عوامل مؤثر بر پديده شيشه‌اي شدن بافت گياهي در شرايط كشت درون‌شيشه‌اي و راهكارهاي مقابله با آن مطالعه موردي: گياه ميخك (Dianthus caryophyllus L.)

Factors Affecting Plant Tissue Hyperhydricity at in vitro Culture and Strategies to Resolve it Case Study: (Dianthus caryophyllus L.)

ﭘﺪﯾﺪه ﺷﯿﺸﻪ اي ﺷﺪن ﻧﺎﺷﯽ از ﺗﻐﯿﯿﺮات ﻧﺎﻣﻄﻠﻮب ﻓﯿﺰﯾﻮﻟﻮژﯾﮑﯽ و ﻣﻮرﻓﻮﻟﻮژﯾﮑﯽ در ﺑﺎﻓﺖ ﮔﯿﺎﻫﯽ اﺳﺖ. در ﺑﺎﻓﺖ ﻫﺎي ﺷﯿﺸﻪ اي ﺷﺪه ﺗﻌﺪاد دﺳﺘﻪ ﺟﺎت آوﻧﺪي، ﺳﻠﻮل ﻫﺎي روزﻧﻪ و ﺳﻠﻮل ﻫﺎي ﮐﻮﺗﯿﮑﻮل ﺑﺮگ ﮐﺎﻫﺶ ﻣﯽ ﯾﺎﺑﺪ و واﮐﻮﺋﻞ ﻫﺎ در ﺳﻠﻮل ﻫﺎي ﻣﺰوﻓﯿﻞ ﺑﺮگ ﺣﺠﯿﻢ ﻣﯽ ﺷﻮﻧﺪ. ﻋﻮاﻣﻞ ﻣﺘﻌﺪدي ﺑﺮ ﭘﺪﯾﺪه ﺷﯿﺸﻪ اي ﺷﺪن ﻣﻮﺛﺮ ﻣﯽ ﺑﺎﺷﺪ ﮐﻪ از ﻣﻬﻢ ﺗﺮﯾﻦ اﯾﻦ ﻋﻮاﻣﻞ ﻧﻮع ﺗﻨﻈﯿﻢ ﮐﻨﻨﺪه ي رﺷﺪ، ﻧﻮع و ﻏﻠﻈﺖ ﺳﯿﺘﻮﮐﯿﻨﯿﻦ ، ﻧﺴﺒﺖ ﯾﻮن ﻧﯿﺘﺮات ﺑﻪ آﻣﻮﻧﯿﻮم در ﻣﺤﻠﻮل ﻏﺬاﯾﯽ، ﻧﻮع و ﻣﻘﺪار آﮔﺎر، ﺗﻬﻮﯾﻪ و ﺗﻌﺪاد دﻓﻌﺎت واﮐﺸﺖ ﻣﯽ ﺑﺎﺷﺪ. اﻓﺰاﯾﺶ ﻏﻠﻈﺖ آﮔﺎر ﺑﻪ ﻋﻠﺖ ﮐﺎﻫﺶ رﻃﻮﺑﺖ ﻣﺤﯿﻂ ﮐﺸﺖ ﺑﺎﻋﺚ ﮐﺎﻫﺶ اﯾﻦ ﭘﺪﯾﺪه ﻣﯽ ﮔﺮدد. درﺣﺎﻟﯿﮑﻪ اﻓﺰاﯾﺶ ﻏﻠﻈﺖ ﻫﻮرﻣﻮن ﺳﯿﺘﻮﮐﯿﻨﯿﻦ و ﮐﺎﻫﺶ ﻧﺴﺒﺖ ﯾﻮن ﻧﯿﺘﺮات ﺑﻪ آﻣﻮﻧﯿﻮم در ﻣﺤﯿﻂ ﮐﺸﺖ ﺑﺎﻋﺚ اﻓﺰاﯾﺶ آن ﻣﯽ ﺷﻮد. ﺣﻀﻮر ﻫﻮرﻣﻮنBA ﻧﺴﺒﺖ ﺑﻪ ﺳﺎﯾﺮ ﺳﯿﺘﻮﮐﯿﻨﯿﻦ ﻫﺎ و ﻫﻤﭽﻨﯿﻦ ﻓﯿﺘﺎژل ﻧﺴﺒﺖ ﺑﻪ ﺳﺎﯾﺮ اﻧﻮاع آﮔﺎر ﺑﺎﻋﺚ اﻓﺰاﯾﺶ ﭘﺪﯾﺪه ﺷﯿﺸﻪ اي ﺷﺪن ﻣﯽ ﺷﻮﻧﺪ. اﺳﺘﻔﺎده از درﭘﻮش ﭘﺎراﻓﯿﻠﻢ ﺑﻪ ﻋﻠﺖ اﻓﺰاﯾﺶ ﺗﺒﺎدﻻت ﮔﺎزي ﺑﺎﻋﺚ ﮐﺎﻫﺶ ﺗﺠﻤﻊ اﺗﯿﻠﻦ و در ﻧﺘﯿﺠﻪ ﮐﺎﻫﺶ ﻣﯿﺰان ﺷﯿﺸﻪ اي ﺷﺪن ﻣﯽ ﮔﺮدد. ﮐﺎرﺑﺮد ﺳﯿﺴﺘﻢ ﺳﺮﻣﺎدﻫﯽ از ﮐﻒ ﻣﯽ ﺗﻮاﻧﺪ ﺑﺎﻋﺚ ﮐﺎﻫﺶ ﻓﻌﺎﻟﯿﺖ ﺗﻤﺎﻣﯽ آﻧﺰﯾﻢ ﻫﺎي آﻧﺘﯽ اﮐﺴﯿﺪاﻧﺖ ﮔﺮدد و ﻣﻨﺠﺮ ﺑﻪ ﮐﺎﻫﺶ ﻣﯿﺰان ﺷﯿﺸﻪ اي ﺷﺪن ﺷﺎﺧﺴﺎره ﻫﺎ ﻣﯽ ﮔﺮدد. ﻟﺬا ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﻧﺘﺎﯾﺞ ﮔﺰارش ﺷﺪه، ﺟﻬﺖ ﮐﺎﻫﺶ ﻣﯿﺰان ﭘﺪﯾﺪه ﺷﯿﺸﻪ اي ﺷﺪن در ﺷﺮاﯾﻂ درون ﺷﯿﺸﻪ اي، ﮐﺎرﺑﺮد ﺳﯿﺘﻮﮐﯿﻨﯿﻦ ﺑﺎ ﻏﻠﻈﺖ ﻫﺎي ﭘﺎﯾﯿﻦ ﺗﺮ، ﺟﺎﯾﮕﺰﯾﻦ ﻧﻤﻮدن BA ﺑﺎ ﺳﺎﯾﺮ ﺳﯿﺘﻮﮐﯿﻨﯿﻦ ﻫﺎي راﯾﺞ و در ﺻﻮرت ﻟﺰوم ﮐﺎرﺑﺮد ﻏﻠﻈﺖ ﻫﺎي ﭘﺎﯾﯿﻦ BA )0/5 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ(، اﺳﺘﻔﺎده از درﭘﻮش ﭘﺎراﻓﯿﻠﻢ، اﻓﺰاﯾﺶ ﻏﻠﻈﺖ آﮔﺎر ﺗﺎ ﺣﺪ ﻗﺎﺑﻞ ﻗﺒﻮل )10 ﮔﺮم در ﻟﯿﺘﺮ(، ﮐﺎرﺑﺮد ﺳﯿﺴﺘﻢ ﺳﺮﻣﺎدﻫﯽ از ﮐﻒ، ﮐﺎﻫﺶ ﺗﻌﺪاد دﻓﻌﺎت واﮐﺸﺖ ﺗﺎ ﺣﺪ اﻣﮑﺎن و ﺳﺎزﮔﺎر ﻧﻤﻮدن ﻫﺮﭼﻪ ﺳﺮﯾﻌﺘﺮ ﮔﯿﺎﻫﭽﻪ ﻫﺎي ﮐﺸﺖ ﺑﺎﻓﺘﯽ ﭘﯿﺸﻨﻬﺎد ﻣﯽ ﮔﺮدد.

