Optimization of in vitro Callus Induction and Somatic Embryogenesis of Horseradish (Armoracia rusticana) Supplemented with 2,4-D and IAA

Horseradish has a male sterility that produces a small amount of seed with a low germination. It then propagates with root cuttings, which is also subject to a limited number of mother rootstocks. Therefore, the mass proliferation of this plant is important through tissue culture, especially somatic embryogenesis protocols. Somatic embryogenesis is a method to develop embryo via plant somatic cells during in vitro culture. The purpose of the present experiment is to find the best method for callus and somatic embryogenesis inducing horseradish, which is carried out in two B5 and NL media in both solid and liquid phases.
 

This research was carry out in the tissue culture lab of the department of horticultural sciences at gorgan university agricultural sciences and natural resources from 2016 to 2019. Calli are developed on callus induction media (B5 + 1 µM 2,4-D + 2 µM kin, NL + 1 µM IAA + 2 µM kin), and then transferred to somatic embryo induction phase (B5 + 3 µM 2,4-D + 4 µM kin, NL + 3 µM IAA + 4 µM kin) for 8 weeks. Elimination of IAA, 2,4-D and kin from induced calli done onto realization phase, and globular, heart and torpedo embryos observed 4 weeks later.
 

Based on the results, the best surface sterilization was achieved by 70% ethanol for 30 seconds and 70% aqueous sodium hypochlorite (v/v) for 60 minutes. The callus and somatic embryo were observed in both solid and liquid B5 and NL media. In the study of callus induction, according to the measurement of appearance quality and the process of callus formation, the growth and increase of callus in solid phase are higher than that of liquid phase. The highest callus induction record is seen in solid B5 medium containing 2 µM Kin and 1 µM 2,4-D. Generally, calli on embryo induction phase are bright green to yellowish and compact consisting of separate clusters of somatic embryonic cells. The results of somatic embryogenesis revealed that globular, heart, torpedo and total embryos in B5 containing 1 µM 2,4-D are more than NL containing 1 µM IAA.

Optimizing the growth of somatic embryos is important for mass propagation of plants that face problems in seed germination or have a long growth period. Due to the success of the production of horseradish somatic embryos, the results of this experiment can provide the possibility of production and propagation of horseradish plants in a short time. Further studies are required to determine the somaclonal variation of horseradish mature somatic embryos and plantlets.