Glimelius, K. 1984. High growth rate and regeneration capacity of hypocotyl protoplasts in some Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 |xM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3-5 days. In these cases 40-50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 \iM) and IAA (0.6 \iiM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 |xM), BA (4.4 \iM) and IAA (0.6 \iM).

Numerous Brassica napus (+) Arabidopsis thaliana somatic hybrids were screened for male sterility and aberrant flower phenotypes. Nine hybrids were selected and backcrossed recurrently to B. napus. The resulting lines displayed stable maternal inheritance of flower phenotypes. Nuclear and organellar genomes were characterized molecularly using RFLP analysis. No DNA from A. thaliana was found in the nuclear genome after six back-crosses, whilst the mitochondrial genomes contained rearranged DNA from both A. thaliana and B. napus. Each line tested had a unique RFLP pattern of the mitochondrial DNA (mtDNA) that remained unchanged between the BC(3) and BC(6) generation. The plastid genomes consisted of B. napus DNA. Five lines of the BC(5) generation were subjected to more comprehensive investigations of growth, morphology and fertility. On the basis of these investigations, the five CMS lines could be assigned to two groups, one represented by three lines displaying reduced vegetative development, complete male sterility, and homeotic conversions of stamens into feminized structures. The second group, represented by the other two lines, were not completely male-sterile but still displayed severely affected flower morphologies. These two lines did not display any reduction in vegetative development. For both groups only stamens and petals suffered from the morphological and functional aberrations, while the sepals and pistils displayed normal morphology. All plants were fully female-fertile. Different rearrangements of the mitochondrial genome disturbed nuclear-mitochondrial interactions and led to various types of aberrant growth and flower development. The existence of numerous CMS lines with different mitochondrial patterns involving a species with a sequenced genome offers new opportunities to investigate the genetic regulation of CMS and its associated developmental perturbations.

Analyses of mitochondrial transcription and in organello translation were performed with the Brassica tournefortii cytoplasm. This cytoplasm causes alloplasmic male sterility when combined with the nuclear genomes of B. napus and B. juncea. Mitochondrial RNA and protein banding patterns were compared between the fertile wild species B. tournefortii, an alloplasmic male-sterile B. juncea line, an alloplasmic male-sterile B. napus line and an alloplasmic B. napus line with restored fertility. The analyses were carried out to identify differences in gene expression and to investigate whether alterations in gene expression accompanied male sterility. A difference in transcription patterns between the fertile B. tournefortii and the alloplasmic lines was found for the atp6 gene. The atp6 region was investigated further, since a similar alteration in atp6 transcription has been observed in two other Brassica cytoplasms which are associated with cytoplasmic male sterility (CMS). The additional longer atp6 transcript detected in the alloplasmic lines in the present study was found to contain an open reading frame (ORF) located downstream of the atp6 gene. DNA sequencing revealed that the ORF, orf263, could encode a protein with a predicted molecular weight of about 29 kDa. In organello analysis detected two proteins of 29 and 32 kDa respectively, which were found only in the alloplasmic lines. Furthermore, the 32 kDa protein accompanied male sterility since it was absent in alloplasmic plants restored to fertility. The protein analysis might indicate that orf263 is translated and causes CMS.

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SummaryHomeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.