Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.)
© The Author(s) 2016
Received: 25 June 2016
Accepted: 14 October 2016
Published: 15 November 2016
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.
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.
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.
KeywordsDedifferentiation DNA marker F1 hybrid in vitro culture Somatic embryo
Transgenic plants play pivotal roles in molecular genetic analysis and crop biotechnology. To obtain transgenic plants, Agrobacterium-mediated transformation techniques have been devised for many crops . In some cases, explants are dedifferentiated in vitro to obtain callus that is subsequently infected with Agrobacterium harboring a recombinant Ti plasmid that will be inserted into plant chromosomes. The transformed cells are subsequently induced to regenerate whole plants.
Sugar beet (Beta vulgaris L.) is generally recalcitrant for dedifferentiation and plant regeneration, although cultivars and breeding lines show different responses to in vitro culture. This finding suggests that dedifferentiation and plant regeneration may be heritable characters and the genes involved in these characters may be scarce in sugar beet populations. In fact, Tomita et al.  investigated 61 sugar beet lines regarding their responses to in vitro culture and found variation in the frequencies of callus formation and somatic-embryo formation; however, the inheritance of these characters was not examined.
The sugar beet line ‘NK-219mm-O’, developed by the Hokkaido Agricultural Research Center (HARC), Japan, is an exceptional genotype that forms callus and regenerates plants very efficiently. These attributes enabled us to develop an Agrobacterium-mediated transformation system for sugar beet [3, 4]. Using this system, molecular analyses of several genes have been completed [5–7]. On the other hand, the shortcomings of NK-219mm-O include its vernalization requirement, i.e. the line must experience a certain duration of low temperatures as a prerequisite for flowering. Examining, for example, flower phenotypes in transgenic sugar beet takes a long time due to the vernalization requirement, which is a rate limiting step for the molecular genetic analysis of sugar beets.
The vernalization requirement in sugar beet is genetically conditioned by a recessive allele of the b gene (bolting) . The dominant allele B makes vernalization unnecessary for flowering. The detailed molecular organization of the B locus was elucidated, and a DNA marker discriminating b from B was reported . If the B gene does not interfere with genes governing callus formation and plant regeneration in NK-219mm-O, it may be possible to breed an annual sugar beet that is suitable for molecular genetic analysis.
First, we wished to determine the inheritance of callus formation and plant regeneration of NK-219mm-O. The parental sugar beet lines were five inbred lines, NK-219mm-O, NK-195mm-O, NK-235mm-O, NK-239mm-O, and NK-294mm-O, all of which were developed by HARC for hybrid breeding using cytoplasmic male sterility (CMS), which is genetically conditioned by male sterility-inducing cytoplasm (S) and a recessive allele of rf (restorer of fertility). A CMS line is produced by repeated backcrossing (more than four times) of a CMS line with a pollen parental line with the rfrf genotype but with normal fertile cytoplasm (N) to secure pollen production. This pollen parental line is called a maintainer or an O-type in sugar beet terminology. As such, a CMS line and its cognate O-type are near-identical in nuclear genotypes but differ in their cytoplasms. CMS and O-type lines are discriminated by suffixes ‘-CMS’ and ‘-O’, respectively. A shared number in the prefix (e.g. ‘219’) indicates that the two lines have nearly identical nuclear genotypes.
Half-diallel table of in vitro-culture response in F1 populations and their parental linesa
a) Frequencies of callus formation (%; mean ± SD)
0.08 ± 0.15
0.93 ± 0.13
0.99 ± 0.04
0.38 ± 0.40
0.97 ± 0.12
0.51 ± 0.49
0.28 ± 0.32
0.75 ± 0.27
0.50 ± 0.43
0.00 ± 0.00
0.35 ± 0.32
0.94 ± 0.11
0.79 ± 0.17
0.93 ± 0.12
0.40 ± 0.24
b) Plant regeneration scores (mean ± SD)
125.00 ± 75.00
139.39 ± 57.63
174.47 ± 26.27
11.91 ± 15.96
100.86 ± 34.78
1.52 ± 5.03
91.13 ± 76.57
41.14 ± 44.30
47.37 ± 45.90
52.74 ± 62.84
113.60 ± 34.91
7.30 ± 12.16
15.52 ± 13.00
6.31 ± 12.08
We next examined the effect of the B gene on in vitro culture. TA-33BB-CMS and TA-33BB-O are annual sugar beet lines developed at the HARC. Pin et al.  isolated the B gene through map-based cloning and showed that it encodes a pseudo-response regulator protein named BvBTC1. We determined part of the nucleotide sequence of BvBTC1 cDNA from TA-33BB-O, and the data indicated that the annual habitat of TA-33BB-O is conditioned by the dominant B allele (Additional file 2: Figure S4).
