بررسی کارآیی تیمارهای تلفیقی گرمادرمانی و کشت مریستم انتهایی بر تولید نهال عاری از سه ویروس آلوده کننده سیب "گوشت سرخ" (Malus pumila Mill.) در شرایط درون شیشه‌ای

نوع مقاله : مقالات پژوهشی

نویسندگان

1 مؤسسه تحقیقات و علوم باغبانی

2 پژوهشگاه بیوتکنولوژی ایران

3 پژوهشکده ملی انگور و کشمش دانشگاه ملایر

چکیده

این پژوهش با هدف تولید نهال عاری از سه ویروس ACLSV، ASPV، ASGV سیب "گوشت سرخ" (Malus pumila Mill.)، در پژوهشگاه بیوتکنولوژی کشاورزی ایران (ABRII) در سال 1394 انجام شد. آزمایش­ها با ارزیابی اثربخشی تیمار‌ گرمادرمانی (0، 7، 14 و 21 روز در دمای 38 درجه سانتی‌گراد) و کشت مریستم انتهایی (در سه اندازه کوچک‌تر از 2/0 میلی‌متر، بین 2/0 تا 7/0 میلی‌متر و بزرگ‌تر از 7/0 میلی‌متر) بر نرخ حذف ویروس از ریزشاخه­ها انجام شد. در ابتدا حضور ویروس­هایACLSV، ASGV و ASPV در نمونه‌های مادری با روش­های آزمون الایزای ساندویچ دو طرفه آنتی بادی و RT-PCR مورد ارزیابی قرار گرفت. سپس تیمارهای گرمادرمانی و کشت مریستم در شرایط درون شیشه‌ای انجام شد. ریزشاخه­های رشد یافته از مریستم توسط الایزا و RT-PCR برای هر سه ویروس بررسی شدند. به­طورکلی نرخ حذف سه ویروس ACLSV (9/25 درصد)، ASGV (4/7 درصد) و ASPV (4/44 درصد) با یکدیگر متفاوت بود. نتایج نشان داد افزایش طول مدت گرمادرمانی و کاهش اندازه مریستم با اثر گذاری متفاوت بر ویروس­های مورد مطالعه باعث افزایش نرخ عاری شدن ریزنمونه­ها از ویروس شده است. افزایش دوره زمانی گرمادرمانی در 21 روز باعث کاهش رشد و تکثیر و حتی از بین رفتن ریزنمونه­ها شد. بنابراین 14 روز گرمادرمانی مؤثرترین تیمار جهت حذف آلودگی ویروس­هایASGV  (11/11درصد)، ASPV (89/88 درصد) و ACLSV (44/44 درصد) از ریزنمونه­های مورد مطالعه بود. در پایان آزمایش نمونه‌هایی که توسط هر دو روش الایزا و RT-PCR سالم تشخیص داده شدند، تکثیر و ریشه‌دار شدند و در شرایط گلخانه­ای سازگار شدند.
 

کلیدواژه‌ها


عنوان مقاله [English]

Culture Efficiency on Virus Elimination from in vitro Shootlets of Red Flesh Apple (Malus pumila Mill.)

نویسندگان [English]

  • N. Kazemi 1
  • A.A. Habashi 2
  • W. Asadi 3
1 Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
2 Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
3 Institute of Grape and Raisin in Malayer University, Iran
چکیده [English]