Hyperhydricity is the development of an undesirable physiological and morphological change in plant tissue. In vitrified plants, the number of vascular bundles, stomata and cuticle cells are reduced and mesophyll cells contain massive vacuole. Several factors influence this phenomenon and the most important of these factors include: type of the plant growth regulator, type and concentration of cytokinin, ratio of ammonium to nitrate in the culture medium, type and concentration of agar, ventilation and number of subculturing. Increasing agar concentrations leads to decreasing this phenomenon due to reducing the moisture of culture medium. Increasing concentration of cytokinins and decreasing the ratio of nitrate to ammonium in the medium cause the occurrence of hyperhydricity. Presence of BA in compare to other types of cytokinins and also phytagel in compare to other type of gelling agent enhance the rate of hyperhydricity. Use of Parafilm cap decreases the amount of this phenomenon due to increasing gas exchange and decreasing accumulation of ethylene. The application of bottom cooling systems can reduce the activity of antioxidant enzymes and therefore cause reduction in the amount of hyperhydricity. By considering the reported results, to reduce the amount of hyperhydricity in vitro, using cytokinins with lower concentration, replacing BA with other commonly used cytokinins and if possible use lower concentrations of BA (0.5 mg/l), using Parafilm as vessel closure cap, increasing the concentration of agar to an acceptable level (10 g/l), using bottom cooling, reducing the frequency of sub-culturing as much as possible and acclimating tissue culture plantlets as soon as possible, is recommended.