We also examined callus formation and the regeneration potential of TA-33BB-O, TA-33BB-CMS, NK-219mm-O x TA-33BB-O, and TA-33BB-CMS x NK-219mm-O. Frequencies of callus formation were 0.1-0.11 for TA-33BB-O and TA-33BB-CMS, and 0.99-1.0 for the two F1 populations (Additional file 2: Figure S5 and Additional file 3: Table S9). Plant regeneration scores were 77-78 for TA-33BB-O and TA-33BB-CMS, and 182-200 for the two F1 populations (Additional file 3: Table S9). We obtained regenerated plants from calli of the two F1 populations (see Additional file 2: Figure S6). The regenerated plants flowered under 24-h day conditions without vernalization. These F1 populations can be propagated as somatic clones by in vitro culture and used for transgenic analyses.
- B (b):
Cytoplasmic male sterility
Hokkaido Agricultural Research Center
Normal fertile cytoplasm
- rf :
Restorer of fertility
Male sterility-inducing cytoplasm
The authors would like to thank Chisato Osawa for technical support. TA is a recipient of JSPS Research Fellowship for Young Scientists.
This work was supported in part by JSPS KAKENHI Grant Numbers 22580001 and 25292001 (TK), and Grant-in-Aid for JSPS Fellows 16 J01146 (TA).
Availability of data and materials
Because plant materials used in this study are breeding materials, they are not freely available. Those who belong to a public sector organization may use these materials by negotiating a contract with HARC.
KT, HT, and TK designed this study. HK, YK and KT developed plant materials. HK and TA performed in vitro culture and genotyping. HK, KT, TA and TK analyzed the data. HK, TA and TK wrote the manuscript. KT and HT performed critical comments on the draft. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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- Wang K. Agrobacterium protocols. 3rd ed. New York: Springer Science + Buisiness Media; 2015.Google Scholar
- Tomita K-i, Hiura S, Tamagake H. Evaluation of the potential for somatic embryogenesis in sugar beet (Beta vulgaris L.) breeding lines and improvement of regeneration efficiency. Plant Biotechnol. 2013;30:479–87.
- Kagami H, Kurata M, Matsuhira H, Taguchi K, Mikami T, Tamagake H, Kubo T. Chapter 27 Sugar Beet (Beta vulgaris L.). In: Wang K, editor. Agrobacterium Protocols. New York: Springer Science + Business Media; 2015. p. 335–47.Google Scholar
- Kubo T. Sugar beet transformation. http://www.agr.hokudai.ac.jp/ikushu/gelab/Sugar_beet_transformation.htm. Accessed 4 Nov 2016.
- Matsuhira H, Kagami H, Kurata M, Kitazaki K, Matsunaga M, Hamaguchi Y, et al. Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.). Genetics. 2012;192:1347–58.View ArticlePubMedPubMed CentralGoogle Scholar
- Matsuhira H, Tamura K, Tamagake H, Sato Y, Anzai H, Yoshida M. High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes. J Biotechnol. 2014;192:215–22.View ArticlePubMedGoogle Scholar
- Kitazaki K, Arakawa T, Matsunaga M, Yui-Kurino R, Matsuhira H, Mikami T, et al. Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris). Plant J. 2015;83:290–9.View ArticlePubMedGoogle Scholar
- Pin PA, Zhang W, Vogt SH, Dally N, Büttner B, Schulze-Buxloh G, et al. The role of a pseudo-response regulator gene in life cycle adaptation and domestication of beet. Curr Biol. 2012;22:1095–101.View ArticlePubMedGoogle Scholar