پرآوری شاخساره درون‌شیشه‌ای گلابی رقم شکری تحت تاثیر عناصر غذایی محیط‌کشت در فضای نرم افزار Design Expert

نوع مقاله : مقالات پژوهشی

نویسندگان

دانشگاه فردوسی مشهد

چکیده

بمنظور بهبود رشد شاخه‌های باززایی شده گلابی رقم شکری آزمایشی در فضای نرم افزار Design Expert در قالب 20 نقطه شامل مقادیر تصادفی از سه فاکتور عناصر غذایی (NH4NO3، آهن و عناصر میکرو) اجرا شد. پاسخ‌ ریزنمونه­ها از نظر تعداد شاخه پرآوری شده، طول شاخه پرآوری شده، تعداد برگ‌، مقدار کلروفیل a برگ، کارتنوئید برگ و قدرت رشدی بعد از دو ماه ثبت گردید. فاکتورهای مورد بررسی در قالب مدل‌های حاصل از تجزیه آماری (مدل‌های خطی، چندجمله‌ای ساده و چند جمله‌ای درجه دوم) بر پاسخ‌های مورد ارزیابی بطور معنی‌داری موثر بودند. افزایش آهن تا یک و نیم برابر و کاهش NH4NO3 به نصف غلظت آن‌ها در محیط کشت MS باعث افزایش تعداد شاخه پرآوری‌شده (43/4 برابر) گردید. NH4NO3، آهن × عناصر میکرو رابطه‌ی خطی منفی با طول شاخه‌ نشان دادند درحالیکه تعداد برگ بطور منفی متاثر از عناصر میکرو بود. با افزایش NH4NO3 از طول شاخه پرآوری شده کاسته شد و از طرف دیگر مقادیر 5/0× و1× محیط کشت MS به ترتیب برای آهن و میکرو باعث افزایش 5/1 برابری در طول شاخه شد. شاخه­های رشد یافته در محیط کشت حاوی 1× مقادیر میکرو در محیط کشت پایه MS به میزان 5/1برابر برگ بیشتری نسبت به مقادیر 2× تولید کردند. محتوی کلروفیل a و کارتنوئید برگ در قالب مدل خطی تحت تاثیر مقادیر آهن (مثبت) و NH4NO3 (منفی) قرار گرفتند. افزایش آهن تا 5/1× برابر و کاهش NH4NO3 تا 5/0× برابر محیط کشت پایه MS باعث افزایش 2 برابری در میزان کلروفیل a و کارتنوئید شد. NH4NO3 و میکرو در قالب رابطه خطی منفی رشد رویشی را تحت تاثیر قرار دادند بطوریکه نصف مقادیر NH4NO3 با افزایش تعداد شاخه پرآوری شده و طول شاخه موجب افزایش قدرت رشدی شاخه تا 5/2 برابر شاهد گردید و کاهش مقادیر میکرو از طریق افزایش طول شاخه بر قدرت رشدی تاثیر مثبت داشت. محیط کشت بهینه شده برای گلابی شکری با استفاده از نرم افزار Design Expert  محیط کشت MS تغییر یافته به ترتیب حاوی 9/0×، 1× و 5/0× مقادیر آهن، میکرو و NH4NO3 بود.

کلیدواژه‌ها


عنوان مقاله [English]

In Vitro Shoot Proliferation of Pyrus communis cv. ʽShekariʼ in Different Medium Nutrition by Design Expert Software

نویسندگان [English]

  • Sajede Karimpour
  • Gholamhossein Davarynejad
  • Mohammad ZakiAghl
  • Mohammadreza Safarnejad
Ferdowsi university of Mashhad
چکیده [English]

