Gametoclonal and Somaclonal Variation among Head Cabbage Androgenic Lines of R 1 and R 2 Generations Obtained from Jaguar F 1 Hybrid

Head cabbage androgenic genotypes of R1 and R2 generations derived from Jaguar F1 donor hybrid were evaluated according to their agroeconomical characters, ability for generative propagation, self-incompatibility, intraline uniformity and stability of morphological characters. Most of R1 and R2 genotypes had desired morphological characters. However, in R1 generation 21 lines showed lack of internal uniformity probably due to the somaclonal variation. Genotypes of R1 and R2 generation from different embryos were more diversified than genotypes derived from the same embryo according to self-incompatibility, ability for seed setting, vegetative period, head shape, internal sump length and mass of head. Rigid selection of androgenic material at R0, R1 and R2 pedigree according to absence of somaclonal variation, agroeconomical traits, self-compatibility and seed setting ability seemed to be crucial for the deriving stable and suitable for the breeding genotypes of head cabbage.


Introduction
Obtaining of androgenic plants from Brassica crops as cabbage, broccoli, cauliflower and Brussels sprout by the use of anther culture technique is widely used as a source of desired genetic diversity (Chiang et al. 1985, Ockendon 1986, 1988, Dore & Boulidard 1988, Chauvin et al. 1993, Górecka et al. 1997, Farnham 1998, Kamiński et al. 1999, Wang et al. 1999, Kamiński et al. 2004, 2005).Androgenic plants with their genetic variation from gametic cells of donor cultivars represent a gametic array each having a different contribution from the parents (Morisson & Evans, 1988).However, anther and microspore cultures are rarely used in cabbage breeding programs probably due to the difficulty in obtaining of androgenic lines with good quality, uniformity and stability in consecutive generations.There are also very few reports with description of practical use of androgenic cabbage genotypes for the creation of commercial cultivars and F 1 hybrids and it practically imposible to determine which cabbage cultivars are based on androgenic parents.Low quality of R 0 androgenic plants and their poor agronomic performance might be caused by the lack of natural selection in the first stages of haploid development as well as by the gametoclonal and somaclonal variation generated itself in plant cell culture (Larkin & Scowcroft 1981).Somaclonal variation that took place during recovery process from in vitro culture, leads to the creation of additional genetic variability with crop improvement potential (Brown & Thorpe, 1995, Evans 1989), but could have a negative effect on the agronomic performance of DH plants, their genetic stability and differentiated ability to generative propagation.This can be the reason why doubled haploid (DH) lines often appear to be inferior in comparison to conventionally obtained inbred lines.Therefore to avoid the undesired source of diversity androgenic material should be multiplied vegetatively or generatively from every single plant (Niemirowicz-Szczytt 1997).Diversified level of intraline uniformity among androgenic Brussels sprout lines of R 2 generation was described by Kamiński (2008), however, the quality and internal uniformity were relatively better than in R 0 and R 1 androgenic populations from Philemon F 1 hybrid (Kamiński 2004(Kamiński , 2005)).Head cabbage is characterized by biennial habit with vernalization period from 8 to 12 weeks, strong depression of inbred lines and usually exhibits diversified and/or high level of self-incompatibility (Thompson 1957, Ockendon 1973, Wallace 1979, Hoser-Krauze 1993, Kamiński 2000, 2001).Cabbage F 1 hybrids are heterozygous according to self-incompatibility and possessed two different s-alleles with four possibly types of domination in pollen and pistil (Haruta 1962, Takayama & Isogai 2003, Stephenson et al 1999, Schopfer et al After evaluation at the field, single plants of R 1 generation with desired agroeconomical characters from internally uniform genotypes were assigned for the generative propagation.For each of selected R 1 genotype ten cuttings were harvested, transplanted, rooted and vernalized.In 2007 twelve R 1 populations obtained from seven androgenic embryos, developed seed stacks and were self pollinated in the greenhouse at open flower and green bud stage.Eight R 1 populations (2.1/31, 2.2/4, 2.2/27, 2.3/14, 2.3/8, 4.2/14, 6.2/7, 6.5/7) were also propagated in 9 m 2 field cages by the use of bees (Osmia rufa) (Table 1).In each cage five vernalized plants of the single androgenic line were planted.Seeds of consecutive R 2 generation were harvested, dried and extracted separately for each of genotype.In 2008 eighteen androgenic genotypes of R 2 generation (twelve obtained in the greenhouse, six from field cages) were evaluated at the field at Research Institute of Vegetable Crops, Skierniewice according to agroeconomical characters.Plants of R 1 (2006) and R 2 (2008) generations developed from seeds in the greenhouse in mid-April.One-month-old seedlings were planted in the field (spacing 50 x 60 cm) in a completely randomized block design with three replications.Each plot consisted of ten plants in one row.The soil type was a pseudopodsolic over loamy sand (1,5 % organic matter, pH 6.5).Fertilization, pest and disease control followed the current recommendations.Plants were harvested gradually from the beginning of August to the end of September when heads reached maturity.Mass, length and width of head and the internal stump length were measured and the head shape (length / width) and internal stump (internal stump length / head height) coefficient were calculated.As a control Jaguar F 1 hybrid was used.Results were subjected to an analysis of variance (ANOVA).The significance of differences among means was evaluated by Newman-Keul's test at α = 0,05.Other morphological characteristics of androgenic population such as: intraline uniformity and length of vegetation period from planting to harvest, were classified separately for each plot.

