بررسي اثر تنظيم كننده هاي رشد گياهي و سويه اگروباكتريوم بر باززايي و تراريختي چند رقم برنج ايراني

Investigation on the effects of plant growth regulators and Agrobacterium strain on regeneration and transformation of some Iranian rice cultivars

ﮐﺸﺖ ﺑﺎﻓﺖ ﯾﮑﯽ از ﻣﻬﻤﺘﺮﯾﻦ ﻣﺮاﺣﻞ ﻣﻬﻨﺪﺳﯽ ژﻧﺘﯿﮏ و ﺳﺎﯾﺮ ﺑﺮﻧﺎﻣﻪ ﻫﺎي اﺻﻼح ﮔﯿﺎﻫﺎن اﺳﺖ. در اﯾﻦ ﭘﮋوﻫﺶ اﺛﺮ ﺗﻨﻈﯿﻢ ﮐﻨﻨﺪهﻫﺎي رﺷﺪي ﻣﺨﺘﻠﻒ ﺷﺎﻣﻞ D-2،4 در 4 ﺳﻄﺢ )1/5، 2، 2/5 و 3 ﻣﯿﻠﯽ-ﮔﺮم در ﻟﯿﺘﺮ( و Kin در ﺳﻪ ﺳﻄﺢ )0، 0/4 و 0/8 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ( ﺑﺮ ﮐﺎﻟﻮسزاﯾﯽ و ﺑﺎززاﯾﯽ ﭼﻬﺎر رﻗﻢ ﺑﺮﻧﺞ اﯾﺮاﻧﯽ ﺷﺎﻣﻞ ارﻗﺎم ﻫﺎﺷﻤﯽ، ﮐﺎﻇﻤﯽ، ﻃﺎرم و ﺷﻔﻖ ﻣﻮرد ﻣﻄﺎﻟﻌﻪ ﻗﺮار ﮔﺮﻓﺖ. اﯾﻦ آزﻣﺎﯾﺶ در ﻗﺎﻟﺐ ﻓﺎﮐﺘﻮرﯾﻞ ﺑﺎ ﻃﺮح ﭘﺎﯾﻪ ﮐﺎﻣﻼ ﺗﺼﺎدﻓﯽ ﺑﺎ ﺳﻪ ﻋﺎﻣﻞ در ﺳﻪ ﺗﮑﺮار اﻧﺠﺎم ﮔﺮدﯾﺪ. ﻧﺘﺎﯾﺞ ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ دادهﻫﺎي ﺣﺎﺻﻞ ﻧﺸﺎن داد ﮐﻪ رﻗﻢ ﺷﻔﻖ ﺑﯿﺸﺘﺮﯾﻦ وزن ﺗﺮ ﮐﺎﻟﻮس )312 ﻣﯿﻠﯽﮔﺮم( و رﻗﻢ ﮐﺎﻇﻤﯽ ﮐﻤﺘﺮﯾﻦ ﻣﯿﺰان وزن ﺗﺮ ﮐﺎﻟﻮس )197 ﻣﯿﻠﯽﮔﺮم( را در ﺑﯿﻦ ارﻗﺎم ﻣﻮرد ﻣﻄﺎﻟﻌﻪ داﺷﺘﻨﺪ. ﺑﻬﺘﺮﯾﻦ ﮐﺎﻟﻮسزاﯾﯽ در ﺗﺮﮐﯿﺐ ﻫﻮرﻣﻮﻧﯽ 0/8 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ Kin و 2/5 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ D- 2،4 ﺑﺪﺳﺖ آﻣﺪ. ﺗﺠﺰﯾﻪ وارﯾﺎﻧﺲ دادهﻫﺎ ﺑﺮاي ﺗﺎﺛﯿﺮ ﻣﺤﯿﻂ ﮐﺸﺖ ﮐﺎﻟﻮسزاﯾﯽ ﺑﻪ ﺑﺎززاﯾﯽ ﻧﺸﺎن داد ﮐﻪ اﺛﺮ ﻫﻮرﻣﻮن ﮐﺎﻟﻮسزاﯾﯽ ﺑﺮ درﺻﺪ ﺑﺎززاﯾﯽ در ﺳﻄﺢ 0/01 ﻣﻌﻨﯽدار اﺳﺖ. اﺛﺮ رﻗﻢ ﻧﯿﺰ در ﻣﯿﺰان ﺑﺎززاﯾﯽ اﺛﺮ ﻣﻌﻨﯽدار داﺷﺖ. ﺑﯿﺸﺘﺮﯾﻦ درﺻﺪ ﺑﺎززاﯾﯽ و ﺗﻌﺪاد ﺷﺎﺧﻪ ﺑﺮاي ﻫﺮ رﯾﺰﻧﻤﻮﻧﻪ از ﮐﺎﻟﻮسﻫﺎي ﺣﺎﺻﻞ از ﺗﯿﻤﺎر ﻫﻮرﻣﻮﻧﯽ 0/8 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ Kin ﺑﻪ ﻫﻤﺮاه 1/5 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ D-2،4 ﺑﺪﺳﺖ آﻣﺪ. ﺑﯿﺸﺘﺮﯾﻦ درﺻﺪ ﺑﺎززاﯾﯽ ﻣﺮﺑﻮط ﺑﻪ رﻗﻢ ﮐﺎﻇﻤﯽ 74% و ﮐﻤﺘﺮﯾﻦ آن ﻣﺮﺑﻮط ﺑﻪ رﻗﻢ ﻃﺎرم 10 ﺑﻮد. ﺑﯿﺸﺘﺮﯾﻦ ﺷﺎﺧﻪزاﯾﯽ ﻣﺮﺑﻮط ﺑﻪ رﻗﻢ ﺷﻔﻖ ﺑﺎ 4/41 ﺷﺎﺧﻪ ﺑﻪ ازاي ﻫﺮ ﮐﺎﻟﻮس و ﮐﻤﺘﺮﯾﻦ آن ﺑﺎ ﯾﮏ ﺷﺎﺧﻪ ﻣﺮﺑﻮط ﺑﻪ رﻗﻢ ﻃﺎرم ﺑﻮد. در ﻣﯿﺎن ﺳﻪ ﺳﻮﯾﻪ EHA105 ،LBA4404، 1-AGL ﺑﮑﺎر ﮔﺮﻓﺘﻪ ﺷﺪه ﺑﺎ ﻧﺎﻗﻞ ﺑﯿﺎن ﮔﯿﺎﻫﯽ pCAMBIA1304 در ﺗﺮارﯾﺨﺘﯽ ﺑﺮﻧﺞ، ﮐﺎﻟﻮسﻫﺎي ﺗﺮارﯾﺨﺘﻪ ﺗﻨﻬﺎ ﺑﺎ اﺳﺘﻔﺎده از ﺳﻮﯾﻪ EHA105 ﺑﺪﺳﺖ آﻣﺪﻧﺪ و ﺑﯿﺎن ژن gus را ﻧﺸﺎن دادﻧﺪ.

Tissue culture is one of the most critical steps in genetic engineering and other breeding programs of plants. In this study, the effect of some factors in regeneration of four rice cultivars, including: Hashemi, Kazemi, Tarom and Shafagh has been studied. In the first experiment the effect of 2,4-D at 4 levels (1.5, 2, 2.5 and 3 mg/l) combination with Kin at three levels (0, 0.4 and 0.8 mg/l) on callus induction and plant regeneration in rice cultivars were studied. After two months calli were transferred to MS medium supplemented with 30g sucrose containing 2.5 mg/l BAP. Analysis of variance showed that Shafagh and Kazemi give the highest and lowest callus respectively. The best callus was obtained in medium supplemented with 0.8 mg/l Kin and 2.5 mg/l of 2,4-D. Analysis of variance showed that plant growth regulators used in callus induction medium, significantly affected the regeneration of plants. Cultivar had a significant effect on the regeneration rate. The highest plant regeneration and number of shoots per explant were obtained from callus derived from medium containing 0.8 mg/l Kin and 1.5 mg/l of 2,4-D. The Kazemi gave the highest percentage (74 %) and Tarom gave the lowest percentage (10%). Among the three strains used for transformation of rice, transgenic callus was obtained only with strain EHA105.