Abstract

Keywords

Introduction

Oilseed rape (Brassica napus, 2n=38, AACC) is an allotetraploid species cumulating the two diploid genomes of B. rapa (2n=20, AA) and of B. oleracea (2n=18, CC). In order to know how these diploid genomes have evolved in an oilseed rape tetraploid structure, it would be of interest to extract the A and C genomes from B. napus. Observations were performed on pollen mother cells (PMCs) at the anaphase I stage of meiosis of haploid oilseed rape (n=19, AC) by Li et al. (1996). These authors described four types of chromosome segregation and the most common was 9 in one pole and 10 to the other. These chromosome numbers might correspond to the haploid progenitors of oilseed rape i.e. A and C genomes. We attempted microspore cultures and sexual crosses from haploid oilseed rape plants. Five different varieties ‘Falcon’, ‘Doublol’, ‘Goeland’, ‘Global’ and ‘Spoke’ were chosen. Cytological observations on meiotic behavior, female and male fertility of haploid plants are reported. After microspore cultures and sexual crosses, the plants obtained were characterized for their chromosome number and isozyme patterns. The efficiency of these methods to extract the oilseed rape original progenitors or to create aneuploid oilseed rape lines, will be discussed.

Characterization of haploid mother plants

Cytological analyses

From the five varieties used ‘Falcon’, ‘Doublol’, ‘Goeland’, ‘Global’ and ‘Spoke’, haploid plants were obtained by microspore culture. Their meiotic behavior was established (table 1) and these five varieties could be classified in two types : the ones with a high percentage of chromosome paired ranging from 65.87 to 76.49% (‘Falcon’, ‘Doublol’, ‘Goeland’, ‘Global’) and the one with a low percentage of chromosome paired, 40.76% (‘Spoke’).

Table 1: Cytogenetic analyses of the five varieties used at the haploid level. In brackets : ranges

Varieties	No of plants observed	2n	No of cells observed	Mean meiotic behavior	% of chromosomes paired
‘Falcon’	4	19	81	6.48I+6.18II+0.05III (3-11) (4-8) (0-1)	65.9
‘Doublol’	6	19	120	4.75I+6.98II+0.05III+0.03IV (1-9) (5-9) (0-1) (0-1)	75.0
‘Goeland’	4	19	80	4.56I+7.01II+0.09III+0.04IV (1-9) (5-9) (0-1) (0-1)	76.0
‘Global’	6	19	103	4.47I+7.03II+0.03III+0.10IV (1-7) (4-9) (0-1) (0-1)	76.5
‘Spok’	6	19	125	11.26I+3.80II+0.02III+0.02IV (5-17) (1-7) (0-1) (0-1)	40.8

Male fertility

Most of the flowers of the haploid plants were male sterile without any stamen, but depending on the greenhouse conditions, some pollen production was observed from narrow stamens. The fertility of this pollen was estimated by using Belling aceto-carmin solution. 0 to 24% of pollen grains were colored by the staining solution.

Female fertility

The mean number of ovules per pistil was estimated by counting the ovules in 87 pistils of haploid plants and 20 in the diploid controls. This ovule mean number was of 30 (from 25.7 to 31.9) for the haploid plants and of 34 (from 31 to 39) for the diploid controls (table 2). The embryo sacks were observed by using the clearing method described by Herr (1971) and modified to oilseed rape by Pellan-Delourme and Renard (1988). About 20% of the embryo sacks from the haploid plant ovules and 97% of those of the diploid controls, were octo-nucleate i.e. with the expected structure of a fertile embryo sack. In the controls, few ovules were mono-nucleate (2.3 %) or stunted (1.2 %) whereas abnormal development was observed from haploid plant ovules, i.e. 57 and 23% of mono-nucleate and stunted ovules, respectively.

Table 2: Ovules and embryo sacks observations octo.: octonucleate sacks; mono. : mono-nucleate sacks

Varieties	No of plants observed	No of pistils observed	Mean number of ovules per pistil	No of embryo sacks observed	% of embryo sacks
					octo	mono	stunted
Falcon haploid	4	14	31.9	140	15.0	66.4	18.6
Doublol haploid	5	23	31.5	231	13.8	54.1	32.0
Goeland haploid	2	7	25.7	90	14.4	60.0	25.6
Global haploid	3	18	32.4	205	41.0	39.0	20.0
Spoke haploid	5	25	28.5	247	15.0	68.0	17.0
Diploid controls	4	20	34.0	256	96.5	2.3	1.2

Microspore cultures of haploid plants

From microspore culture, we observed that 1 to 11% of haploid plant microspores were identical in shape and size to diploid plant microspores. Embryos were obtained and 289 seedlings were analysed by flow cytometry (Eber et al., 1997) in order to estimate their chromosome number (table 3).

