Effects of GA₃ and KNO₃ on the Germination and DNA Content of Flax (Linum usitatissimum L.).)

Germination rate of seeds has been enhanced either by exogenous application of growth hormone or by seed priming. Batista et al. [1] recorded that seeds of pepper primed with GA₃ at 200 mg/L concentration before germination facilitated the rate of germination unlike the unprimed seeds. Also, seeds of Amaranthus cruentus pretreated with BAP have shown to have increased germination rate [2]. Exogenous application of hormones and salts have been recorded as promising in removing the factors that led to dormancy in seeds [3,4]. Although there may be some exceptions where application of some growth hormones may not enhance germination; Wall et al. [5] stated that exogenous application of GA₃ did not enhance germination in Pyxidanthera brevifolia.

Seeds act as sensors to changes to their immediate environment and hence are adjustable to range of environmental factors. Therefore, most seeds have evolved different means of sensing when the environment is convenient to complete germination [6- 8], the adjustments create differential seasons for germination to take place. BAP (6-Benzylaminopurine) is an aromatic cytokinin and among the growth stimulants has been involved in seedling enhancement under stress such as; low temperature, salinity and water stress. There is a link between stress and hormonal balance; the pressure stimulates the release of inhibitors and causes a decline in the amount of endogenous growth stimulants, therefore when there is an external application of this growth hormone, it induces the event responsible for uptake of water [9]. Gibberellic acid (GA₃) is actively involved in enhancing germination of quite a number of plant species [10-13]. Therefore, application of GA₃ is a regular method for releasing seed dormancy [14]. Gibberellin can release dormancy in some seeds that normally require cold (stratification) or light to complete germination [15-17]. Potassium nitrate (KNO₃) is a known chemical treatment used to enhance germination; the common application is the use of 0.1-0.2% KNO₃ solutions, which are recommended by some International analysts for regular seed germination test of various species [18,19]. It was observed to be effective in improving the seeds of watermelon germination when subjected to the treatment under unfavourable condition [3]. This research focuses on the germination and DNA studies of Flax (Linum usitatissimum L.) seed treated with growth stimulants.

The matured flax (Linum usitatissimum L.) seeds (Plate 1) were obtained from fruit garden Port Harcourt, Nigeria. The seeds were properly identified by the Curator at the Herbarium Unit of Department of Plant Science and Biotechnology, University of Port Harcourt. Viability test was carried out on the seeds to ascertain its viability; hence, non-viable seeds were discarded. The viable seeds were surface sterilized with ethanol for five (5) minutes and rinse with distilled water. Germination studies were first carried out both under light and dark condition.

The growth stimulants used in the study were gibberellic acid (GA₃-350 g/mol) and potassium nitrate (KNO3 -101.1 g/mol). The concentrations (1 mM, 5 mM and 10 mM) of these growth stimulants were prepared for each growth stimulants, respectively. Water was used as the Control treatment. These concentrations were used to pretreated flax seeds with 20-seeds per batch. Each treatment was replicated five times. The seeds were germinated under room temperature of 25 °C, monitored daily and the process lasted for 14 days. Germination percentage of seeds taken for each treatment. Also, the DNA concentration of the radicle with the highest germination count across treatments were assessed using a Quick DNA miniprep kit for isolation of the total DNA from the radicle sample of flax ensuring that there was no contamination with RNA.

The data obtained from the study were subjected to statistical analysis using SAS 9.1.3 version (Plate 1).

The percentage germination of flax seed germinated under light and dark conditions are presented in Figure 1. The dark condition promotes the germination of flax seed than under light condition. However, the results of the germinations indicated low germinations.

The percentage germination of flax seeds treated with different concentrations of GA₃ and KNO₃ are presented in Figure 2. The study showed that GA₃ and KNO₃ treatments enhanced the germination of flax seed when compared to Control treatment. Across the treatments, GA₃ gave the highest germination percentage with 1 mM concentration been the highest. Also, as the concentration of GA₃ and KNO₃ increases (1-10 mM), the germination percentage reduces with 1 mM concentration producing the highest germination count. The DNA concentration of the flax radicle that produced these highest germination percentage in the different treatments are: GA₃ (291.50 ng/μl), KNO₃ (47.77 ng/μl) and Control (47.33 ng/μl). The analysis of variance (ANOVA) showed that treatments are significant at p-value (0.0001) < 5% significant level for flax seed. Also, the multiple comparisons using least significant difference (LSD) showed that the Control is significantly different at 5% significant level from GA₃.