Introduction: Micropropagation is important for both multiplication and preservation of a wide range of nursery plants, including many fruit crops. A number of studies exist on optimization of growth in in vitro condition for one or two cultivars, but often these results cannot be used for the other genotypes because individual cultivars may differ greatly in their requirements. Therefore, genotype-specific medium are usually empirically developed for many plants including pear. Pear cultivars and species are often recalcitrant to tissue culture manipulations and Murashige and Skoog (23) (MS) basal nutrient medium at full or half strength or with slight modifications is the most media were used. The QL, DKW, and WPM media are also used and they differ mostly in types or amounts of calcium and nitrogen in compared with MS. Developing growth media for specific and unique cultivars is complex and time-consuming. Currently, improved experimental design and using statistical softwares allow much more efficient approaches to be utilized for the improvement of micropropagation media and conditions. Improving of growth medium for in vitro propagation of plants depends on type and quantities of mineral nutrients and plant growth regulators as important ones. The existence of statistical softwares to manage effective factors is very needed to access an optimized growth medium for in vitro propagation of plants. Design Expert is used as auxiliary software to identify essential factors in in vitro culture. Since the in vitro proliferation parameters of Pyrus communis cv. ʽShekariʼ need to optimize for growth better, we were designed and performed a multifactor surface response experiment by Design Expert software to following two goals. First, to find optimized amount of some elements in medium and second, to show the response surface method can be useful for improving in vitro culture.
Materials and Methods: One experiment was designed by Design Expert software and was performed to improve in vitro proliferated shoots of Pyrus communis cv. ʽShekariʼ. Shoots were grown in a modified MS medium (supplement with 1 mg l-1 of N6-benzyladenine) were used for this experiment. The experiment was included 20 model points randomly based on three nutritional factors: NH4NO3 (0.5-1.5×), Fe (0.5-1.5×) and micro nutrients (1-2×) in different concentration of their MS amounts. Media enriched with sucrose (30 g l-1) and agar (8 g l-1) after pH adjustment at 5.7. Cultures were grown at 25°C under a 16-h photoperiod with 70–90 μM m-2 s-1 irradiance provided by a combination of cool- and warm-white fluorescent bulbs and were transferred to new medium every 3 weeks. Several responses were recorded after two months: Proliferated shoot number, proliferated shoot length (cm), total leaf number, leaf chlorophyll a (mg g-1), leaf carotenoids (mg g-1), and vegetative growth (cm). Responses for each point were the mean of 5 replicates. Experimental design, model evaluation, and analysis were done by Design-Expert® 8 (2010) software and the highest-order polynomial model that was significant for each response was used for ANOVA.
Results and Discussion: Factors statistically were significant for responses according to ANOVA in linear, 2FI and quadratic models. Reduced NH4NO3 (×0.5) and enhanced Fe (×1.5) induced the higher number of proliferated shoots up to 4.43 folds of control according a quadratic model. NH4NO3 and Fe×Micro had negative liner relationships with shoot length, while leaf number negatively was affected by micros. Fe and NH4NO3 were effective factors on leaf chlorophyll a and carotenoids contents. Increasing Fe (×1.5) and decreasing NH4NO3 (×0.5) led to 2 folds higher production of chlorophyll a and carotenoids. Vegetative growth of Pyrus communis cv. ʽShekariʼ  in a quadratic-order method (negatively controlled by NH4NO3 and micros) increased by high values of proliferated shoot number and shoot length induced by reduced NH4NO3 (×0.5). Optimized amount of three studied factors based on two important responses, maximum amount of proliferated shoot number and length, were 0.9, 1 and 0.5× for Fe, micro and NH4NO3 in MS medium, respectively.
Conclusions: Design Expert software and response surface method were used successfully for in vitro optimizing of Pyrus communis cv. ʽShekariʼ regenerated shoots. Fe, Micro and NH4NO3, were the effective factors for shoot regeneration responses in linear, 2FI and quadratic models. The multifactor investigation in surface response design will enable us to predict an optimal medium for several effective factors and estimate suitable responses. Outputs of these types of experiments provide a suitable background to increase optimization accuracy for future experiments.

کلیدواژه‌ها [English]

