The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii.

Dmitry Miroshnichenko,Sergey Dolgov,Anna Klementyeva

doi:10.3390/plants10122620

Abstract

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Highlights

IntroductionT. timopheevii is actively involved in modern breeding programs to create introgression lines and increase the genetic diversity of modern wheat due to its partial homology with the common wheat genome (BBAu Au DD) [1,2]
In recent decades, wheat species of the Timopheevii group with the G genome, such as Triticum timopheevii Zhuk. (2n = 4x = 28; At At GG) and its wild ancestor T. araraticumJakubz., have received considerable attention as promising sources of immune genes
The effect of various daminodize concentrations on somatic embryogenesis and plant regeneration was assessed in combination with the auxin 2,4-D, which is commonly used for somatic embryogenesis in cereals

Summary

Introduction

T. timopheevii is actively involved in modern breeding programs to create introgression lines and increase the genetic diversity of modern wheat due to its partial homology with the common wheat genome (BBAu Au DD) [1,2]. T. timopheevii has been shown to be a valuable donor for resistance genes to wheat fungal diseases [3,4,5], environmental stresses [6], and modified protein content [7]. Genetic engineering and genome editing may serve as alternative means with which to study the functions of candidate genes and their regulatory elements, which control important traits of wheat with a G genome prior to introgression. Genetic transformation and genome editing systems require several key components Genetic engineering and genome editing may serve as alternative means with which to study the functions of candidate genes and their regulatory elements, which control important traits of wheat with a G genome prior to introgression. 4.0/).

Methods

Results

Conclusion