The stimulatory effect of GA₃ on germination of flax seed is in line with the work of other researchers [10-14]. The study showed that GA₃ enhanced germination compared to KNO₃ and Control. Again, the KNO₃ treatments did not enhanced percentage germinations over Control. Similar reasons were adduced by Bewley et al. [20] that when the damage of the nucleic acids, organelles, enzymes and membranes occurred during storage and maturation by the dry seeds, exceed the ability of the imbibed seeds to repair the systems; then the functional systems start to deteriorate and eventually lead to death which terminate germination. On the other hand, KNO₃ treatment shortens the time spent for germination when control of physical environment only is a factor. This was observed by Demir, et al. [3] to be effective in improving germination in the seeds of watermelon subjected to unfavourable growth condition.According to Alonso Ramirez et al. [11], the exogenous application of GA₃ on Arabidopsis seeds in an unfavourable condition greatly enhanced its germination. Potassium nitrate (KNO₃) is a known chemical treatment that enhance germination [3].

Seeds of flax treated with 1 mM GA3 probably had made for fast DNA repair when compared to the Control treatment. In comparing the DNA contents with germination percentage, the difference in the trend may be attributed to the type of seeds and the responses of the seeds to the conditions of the treatments. This was also observed by Liu et al. [21], who documented the effect of osmopriming on DNA synthesis in tomato seeds.

The study has shown that 1 mM concentration of GA₃ and KNO₃ enhances the germination. It also reveals that as the concentration of the growth stimulants increases the germination percentage decreases. Again, the DNA concentration of the flax radicle depends on the chemical used in treating the seeds of flax.

None

No conflict of interest.

