Keller, 1995).

Selecting cultivars As soon as individual plants have been obtained, which represent the genotype of the desired cultivar, the third phase commences in the breeding program with intensive testing for final cultivar selection. This is the phase in which modern biotechnologies have produced m a x i m u m benefit and a rapid progress in selection at both the phenotypic and the genotypic levels.

430 At phenotypic levels, yield is still and will continue to be determined largely in field tests over several years and locations. Here, nursery machinery during the last two decades h a s made remarkable progress. Plot seeders, e.g. Oyjord system, or single seed planters guarant ee the possible best positioning of the seed in the seedbed, a nd plot combines the desire for lowloss, clean seed harvest with highly computerized yield dat a assessment. For prescreening of disease resistance, greenhouse tests with artificial inoculation of y o u n g seedlings u n d e r well controlled conditions yield useful results for, e.g., Verticillium dahliae, Alternaria brassicae, Sclerotinia sclerotiorum or even P h o m a lingam. Last but not least the revolutionary success of seed quality improvement is d e p e n d e n t on the development of quick, cheap and sufficiently reliable qualitative analytical tests as well as s u b s e q u e n t accurate quantitative ones described in detail by R6bbelen and Thies (1980a, b). Meanwhile, new physical e q u i p m e n t h a s been developed and n o w - in addition to NMR for the estimation of oil content - NIR spectroscopy allows for a s i m u l t a n e o u s determination of seed humidity, oil, protein and glucosinolate co n ten ts as well as t hat of oleic acid and possibly other fatty acids, each within s o m e w h a t different but mostly satisfying accuracy (Reinhardt, 1992; Tillmann, 1997; Velasco and Becker, 1998). Taken together accounts for an e n o r m o u s l y increased selection intensity in B r a s s i c a oilseed breeding. At genotypic levels, DNA p o l y m o r p h i s m s today offer m a r k e r s to assist in selection for i m p o r t a n t traits as well as in the identification and differentiation of genetic lines a n d cultivars. For example, using DNA-markers virus resistance can be identified without employment of infective insect vectors and prior to the expression of phenotypic symptoms, e.g. in breeding against the turnip yellow virus of rapeseed, B. n a p u s (Graichen, 1994). Restriction fragment length polymorphism (RFLP) m a p s have been produced by several groups of researchers, after the first m a p was published by Landry et al. (1991) for spring rape, B. n a p u s (Chyi et al., 1992; Uzunova et al., 1995). The u s ef u ln es s of these molecular m a p s increases quickly with the density of linked loci as well as with the n u m b e r of important agronomic traits positioned on the map. For this purpose, cDNA probes may be as valuable to associate particular probes with agronomic traits of interest as detailed phenotypic information from the segregating population is u s e d for molecular mapping. With these data, positions may also be m arked for quantitative trait loci (QTL) which are of particular interest in breeding (Weissleder et al., 1996). Like with isozymes, RFLPs and also non-radioactive RAPD-markers permit the introgression of genes, which are difficult to m e a s u r e in their phenotype to be followed. They also offer the chance of pyramiding genes, e.g., for fatty acid synthesis or disease resistance, where phenotypically one gene m a s k s the effect of a n o t h e r (for example see Dion et al., 1995; J o u r d r e n et al., 1996; Kuginuki et al., 1997; Chevre et al., 1997; Voorrips et at., 1997; J e a n et al., 1997). Ultimately, molecular m a r k e r s may provide additional characters for the identification of cultivars for registration and prosecution of property rights (e.g., Mailer et al., 1994; Lee et al., 1995).

431 One aspect rarely considered when describing breeding operations, t h o u g h increasingly important, is the "logistics" of a breeding program (Buzza, 1995). While for winter B. n a p u s in Europe the evaluation of breeders' lines takes u p almost the whole year from sowing in A u g u s t to harvest in following July, spring type and other Brassica species, or more favorable areas where the growing season is b r o u g h t to an earlier end, m a y allow for an extra generation outside the normal season. This can occur in growth rooms or greenhouses, b u t also sometimes m o s t efficiently in "off-season" or "contra-season" (other hemisphere) nurseries.

Breeding m e t h o d s The general usage of the term breeding m e t h o d refers to the overall working program which the breeder p u r s u e s from the initial identification of appropriate source materials until the ultimate release of the new cultivar (Schnell, 1982a). The strategy employed thereby is strongly determined by the mode of reproduction of the species in question and the type of variety to be bred accordingly. In the outbreeding B. oleracea, new "population cultivars" of, e.g., cabbage which were previously raised by m a s s selection, were generated by m e a n s of single plant selection and consecutive progeny testing. The gain in selection could be increased by vegetative m a i n t e n a n c e of the female stock plants, a nd clones of these were planted together in an outcrossing plot as soon as sufficient performance d a t a h a d been raised for their effective selection (Rundfeldt, 1960). On the other h a n d , al t hough the amphidiploid species B. n a p u s a n d B. j u n c e a u n d e r field conditions always undergo some outcrossing, pedigree breeding has been m o s t widely employed to develop "line cultivars". The procedure is similar to t h a t in the self pollinating cereals; b u t B. n a p u s a n d B. j u n c e a differ from these in two import a n t aspects: Firstly, they have a m u c h higher multiplication rate per generation (ca. 1000 : 1) and secondly, they have a p l a n t - t o - p l a n t outcrossing rate ranging from as low as 5 % to m u c h beyond 30 %. Therefore, replicated progeny testing can begin early in the F3 and certain levels of heterosis can be captured from the initial cross and retained in s u b s e q u e n t generations. From this, breeding of improved populations as "synthetic cultivars" was considered b u t little use h a s been made of this m e t h o d in the alloploid species. However, in the self incompatible diploids, mainly B. rapa, population improvement by r e c u r r e n t selection has been an efficient way to develop high yield cultivars (Downey a nd Rakow, 1987), and early progress by this m e a n s h a s been reported in vegetable breeding of cabbage cultivars (Rundfeldt, 1960). Of course, full exploitation of heterosis always requires a complete fertilization control as u s e d in the breeding of conventional "hybrid cultivars"; B. n a p u s cultivars of the latter type are j u s t now (1997) entering seed markets.