  • Growth medium
  • In vitro propagation
  • Mineral Nutrition
  • Pear
  • Surface response design
1. Al Maarri K., Duron M., Arnaud Y., and Miginiaaac E. 1986. Etude comparative de l’aptitude a la micropropagation, par culture de meristemes in vitro, du poiriers juveniles issus de semis de ‘Passe Crassane’. C.R. Acad. Agric. Fr., 72:413-421.
2. Bell R.L., and Reed B.M. 2002. In vitro tissue culture of pear: advances in techniques for micropropagation and germplasm preservation. Acta Horticulturae, 596:412–418.
3. Bell R.L., Srinivasan C., and Lomberk D. 2009. Effect of nutrient media on axillary shoot proliferation and preconditioning for adventitious shoot regeneration of pears. In Vitro Cellular & Developmental Biology – Plant., 45:708–714
4. Bennett W.F.1993. Nutrient deficiencies and toxicities in crop plants. APS Press, Minneapolis, MN, 202 pp.
5. Chenard, C.H., Kopsell, D.A., and Kopsell, D.E. 2005. Nitrogen Concentration Affects Nutrient and Carotenoid Accumulation in Parsley. Journal of Plant Nutrition, 28(2): 285-297.
6. Chevreau E., and Skirvin R.M. 1992. Pear. In: Hammerschlag F.A., Litz R.E. (ed.): Biotechnology of perennial fruit crops. Biotechnology in Agriculture. CAB International, Wallingford, 8: 263–276.
7. Dere S., Gunes T., and Sivaci R.1998. Spectrophotometric determination of chlorophyll a, b and total carotenoid content of some algae species using different solvent. Botany, 22 (1): 13-17.
8. Design-Expert. 2010. Stat-Ease, Inc., Minneapolis.
9. Domonkos M., Kis Z., and Gombos B. 2013. Ughy Carotenoids, versatile components of oxygenic photosynthesis. Prog. Lipid Res., 52: 539-561.
10. Engelsberger W.R., and Schulze W.X. 2012. Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen starved Arabidopsis seedlings. Plant Journal, 69: 978–995.
11. Evens T.J., and Niedz R.P. 2008. ARS-Media: Ion Solution Calculator, U.S. Horticultural Research Laboratory, Ft. Pierce, FL 34945 USA
12. Gago J., Martinez-Nú´nez L., Landin M., and Gallego P.P. 2010.Artificial neural networks as an alternative to the traditional statistical methodology in plant research. Journal of Plant Physiology, 167: 23–27.
13. Gallego P.P., Gago J., and Landin M. 2011. Artificial neural networks Technology to model and predict plant biology process. p. 197–216. In K. Suzuki, Artificial Neural Networks-Methodological and Biomedical Applications, (Croatia: Intech Open Access Publisher).
14. Greenway M.B, Phillips I.C., Lloyd M.N., Hubstenberger J.F., and Phillips G.C. 2012. A nutrient medium for diverse applications and tissue growth of plant species in vitro. In Vitro Cellular & Developmental Biology – Plant, 48:403–410
15. Hermans C., Vuylsteke M., Coppens F., Cristescu S.M., Harren F.J., Inze D., and Verbruggen N. 2010. Systems analysis of the responses to long-term magnesium deficiency and restoration in Arabidopsis thaliana. New Phytologist, 187:132–144.
16. Hashimoto H., Uragami C., and Cogdell R.J. 2016. Carotenoids and photosynthesis Subcell. Biochem, 79: 111-139.
17. Hsu W., and Miller G.W. 1969. Copro-porphyrinogenase in tobacco (Nicotiana tabacum L.). Biochem, 1 (117): 215-220.
18. Ivanova M., and Van Staden J. 2009. Nitrogen source, concentration, and NH4+:NO3− ratio influence shoot regeneration and hyperhydricity in tissue cultured Aloepolyphylla. Plant Cell, Tissue and Organ Culture, 99: 167–174.
19. Karimpour S., Davarynejad G.H., Bagheri A., and Tehranifar A. 2013. In Vitro Establishment and Clonal Propagation of Sebri Pear Cultivar. Journal of Agricultural Science and Technology, 15: 1209-1217.
20. Li B.H., Li Q, Xiong L.M., Kronzucker H.J., Kramer U., and Shi W.M. 2012. Arabidopsis plastid AMOS1/EGY1 Integrates abscisic acid signaling to regulate global gene expression response to ammonium stress. Plant Physiol, 160: 2040-51.
21. Lloyd G., and McCown B. 1981. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. The International Plant Propagators' Societ, 30:421-427.
22. Miller G.W., Pushnik J.C. and Welkie G. 1984. Iron chlorosis a worldwide problem, the relation of chlorophyll biosynthesis to iron. 1. Plant Nutrition, 7: 1-22.
23. Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15:473–497
24. Nakajima I., Ito A., Moriya S., Saito T., Moriguchi T., and Yamamoto T. 2012. Adventitious shoot regeneration in cotyledons from Japanese pear and related species. In Vitro Cellular & Developmental Biology – Plant, 48:396–402