1. Singh D, Sharma RR (2007) Postharvest diseases of fruit and vegetables and their management. In: Prasad D (Edn.), Sustainable Pest Management, Daya Publishing House, New Delhi, India.
2. Tomassini A, Selia L, Raiola A, D Ovidio R, Favaron F (2009) Characterization and expression of Fusarium graminearum endopolygalacturonases in vitro and during wheat infection, Journal of Plant Pathology 58(3): 556-564.
3. Torimiro, N. and Okonji REA (2013) Comparative Study of Pectinolytic Enzyme Production by Bacillus Species. African Journal of Biotechnology 12: 6498-6503.
4. Barth M, Hankinson TR, Zhuang H, Breidt F (2009) Microbiological spoilage of fruits and vegetables. Journal of Food Microbiology and Food Safety 1(6): 135-174.
5. Mukesh Kumar DJ, Saranya GM, Suresh K, Andal Priyadharshini D, Rajakumar R, et al. (2012) Production and Optimization of Pectinase from Bacillus sp. MFW7 using Cassava Waste. Asian Journal of Plant Science and Research 2: 369-375.
6. Bayoumi RA, Yassin HM, Swelim MA, Abdel All EZ (2008) Production of Bacterial Pectinase(S) from Agro-Industrial Wastes Under Solid State Fermentation Conditions. Journal of Applied Sciences Research 4: 1708-1721.
7. Jayashankar NP, Graham PH (1970) An agar plate method for screening and enumerating pectinolytic microorganisms. Can J Microbiol 16(10): 1023
8. Maldonado MC, Strasser de Saad AM (1998) Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid-state systems, J Ind Microbiol Biotechnol 20: 34-38
9. Breeds R, Murray EGD and Smith NR (1957) Bergey’s manual of Determinative Bacteriology (7^th edn), The Williams and Wilkins Company, Baltimore USA.
10. Olutiola PO, Famurewa O, and Sonntag HG (1991) An Introduction to General Microbiology: A practical Approach. (1^st edn) Heidelberg, Germany. pp. 267.
11. Li Q, Coffman AM, Ju LK (2015) Development of reproducible assays for polygalacturonase and pectinase. Enzyme Microb Technol 72: 42-48.
12. Nelson N (1944) A photometric adaptation of the Somogyi method for determination of glucose. J Biol Chem 153: 375-380.
13. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195: 19-23.
14. Ayers WA, Papavizas GC, Diem A (1966) Polygalacturonate trans-eliminase production by Rhizoctonia solani. Phytopathol 56: 1006-1011.
15. Whitaker JR (1990) Microbial pectinolytic enzymes. In: Fogarty WM, Kelly CT, editors. Microbial enzymes and biotechnology. 2nd ed. London: Elsevier Science Ltd pp. 133-176.
16. Banu RA, Devi KM, Gnanaprabhal GR, Pradeep BV, Palaniswamy A (2010) Production and characterization of pectinase enzyme from Penicillin chrysogenum. Ind J Sci Technol 3(4): 377-381.
17. Namasivayam E, Ravindar JD, Mariappan KA, Kumar M (2011) Production of extracellular pectinase by Bacillus cereus isolated from market solid waste. J Bioanal Biomed 3: 070-075.
18. Vibha B, Neelam G (2010) Exploitation of microorganisms for screening of pectinase from environment.8^th International Conference. Globelics.
19. Hirose N, Kishida M, Kawasaki H, Sakai T (1999) Purification and Characterization of an Endo Polygalacturonase from a Mutant of Saccharomyces cerevisiae. Biosci Biotechnol Biochem 63(6): 1100-1103.
20. Kashyap DR, Chandra S, Kaul A, Tewari R (2000) Production, purification and characterization of pectinase from a Bacillus sp. DT7. World J Microbiol Biotechnol 16: 277-282.
21. Saad Naïma, Briand M, Gardarin C, Briand Y, Michaud Philippe (2007) Production, purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894. Enzyme and Microbial Technology 41: 800-805.
22. Jayani RS, Saxena S, Gupta R (2005) Microbial pectinolytic enzymes: a review. Proc Biochem 40(9): 2931-2944.
23. Freitas PM, Martin N, Silva D, da Silva R, Gomes E (2006) Production and partial characterization of polygalacturonases.
24. Silva Dênis, Martins Eduardo da Silva, Silva, Roberto da, Gomes Eleni (2002) Pectinase production by Penicillium viridicatum RFC3 by solid state fermentation using agricultural wastes and agro-industrial by-products. Brazilian Journal of Microbiology 33(4): 318-324.
25. Zhang CH, Li ZM, Peng XW, Jia Y, Zhang HX, et al. (2009) Separation, purification and characterization of endopolygalacturonase from a newly isolated Penicillium axalicum. Chinese Journal of Process Engineering 9: 242-249.
26. Freitas Paula Mendes de, Martin, Natalia Silva, Dênis Silva, Roberto da, Gomes Eleni (2006) Production and partial characterization of polygalacturonases produced by thermophilic Monascus sp N8 and by thermotolerant Aspergillus sp N12 on solid-state fermentation. Brazilian Journal of Microbiology 37(3): 302-306.
27. Nighojkar Sadhana, Phanse Yashdeep, Sinha Divya, Nighojkar Anand, Kumar Anil (2006) Production of polygalacturonase by immobilized cells of Aspergillus niger using orange peel as inducer. Process Biochemistry 41: 1136-1140.
28. Ahlawat S, Dhiman SS, Battan B, Mandhan RP, Sharma J (2009) Pectinase production by Bacillus subtilis and its potential application in biopreparation of cotton and micropoly fabric. Process Biochem 44(5): 521-526.
29. Beg QK, Bhushan B, Kapoor M, G Hoondal (2000) Production and Characterization. Thermostable Xylanase and Pectinase from Streptomyces Sp. QG-11-3. Journal of Industrial Microbiology and Biotechnology 24: 396-402.
30. Fawole OB, Odunfa SA (1992) Pectolytic moulds in Nigeria, fermentation using agricultural residues and agro-industrial by products. Brazilian Journal of Microbiology 33: 318-324.