In Vitro Callus Formation And Regeneration Of Juniperus seravschanica Kom. In Different Culture Media

After pines, Ors (Juniperus) is the second most diverse genus of Coniferals, consisting of approximately 75 species and comprising the majority of evergreen shrubs and trees. Several studies have pointed out the importance of the genus in soil stabilization, and its ornamental, medicinal, and industrial applications. Ors or sarve kuhi (Juniperus seravschanica) has a wide distribution in Kerman province. However, in most areas, seedlings or young plants have very little distribution, which indicates the difficulty of seed germination due to various reasons. Also, rooting of its microshoots in micro culture techniques is considered to be difficult due to poor development and low frequency of rooting. Therefore, to overcome these problems, it needs to optimize an in vitro shoot multiplication and root induction and growth protocol. The purpose of this study was in vitro culture of apical shoots to induce their multiplication and rooting. Further, seeds were planted on micro culture media to study seed germination, callus induction and shoot and root regeneration from calli. The studied species was a population of J. seravschanica grown in Glochar (Raber, Kerman province).

Young shoots were collected from Glochar reserve of Rabor city (Kerman province) and kept at 4°C temperature for 3-7 days. The apical shoots with a size of 1.5 to 1.5 cm were separated and after sterilization, were cultured in woody plant medium (WPM) and Morashig and Skoog (MS) culture media without hormones to establish the samples. For shoot multiplication and growth, all samples were transferred to the medium containing plant growth regulators with optimized levels. Young shoots were transferred to rooting media (WPM, MS and ½MS) with different levels of IBA hormones. Seeds were separated from mature female cones, and full seeds (there are many empty seeds in cones) were exposed to cold treatment for 1-2 weeks. Then, they were sterilized and cultivated in MS (containing hormone) medium to study seed germination and callus formation from embryos. Callus regeneration and shoot formation and their rooting were investigated under appropriate hormone concentrations.

In the WPM medium, the highest percentage of shoot proliferation (40%) was obtained in the combined treatment of 3 mg/L BAP and 2 mg/L NAA. The shoot proliferation percentage was higher in MS media than WPM with 57% in concentrations of 0.2 and 3 mg/L of BAP and NAA, respectively. In both media, Proliferated shoots did not form any root in both media until four months after planting. The highest percentage of seed germination in MS medium was 33% at the concentrations of 5 mg/L BAP and 0.2 mg/L NAA. The highest rate of callus formation (40%) from embryos (seeds), as explants, was recorded in MS culture medium at the concentration of 0.2 and 3 mg/L of BAP and NAA, respectively. Calli produced shoots in in 3 mg/L BAP and 2 mg/L NAA in WPM medium. Root formation was not observed until six months, but about 10% of these regenerated shoots produced roots eight months after shoot proliferation.

It seems that various factors such as genotype, polyploidy or hybrid formation, slow plant growth (which is common in coniferal), or secondary compounds are the reasons for the low rate of regeneration, especially rooting. Also, the duration of cultivation can be effective in rooting.