تاثير غلظت هاي مختلف عناصر غذايي محيط كشت بر بهينه سازي القاي ريشه هاي مويين خرفه

Effect of different concentrations of medium nutrients on optimization of hairy root induction in purslane

اﻟﻘﺎي رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ ﺑﻪوﺳﯿﻠﻪ Agrobacterium rhizogenes روش ﻣﻮﺛﺮي ﺑﺮاي اﻓﺰاﯾﺶ ﺗﺮﮐﯿﺒﺎت داروﯾﯽ ﮔﯿﺎه ﺧﺮﻓﻪ اﺳﺖ. رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ ﻋﻼوه ﺑﺮ ﻣﯿﺰان رﺷﺪ زﯾﺎد، از ﭘﺎﯾﺪاري ژﻧﺘﯿﮑﯽ و ﺑﯿﻮﺷﯿﻤﯿﺎﯾﯽ ﺑﺮﺧﻮردارﻧﺪ. ﺧﺮﻓﻪ ).Portulaca oleracea L( ﯾﮑﯽ از ﮔﯿﺎﻫﺎن داروﯾﯽ ارزﺷﻤﻨﺪ ﻣﯽﺑﺎﺷﺪ و داراي ﻣﺘﺎﺑﻮﻟﯿﺖﻫﺎي ﺛﺎﻧﻮﯾﻪ ارزﺷﻤﻨﺪي از ﺟﻤﻠﻪ دوﭘﺎﻣﯿﻦ و ﻧﻮرآدرﻧﺎﻟﯿﻦ اﺳﺖ. در اﯾﻦ ﭘﮋوﻫﺶ رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ ﺧﺮﻓﻪ ﺑﺎ اﺳﺘﻔﺎده از Arobacterium rhizogenes ﺳﻮﯾﻪي A4 اﻟﻘﺎ ﺷﺪ. ﻫﻤﭽﻨﯿﻦ ﺗﺎﺛﯿﺮ ﻏﻠﻈﺖﻫﺎي ﻣﺨﺘﻠﻒ ﻋﻨﺎﺻﺮ ﻏﺬاﯾﯽ ﻣﺎﮐﺮو ﻣﺤﯿﻂﮐﺸﺖ )2x ،1/5x ،1x ،0/5x و 2/5x ﺑﺮاﺑﺮ ﻏﻠﻈﺖ ﻋﻨﺎﺻﺮ در ﻣﺤﯿﻂﮐﺸﺖ MS ﭘﺎﯾﻪ( ﺑﺮ درﺻﺪ اﻟﻘﺎي رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ و ﻣﯿﺰان ﻓﻨﻮل و ﻓﻼوﻧﻮﺋﯿﺪ ﮐﻞ در رﯾﺸﻪﻫﺎ ﺑﺮرﺳﯽ ﺷﺪ. ﺑﻪﻣﻨﻈﻮر ﺗﺎﺋﯿﺪ ﺗﺮارﯾﺨﺘﯽ رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ، وﺟﻮد ژن rolB در رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ، از روش PCR و ﭘﺮاﯾﻤﺮﻫﺎي ژن rolB اﺳﺘﻔﺎده ﺷﺪ. ﻧﺘﺎﯾﺞ ﺑﻪدﺳﺖ آﻣﺪه ﻧﺸﺎن داد ﮐﻪ ﺑﯿﺸﺘﺮﯾﻦ درﺻﺪ اﻟﻘﺎي رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ در ﻏﻠﻈﺖ 0/5x ﭘﺘﺎﺳﯿﻢ ﻧﯿﺘﺮات )80درﺻﺪ( ﻣﺸﺎﻫﺪه ﺷﺪ. ﻏﻠﻈﺖ 0/5x ﭘﺘﺎﺳﯿﻢ ﻧﯿﺘﺮات ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﻓﻨﻮل ﮐﻞ )4/52 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﮔﺮم وزن ﺧﺸﮏ( را داﺷﺖ. ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﻓﻼوﻧﻮﺋﯿﺪ ﮐﻞ )18/2 و 17/02 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﮔﺮم وزن ﺧﺸﮏ( ﺑﻪﺗﺮﺗﯿﺐ ﻣﺮﺑﻮط ﻏﻠﻈﺖ 0/5x و 1x ﭘﺘﺎﺳﯿﻢ ﻧﯿﺘﺮات ﺑﻮد. ﺗﺎﺋﯿﺪ ﻣﻮﻟﮑﻮﻟﯽ رﯾﺸﻪﻫﺎي ﻣﻮﯾﯿﻦ ﺗﺮارﯾﺨﺘﻪ ﺗﻮﺳﻂ واﮐﻨﺶ PCR اﻧﺠﺎم ﺷﺪ.

Hairy root induction by Agrobacterium rhizogenes is a reliable method to increase P. oleracea medicinal compounds. The hairy roots in addition to high growth rates are genetically and biochemically stable. Purslane (Portulaca oleracea L.) is one of the valuable medicinal plants and has important secondary metabolites such as dopamine and noradrenaline. In this study, hairy roots of Purslane were produced by using Agrobacterium rhizogenes strain A4 and different concentrations of macro elements (0.5x, 1x, 1.5x, 2x and 2.5x concentrations of MS base medium) were investigated. For confirming hairy roots transformation, the presence of the rolB gene in the hairy roots was performed by PCR and using rolB gene's specific primers. The results show that maximum hairy root induction was observed by using 0.5x KNO3 (80 percentage). The highest phenolic content was obtained by using 0.5x KNO3 (4.52 mg/g DW) and also maximum flavonoid content, (18.2 and 17.02 mg/g DW respectively) was belonged to 0.5x and 1x KNO3. Molecular confirmation of transgenic hairy roots was done with PCR reaction