Introduction: Apple (Malus spp.) is one of the most economically important fruit crop worldwide. This crop is highly affected by various virus infections, leading its considerable devastation and eventually results in yield loss in the whole world. Among effective viruses, Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), and Apple chlorotic leaf spot virus (ACLSV) which have been firstly characterized in apple (Malus domestica), play important roles in altering the plant defense mechanism, leading a low performance. According to the references, heat treatments can reduce the movement of virus particles into the apical meristem through inhibiting viral RNA synthesis, so high temperature over a long period is an efficient method for virus elimination. In additions, meristem culture is also a common method to eradicate viruses from horticultural plants. This study was done to produce virus-free (ACLSV,  ASPV,  ASGV) apple (Malus pumila Mill.) plantlets.
Materials and Methods: The effect of different thermotherapy duration (0, 7, 14 and 21 days at 38 °C) and the sizes of apical meristems for meristem culture(less than 0.2 mm, between 0.2 and 0.7 mm and larger than 0.7 mm) was assessed on virus eradication. Our plant material was a genotype of red flesh apple belongs to Budagovsky Bud.9 (Malus+pumila) "Niedzwetzkyana" of Rosaceae family from Shahroud, that has high levels of important phytochemicals like antioxidants, flavonoids and anthocyanins in its cortex, which in addition to attractiveness, creating special healing properties in some disease. At first the presence of ACLSV, ASGV and ASPV were checked in mother samples by ELISA and RT-PCR methods. Then we performed the thermotherapy treatments and later meristems were cultivated and grown in vitro. Regenerated shoots from meristem were tested by ELISA and RT-PCR methods for all three viruses. Samples that were diagnosed virus-free by both techniques, were proliferated, rooted and transferred into the pots to be used for later propagation and establishment of the mother orchard.
Results and Discussion ELISA results for the presence or absence of ACLSV, ASGV and ASPV were indeterminate; they were neither negative nor positive for each virus, indicating ELISA was not an accurate method to study virus infections in our samples. Therefore, careful examination of initial infection of samples was performed by RT-PCR. Examining mother samples by RT-PCR showed that all samples were infected by ASG, ASP and ACLS viruses.
Results of RT-PCR testing suggested that the number of days in thermotherapy and the size of meristem had a significant effect (P<0.01) on the ACLSV, ASGV and ASPV elimination. Increasing in the duration of the thermotherapy decreased the survival rate of the explants and it was difficult to acquire virus-free explants after 21 days treatments, because all the explants were destroyed in this treatment. The relation between the size of meristem and its survival has been examined in different studies, whose results are consistent with the present research and confirms that increasing the size of the meristem and the number of leaf primordium, will increase the vitality. However, it should be noted that larger meristems are more susceptible to viral contamination. In the previous study, the percentage of ASGV and ACLSV elimination was significantly influenced by the size of the meristem and the duration of the thermotherapy. In the same study, ACLSV, ASGV and ASPV could not be detected in plants grown from meristems smaller than 1 mm after 35 days of thermotherapy. They also showed that virus eradication was happened after shorter period of thermotherapy if smaller meristems were cultured (0.7 mm). The results of our study were consistent with these observations; ACLSV, ASGV and ASPV appeared to be eliminated more frequently from the smallest meristem treatment (smaller than 0.2 mm) compared to the other treatments (0.2-0.7 mm and larger than 0.7 mm). Elimination rates of ACLSV, ASGV and ASPV were different in each case and ASPV-free frequency obtained in regenerated shoots treatment was higher than other viruses (ASGV and ACLSV). This observation was similar to the results of some previous studies. It could be due to differences in the morphology of the viruses and transmission factors in plant cells that affect viruses, which has been investigated and confirmed in previous studies.
Meristem culture alone, without other treatments, had very low efficiency in virus elimination from explants. The best results of virus eradication from apple explants was obtained after 14 days of thermotherapy, that we had 44.44%, 11.11% and 88.89% of virus-free samples from ACLSV, ASGV and ASPV, respectively. Generally, our results showed that by increasing the thermotherapy duration, the elimination of all three types of viruses was increased. Many researchers also found that thermotherapy is a very effective treatment in virus eradication.
Conclusion: Results showed that increasing the duration of thermotherapy (14 days) and reducing the size of cultivated meristem (smaller than 0.2mm) increased the elimination rate of all three viruses (ACLSV، ASPV، ASGV) from apple explants.

کلیدواژه‌ها [English]