3.1
Seed productivity of R 0 /R 1 head cabbage androgenic population obtained from Jaguar F 1 hybrid was diversified and ranged from 0.1 seeds/silica for line 2.3/14 to 6.15 seeds/silica for 4.2/15 line after pollination at the bud stage (Table 2).The average seed setting at bud pollination for all genotypes (2.42 seed/silica) was five-fold higher than for open flower pollination (0.53 seed/silica).The average seed set of six genotypes from 4,2 embryo was higher both in bud (4.0 seed/silica) and in open flower stage (1.09 seed/silica), than from other androgenic embryos.Eight genotypes (2.2/4, 2.2/7, 2.2/27, 2.3/8, 4.2/4, 4.5/25, 6.5/1, 6.5/2) did not set seeds after open flower pollination according to the high level of self incompatibility.For all 10 androgenic lines resulted from 6,5 embryo, bud pollination was 10 fold more effective (2.59 seed/silica) than after pollination at opened flower stage (0.22 seed/silica).The ability of seed propagation of androgenic R 1 genotypes from the same embryo was also highly diversified.The highest differences in seed set effectiveness for androgenic genotypes obtained from 4,2 embryo ranged from 0.00 seeds/silica for line 4.2/4 to 3.00 seed/silica for line 4.2/14 when pollinated at open flower stage.The highest differences in seed setting after bud pollination was observed for 2,1 embryo between plant 2.1/12 (0.39 seed/silica) and 2.1/33 (6.00 seed/silica).Androgenic R 1 generation, was also characterized by differentiated ability of seed setting in 2007 (Table 3).In the greenhouse, seed setting of R 1 /R 2 pedigree ranged from 0.05 seed/silica (6.5/7 line) to 1.25 seed/silica (2.2/7 line) for open pollination and from 0.27 seed/silica (2.2/15 line) to 3.14 seed/silica (2.1/31 line) for bud pollination.Two androgenic lines (2.1/31, 2.2/27) propagated at the field, that did not set seeds, were characterized by the high level of self-incompatibility. Two lines (6.2/7, 4.2/14) that set the highest mass of seeds (4.2 g, 2.6 g/plant) were self compatible.Four genotypes (2.2/4, 2.3/14, 2.3/8, 6.5/7) set the average seed yield from 0.1 to 0.5 g/plant and were partially self-compatible.
In both years of field experiment (2006,2008), androgenic genotypes of R 1 generation and 16 androgenic R 2 lines had lower head mass than cv.'Jaguar F 1 ' probably according to inbreeding depression (Table 4, 5).Only in 2008 two lines of R 2 populations: 2.2/4 (3.48 kg) and 2.1/31 (3.58 kg) had similar yield to 'Jaguar F 1 ' (3.22 kg).Androgenic R 1 lines were not significantly diversified as to the mass of head, with the exception of 4.5/25 line (2.47 kg) that yielded better than 4.2/17 (1.47 kg).More significant differences according to head mass between androgenic genotypes of head cabbage were observed in R 2 generation as to inbreeding depression probably.Three androgenic R 2 lines (2.2/4, 6.2/7, 4.2/14), propagated by sib-pollination in the field cages, yielded better than those propagated by self pollination in the greenhouse.Two other lines 6.5/7 and 2.3/8 had higher mass of head when self pollinated at the greenhouse and for 2.3/14 line the mass of head were similar for both methods of propagation (Table 5).
The head shape coefficient of androgenic R 1 lines ranged from flattened 0.81 (line 6.5/19) to elongated 1.2 (line 4.2/4) (Table 3).R 1 genotypes from the same embryo had similar head shape and did not differed significantly from each other according to that trait.Genotypes from 6.5 and 5.2 embryos had more flattened shape of head (0.81-1.0) than genotypes derived from 4.2 and 4.5 embryos (1.02-1.2),while genotypes from 2.2, 13.1 and 2.1 embryo were rounded (0.97-1.11) similarly to Jaguar F 1 donor cultivar.In consecutive R 2 generation, head cabbage lines were more uniform according to that trait as most genotypes were rounded and were similar to 'Jaguar F 1 ' or slightly elongated.Only 4.2/14 and 4.2/14i R 2 genotypes were significantly more elongated (1.29, 1.33) than all other populations.(Table 5).The way of propagation of R 2 androgenic lines had no influence on the head shape.
21 lines of androgenic R 1 generation were not uniform according to several other morphological characters such as waxiness, color and blistering of leaves, earliness and shape of head.Twelve from 39 R 1 genotypes were characterized by the lack of intraline uniformity according to more than one trait, nine lines were not uniform according to one of investigated characters and only nineteen R 1 lines evaluated at the field were internally uniform (Table 4).In contrary to R 1 generation, androgenic R 2 lines were generally characterized by internal uniformity with the exception of 6.2/7 genotype (Table 4).

