Priming edilmiş endüstriyel kenevir (Cannabis sativa L.) tohumlarının in vitro çimlenmesinin optimize edilmesi

Öz

Endüstriyel kenevir (Cannabis sativa L., Cannabaceae) bitkisinin in vitro çimlenmesi, genom düzenleme gibi biyoteknolojik araçların uygulanması için oldukça önemlidir. Bu nedenle, tohumlar yüzey sterilize edildikten sonra 24, 48 ve 72 saat boyunca hidrojen peroksit (H2O2) ve steril distile su (dH2O) ile priming uygulama yapılmıştır. Daha sonra tohumlar, 1.0 mg/L BAP ve 200 mg/L antibiyotik ile zenginleştirilmiş Murashige ve Skoog (MS) ortamı üzerinde kültüre alınmıştır. Tohumların H2O2 ile yapılan priming sonucunda daha iyi sonuç verdiğini ve %100 çimlenme olduğu gözlemlenmiştir. Buna karşı hidropriming ile yapılan çalışmada 30-52,5 çimlenme gözlenirken, en iyi sonucun 48 saatlik priming sonucu olduğu kaydedilmiştir. Sürgün sayılarına bakıldığında en fazla 1,98 sürgün, %3,0 H2O2 ve 72 saatlik priming süresi kombinasyonundan elde edilmiştir. Sonuçlar ayrıca kutu grafiği, normal grafikler, kontur grafikleri ve yüzey grafikleri gibi farklı istatistiksel grafikler oluşturularak da analiz edilmiştir. Normal grafikler, H2O2 konsantrasyonunun her iki çıktı değişkenindeki önemini açıkça göstermiştir. Kontur ve yüzey grafikleri ise çıktı verilerini farklı alt gruplara ayırmış ve sonuçları doğrulanmıştır.

Anahtar Kelimeler

• Çimlenme
• endüstriyel kenevir
• in vitro
• priming
• istatistiksel analiz

Optimizing in vitro germination of primed industrial hemp (Cannabis sativa L.) seeds

Öz

Germination under in vitro conditions for industrial hemp (Cannabis sativa L., Cannabaceae) is highly significant for the application of biotechnological tools like genome editing. Therefore, seeds were surface sterilized followed by priming with hydrogen peroxide (H2O2) and sterile distilled water (dH2O) for 24, 48, and 72 h. The primed seeds were inoculated on the Murashige and Skoog (MS) medium enriched with 1.0 mg/L benzyl amino purine (BAP) and 200 mg/L antibiotics. Exposing seeds to H2O2 was superior and 100% germination was observed. Whereas hydropriming resulted in 30-52.5% germination with a maximum of 48h priming time. The results on shoot counts revealed a maximum of 1.98 shoots from the combination of 3.0% H2O2 and 72h priming time. The results were also analyzed by constructing different statistical plots like box plots, normal plots, contour plots, and surface plots. The normal plots exhibited the significance of H2O2 concentration on both output variables. Whereas contour and surface plots classified the output data into different sub-groups and confirmed the results.

Anahtar Kelimeler

• Germination
• industrial hemp
• in vitro
• priming
• statistical analysis

Kaynakça

1. Aasim M, Ali SA, Altaf MT, Ali E, Nadeem MA, Baloch FS (2023). Artificial neural network and decision tree facilitated prediction and validation of cytokinin-auxin induced in vitro organogenesis of sorghum (Sorghum bicolor L.). Plant Cell, Tissue and Organ Culture (PCTOC) 1-14.
2. Aasim M, Katırcı R, Akgur O,Yildirim B, Mustafa Z, Nadeem MA,Yılmaz G (2022). Machine learning (ML) algorithms and artificial neural network for optimizing in vitro germination and growth indices of industrial hemp (Cannabis sativa L.). Industrial Crops and Products 181: 114801.
3. Abass SM, Mohamed HI (2011). Alleviation of adverse effects of drought stress on common bean (Phaseolus vulgaris L.) by exogenous application of hydrogen peroxide. Bangladesh Journal of Botany 41: 75-83.
4. Anawar H, Canha N, Freitas M, Santa Regina I, Garcia-Sanchez A (2011). Effects of Different Drying Processes on the Concentrations of Metals and Metalloids in Plant Materials. J Radioanal Nucl Chem 289: 29-34.
5. Chandran H, Meena M, Barupal T, Sharma K (2020).Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnology Reports 26: e00450.
6. Hossain MA, Bhattacharjee S, Armin SM, Qian P, Xin W, Li HY (2015). Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: insights from ROS detoxification and scavenging. Frontiers in Plant Science 6: 420. Karatas M, Dogan M, Emsen B, Aasim M (2015). Determination of in vitro free radical scavenging activities of various extracts from in vitro propagated (Ceratophyllum demersum L.) Fresenius Environmental Bulletin 24(9a): 2946-2952.
7. Karataş M, Aasim M, Dazkirli M (2016). Influence of light-emitting diodes and benzylaminopurin on adventitious shoot regeneration of water hyssop (Bacopa monnieri (L.) Pennell) in vitro. Archives of Biological Sciences 68(3): 501-508.
8. Katirci R (2015). Statistical approach to optimizing a Zn-Ni bath containing ed and tea. Surface Review and Letters 22: 1550015.

9. Ramazan K, Uğur Y (2014). Statistical Studies of Zn-Ni Alloy Coatings Using Non-Cyanide Alkaline Baths Containing Polyethyleneimine Complexing Agents. Trans IMF 92: 245-252.
10. Murashige T, Skoog F (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.
11. Paparella S, Araújo SS, Rossi G, Wijayasinghe M, Carbonera D, Balestrazzi A (2015). Seed priming: state of the art and new perspectives. Plant Cell Reports 34: 1281-1293.
12. Salah SM, Yajing G, Dongdong C, Jie L, Aamir N, Qijuan H (2015). Seed priming with polyethylene glycol regulating the physiological and molecular mechanism in rice (Oryza sativa L.) under nano-ZnO stress. Scientific Reports 5: 14278.
13. Salentijn EMJ, Zhang Q, Amaducci S, Yang M, Trindade LM (2014). New developments in fiber hemp (Cannabis sativa L.) breeding. Industrial Crops and Products 68: 32-41.
14. Sorokin A, adav NS, Gaudet D, Kovalchuk I (2021). Development and standardization of rapid and efficient seed germination protocol for (Cannabis sativa ) Bio-protocol 11(1): e3875-e3875.
15. Tremlová B, Mikulášková HK, Hajduchová K, Jancikova S, Kaczorová D, Ćavar Zeljković S, Dordevic D (2021). Influence of technological maturity on the secondary metabolites of hemp concentrate (Cannabis sativa L.). Foods 10(6): 1418.
16. Ventura L, Dona M, Macovei A, Carbonera D, Buttafava A, Mondoni A (2012). Understanding the molecular pathways associated with seed vigor. Plant Physiology and Biochemistry 60: 196-206.
17. WojtylaŁ, Lechowska K, Kubala S, Garnczarska M (2016). Different modes of hydrogen peroxide action during seed germination. Frontiers in plant science 7: 66.
18. Zhang W, Wang XM, Rong FAN, Yin GX, WANG K, Du LP, Xiao LL, Ye XG (2015). Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos. Journal of Integrative Agriculture 14(1): 11-19.