Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

Wenting Liu,Zongsuo Liang,Susan Sibbald,Lining Tian,Xinhua Wang,David Hunter,Ive De Smet

doi:10.1371/journal.pone.0056699

Wenting Liu, Zongsuo Liang + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0056699

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Abstract

Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species.

Highlights

Introduction of new traits into plants via genetic transformation has become an important technology for plant improvement
Transgenic alfalfa plants were obtained via Agrobacteriummediated transformation using the method well established in our laboratory [37]
Somatic embryos were efficiently induced from different transgenic alfalfa plants (Figure 1A)

Summary

Introduction

Introduction of new traits into plants via genetic transformation has become an important technology for plant improvement. Induction of somatic embryos from transgenic plants and the use of artificial seeds may provide a new system for transgene preservation. We report stable transgene preservation and faithful expression of a transgene in plants developed from dried somatic embryos in alfalfa. The DNA constructs for plant transformation The uid gene coding for b-glucuronidase (GUS) [26] was used as the reporter of transgene expression in the study.

Results

Conclusion