3.2
Smaller head mass of androgenic R 1 and R 2 lines in comparison to Jaguar F 1 donor cultivar, confirmed the presence of strong inbreeding depression, characteristic for androgenic lines of cabbage obtained from 'Kamienna Glowa' (Kamiński 2000) and for traditionally derived inbred genotypes.Relatively lower ability for seed propagation both for bud and open-flower pollination among androgenic genotypes obtained from Jaguar F 1 hybrid in comparison to traditionally derived inbred lines described for cabbage plants by Dickson and Wallace (1986), can be explained not only by the occurrence of inbreeding depression typical for DH lines but also by the somaclonal variation.Difficulties with generative propagation of androgenic Brussels sprout genotypes described by Kamiński et al. (2005) may decrease the effectiveness of anther culture as a source of new genetic diversity.Low number of R 1 seeds obtained from doubled haploid plants showed that somaclonal variation in R 0 population critically affected the ability for generative propagation of androgenic head cabbage plants from Jaguar F 1 hybrid.
Lack of intraline uniformity among most of androgenic R 1 lines was probably caused by somaclonal variation that also affect negatively the quality of DH lines (Brown & Thorpe 1995, Larkin & Scowcroft 1981, Niemirowicz-Szczytt 1997).R 0 genotypes derived from the same embryo should be theoretically identical according to their genome and their phenotypic expression without somaclonal variation (Niemirowicz-Szczytt 1997).High interline variability among DH lines originating from several clones among cabbage genotypes was also recorded by Dore & Boulidard (1988) whereas other lines were equivalent to pure lines from pedigree selection.Obtained in this paper results suggests that somaclonal variation among head cabbage plants of R 0 and R 1 generations may significantly decreased the value of cabbage androgenic genotypes from Jaguar F 1 hybrid.Somaclonal variation among head cabbage androgenic genotypes may have influence on the evaluation of internal homozygousity by the use of molecular markers.Androgenic lines of R 1 generation with high level of somaclonal variation might have been accessed as heterozygotes obtained from somatic tissue by mistake (Kamiński et al. 2003).Presence of somaclonal variation among androgenic cabbage plants required a special approach to eliminate genotypes with the lack of internal uniformity in respect of their utility for the breeding purposes.Strong and attentive selection of internally uniform genotypes of R 1 generation for desired commercial traits will effectively lead to obtain more stable and uniform R 2 generation.For that reason, head cabbage cultivars with their biennial habit may require two or more additional years of testing of androgenic material in respect of their genetic stability.
Wang M., Farnham M. W.,& Nannes J. S. P. (1999).Ploidy of broccoli regenerated from microspore versus anther culture.Plant Breeding, 118, 249-252.Means followed by the same letter are not significantly different at α=0,05 Intraline uniformity: 1 -lines uniform, 2 -lack of uniformity according to one trait, 3 -lack of uniformity according to more than one trait Means followed by the same letter are not significantly different at α=0,05 Intraline uniformity: 1 -lines uniform, 2 -lack of uniformity according to one trait, 3 -lack of uniformity according to more than one trait

Table 1 .
Androgenic head cabbage plants from Jaguar F 1 hybrid in consecutive generations, Skierniewice, Poland

Table 2 .
Seed setting of R 1 generation obtained from head cabbage androgenic R 0 plants from Jaguar F 1 donor

Table 3 .
Seed setting of R 2 generation obtained from head cabbage androgenic R 1 genotypes from Jaguar F 1 donor cultivar.Skierniewice 2007