Table 3 :Estimation of chromosome number of seedlings obtained by microspore culture of haploid plants

range of chromosome numbers	Number of plants observed	% of plants
18-25	208	72.0
35-42	70	24.2
48-49	3	1.0
56-57	4	1.4
71-76	4	1.4

Most of the plants produced (71.97%) were distributed in the class of chromosome number ranging from 18 to 25. However, a high frequency of plants had a chromosome number close to the one of diploid oilseed rape i.e. 2n=38.

Isoenzyme analyses

Plants obtained from microspore culture of haploid cultivars were characterized with ten isoenzymatic systems using the standard starch-electrophoresis method : phosphoglucoisomerase (PGI), triosephosphate isomerase (TPI), phosphoglucomutase (PGM), aconitase (ACO), 6-phosphogluconate dehydrogenase (6-PGD), leucine aminopeptidase (LAP) and glutamate oxaloacetate transaminase (GOT). The six first isozyme systems (PGI, TPI, PGM, ACO 6-PGD and LAP) were studied according to the method described by Chèvre et al.(1995). GOT was separated on a tris citrate/ lithium-borate buffer pH 8.3 (Shield et al., 1983) and stained as reported by Vallejos (1983). These ten isoenzyme systems allowed to observe 36 loci.

None of the plant had only the bands specific of one genome of the oilseed rape parental species (A or C). Only 0 to 3 deletions per plant were observed (table 4).

Table 4 : Deletions of A or C bands at 36 loci revealed from 10 isoenzymatic systems (PGI, TPI, PGM, ACO, 6-PGD, LAP and GOT) from plants obtained by microspore culture of haploid lines.

Varieties	No of plants observed	No of plants with 1 deletion	No of plants with 2 deletions	No of plants with 3 deletions
‘Falcon’	51	12	13	0
‘Doublol’	59	13	2	3
‘Goeland’	3	1	0	0
‘Global’	9	1	3	1
‘Spoke’	9	4	3	0

Among the 131 plants studied, the most frequent deletions occurred for Got (22 plants), Aco3 (17 plants) and Pgi2 (12 plants) loci.

Sexual Crosses

Selfings at the bud stage were performed on 601 pistils of haploid plants and 819 seeds were obtained. Crosses between two haploid plants were realized on 40 pistils and 46 seeds were yielded. Haploid plants were also crossed to diploid plants and on 10 pistils pollinated 19 seeds were obtained.

Chromosome number of the plants obtained from sexual crosses were estimated by flow cytometry. For the three types of progenies (selfing of haploid plants, haploid plant crossed to another haploid plant, haploid plant crossed to diploid plant), 91.38% of the plants had 38 chromosomes.

No isozyme locus deletion was detected.

Discussion

We observed that the oilseed rape varieties can be classified in two types according to their meiotic behavior at the haploid stage. This result was already reported by Renard and Dosba (1980).

None of the seedlings produced by microspore culture from haploid oilseed rape mother plants had only B. rapa or B. oleracea isozyme loci. So it is likely that the 71,97% of the plants which had a chromosome number ranging from 18 to 25, were produced from unreduced gametes of haploid oilseed rape (AC, n=19). Similarly, the seedlings presenting a chromosome number close to the one of oilseed rape probably resulted from chromosome doubling from unreduced gametes. Such gametes probably correspond to the mainly mono-nucleate embryo sacks and the normal size of microspores and pollen grains observed in haploid mother plants. The efficiency of unreduced gametes was confirmed by the production of mainly 2n=38 plants after selfing or crosses. The establishment of the meiotic behavior of the plants produced from microspore culture is in progress.

So, the method proposed does not seem to be efficient to extract A or C genome of oilseed rape. However, isozyme loci deletions in some plants, whatever their chromosome number, indicated that chromosome rearrangements occurred before microspore production ; this result will be confirmed by analyzing molecular markers well spread on the oilseed rape genetic map (Foisset et al. 1995). Then, microspore culture could be an interesting method to generate either chromosome rearrangements or aneuploid lines. Monosomic (2n=37) or nullisomic (2n=36) lines will be preferentially studied as they could be an efficient tool to allocate one linkage group of the oilseed rape genetic map to a specific chromosome.

References

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