432

Breeding of line cultivars Although there are m a n y variations according to the genetic materials to work with, the type a n d inheritance mode of the c h a r a c t e r s to be improved, the available technical a n d financial inputs, or j u s t to the personal preference of the breeder, the m e t h o d of pedigree breeding for B. n a p u s oilseed cultivars essentially proceeds as follows (Figure 13.2): 1. Select p a r e n t a l p l a n t s (P1 a n d P2) with the view to combine the desirable traits from each. 2. Grow F1 p l a n t s in the g r e e n h o u s e u n d e r selfing (to save one year) a n d vernalize artificially (if winter form). 3. Raise spaced p l a n t s of F2 in the field a n d h a r v e s t vigorous, healthy single p l a n t s in a sufficient n u m b e r (to satisfy the expected recombination rate). 4. S u p p l e m e n t preceding p l a n t selection d u r i n g the season in the F2 plot with selection tests d u r i n g winter for quality a n d resistance p a r a m e ters of F3 seeds a n d seedlings, respectively. 5. Raise A-lines from selected F3 plants, bag for self-pollination (where needed) a n d confirm the selection traits. 6. Again raise A-lines from selected F4 plants, bag for selfing a n d after h a r v e s t of the selfed, b u l k all rest p l a n t s per plot. 7. C o n t i n u e A-line sowing a n d single plant selection for field performance within a n d between plots t h r o u g h Fs a n d F6 generations. 8. C o n d u c t replicated p e r f o r m a n c e trials with the F4 bulked seed at several different locations for two successive years. 9. Considering these results, b u l k h a r v e s t the best type-identical, hom o g e n o u s F6 plots belonging to the s a m e F4 single p l a n t descent to obtain a first lot of breeder's seed of the new candidate cultivar. 10. Finally, increase this F7 seed u n d e r isolation to produce the foundation seed, of which part is u s e d for the official trials aimed to secure Plant Breeder's Rights as well as to entitle to seed commercialization. The r e m a i n i n g seed is held in reserve for future cultivar m a i n t e n a n c e a n d multiplication needs. Alternatives to this pedigree m e t h o d are the d e v e l o p m e n t of doubled haploid (DH) lines or the single seed d e s c e n d (SSD) m e t h o d . From a genetical point of view these two m e t h o d s are very similar: both are rapid a p p r o a c h e s to reach homozygosity w i t h o u t a n y selection. In experimental comparisons, DH a n d SSD p o p u l a t i o n s from the s a m e crosses were similar in m e a n a n d variance of the lines (Charne a n d Beversdorf, 1991; S t r i n g h a m and Thiagarajah, 1991). Application of microspore culture saves time, b u t a more intensive selection is required in later g e n e r a t i o n s and, therefore, the time saving effect c a n be smaller t h a n s o m e t i m e s expected. In winter oilseed rape, the

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Figure 13.2 Breeding of line cultivars using pedigree selection of oilseed winter rape, Brassica napus, as applied in Europe (after Downey and R6bbelen, 1989).

434 u s e of d o u b l e d h a p l o i d s m a y r e d u c e the time to develop a new cultivar by one or two y e a r s ( P a u l m a n n a n d F r a u e n , 1991; F r a u e n , 1994). However, the additional y e a r s required for pedigree selection can be u s e d for a better evaluation of the y e a r to year variation of the material. Today double haploids are u s e d in m a n y breeding p r o g r a m s , b u t systematic r e s e a r c h on their efficiency including relative costs c o m p a r e d to o t h e r m e t h o d s is rare. Theoretically, the u s e of DH lines s h o u l d be less efficient in crosses, where one of the p a r e n t s is not a d a p t e d or lacks required quality traits. In s u c h material pedigree selection in early segregating gene r a t i o n s is relatively easy, w h e r e a s u n s e l e c t e d DH lines would contain a large p r o p o r t i o n of u n a c c e p t a b l e types. If, on the contrary, crosses a m o n g relatively n a r r o w elite material are performed, visual selection is m u c h easier a m o n g u n i f o r m DH lines t h a n a m o n g segregating progenies in a pedigree program. There is still some d e b a t e on the q u e s t i o n of which generation s h o u l d be u s e d for the p r o d u c t i o n of DH lines. In m o s t c a s e s F1 derived DH lines are u s e d to get a m a x i m a l gain in time, b u t DH p r o d u c t i o n from F2 or F3 p l a n t s will allow for more r e c o m b i n a t i o n a n d for some preselection of the material as well.

