بهينه‌سازي رشد گياهچه و بررسي توليد تاكسان در كشت درون‌شيشه‌اي سرخدار (Taxus baccata L.)

Seedling growth optimization and investigation of taxane production in Taxus baccata L. via in vitro culture

ﺳﺮﺧﺪار ).Taxus baccata L( ﺑﻪﻋﻠﺖ دارا ﺑﻮدن ﺗﺮﮐﯿﺒﺎت ديﺗﺮﭘﻨﻮﺋﯿﺪي ﺑﺎ اﺛﺮات ﺿﺪ ﺳﺮﻃﺎن، از ﻣﻬﻢﺗﺮﯾﻦ ﮔﯿﺎﻫﺎن داروﯾﯽ ﺷﻨﺎﺧﺘﻪ ﺷﺪه در ﺟﻬﺎن اﺳﺖ ﮐﻪ در ﻣﻌﺮض اﻧﻘﺮاض ﻗﺮار دارد. در اﯾﻦ ﺗﺤﻘﯿﻖ ﮐﻪ در ﭘﮋوﻫﺸﮑﺪه ﮔﯿﺎﻫﺎن داروﯾﯽ داﻧﺸﮕﺎه ﺷﻬﯿﺪ ﺑﻬﺸﺘﯽ ﺻﻮرت ﭘﺬﯾﺮﻓﺖ، اﺑﺘﺪا اﺛﺮ ﻧﻮع ﻣﺤﯿﻂ ﮐﺸﺖ )MS٬MS 1/2 و NN(، ﺟﺎذبﻫﺎي ﻓﻨﻮﻟﯽ )زﻏﺎل ﻓﻌﺎل و PVP( و ﺗﻨﻈﯿﻢ ﮐﻨﻨﺪهﻫﺎي رﺷﺪ ﮔﯿﺎﻫﯽ )GA3 ،IBA و BA( در ﻗﺎﻟﺐ ﻃﺮح ﻓﺎﮐﺘﻮرﯾﻞ ﺑﺎ ﺳﻪ ﺗﮑﺮار، ﺑﺎ ﻫﺪف ﺑﻬﺒﻮد ﺟﻮاﻧﻪزﻧﯽ و رﺷﺪ ﮔﯿﺎﻫﭽﻪﻫﺎي ﺳﺮﺧﺪار در ﮐﺸﺖ درونﺷﯿﺸﻪاي ﺑﺮرﺳﯽ ﮔﺮدﯾﺪ. در اداﻣﻪ ﺗﻮﻟﯿﺪ ﺗﺎﮐﺴﺎن در ﮔﯿﺎﻫﭽﻪﻫﺎﯾﯽ ﮐﻪ ﻃﯽ ده ﻣﺎه در ﻣﺤﯿﻂ اﻧﺘﺨﺎﺑﯽ رﺷﺪ ﮐﺮده ﺑﻮدﻧﺪ، اﻧﺪازهﮔﯿﺮي ﺷﺪ. ﻣﺤﯿﻂ ﮐﺸﺖ NN ﺑﻪدﻟﯿﻞ ﻏﻠﻈﺖ ﻧﻤﮏﻫﺎي ﭘﺎﯾﻪ ﮐﻢﺗﺮ در ﻣﺤﯿﻂ ﮐﺸﺖ ﺗﺄﺛﯿﺮ ﻣﻌﻨﯽداري ﺑﺮ درﺻﺪ ﺟﻮاﻧﻪزﻧﯽ ﻧﺪاﺷﺖ، وﻟﯽ ﻣﻨﺠﺮ ﺑﻪ اﻓﺰاﯾﺶ ﻃﻮل رﯾﺸﻪ و درﺻﺪ روﯾﺶ ﻋﺎدي ﮔﯿﺎﻫﭽﻪ ﮔﺮدﯾﺪ. ﻫﻤﭽﻨﯿﻦ اﺳﺘﻔﺎده از ﺟﺎذبﻫﺎي ﻓﻨﻮﻟﯽ ﻣﻮﺟﺐ اﻓﺰاﯾﺶ ﺑﯿﺶﺗﺮ ﻃﻮل رﯾﺸﻪ و ﺳﺎﻗﻪ و ﺑﻬﺒﻮد ﻧﺮخ روﯾﺶ ﻋﺎدي ﮔﯿﺎﻫﭽﻪ ﮔﺮدﯾﺪ. ﺑﻪﻋﻼوه ﺗﺮﮐﯿﺐ ﻫﻮرﻣﻮﻧﯽ IBA × GA)0/2 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻟﯿﺘﺮ ﺟﯿﺒﺮﻟﯿﮏ اﺳﯿﺪ × 0/01 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻟﯿﺘﺮ اﯾﻨﺪول ﺑﻮﺗﯿﺮﯾﮏ اﺳﯿﺪ( ﺑﯿﺶﺗﺮﯾﻦ ﺗﺄﺛﯿﺮ را ﺑﺮ ﻣﯿﺰان روﯾﺶ ﻋﺎدي ﮔﯿﺎﻫﭽﻪﻫﺎ داﺷﺘﻪ و ﺑﯿﺶﺗﺮﯾﻦ ﻃﻮل رﯾﺸﻪ ﺑﺎ 0/1 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻟﯿﺘﺮ اﯾﻨﺪول ﺑﻮﺗﯿﺮﯾﮏ اﺳﯿﺪ و ﺣﺪاﮐﺜﺮ ارﺗﻔﺎع ﺳﺎﻗﻪ از ﺗﺮﮐﯿﺐ ﻫﻮرﻣﻮﻧﯽ BA × GA )0/2 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻟﯿﺘﺮ ﺟﯿﺒﺮﻟﯿﮏ اﺳﯿﺪ × 2 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻟﯿﺘﺮ ﺑﻨﺰﯾﻞ آدﻧﯿﻦ( ﺑﻪدﺳﺖ آﻣﺪ. ﻫﻤﭽﻨﯿﻦ ﻣﯿﺰان ﺗﺎﮐﺴﺎنﻫﺎ در اﻧﺪام ﻫﻮاﯾﯽ ﺑﯿﺶﺗﺮ از رﯾﺸﻪ ﺑﻮده و ﺑﯿﺶﺗﺮﯾﻦ ﺗﺎﮐﺴﺎن در اﻧﺪام ﻫﻮاﯾﯽ 10- دي اﺳﺘﯿﻞ ﺑﺎﮐﺎﺗﯿﻦ  و در رﯾﺸﻪ ﺗﺎﮐﺴﻮل و 10- دي اﺳﺘﯿﻞ ﺗﺎﮐﺴﻮل ﺑﻮد ﮐﻪ ﺣﺪاﮐﺜﺮ ﻣﻘﺪار آنﻫﺎ در ﻫﻔﺘﻪ ﭼﻬﻠﻢ ﺑﻪدﺳﺖ آﻣﺪ. در ﮐﻞ ﻧﺘﺎﯾﺞ اﯾﻦ ﺗﺤﻘﯿﻖ ﺣﺎﮐﯽ از ﺗﺄﺛﯿﺮ ﻣﺜﺒﺖ اﺳﺘﻔﺎده از ﺗﻨﻈﯿﻢﮐﻨﻨﺪهﻫﺎي رﺷﺪ، زﻏﺎل ﻓﻌﺎل و ﮐﺎﻫﺶ ﻏﻠﻈﺖ ﻧﻤﮏﻫﺎي ﻣﺤﯿﻂ ﮐﺸﺖ ﺑﺮ رﺷﺪ رﯾﺸﻪ و ﺗﻮﺳﻌﻪ ﻋﺎدي ﮔﯿﺎﻫﭽﻪ ﺑﻪﻣﻨﻈﻮر ﺑﻬﯿﻨﻪﺳﺎزي ﺗﮑﺜﯿﺮ ﮔﯿﺎﻫﭽﻪﻫﺎي ﺳﺮﺧﺪار در ﮐﺸﺖ درونﺷﯿﺸﻪاي ﺑﻮد. ﻫﻤﭽﻨﯿﻦ ﻧﺸﺎن داده ﺷﺪ ﮐﺸﺖ ﺳﻠﻮﻟﯽ ﺣﺎﺻﻞ از اﻧﺪام ﻫﻮاﯾﯽ ﻧﺴﺒﺖ ﺑﻪ ﮐﺸﺖ رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ ﮔﺰﯾﻨﻪ ﺑﻬﺘﺮي در ﺟﻬﺖ ﺗﻮﻟﯿﺪ ﺗﺎﮐﺴﺎنﻫﺎ در ﺷﺮاﯾﻂ آزﻣﺎﯾﺸﮕﺎﻫﯽ اﺳﺖ.

