اثرات محلول‌پاشی محرک‌های رشدی بر ویژگی‌های مورفوفیزیولوژیکی و اسانس گیاه دارویی زرین گیاه (Dracocephalum kotschyi Boiss.)

Introduction
The environmental factors cause changes in the growth, quantity, and quality of active substances (such as alkaloids, glycosides, steroids and volatile oils) in the medicinal and aromatic plants. Among environmental stresses, water deficit stress plays an important role in the biosynthesis of secondary metabolites in medicinal and aromatic plants in arid and semi-arid regions like Iran. Dracocephalum kotschyi Boiss. commonly known as Badrandjboie-Dennaie and Zarrin-Giah called in Farsi, belongs to the family Lamiaceae, is one of the endangered endemic species in Iran. The main compounds of the essential oil from D.kotachyi are Monoterpene hydrocarbons category (alpha-pinene, gamma-terpinene, neral, geranial, nerol, geraniol, limonene, carvacrol and thymol), Oxygenated monoterpenes category (alpha-campholene aldehyde), Sesquiterpenes (caryophyllene) and geranyl acetate from Acetates of carboxylic acids category. The extreme harvesting, limited distribution, and no commercial farming of D.kotachyi has led to the danger of its extinction which is one of the most important challenges. Therefore, this study was done to evaluate the elicitors (organic and alcoholic) on the morpho-physiological characters and essential oil quantity and quality of D.kotschyi to determine and introduce the best elicitor.
 
Materials and methods
This investigation was done in biennial research  from spring (May) 2022 to fall (September) 2023 at the Research Farm of Islamic Azad University Branch of Shahrekord, Iran (latitude. 32°20′ N, longitude. 50°51′ E, altitude. 2061 m). Based on the Köppen climate classification, the climate of the area of study is classified as cold and semiarid. Seeds of D.kotschyi Boiss. (Lamiaceae) were obtained from Forest and Rangeland Institute, Iran. Firstly, the seeds were sterilized and sown on May 2022 and 2023 under greenhouse condition (25^0C, light 12/12 day/night, 65% RH). After about 45-50 days from sowing, when the seedlings had 4-6 true leaves with 8-10 cm tall, were transplanted in the experimental field on 20 May.The present study was conducted in a randomized complete block design (RCBD) with three replications to investigate the effect of foliar application of different elicitors of organic [chitosan (0.25 and 0.5 gr.l^-1, salicylic acid (1.5 and 3 mM), phenylalanine (1000 and 2000 mg.l^-1)] and alcoholic [(ethanol (10% and 20%), methanol (10% and 20%), acetone (10% and 20%)] and control level (without any spraying) on the morpho-physiological characters and essential oil quantity and quality of D.kotschyi  In each year, treatments were applied in stage of V₄-V₈ and the sampling was performed in flowering stage. The essential oils were extracted by hydro-distillation and analyzed using GC/MS and GC-FID.
 
Results and discussion
According to obtained results, applied organic and alcoholic elicitors significantly (α=1% and 5%) influenced the morpho-physiological characters and essential oil content/composition of D.kotschyi, however the assayed traits showed slightly variation during the studied years. In two years, the highest shoot dry matter (140.3-145.1 g.plant^-1) and chlorophyll content (2.51-2.55 mg.g^-1 DW) was obtained in salicylic acid (3 mM) and phenyl alanine (1000 mg.l^-1) treatments respectively. In most of measured characters, plants treated with salicylic acid (3 mM), phenyl alanine (1000 mg.l^-1) and ethanol (20 %) were in the same group. Amounts of some characters in plants treated with methanol and acetone lesser than control plants. The main constituents of essential oil were Neral, Alpha-Pinene, Limonene, Geranial and Geraniol (alcoholic monoterpenes) that made the most components of essential oil plants. The organic elicitors effect especially salicylic acid on increasing the total phenol content was higher than the alcoholic ones. Applied elicitors showed significant effects on essential oil content and composition of D.kotschyi and the highest essential oil content (1.12-1.18 %) was obtained from the treated plants by salicylic acid (3 mM). According to the biennial results of the chemical analysis of the essential oils from D. kotschyi by GC/MS and GC-FID, the most important chemical compounds that determine the quality of D. kotschyi essential oil including α-pinene (21.32-29.22%), geranial (8.34-15.78%), neral (6.43-13.1%), limonene (4.33-8.76%), geraniol (1.08-2.71%), geranyl acetate (4.11-8.91%), α-campholene aldehyde (1.32-3.77%) were identified. α-Pinene belonging to monoterpene hydrocarbons was the predominant constituent of the D. kotschyi. The most correlation indices were obtained between essential oil content with shoot dry matter and alpha-pinene.
 
Conclusions
Foliar application of salicylic acid and phenyl alanine significantly increased the monoterpene alcohols compounds in essential oils of D.kotschyi so that the content of these compounds in treated plants by organic elicitors 58% was more than the control plants. Finally, the application of salicylic acid at 3 mM can be a good strategy to improve morpho-physiological characters and essential oil quantity and quality of D.kotschyi in cold and semi-arid climates.