Breeding of population cultivars In the cross pollinated B r a s s i c a species v a r i o u s m e t h o d s of population i m p r o v e m e n t are used. In m a n y c a s e s m a s s selection h a s been successful, b u t this simple m e t h o d is more a n d more replaced by a d v a n c e d types of rec u r r e n t selection (Downey a n d Rakow, 1987). In B. rapa, where self incompatibility e n s u r e s a high degree of heterozygosity u n d e r open pollination, s y n t h e t i c s can be p r o d u c e d by mixing two or at m o s t three p a r e n t a l c o m p o n e n t s . The ' S y n - l ' seed borne from a two c o m p o n e n t synthetic will be c o m p o s e d 25 % of each of the p a r e n t a l s and 50 % hybrids, which provides for a n equal c h a n c e of cross between a n d within lines or h y b r i d s a n d t h u s g u a r a n t e e s stable gene frequencies. This procedure h a s been successfully applied in the first B. r a p a s y n t h e t i c s (Hysyn 100 and H y s y n 110), which were registered in C a n a d a in 1994. Even in p r e d o m i n a n t l y self pollinated species, the breeding of population cultivars was s u g g e s t e d to m a k e at least partial use of the heterosis. Already in the 1970s, before efficient s y s t e m s of fertilization control became available for hybrid breeding in B. n a p u s , breeding of synthetic populations (Figure 13.3; Schnell, 1982b) h a d been proposed. S c h u s t e r (1982) had discovered t h a t if two or more lines or cultivars h a d been grown in a mixed s t a n d (Syn-0), the next generation (Syn-1) will exhibit some of the heterosis of seed yield expected from F1 hybrids. This ' S y n - l ' p o p u l a t i o n will be composed of the c o m p o n e n t p a r e n t a l lines a n d all possible F1 h y b r i d s between them. Leon (1987, 1991) f u r t h e r d e t e r m i n e d t h a t the recovered yield increases over the calculated p a r e n t a l m e a n s were c a u s e d by heterozygosity a n d heterogeneity of this mixture as well a n d t h a t not only yield height b u t also

435 0

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Topcross

GCA yield test

f

Combining cross (Syn-0) Syn-1

t Syn-2

Figure 13.3 Breeding of synthetic cultivars of oilseed rape using inbred lines (Becker, 1993).

436 yield stability increased over years and locations (Schnell and Becker, 1986). The process can be c o n t i n u e d by sowing 'Syn-1' seed to produce 'Syn-2' seed and so on. But the genetic composition of the consecutive generations will change with the n u m b e r of parental lines used and the outcrossing rates between t h e m until the population ha s reached its equilibrium (Geiger, 1982). This led to debates a b o u t how synthetic cultivars should be constructed (Becker, 1982) a nd m a i n t a i n e d (Geiger, 1982) and which synthetic generation should be u s e d for commercial oilseed p r o d u c t i o n s (Becker, 1989) in order to secure a sufficient degree of heterosis. The proportion of hybrids might be increased by selecting parental lines with a high level of self sterility (Hackenberg and K6hler, 1996) or by initiating the synthetic with h a n d crossed F1 seed. So far, m os t of the efforts on utilizing heterosis in oilseed rape, B. n a p u s , are d~rected worldwide to establishing true hybrid cultivars. But this can only be achieved at a great expense and application of the m u c h simpler a p p r o a c h of developing synthetic cultivars should be considered in those cases where the production size and therewith the ret urn of economic investment will be restricted, such as in certain oleochemical or specialized technical oil m a r k e t s (Becker et al., 1998).

Breeding of hybrid cultivars For breeding of hybrid cultivars, three principal requirements have to be fulfilled: (1) there m u s t be a sufficient a m o u n t of heterosis; (2) a system for large scale production of hybrid seed m u s t be available; and (3) an efficient method to identify hybrids with high combining ability. A n u m b e r of studies have shown t h a t there is a considerable heterosis for yield in the oilseed B r a s s i c a species (Table 13.5; Becker, 1987; McVetty, 1995). However, in practical breeding programs, m a n y interesting lines are related by pedigree. In order to maximize heterosis it will be necessary in the long r u n to develop different gene pools. In B. n a p u s it has been suggested to use the diversity between spring and winter type (Diers and Osborne, 1994; Knaak, 1996) or to use resynthesized rapeseed (Becker and Engqvist, 1995). The problem for al m os t two decades has been to find a reliable system for fertilization control, which allows for the production of hybrid seed. Finally, extensive worldwide research and development have led to several different s y s tems which are presently being introduced with first F1 hybrid cultivars in the leading rape growing countries and now have to prove their suitability (also see Downey and Rimmer, 1993; Stiewe et al., 1995; and chapter 6). In principle, three different genetic m e c h a n i s m s exist to produce hybrid seed: (1) self incompatibility (SI), (2) nuclear male sterility (NMS), and (3) cytoplasmic male sterility (CMS). B r a s s i c a is probably the only crop eliminated. Inbreds m a y be obained from in vitro anther- or microspore culture as doubled haploids (Keller and Armstrong, 1983) or by selfing of about five generations. In the latter case, stigmas are h a n d pollinated either several (5-10) days prior to anthesis, when the self incompatibility barrier in the

