Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.).

BACKGROUND: Obtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line 'NK-219mm-O' is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis. FINDINGS: Five sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross, the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat. CONCLUSIONS: Efficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.

Suppressed expression of choline monooxygenase in sugar beet on the accumulation of glycine betaine.

Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of choline. Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation.

Sugar beet (Beta vulgaris L.).

Creating transgenic plants is invaluable for the genetic analysis of sugar beet and will be increasingly important as sugar beet genomic technologies progress. A protocol for Agrobacterium-mediated transformation of sugar beet is described in this chapter. Our protocol is optimized for a sugar beet genotype that performs exceptionally well in tissue culture, including the steps of dedifferentiation, callus proliferation, and regeneration. Because of the infrequent occurrence of such a genotype in sugar beet populations, our protocol includes an in vitro propagation method for germplasm preservation. The starting materials for transgenic experiments are aseptic shoots grown from surface-sterilized seed balls. Callus is induced from leaf explants and subsequently infected with Agrobacterium. Plantlets are regenerated from transgenic callus and vernalized for flowering, if necessary. The efficiency of transformation was quite high; in our laboratory, the culture of only ten leaf explants, on average, generated one transgenic plant.

High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes.

Levan, a type of fructan, is an oligomer or polymer with mainly a ß(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of ß(2,6)-linked levans (about 20 to 75mgg(-1) FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure ß(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms.

Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.).

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Preferential accumulation of betaine uncoupled to choline monooxygenase in young leaves of sugar beet--importance of long-distance translocation of betaine under normal and salt-stressed conditions.

It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.