Controlling the barriers of cloning mature Picea abies (L.) H. Karst. via tissue culture and co-cultivation with Agrobacterium rhizogenes

Abstract

Providing an improved protocol for cloning of Picea abies (L.) H. Karst. mature trees via Agrobacterium rhizogenes. In reforestation programs, rejuvenation of superior genotypes is of great importance in helping juvenile rootstock production. In this regard, the use of plant tissue culture to produce axillary and/or adventitious shoots from explants of mature trees has been the objective of many researchers. There are a few reports about the successful in vitro rooting of the regenerated shoots from explants of mature trees. There is a very low rate of natural rooting in conifers, between 1 and 5%. Furthermore, they are still hard to root even when auxins are applied. In this study, in vitro propagation of Norway spruce explants and its rooting improvement by A. rhizogenes with the aim of receiving an efficient clonal propagation method was investigated. The effects of BA (0.5, 1, 1.5, and 2 μM) and TDZ (0.0025, 0.025, 0.25, and 2.5 μM) were examined on axillary shoot development from shoot-tip explants of the Norway spruce. The effects of three strains of Agrobacterium rhizogenes (K599, AR15834 and GMI9534) were also examined on enhancement of the root induction. According to the results, after the treatment of 1.5 μM BA, 87.5% of shoot-tip explants produced axillary shoots (the highest percentage among treatments), and also the maximum mean number of regenerated shoots per explant (6.5) was achieved in this treatment. Furthermore, the induction of rooting was performed using IBA (7.5 µM) and IAA (0.5 or 1 μM) in the WPM medium along with keeping the explants in the darkness at 21 °C, which yielded 37.5% and 12.5% rooting, respectively. Moreover, the application of coumarin along with IAA caused an increase of 22% in rooting, compared to the use of IAA alone. Two strains, K599 and AR15834, succeeded in improving the percentage of rooting, with the highest being 54.2% as a result of using the K599 strain alongside 7.5 μM IBA. The proposed treatment can be used efficiently for the propagation of Norway spruce in landscaping and/or reforestation programs.