437 stigma has not yet been developed (Tatebe, 1951), or after previous spraying of the open flowers with a solution of 3% NaC1 (Monteiro et al., 1988). After e s t a b l i s h m e n t of the SI lines, their m a s s propagation is usual l y performed by in vitro microcloning (Clare and Collin, 1973) or by cultivation of the flowering self pollinated plants at an increased concentration (ca. 5 %) of CO2 gas (Nakanishi et al., 1969; Palloix et al., 1985), which reduces the SI reaction to such an extent t h a t self pollen can attain fertilization and seed set. Another method for m a s s propagation in inbred lines is based on the use of near isogenic sublines which only differ with regard to their S-allele constitution, i.e. $1S1 v e r s u s $2S2 and $3S3 v e r s u s 8484 ( Figure 13.4a). These sublines are easily crossed by insect pollination giving rise to very uniform and incompatible S1S2 a n d $3S4 parental lines which are directly u s e d for hybrid seed production (Kuckuck, 1979). Occasionally, high t e m p e r a t u r e s , high air h u m i d i t y or similar environmental anomalies may d i s r u p t SI functions. Therefore, seed lots harvested for F1 plant cultivation are routinely tested for purity of their hybrid character. All seeds, which may have formed within the female by selfing or intracrossing will generate weak inbred plants, which of course are undesired. The proportion of these seeds is determined by use of morphological seedling m a r k e r s or by biochemical markers, s u c h as isozymes or PCR-markers (Nijenhuis, 1971). From experience, the off-types are mainly contained in the small seed fraction. Meanwhile, the combination of breeding procedures and seed production techniques in cabbage regularly produce almost 100 % hybridity in the commercial seed of m o s t cultivars. Hybrid development t h r o u g h SI h a s also been a t t e m p t e d in oilseed breeding with B. n a p u s (McVetty, 1995; O d e n b a c h a n d Beschorner, 1995), although this species is normally self compatible. But SI alleles can be selected within given rapeseed populations or be introduced from B. o l e r a c e a or B. r a p a a n d applied to produce hybrids. Both d o m i n a n t or recessive SI can be u s ed (Ekuere et al., 1995; Kott, 1995). Nuclear male sterility (NMS) is c o m m o n in all plants, b u t its use in breeding is normally restricted by the fact t h a t NMS lines can not be maintained. They have to be pollinated by a fertile line and the progeny will segregate 1:1 for sterile a nd fertile plants. In China, NMS hybrids are produced after removing the fertile plants by h a n d (Fu et al., 1997). Alternatively, a two-gene s y s t e m is also in use in China, which allows the production of completely male sterile lines (Renard et al., 1997). By transgenic m eans, the Belgian c o m p a n y Plant Genetic Systems (PGS) h a s developed the "Seedlink" system (de Both, 1995), where a nuclear sterility gene is linked to a gene for herbicide tolerance; hence, the fertile plants can easily be removed from the propagation fields by spraying with the respective herbicide (see chapt er 6). Cytoplasmic male steriliW (CMS) is a m o s t elegant genetic system of fertilization control. It ha s been found to occur whenever a species was under intensive research (K~ck and Wricke, 1995). The system consists of three

438 c o m p o n e n t s : the A-line, i.e. the male sterile seed p a r e n t carrying a cytoplasmic (mitochondrial) genome which codes for a dysfunction of pollen d e v e l o p m e n t (Stiewe et al., 1995), the fertile B-line to m a i n t a i n the A-line by pollination, a n d the R-line to act as the fertility restorer a n d heterotic partner to the sterile A-line (Figure 13.4b). The respective genic c o m p o n e n t s , i.e. the pollen sterility allele(s) a n d the fertility restorer allele(s) as well as the c o r r e s p o n d i n g c y t o p l a s m are i n t r o d u c e d into existing a d a p t e d cultivars a n d high yielding breeder's lines by m e a n s of backcrossing. In m a n y cases, restorer alleles exist in the actual breeding materials a n d good combining ability m a y easily be selectable within the broad general gene pool. Once the s y s t e m is established, f u r t h e r p r o g r e s s in new hybrid cultivars d e p e n d s on a n a p p r o p r i a t e genetic i m p r o v e m e n t of the hybrid partners, i.e. the A- a n d the R-lines. The breeding of R-lines m a y essentially occur by pedigree breeding as outlined above. Lines of this gene pool with satisfying p e r f o r m a n c e will be exposed to additional tests of c o m b i n i n g ability with corr e s p o n d i n g A-lines. On the other h a n d , A-line i m p r o v e m e n t in a CMS system is a m u c h more t e d i o u s exercise, not only b e c a u s e the first A-line u s u a l l y h a d been j u s t one r a n d o m genotype, b u t also b e c a u s e the development of this pool involves the parallel t r e a t m e n t of both the sterile seed p a r e n t a n d the c o r r e s p o n d e n t fertile m a i n t a i n e r lines (Figure 13.5). Recently estimates of c o m b i n i n g ability have been raised t h r o u g h m o l e c u l a r analysis of genomic (DNA) r e l a t i o n s h i p s between the c a n d i d a t e cultivars or breeding lines by principle c o m p o n e n t a n a l y s i s or by d e n d r o g r a m s . S u c h d a t a m a y provide the breeder with useful e s t i m a t e s for g r o u p i n g his gene pool into heterotic groups for u s e in h y b r i d breeding (Knaak a n d Ecke, 1995; Becker a n d Engqvist, 1995; Voss et al., 1996). In oilseed spring rape, hybrid cultivars have been available since the end of the 1980s m a i n l y based on the 'Polima' CMS s y s t e m (McVetty et al., 1995). In winter type rapeseed, however, it is more difficult to obtain stable CMS. In addition to the 'Polima' s y s t e m (Bartkowiak-Broda, 1995) the 'oguINRA' s y s t e m a n d several other s y s t e m s (Stiewe et al., 1995; P r a k a s h et al., 1995) are in use. In the 'ogu-INRA' system, all presently available restorer lines generate a too high glucosinolate c o n t e n t due to linkage of the restorer gene with a gene for glucosinolate b i o s y n t h e s i s (Delourme et al., 1995). But in cabbage breeding, where the F1 hybrid is h a r v e s t e d as a vegetative produce a n d its p r o d u c t i o n is not d e p e n d e n t on fertility restoration, the oguCMS is well accepted a n d u s e d as a valuable hybridizing system (ClauseSemences, 1994; de Melo et al., 1994). To avoid the p r o b l e m s of high glucosinolate c o n t e n t in oilseed rape production, u n r e s t o r e d 'ogu-INRA' F1 h y b r i d s are mixed with a certain percentage of fertile pollinator plants. S u c h 'composite h y b r i d s ' have been employed in E u r o p e for several years, b u t with i n c o n s i s t e n t results. Under favorable conditions d u r i n g flowering they m a y have an additional advantage due to a positive "sterility effect" b e c a u s e of the energy saved for not producing pollen; b u t in a n u n f a v o r a b l e e n v i r o n m e n t s o m e t i m e s poor seed set can be ob-

