Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

Abstract

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars.

Fig 1. Endogenous hormone contents in calli.

Endogenous hormone contents of GA1, IAA, iP, tZ, and ABA were determined in calli cultured under a 16-h photoperiod and continuous darkness during callus-induction. Error bars represent standard errors (n = 3). *, **: Significant difference between light conditions at P<0.05 and P<0.01, respectively.

Fig 2. Effects of exogenous ABA and fluridone on callus growth.

Immature embryos were cultured with ABA (5 μM) or fluridone (0.5 μM) in different light conditions (16-h photoperiod and continuous darkness) in Kanto Nijo-5.

Fig 3. Effects of exogenous ABA on shoot regeneration.

Percentages of green and albino shoot regeneration in calli cultured with or without ABA during callus-induction in different light conditions (16-h photoperiod and continuous darkness). Error bars represent standard errors (n = 3). Bars with different letters show a significant difference between total regeneration percentages (green and albino) by Duncan’s multiple range test (P<0.05).

Fig 4. Effects of fluridone on shoot regeneration.

Percentages of green and albino shoot regeneration in calli cultured with or without fluridone in different light conditions (16-h photoperiod and continuous darkness). Error bars represent standard errors (n = 3). Bars with different letters show a significant difference between total regeneration percentages (green and albino) by Duncan’s multiple range test (P<0.05).

Fig 5. Effects of light conditions on endogenous hormone contents in calli under varied fluridone concentrations.

Endogenous hormone contents of IAA, iP, tZ, and ABA were determined in calli cultured with or without fluridone in different light conditions (16-h photoperiod and continuous darkness). Error bars represent standard errors (n = 3). **: Significant difference between light conditions at P<0.01.

Fig 6. Relative amounts of transcripts of HvABA8’OH-1 and HvNCED1 in calli.

Transcripts were determined in calli cultured under different light conditions (16-h photoperiod and continuous darkness). Error bars represent standard errors (n = 3). *, **: Significant difference between light conditions at P<0.05 and P<0.01, respectively.