effect of gamma rays on sophora davidii and detection of dna...

Research ArticleEffect of Gamma Rays on Sophora davidii and Detection of DNAPolymorphism through ISSR Marker

PuchangWang1 Yu Zhang1 Lili Zhao2 Bentian Mo1 and Tianqiong Luo1

1Guizhou Institute of Prataculture Guiyang 550006 China2College of Animal Science Guizhou University Guiyang 550025 China

Correspondence should be addressed to Lili Zhao zhaolili_0508163com

Received 14 November 2016 Accepted 28 February 2017 Published 22 May 2017

Academic Editor Marco Bazzicalupo

Copyright copy 2017 Puchang Wang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sophora davidii (Franch) Kom ex Pavol is an important medicinal plant and a feeding scrub with ecological value The effects ofdifferent gamma irradiation doses (20ndash140Kr) on seed germination and seedling morphology were investigated in S davidii andintersimple sequence repeat (ISSR) markers were used to identify the DNA polymorphism among mutants Significant variationswere observed for seed germination stem diameter and number of branches per plant The improved agronomic traits such asstem diameter and number of branches per plant were recorded at 80Kr dose and 20Kr dose for seed germination ISSR analysisgenerated in total 183 scorable fragments of which 94 (5137) were polymorphic The percentage of polymorphism ranged from1429 to 9333 with an average of 4569 Jaccardrsquos coefficients of dissimilarity varied from 06885 to 1000 indicative of the levelof genetic variation among the mutants The constructed dendrogram grouped the entities into five clusters Consequently it wasconcluded that gamma rays irradiation of seeds generates a sufficient number of induced mutations and that ISSR analysis offereda useful molecular marker for the identification of mutants

1 Introduction

Sophora davidii (Franch) Kom ex Pavol is an importantleguminous scrub which has significant economic values andhas been widely used as fodder as herbal medicine and asa nectar source [1 2] It is also a kind of pioneer plant inkarst rocky desertification areas of southwestern China foreco-economical consideration The reason for its applicationin restoring vegetation and ecological engineering is simplythat this species could tolerate extremely dry environmentswith deeper root systems smaller leaf sizes and greateraccumulating ability of soluble sugars in leaves [3] S davidiihas drawn wide attention of many scientists due to its highadaptability among diverse native species in karst rockydesertification areas However complex local microclimatesand habitat fragmentation in karst areas have made thisspecies suffer serious risk of genetic diversity reductionSo species conservation and improvement are the majorproblems for its reasonable utilization Induced mutationhas been an important technique of cultivars improvementand has been highly effective in enhancing natural genetic

resources especially for unusual plants and in the selectionof mutants with desirable agronomic characteristics [4]And more than 2700 varieties derived via mutations of 170different species throughout the world have been releasedofficially [5]

Induced mutations contribute to genetic variabilitymainly by increasing DNA polymorphism [6] A great num-ber of DNA markers have been broadly used to estimategenetic diversity genetic stability the genetic characteriza-tion and the identification and screening of mutant plantssuch as maize [7] soybean [8] faba bean [9] potato [10]Curcuma alismatifolia Gagnep [11] and Acorus calamus L[12]

However molecular characterization of mutation eventsinduced by irradiation treatments in Sophora is limited Theaim of our study was to develop a suitable technique formutation breeding of S davidii cultivars using gamma radia-tion For this purpose we investigated the effect of differentdoses (20ndash140Kr) of gamma irradiation on seed germinationand seedling morphological traits of S davidii and identified

HindawiBioMed Research InternationalVolume 2017 Article ID 8576404 6 pageshttpsdoiorg10115520178576404

2 BioMed Research International

Table 1 Effects of gamma radiation on seed germination percentage and morphological traits of S davidii at seedling stage

Dose SG () LL (cm) LW (cm) SH (cm) BL (cm) SD (mm) NBControl 4000 plusmn 100b 148 plusmn 036a 069 plusmn 017a 2711 plusmn 331a 1156 plusmn 189a 288 plusmn 032c 967 plusmn 153ab

20Kr 4567 plusmn 321a 148 plusmn 032a 067 plusmn 018a 2707 plusmn 416a 1042 plusmn 255a 311 plusmn 032c 933 plusmn 208ab

40Kr 3967 plusmn 252b 149 plusmn 025a 070 plusmn 012a 2543 plusmn 589a 1041 plusmn 233a 333 plusmn 028bc 1033 plusmn 152ab

60Kr 3367 plusmn 115c 142 plusmn 029a 061 plusmn 019a 2310 plusmn 439a 1009 plusmn 402a 386 plusmn 029ab 967 plusmn 115ab

80Kr 3133 plusmn 115c 136 plusmn 034a 055 plusmn 021a 2321 plusmn 463a 946 plusmn 252a 393 plusmn 047a 1200 plusmn 265a

100Kr 2267 plusmn 231d 131 plusmn 030a 051 plusmn 020a 2285 plusmn 448a 852 plusmn 281a 323 plusmn 021c 967 plusmn 115ab