439

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Figure 13.4 a) above: Self incompatibility (SI) based hybrid seed production of cabbage (Kuckuck, 1979). b) below: Cytoplasmic male sterility (CMS) based hybrid seed production of oilseed rape (Buzza, 1995).

440

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441 served ( F r a u e n a n d Baer, 1996). Alternatively, t h r e e - w a y h y b r i d s c a n be prod u c e d b e t w e e n a CMS m o t h e r line a n d a n F1 as the male c o m p o n e n t being heterozygote for the r e s t o r e r gene. S u c h t h r e e - w a y h y b r i d s segregate into u n r e s t o r e d p l a n t s with low glucosinolate a n d r e s t o r e d p l a n t s with h i g h e r glucosinolate in a 1:1 ratio. Another male sterility system, n a m e d MSL (Male Sterility Lembke), h a s been developed by the private G e r m a n b r e e d i n g c o m p a n y NPZ-Lembke. This s y s t e m h a s its origin in a sterile p l a n t selected as s p o n t a n e o u s m u t a n t in NPZ n u r s e r i e s in 1983. In a large s c r e e n i n g p r o g r a m m a i n t a i n e r a n d r e s t o r e r genes could be selected. The MSL s y s t e m c a n be c h a r a c t e r i z e d as follows ( P a u l m a n n a n d F r a u e n , 1997): -

-

-

M a i n t a i n e r genes m u s t be selected carefully in a u n i q u e c y t o p l a s m in the a p p r o p r i a t e genetic b a c k g r o u n d . This m a i n t a i n e r m a t e r i a l is the p r o p e r t y of NPZ-Lembke only. Restorer genes are of f r e q u e n t o c c u r r e n c e ; are f u n c t i o n a l restorers.

m o s t released cultivars

Selection for modifier genes is n e c e s s a r y to obtain a n e n v i r o n m e n t a l ly stable male sterility.

The m a i n a d v a n t a g e of the MSL s y s t e m is t h a t r e s t o r a t i o n b e c o m e s only a s e c o n d a r y p r o b l e m since all breeding m a t e r i a l s c a n be directly u s e d as restorers. The v a r i o u s hybridization s y s t e m s differ in their practicability to p r o d u ce hybrid seed. A practical a d v a n t a g e of SI s y s t e m s is t h a t the h y b r i d seed c a n be p r o d u c e d by simply growing a m i x t u r e of the two c o m p o n e n t s . Using male sterility the two c o m p o n e n t s m u s t be grown in stripes a n d only the male sterile c o m p o n e n t harvested. This seed p r o d u c t i o n s y s t e m at h a r v e s t requires a d d i t i o n a l a t t e n t i o n a n d labor a n d , moreover, only t h o s e p a r t s of the field p r o d u c e h y b r i d seed, where the A-lines are raised. Therefore, it is u n d e r d i s c u s s i o n w h e t h e r the male sterile c o m p o n e n t s h o u l d be grown in a m i x t u r e with a small p e r c e n t a g e of the p a r e n t a l c o m p o n e n t a n d all p l a n t s harvested. In this case, the h y b r i d seed would c o n t a i n a certain a m o u n t of the p a r e n t a l genotype, b u t seed p r o d u c t i o n would be m u c h facilitated. S u c h a s y s t e m is s u c c e s s f u l l y u s e d in E u r o p e for h y b r i d rye a n d is u n d e r investigation for c a n o l a in C a n a d a (Renard et al., 1997). Cultivars p r o d u c e d in this way are a p o p u l a t i o n consisting m a i n l y of h y b r i d s with a small p e r c e n t a g e of the male p a r e n t a l line. This type of h y b r i d h a s some similarities with the above m e n t i o n e d composite hybrid cultivars. However, for composite h y b r i d s in a first step 100 % n o n - r e s t o r e d male sterile F1 h y b r i d s are p r o d u c e d , a n d these in a s e c o n d step, are artificially mixed with a n y pollinator, w h i c h m u s t not be genetically related to the hybrid. So far, r e s e a r c h in hybrid b r e e d i n g h a s b e e n very m u c h focused on the d e v e l o p m e n t of a reliable fertilization control. After this s y s t e m is available, it is n e c e s s a r y to optimize all steps of the m e t h o d to identify the b e s t c o m b i n a -