Yew (Taxus baccata L.) has diterpenoid compounds with anti-cancer effects therefore it is one of the most well-known medicinal plants that is in danger of extinction. In this study, which was conducted at the Research Institute of Medicinal Plants of Shahid Beheshti University, the effect of culture medium (MS, 1/2MS and NN), phenolic absorbents (PVP and activated charcoal) and plant growth regulators (IBA, GA3 and BA) was investigated in a factorial design with three replications, with the aim of improving germination and growth of seedlings in in vitro culture. In the following, taxane production was measured in plantlets that were grown in select medium for ten months. According to our results, NN culture medium due to lower concentration of basal salts had no significant effect on germination percentage but increased root length and normal seedling growth percentage. Also supplementing the media with phenolic absorbent extended root length and shoot height as well as increasing normal seedling growth rate. In using PGRs, the highest normal seedling growth rate, shoot height and root length was obtained respectively from IBA × GA3 (0.2 mg/l gibberellic acid × 0.01 mg/l indole butyric acid), IBA × BA (0.2 mg/l gibberellic acid × 2 mg/l benzyl adenine) and 0.1 mg/l indole butyric acid, while PGRs had no significant differences in germination rate. Also, the amount of taxan in the aerial part was higher than the root and the highest taxan in the aerial part was 10-deacetylbaccatinIII and in the root were taxol and 10-deacetyltaxol, the maximum of all was achieved at the end of the experiment. Overall, the results indicate the positive effect of activated charcoal, PGRs and reducing culture medium macronutrients on root length, shoot height and the normal growth process of Taxus baccata seedling, in order to increase the yeild of yew propagation under in vitro culture condition. The cell culture obtained from the aerial part is much better than the hairy root cell culture.