120Kr 2000 plusmn 200d 133 plusmn 031a 047 plusmn 019a 1984 plusmn 524a 770 plusmn 292a 279 plusmn 029c 733 plusmn 058b


Each value was expressed as mean plusmn standard deviation SG seed germination LL leaf length LW leaf width SH seedling height BL branch length SD stemdiameter NB number of branches per plant Within columns means plusmn standard deviation followed by the same letter are not significantly different at 119875 =005

DNApolymorphism among themutants through intersimplesequence repeat (ISSR) marker analysis

2 Materials and Methods

21 Plant Source Irradiation and Morphology Study Theseeds of Sophora davidii (Franch) Kom ex Pavol werecollected in October 2013 from a karst mountain region(105∘381015840N 25∘551015840E) in Guanling Town Guizhou China Allseeds were dried under sunshine and then stored in a cooldry location Prior to radiation the moisture content of allseeds was equilibrated by placing them inside paper bagsstored in a calcium oxide-dried container

Then the seeds were irradiated with different doses (2040 60 80 100 120 and 140Kr) of 60Co-120574 in October 2013 atthe Institute of Biological and Nuclear Technology GuizhouProvince Academy of Agricultural Sciences China

Immediately after radiation 200 treated seeds were sownat a depth of 1 cm with a mixed medium of river sandred soil and farm yard manure at the ratio of 3 2 1 inplastic pots (23 times 27 cm 6 cm in height) After a month ofgermination the number of germinated seeds was countedand expressed as percentage of the total number of seedsplanted Ten seedlings from each treatmentwere transplantedto experimental field Morphological traits including leaflength leaf width plant height branch length stem diameterand number of bunches per plant were recorded twomonthslater after transplantation

22 DNA Extraction and ISSR-PCR Amplification The freshleaf material was harvested from the three-month-old plantstreated with gamma rays DNA was extracted using a mod-ified CTAB method [13] and dissolved in 1x TE butter forsubsequent use ISSR-PCR amplifications were performedin a GeneAmp PCR System 9700 DNA Thermal Cycler(PerkinElmer USA) with cycling profiles 4min at 94∘Cfollowed by 35 cycles of 30 s at 94∘C 30 s at 50∘C and 1min at72∘C and endingwith 10min at 72∘C 50 primers (Biotechnol-ogy Laboratory InnerMongolia AgricultureUniversity) werescreened initially to identify well-amplified polymorphicbands among the populations Of the 50 primers tested 20produced bright clear and reproducible bands

PCR was carried out in a total volume of 20 120583L whichincluded 50 ng of template DNA 2 120583L 10x PCR buffer (Mg2+Plus) 2 120583LMgCl

2(25mM) 10 120583L dNTPs mixture (25mM)

10U of Taq polymerase (TaKaRa Biotechnology (Dalian)Co Ltd Dalian China) and 04120583M primers (ShanghaiSangon Biological Engineering Technology and Service CoLtd Shanghai China) Amplification products along with aGeneRuler100 ladder (Fermentas UAB Inc) were separatedvia electrophoresis on 15 (wv) agarose gels with 05x TBEbuffer at 120V for 3-4 h stained with ethidium bromide(01mg120583L) They were then photographed with an Epsondigital still AF camera Negative controls which lackedtemplate DNA were also included in each PCR set to test forpossible contamination

23 Data Statistical Analysis Data of seed germination andmorphological traits were analyzed using one-way ANOVAwith SPSS software package (170) Data of ISSR markeranalysis were scored as discrete variables using ldquo1rdquo forpresence and ldquo0rdquo for absence of bands for each of the primerpairs The faint and unclear bands were not consideredfor data scoring The binary data generated were used toestimate levels of polymorphismby dividing the polymorphicbands by the total number of scored bands A dendrogrambased on Jaccardrsquos similarity coefficients was constructed byusingUnweighted Pair GroupMethodwith ArithmeticMean(UPGMA) with the SHANmodule of NTSYS-PC 20 to showphenetic representation of genetic relationships as revealedby the similarity coefficient

3 Results

31 Seed Germination and Seedling Morphological Traits inGamma Rays Treated Plants In this study we measured theeffect of different doses (control 20 40 60 80 100 120and 140Kr) of gamma irradiation on seed germination andseedling morphological traits The results (mean plusmn standarddeviation) are given in Table 1 Repeated measures ANOVArevealed a significant positive influence of the gamma irra-diation on seed germination stem diameter and number ofbranches per plant (119875 lt 005)

BioMed Research International 3

Table 2 List of primers number of amplified products polymorphic bands and polymorphism percentage

