Abstract
Male sterile and seedless characters are highly desired for citrus cultivar improvement. In our breeding program, a male sterile cybrid pummelo, which could be considered as a variant of male fertile pummelo, was produced by protoplast fusion. Herein, ecotopic stamen primordia initiation and development were detected in this male sterile cybrid pummelo. Histological studies revealed that the cybrid showed reduced petal development in size and width, and retarded stamen primordia development. Additionally, disorganized cell proliferation was also detected in stamen-like structures (fused to petals and/or carpel). To gain new insight into the underlying mechanism, we compared, by RNA-Seq analysis, the nuclear gene expression profiles of floral buds of the cybrid with that of fertile pummelo. Gene expression profiles which identified a large number of differentially expressed genes (DEGs) between the two lines were captured at both petal primordia and stamen primordia distinguishable stages. For example, nuclear genes involved in nucleic acid binding and response to hormone synthesis and metabolism, genes required for floral bud identification and expressed in particular floral whorls. Furthermore, in accordance with flower morphology of the cybrid, expression of PISTILLATA (PI) was reduced in stamen-like structures, even though it was restricted to correct floral whorls. Down-regulated expression of APETALA3 (AP3) coincided with that of PI. These finding indicated that, due to their whorl specific effects in flower development, citrus class-B MADS-box genes likely constituted ‘perfect targets’ for CMS retrograde signaling, and that dysfunctional mitochondria seemed to cause male sterile phenotype in the cybrid pummelo.
Highlights
In the past two decades, somatic hybridization have been a powerful tool in citrus breeding for specific traits such as seedlessness and disease resistance, and numerous somatic hybrids have been produced for both scion and rootstock improvement [1,2,3,4,5]
Histological sections revealed that floral bud morphology of Guoqing No 1 (G1)+HBP and HBP remained unchanged until stamen primordia emerged (Figure 1B, F)
Male Sterile Cybrid G1+HBP In this study, we successfully identified 54 (46 for induced, 8 for repressed) and 247 (212 for induced and 35 for repressed) differentially expressed genes (DEGs) at 0.05% significance level that were changed at least two fold during petal primordia and stamen primordia stages in G1+HBP compared with HBP (Table S1)
Summary
In the past two decades, somatic hybridization have been a powerful tool in citrus breeding for specific traits such as seedlessness and disease resistance, and numerous somatic hybrids have been produced for both scion and rootstock improvement [1,2,3,4,5]. Recovered cybrids are generally composed of the nuclear genome of the leaf parent, the mitochondrial genome of the embryogenic callus/suspension parent, and a randomly inherited chloroplast genome [7,8]. Considering the possibility of targeted mitochondrial genome transfer and the importance of seedless trait for fresh citrus market, we put forward a strategy to transfer male sterile and seedless trait by somatic cybridization in efforts to reduce the seed content of important cultivars [9]. Diploid cybrids were generated [10] by transferring the male sterile cytoplasm of Satsuma mandarin (Citrus unshiu Marc.), a seedless variety with CMS character [11], to seedy citrus cultivars. An interesting diploid cybrid (G1+HBP) was regenerated with the nuclear genome and chloroplast DNA from the leaf parent Hirado
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