442 tion. Selection of the b e s t cross c o m b i n a t i o n c a n be m a d e in three steps: (1) selection a m o n g the c o m p o n e n t s d u e to their p e r s e performance, (2) selection a m o n g test c r o s s e s for high general c o m b i n i n g ability, a n d (3) selection a m o n g h y b r i d s for b o t h general a n d specific c o m b i n i n g ability. Selection for line p e r s e p e r f o r m a n c e is more i m p o r t a n t in r a p e s e e d t h a n in completely o u t c r o s s i n g crops, like rye a n d maize, b e c a u s e of its relatively lower level of heterosis (Geiger, 1990). If only a limited n u m b e r of sterile testers is available, as is u s u a l in the beginning of h y b r i d breeding p r o g r a m s , it m a y be a r e a s o n a b l y short term strategy to c o n c e n t r a t e on selection of superior pollinators; the seed compon e n t of the hybrid c a n t h e n be directly u s e d as the male sterile tester. But in the long r u n it would be more efficient to u s e a s y s t e m a t i c a p p r o a c h to improve both gene pools for both GCA a n d SCA in two steps like in maize breeding (Figure 13.5).

Breeding results The p r o d u c t i o n of cabbage, B. oleracea, h a s profited considerably from hybrid breeding. The benefits not only consist in i n c r e a s e d yields a n d better targeted c h a r a c t e r c o m b i n a t i o n s , b u t p a r t i c u l a r l y in a better uniformity of the p l a n t s a n d their p r o d u c t s . O p t i m u m m a r k e t qualities as required today can only be o b t a i n e d from F1 hybrid cultivars, which, therefore, by now are the only ones p r o d u c e d in cabbage by all major seed c o m p a n i e s . The application of SI in c a b b a g e breeding h a s been pioneered a n d led to first economic s u c c e s s in J a p a n . Already by 1951 J a p a n e s e h y b r i d s h a d been licenced a n d sold in the seed m a r k e t , with two self incompatible genotypes as p a r e n t a l lines (Yoshikawa, 1993). Similar a p p r o a c h e s are employed in cabbage breeding worldwide. But also in oilseed rape, the first commercial hybrids b a s e d on the SI s y s t e m are m a r k e t e d in C a n a d a a n d China. Worldwide, h y b r i d oilseed rape cultivars m a i n l y rely on the Polima CMS system. The c o u n t r y pioneering in hybrid r a p e s e e d p r o d u c t i o n is China, where in 1996 a b o u t 1.5 million h a were sown with hybrids, which is a b o u t 22 % of the total acreage. In C a n a d a in 1997, the p e r c e n t a g e of hybrids was still less t h a n 10 % of the area. In E u r o p e in 1997, a b o u t 2 2 0 . 0 0 0 composite h y b r i d s b a s e d on the o g u - I N R A s y s t e m were grown, mainly in F r a n c e (Ren a r d et al., 1997). The first spring r a p e s e e d cultivars u s i n g the transgenic "Seedlink" s y s t e m from the Belgian PGS c o m p a n y were released in 1997 in C a n a d a (Renard et al., 1997). However, only with the MSL s y s t e m of CMS, the first fully r e s t o r e d winter r a p e s e e d hybrid cultivars, i.e. 'Joker' a n d 'Pronto', were released in G e r m a n y in 1995, a n d in 1 9 9 6 / 9 7 a b o u t 30 000 h a were p l a n t e d with t h e s e h y b r i d s (Frauen a n d Baer, 1996). Oilseed rape b r e e d i n g since the early 1960s h a d been s h a p e d by the need to improve the quality of the oil a n d meal. The first low erucic acid spring rape cultivar 'Oro' (1966) was developed in C a n a d a from a cross be-

443 tween 'Nugget', a n Argentinian selection, a n d a low erucic selection from the G e r m a n accession 'Liho' (Limburger Hof, BASF), a n d seed of 'Oro' was generously d i s t r i b u t e d to b r e e d e r s all over the world to s t a r t domestic quality breeding p r o g r a m s . Similarly, 'Span' (1971) was developed in C a n a d a by selection for low erucic acid c o n t e n t in B. rapa from the initial Polish accession a n d the cultivar 'Arlo'. Low glucosinolate p l a n t s were first selected from 'Bronowski', a cultivar from Poland i n t e r m e d i a t e between spring a n d winter type, which interestingly e n o u g h belonged to the s a m e gene pool as 'Liho', the d o n o r of the zero erucic character. Worldwide all of the 0 0 - r a p e s e e d cultivars were developed from this origin. 'Erglu' in G e r m a n y a n d 'Tower' in C a n a d a were the first spring 00-types released in 1973, a n d 'Ledos' was the first winter 00-type licensed in G e r m a n y 1976; b u t only with 'Librador' (1981) first p r o d u c t i o n scales were r e a c h e d in 0 0 - w i n t e r rape. Despite of the u n d e n i a b l e s u c c e s s of breeding r a p e s e e d with 00-quality s t a n d a r d , it m u s t be noticed t h a t with each quality change, the first cultivars released were lower in seed a n d oil yields t h a n the earlier low quality forms, a n d it always took m u c h effort a n d several y e a r s to r e a c h a n d later s u r p a s s the former level of p e r f o r m a n c e (see Figure 13.6; alike for C a n a d i a n cultivars see Downey a n d R6bbelen, 1989). This lagging of the quality forms occurred for several r e a s o n s , the m o s t i m p o r t a n t being the drain of selection intensity from yield to quality c h a r a c t e r s ( S a u e r m a n n , 1988; R6bbelen 1989), reflecting breeding priorities a n d capacity limits r a t h e r t h a n physiological constraints. On the contrary, the increasing a t t e n t i o n which r a p e s e e d breeding received from the s e n s a t i o n a l conversion of seed quality, s u p p o r t e d the worldwide s e a r c h a n d exchange of g e r m p l a s m conditioning new, more productive plant ideotypes with higher h a r v e s t index a n d better suitability for m e c h a n i c a l h a r v e s t i n g (Leitzke, 1975; R6bbelen a n d Leitzke, 1974). However, with the e n o r m o u s e x p a n s i o n of acreage, s t r e s s factors increased in n u m b e r a n d intensity. Therefore, after e s t a b l i s h i n g the 00-quality level in winter rape in the 1970s in Europe, the prime of selection w a s aimed at p l a n t resistance. Considerable progress was a t t a i n e d in r e s i s t a n c e a g a i n s t fungal diseases. In p a r t i c u l a r Phoma resistance, which h a d allowed for the u n u s u a l l y wide d i s t r i b u t i o n of the F r e n c h cultivar 'Jet Neuf in the early 1980s, was successfully i n t r o d u c e d into m o s t of the i m p o r t a n t winter rapeseed cultivars. I m p r o v e m e n t s in resistance a g a i n s t Cylindrosporium, Alternar/a a n d Sclerotinia are perceptible, too, in the recent official list of r a p e s e e d cultivars in G e r m a n y (Anonymous, 1996). Breeding p r o g r e s s is also reflected by a strong increase in the n u m b e r of new breeders' lines e n t e r i n g official cultivar tests as in G e r m a n y each year. In winter rape for seed production, there were only 2 c a n d i d a t e s in 1968, b u t there were 47 in 1982, a n d a l m o s t a double n u m b e r is now s u b m i t t e d by the breeders. Correspondingly, the cultivar s p e c t r u m c h a n g e s quickly with a life time of a b o u t 4-6 y e a r s of economic i m p o r t a n c e for m a n y of t h e m (R6bbelen, 1994). For these r e a s o n s , for the individual breeder an a p p r o p r i a t e re-