  • Apple chlorotic leaf spot virus
  • Apple stem grooving virus
  • Apple stem pitting virus
  • ELISA
  • RT-PCR
1- Adams M.J., Antoniw J.F., Bar Joseph M., Brunt A.A., Candresse T., Foster G.D., Martelli G.P., Milne R.G., and Fauquet C. M. 2004. The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Archives of Virology 149(8): 1045-1060.
2- Asghar A., and Singh G. 2007. Production of Indian citrus ringspot virus free plants of Kinnow employing chemotherapy coupled with shoot tip grafting. Journal of Central European Agriculture 8(1).
3- Cies´lin´ska M. 2002. Elimination of Apple chlorotic leaf spot virus (ACLSV) from pear by in vitro thermotherapy and chemotherapy. Acta Horticulture 596: 481-484.
4- Deng X.Y., Hong N., Hu H.J., and Wang G.P. 2004. Detection of latent viruses in Pyrus pyrifolia by IC-RT-PCR and TC-RT-PCR. Journal of Fruit Science 21: 569-572.
5- Etherton P. 2002. Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer, Journal of Medicine 113: 71–88.
6- Gambino G., Di Matteo D., and Gribaudo I. 2009. Elimination of Grapevine fanleaf virus from three Vitis vinifera cultivars by somatic embryogenesis. European Journal of Plant Pathology 123(1): 57-60.
7- Hadidi A., and Barba M. 2011. Economic impact of pome and stone fruit viruses and viroids. Virus and Virus Like Diseases of Pome and Stone Fruits 1(8).
8- Hu G.J., Hong N., Wang L.P., Hu H.J., and Wang G.P. 2012. Efficacy of virus elimination from in vitro-cultured sand pear (Pyrus pyrifolia) by chemotherapy combined with thermotherapy. Crop Protection 37: 20-25.
9- Hu G., Dong Y., Zhang Z., Fan X., Ren F., and Zhou J. 2015. Virus elimination from in vitro apple by thermotherapy combined with chemotherapy. Plant Cell, Tissue and Organ Culture (PCTOC) 121(2): 435-443.
10- Kazemi N., Amiri M.E., and Jafarkhani Kermani M. 2013. Hormonal optimization of medium for multiplication of rare apple genotype of Red Flesh. Thesis for M. Sc. in Horticulture. University of Zanjan.
11- Kazemi N., Zaree Nahandi F., Habashi A.A., and Asadi W. 2019. Molecular Assessment of Chemotherapy and Meristem Culture Efficiency for Production of Seven Cultivars of Virus-Free Pear (Pyrus communis L.). Journal of Agricultural Crops Production 21(1): 107-118.
12- Komorowska B., Malinowski T., and Michalczuk L. 2010. Evaluation of several RT-PCR primer pairs for the detection of Apple stem pitting virus. Journal of Virological Methods 168(1-2): 242-247.
13- Manganaris G.A., Economou A.S., Boubourakas I.N., and Katis N.I. 2003. Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine. Plant Cell Reports 22(3): 195-200.
14- Masoomi-Aladizgeh F., Jabbari L., Khayam Nekouei R., and Aalami A. 2016. A simple and rapid system for DNA and RNA isolation from diverse plants using handmade kit. Protoc Exch.
15- Mathioudakis M.M., Maliogka V.I., Dovas C.I., Paunović S., and Katis N.I. 2008. Reliable RT‐PCR detection of Apple stem pitting virus in pome fruits and its association with quince fruit deformation disease. Plant Pathology 58(2): 228-236.
16- Menzel W., Jelkmann W., and Maiss E. 2002. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. Journal of Virological Methods 99(1-2): 81-92.
17- Panattoni A., Luvisi A., and Triolo E. 2013. Elimination of viruses in plants: twenty years of progress. Spanish Journal of Agricultural Research (1): 173-188.
18- Paprstein F., Sedlak J., Polak J., Svobodova L., Hassan M., and Bryxiova M. 2008. Results of in vitro thermotherapy of apple cultivars. Plant Cell, Tissue and Organ Culture 94(3): 347-352.
19- Paprštein F., Sedlak J., Svobodova L., Polak J., and Gadiou S. 2013. Results of in vitro chemotherapy of apple cv. Fragrance. Horticultural Science (Prague) 40: 186-190.
20- Ramgareeb S., Snyman S.J., Van Antwerpen T., and Rutherford R.S. 2009. Elimination of virus and rapid propagation of disease-free sugarcane (Saccharum spp. cultivar NCo376) using apical meristem culture. Plant Cell, Tissue and Organ Culture (PCTOC) 100(2): 175-181.
21- Retheesh S.T., and Bhat A.I. 2010. Simultaneous elimination of Cucumber mosaic virus and Cymbidium mosaic virus infecting Vanilla planifolia through meristem culture. Crop Protection 29(10): 1214-1217.
22- Sareila O., Hohkuri M., Wahlroos T., and Susi P. 2004. Role of viral movement and coat proteins and RNA in phloem‐dependent movement and phloem unloading of tobamoviruses. Journal of Phytopathology 152(11‐12): 622-629.
23- Tan R., Wang L., Hong N., and Wang G. 2010. Enhanced efficiency of virus eradication following thermotherapy of shoot-tip cultures of pear. Plant Cell, Tissue and Organ Culture (PCTOC) 101(2): 229-235.
24- Wang L., Wang G., Hong N., Tang R., Deng X., and Zhang H. 2006. Effect of thermotherapy on elimination of Apple stem grooving virus and Apple chlorotic leaf spot virus for in vitro-cultured pear shoot tips. HortScience 41(3): 729-732.
25- Wang Q., Cuellar W.J., Rajamaki M.L., Hirata Y., and Valkonen J.P. 2008. Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips. Molecular Plant Pathology 9(2): 237-250.