25. Nedelcheva S. 1986. Effect of inorganic components of the nutrient medium on in vitro propagation of pears. Genetics Selection Evolution, 19:404-406.
26. Nezami Alanagh E., Garoosi GH-A., Hadad B., Maleki S., Landin M., and Gallego P.P. 2014. Design of tissue culture media for efficient Prunus rootstock micropropagation using artificial intelligence models. Plant Cell, Tissue and Organ Culture, 117:349 -359.
27. Niedz R.P., and Evens T.J. 2006. A solution to the problem of ion confounding in experimental biology. Nature Methods, 3:417.
28. Niedz R.P., and Evens T.J. 2007. Regulating plant tissue growth by mineral nutrition. In Vitro Cellular & Developmental Biology – Plant, 43:370–381.
29. Niedz R.P., Hyndman S.E., and Evens T.J. 2007. Using a Gestalt to measure the quality of in vitro responses. Scientia Horticulturae, 112:349–359.
30. Niedz R.P., Hyndman S.E., Evens T.J., and Weathersbee A.A. 2014. Mineral nutrition and in vitro growth of Gerbera hybrida (Asteraceae). In Vitro Cellular & Developmental Biology – Plant, 50: 458.
31. Pandey N. 2018. Role of Plant Nutrients in Plant Growth and Physiology (Chapter 2). M. Hasanuzzaman et al. (eds.), Plant Nutrients and Abiotic Stress Tolerance, 51-93.
32. Preece J. 1995. Can nutrient salts partially substitute for plant growth regulators?. Plant Tissue Culture and Biotechnology, 1:26–37.
33. Quoirin M., and Lepoivre P. 1977. Etude de milieux adaptes aux cultures in vitro de Prunus. Acta Horticulturae, 78:437-442.
34. Ramage C.M, and Williams R.R. 2002. Mineral nutrition and plant morphogenesis. In Vitro Cellular & Developmental Biology – Plant, 38:116–124.
35. Reed B.M, DeNoma J.S., Wada S., and Postman J.D. 2012. Micropropagation of pear (Pyrus sp). Chapter 1. p. 3–18.In: Lambardi M., Ozudogru E.A., Jain S.M. (eds) Protocols for micropropagation of selected economically-important horticultural plants. Humana, New York.
36. Reed B.M., Sarasan V., Kane M., Bunn E., and Pence V. 2011. Biodiversity conservation and conservation biotechnology tools. In Vitro Cellular & Developmental Biology – Plant. 47:1–4
37. Reed B.M., Wada S., DeNoma J., and Niedz RP. 2013a. Improving in vitro mineral nutrition for diverse pear germplasm. In Vitro Cellular & Developmental Biology – Plant, 49:343-355.
38. Reed B.M., Wada S., DeNoma J., and Niedz RP. 2013b. Mineral nutrition influences physiological responses of pear in vitro. In Vitro Cellular & Developmental Biology – Plant, 49:699-709.
39. Sanchez-Zabala J., Gonz_alez-Murua C., and Marino D. 2015. Mild ammonium stress increases chlorophyll content in Arabidopsis thaliana. Plant Signaling & Behavior, 10 (3): 9915961-3.
40. Sathyanarayana B.N., and Blake J.1994. The effect of nitrogen sources and initial pH of the media with or without buffer on in vitro rooting of jackfruit (Artocarpus heterophyllus Lam). P. 77-82. In P. J. Lumsden, J.R. Nicholas and W.J. Davies. Physiology Growth and Development of Plants in Culture. Dordrecht: Kluwer.
41. Spiller S.C., Castelfranco A., and Castelfranco P. 1982. Effects of iron and oxygen on chlorophyll biosynthesis. I. In vivo observations of iron and oxygen - deficient plants. Plant Physiol, 69: 107-111.
42. Takatsuji H. 1999. Zinc finger proteins: the classical zinc finger emerges in contemporary plant science. Plant Mol Biol 39:1073–1078.
43. Tang H, Luo Y., and Liu C. 2008. Plant regeneration from in vitro leaves of four commercial Pyrus species. Plant, Soil and Environment, 54 (4): 140–148.
44. Thakur A., and Kanwar J.S. 2008. Micropropagation of ‘wild Pear’ Pyrus pyrifolia (Burm F.) Nakai.I. explant establishment and shoot multiplication. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 36 (1): 103-108.
45. Wada S., Niedz R.P., and Reed B.M. 2013. Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species. In Vitro Cellular & Developmental Biology - Plant, 49:356–365.
46. Wada S., Maki Sh., Niedz R.P., and Reed B.M. 2015. Screening genetically diverse pear species for in vitro CaCl2, MgSO4 and KH2PO4 requirements. Acta Physiologiae Plantarum, 37: 63.
47. Wada S., Niedz R.P., DeNoma J., and Reed B.M. 2013. Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation. In Vitro Cellular & Developmental Biology – Plant, 49:356–365.