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445 t u r n of his financial i n v e s t m e n t is only g u a r a n t e e d by the p r e s e n t l y large total r a p e s e e d acreage in the relevant regions, which his cultivars m a y share. Today, the m a i n activities in r a p e s e e d breeding worldwide c o n c e r n the e s t a b l i s h m e n t of F1 hybrid cultivars (Renard et al., 1997). The increase of yield expected from the additional full u s e of heterosis gives promise to produce the a t t a i n e d high p r o d u c t quality of r a p e s e e d in a c o n s i d e r a b l y higher quantity, which will a d d to the superiority of this vegetable oil production. Although only initial c o m b i n a t i o n s are available for commercial production, hybrid cultivars like 'Joker' a n d 'Pronto' have confirmed their yield benefit from heterosis in farmers' fields in 1996 (Table 13.5). While at first there was only one application for concession of license a n d release of F1 hybrid cultivars in G e r m a n y in 1993 a n d three in 1994, far more t h a n half of all cultivar c a n d i d a t e s for the G e r m a n official tests in 1996 were of F1 hybrid type. This alone d e m o n s t r a t e s the powerful a d v e n t of hybrid cultivars in rapeseed, as takes place today in all rape growing c o u n t r i e s of the world.

Future d e v e l o p m e n t s From their experience a n d s u c c e s s a t t a i n e d to date, p l a n t b r e e d e r s expect o p p o r t u n i t i e s for c o n t i n u e d genetic i m p r o v e m e n t of B r a s s i c a crops by straight forward selection to be a l m o s t as p r o m i s i n g today as w h e n the quality i m p r o v e m e n t p r o g r a m was first initiated three d e c a d e s ago. Most of the breeding objectives will be the s a m e as in those early days. However, with the r e m a r k a b l e e x p a n s i o n of breeding tools, in p a r t i c u l a r those derived from biotechnology a n d m o l e c u l a r genetics, the breeder will be able to optimize his classical p r o c e d u r e s considerably as is displayed t h r o u g h o u t this monog r a p h a n d roughly s u m m a r i z e d in Table 13.6. Seed yield, p r o d u c t i o n stability, a n d p r o d u c t quality will r e m a i n the pillars of the r a p e s e e d industry. In the details, however, new objectives will be m a d e accessible to breeding a n d p r o d u c t i o n of B r a s s i c a . Herbicide tolerance as well as disease a n d pest resistance p r o d u c e d by alien gene t r a n s f e r will have a n economic i m p a c t on all B r a s s i c a crops including vegetable cultivars. Tayloring seedoil composition for new technical a n d oleochemical u s e s will be as attractive as "molecular farming" of high value materials s u c h as p o l y h y d r o x y b u t y r a t e , drugs, or horm o n e s (see Table 13.4). This overall confidence in future breeding progress h a s general as well as specific reasons. In general, the rapidly e x p a n d i n g world p o p u l a t i o n will heavily d e p e n d on increased supplies of p l a n t p r o d u c t s . Life on e a r t h is driven by p l a n t phot o s y n t h e s i s which provides the p r i m a r y energy a n d raw m a t e r i a l s for all biological s y n t h e s e s . Man, like all a n i m a l s , is absolutely d e p e n d e n t on a sufficient global level of p l a n t productivity in order to satisfy his n e e d s for food, for direct c o n s u m p t i o n or after refinement by a n i m a l h u s b a n d r y , as well as for all renewable supplies of i n d u s t r i a l raw m a t e r i a l s a n d even organic energy sources. D e m a n d s rapidly multiply with growing world p o p u l a t i o n a n d p r e t e n t i o n s of prosperity. On the other h a n d , crop p r o d u c t i o n increase is

446 T a b l e 13.5 Performance of different types of cultivars of winter rapeseed, Brassica napus; selected data from the official tests in Germany in 1995

and 1996 from a total of 23 locations (*only 9 in 1996 and ** 12 in 1995, respectively) for grain and oil yield relative to the standard cultivars Falcon and Lirajet (= 100).

