Sugar beet transformation

A. Background: dissemination of Tamagake protocol

Hideto Tamagake developed a protocolof sugar beet transformation by himself. Initially, his protocol included protoplastisolation and electroporation but these were later replaced by Agrobacterium infection of callus. Theprotocol was released as research reports of agricultural experimental station^{1,2, 3}. Several research groups in Japan noticed this protocol and examinedwhether it works in their labs.

B. Research papers using Tamagake protocol

Hiroyo Kagami and colleagues modifiedsome (minor) points of the original protocol for the adaptation to their lab. Thedetails of the Kagami version have been published⁴, but this paper hassome erroneous descriptions that were pointed out by several researchers after thepublication (see below). The Kagami version was used to generate the transgenicsugar beet to analyze restorer-of-fertilitygene for Owen cytoplasmic male sterility⁵.

There are another version ofprotocol that are slightly different from the Kagami version because eachresearch group modified the original Tamagake protocol to adapt their lab. HiroakiMatsuhira and colleagues reported biotechnological application of transgenicsugar beets⁶ that were made by the Tamagake protocol. Ken-ichi Tomitaand colleagues investigated the effect of genotype in the callus formation andplant regeneraton⁷ by the Tamagake protocol.

C. Qs and As of the protocol

Several questions about the Kagamiversion⁴ have been asked since its publication. The questions andanswers that are informative for the people who want to know more about theKagami version are given below:

i. Is composition of culture medium correct?

In Modified Murashige-and-Skoog(mMS) medium described on page 338, subheading 2.4.2;
MnSO₄·H2O is correct instead of MnSO₄·4H2O;
Na₂EDTA·2H₂O is correct instead of Na₂·EDTA;and
30 g/L sucrose is correct instead of 0.3 % w/v sucrose.

ii. Are both gellan gum and agar necessary in thisprotocol?

Gellan gum can be replaced withagar, according to our experience. Gellan gum solidified medium is visuallyclear, making easy recognition of bacterial contamination possible. But theopposite (replacement of agar with gellan gum) is not recommended because ofthe occurrence of less healthy plantlet.

iii. Is New Medel necessary in this protocol?

The primary reason why we use NewMedel is easy to handle. Cutting into pieces is unnecessary for New Medel. Inour experimental condition, longevity of New Medel is better than parafilm. NewMedel may, therefore, be replaced with parafilm.

iv. Meropenem is expensive.

We use Meropen (Dainippon SumitomoPharma), which is a medical product for injection or intravenous drip. Meropenis cheaper than meropenem for research use in Japan.

¹Tamagake H (1999) Efficient and stabletissue culture technique suitable for gene transfer in sugarbeet. In: NewResearch Results for HokkaidoRegion in 1998 (H10,Atarashi-kenkyuseika, Hokkaido-chiki),Hokkaido National Agricultural Experiment Station, Sapporo, pp 38-40 (inJapanese).

²Tamagake H (1999) Establishment of tissueculture methods for introducing a gene into sugar beet. Attachment for the meeting ofAgricultural Experimental Stations in Hokkaido, H10. (in Japanese).

³Tamagake H (2002) Establishment of aprotocol of sugar beet transformation. Attachment for the meeting of AgriculturalExperimental Stations in Hokkaido, H13. (in Japanese).

⁴Kagami H, Kurata M, Matsuhira H, TaguchiK, Mikami T, Tamagake H, Kubo T, Chapter 27 Sugar Beet (Beta vulgaris L.), In: (Kan Wang ed.) Agrobacterium Protocols,Methods in Molecular Biology, vol. 1223, pp. 335-347, DOI10.1007/978-1-4939-1695-5_27, Springer Science+Business Media, New York, 2015.

⁵Matsuhira H, Kagami H, Kurata M, KitazakiK, Matsunaga M, Hamaguchi Y, Hagihara E, Ueda M, Harada M, Muramatsu A,Yui-Kurino R, Taguchi K, Tamagake H, Mikami T, Kubo T, Unusual and typicalfeatures of a novel restorer-of-fertilitygene of sugar beet (Beta vulgarisL.), Genetics,192：1347-1358, 2012.

⁶Matsuhira H, Tamura K, Tamagake H, Sato Y,Anzai H, Yoshida M, High production of plant type levan in sugar beettransformed with timothy (Phleum pratense)6-SFT genes, Journal of Biotechnology, 192: 215-222, 2014.

⁷Tomita K, Hiura S, Tamagake H, Evaluationof the potential for somatic embryogenesis in sugar beet (Beta vulgaris L.) breeding lines and improvement of regenerationefficiency, Plant Biotechnology, 30: 479-487, 2013.

Dec. 24, 2014