Cultivar

Released

Grain

Oil

Line cultivars

Falcon Liraj et Express Zenith

1989 1989 1993 1997

98 102 103 109

98 102 109 115

1996 1996

103 * 120

104" 124

1995 1995 1996

107" 119"* 119

109" 122"* 119

40,6

16,8

Composite hybrid eultivars

Synergy Life Restored hybrid cuitivars

Joker Pronto Panther Absolute yield (in dt/ha = 100)

where all three m e c h a n i s m s are successfully used to produce commercial cultivars. Self incompatibility (SI) exists in the diploid B r a s s i c a species and is commercially used to produce hybrid cultivars in B. oleracea. In all cruciferous species, SI is u n d e r sporophytic control (see chapter 5), i.e. both Salleles of the male determine inability of the principally fertile pollen grains to penetrate the stigma and reach the egg cell of the female receptor plant carrying one of these S-alleles in common (Hinata et al., 1994) The first step of an SI based hybrid breeding programme consists in the production of inbred lines with stable SI expression u n d e r all relevant environments. Lines with pseudocompatibility and offspring including self fertile individuals, which occur rather frequently in cauliflower and early red cabbage, m u s t be

447 T a b l e 13.6 Breeding goals for future oilseed rape cultivars.

Preferably used procedures: CS MMGT IC QA-

Classical selection by phenotype Molecular marker use for DNA analysis Gene transfer and gene construction In vitro cell and tissue culture Instrumental quality analytics

1. Increase of grain yield

Broadening of the genetic base by, e.g., interspecific crosses (IC, CS) Improved precision of selection by using - doubled haploid lines (IC, CS), and - molecular markers for selection by genotype, i.e. at the DNA level, rather than by phenotype (MM) Hybrid parents with better combining ability by determination of genetic distances (MM), and establishment of complementary gene pools (MM, CS) 2. Improvement of yield stability

Improved disease resistance (MM, IC, CS, GT) Improved winter hardiness, shattering and lodging resistence (CS, GT) Protection against insect damage (GT) Herbicide tolerance (GT) Improved nutrient efficiency (CS, IC, GT) 3. Improved seed quality

Higher oil content (QA, CS, MM) Higher protein content (QA, CS, MM) Altered fatty acids profile (GT, CS, MM) Synthesis of new fatty acids (GT) Synthesis of new storage proteins (GT)

448 limited by worldwide decline of available arable l a n d a n d by enlarging recognition of the b u r d e n w h i c h intensive a g r i c u l t u r a l land u s e s impose on n a t u ral e c o s y s t e m s . Genetic i m p r o v e m e n t is the only m e a n s to a t t a i n more effective p l a n t p r o d u c t i o n not b a s e d on additional e x t e r n a l i n p u t s b u t on a more efficient t r a n s f o r m a t i o n of solar irradiation a n d i n t e r n a l regulation of metabolic f u n c t i o n s in the crop p l a n t s p e r se. This is the crucial c h a n c e for coming h u m a n g e n e r a t i o n s to s u s t a i n the essential conditions for their life in the future. After the i n d u s t r i a l revolution by c h e m i s t r y a n d physics the 21 st will be the "century of biology" a n d p l a n t breeding will be a major application of all forthcoming scientific a n d technical d e v e l o p m e n t s in this field. In the s a m e context, Brassica crops offer exceptional progress by furt h e r breeding. Brassica oilseeds have gained worldwide a t t e n t i o n in recent decades, a n d a highly potential p l a n t breeding i n d u s t r y h a s been established in m u l t i n a t i o n a l as well as in m e d i u m sized private c o m p a n i e s in all rape growing countries. T h e s e are able to actively utilize m o d e r n biotechnological knowledge a n d discoveries, which h a s been a special d o m a i n of the brassicas. Brassica species have pioneered in the d e v e l o p m e n t of in vitro culture t e c h n i q u e s for m a s s propagation, for microspore c u l t u r e to p r o d u c e doubled haploid lines, a n d for wide hybridization by p r o t o p l a s t fusion. Various gene t r a n s f e r m e t h o d s are highly effective in Brassica a n d information on u s a b l e genes is rapidly e x p a n d i n g s u p p o r t e d by early scientific cooperation worldwide as well as by r e c e n t benefit of extensive r e s e a r c h into the genetic model p l a n t Arabidopsis thaliana, which, b a s e d on genetic s y n t e n y paves the way for even more innovative genetic t r e a t m e n t of the related b r a s s i c a s . There is ample a c c e s s to genetic diversity by classical m e t h o d s , too, a n d m u c h rem a i n s to be exploited in both e s t a b l i s h e d Brassica crops a n d in m a n y allied wild species. Last, b u t not least, m a r k e t s are r e a d y to a b s o r b not only large quantities, b u t also a wide palette of very different quality p r o d u c t s derived from Brassica species, s u c h as vegetables, seedoils as well as seed meals for food, feed, a n d non-food uses, the latter e x t e n d i n g as far as to biodiesel fuels. In all i n s t a n c e s , p l a n t breeding will be pivotal. The preceding c h a p t e r h a s hopefully been convincing t h a t its s u c c e s s is already well programed.

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