Number Primers Sequence (51015840ndash31015840) TAP NPB PPB ()1 UBC857 AGAGAGAGAGAGAGAGYG 17 12 70592 UBC818 CACACACACACACACAG 7 2 28573 UBC850 GTGTGTGTGTGTGTGTYC 9 4 44444 UBC840 GAGAGAGAGAGAGAGAYT 10 2 20005 UBC812 GAGAGAGAGAGAGAGAA 7 1 14296 ISSR04 GAGAGAGAGAGAGAGACT 10 2 20007 ISSR11 ACACACACACACACACTG 11 8 72738 ISSR21 GAGAGAGAGAGAGAGATG 7 2 28579 ISSR25 ACACACACACACACACCT 10 4 400010 ISSR24 ACACACACACACACACCA 13 7 538511 ISSR36 GCGTCTCTCTCTCTCTC 8 2 250012 ISSR47 AGAAGAAGAAGAAGAAGAAGA 7 4 571413 ISSR02 AGAGAGAGAGAGAGAGCC 12 10 833314 ISSR05 GAGAGAGAGAGAGAGACC 7 5 714315 ISSR06 GAGAGAGAGAGAGAGACG 12 9 750016 ISSR08 GTGTGTGTGTGTGTGTCC 15 14 933317 ISSR12 CCCTCCCTCCCTCCCT 3 1 333318 ISSR14 CTTCACTTCACTTCA 7 3 428619 ISSR10 ACACACACACACACACTA 4 1 250020 ISSR15 GGAGAGGAGAGGAGA 7 1 1429

Total 183 94 91375Mean 915 47 4569

TAP total amplified products NPB number of polymorphic bands PPB percentage of polymorphic bands

Seeds untreated with gamma rays showed higher ger-mination rate than the irradiation treatment except for the20Kr dose treatment with germination percentage of 45In the treated seeds the germination decreased significantly(119875 lt 005) as the irradiation doses of gamma rays increasedto 120Kr while the 140Kr treatment showed an increasedgermination of 2033 Leaf length and leaf width rangedfrom 123 to 149 cm and from 046 to 070 cm respectivelyThe maximum of leaf length (149 cm) and the maximumof leaf width (072 cm) were obtained at plants treated with40Kr dose and the minimum of leaf length (123 cm) and theminimum of leaf width (046 cm) occurred at 140Kr dose ingamma rays treated plants

Only the seedling height showed a constant decline asthe irradiation doses increased though not reaching thesignificant level Similar to the effect on germination thebranch length decreased gradually as the irradiation doseincreased up to 120Kr and showed a slight rise at 140Krtreatment The maximum stem diameter (393mm) andnumber of branches per plant (12) occurred at 80Kr dosetreatment while the minimum stem diameter (279mm) andnumber of branches per plant (733) were observed at 120Krdose treatment

32 ISSR Marker Analysis in Gamma Rays Treated Plants Inour research the differences among the gamma irradiationmutants were examined by using ISSR marker 20 primerswere selected for amplification and data scoring (Table 2)

the results were analyzed for identifying genetic diversity ingamma rays treated plants

A total of 183 bands were scored of which 94 werepolymorphic The number of bands generated per primervaried from 3 to 17 and a minimum of 3 bands was generatedby the primer ISSR12 while the maximum of 17 bands wasscored with UBC857 followed by ISSR08 which produced15 bands (Table 2) The percentage of polymorphism wasranged from 1429 to 9333 with an average polymorphismpercentage of 4569 Primer ISSR08 produced relativelyhigh number of bands and the vast majority of these bandswere polymorphic showing 9333 of polymorphism (about14 polymorphic bands) while UBC812 and ISSR15 producedrelatively low number of bands and showed the lowestpercentage of polymorphism of 1429 (only 1 polymorphicband)

The genetic similarity based on ISSR data ranged from06885 to 1000 with an overall mean of 07884 (Table 3)All the treatments revealed the maximum genetic diversitywith combination of controlThe dendrogram obtained fromUPGMA analysis of genetic similarity based on the ISSRmarker is presented in Figure 1 The dendrogram shows theformation of five main groups of mutants The dendrogramindicated five distinct clusters the first cluster comprisedthree treatments namely control 40Kr and 80Kr thesecond cluster comprised two treatments namely 120Krand 140Kr and the other three clusters included 100Kr60Kr and 20Kr respectively indicating their higher geneticdistinctness from other treatments of gamma rays According


Table 3 Distance matrix based on Jaccardrsquos coefficients

Control 20Kr 40Kr 60Kr 80Kr 100Kr 120Kr 140KrControl 1000020Kr 07596 1000040Kr 08579 07814 1000060Kr 07377 07486 08033 1000080Kr 08361 07705 08251 08251 10000100Kr 07814 07596 08033 07486 08142 10000120Kr 08087 06885 08197 07432 08306 07650 10000140Kr 08415 07541 08087 07869 07978 07541 08251 10000

075 077 079 080 082 084 086

Control

120Kr

140Kr

80Kr

100Kr

40Kr

60Kr20Kr

Figure 1 Dendrogram representing the morphological variationamong seven irradiatedmutants (20 40 60 80 100 120 and 140Kr)and the control based on similarity coefficients of ISSR data

to the dendrogram obtained 20Kr 60Kr and 100Kr weremore distant to control than to other treatments

4 Discussion

41 Germination and Morphological Traits in the GammaRays Treated Plants Many researchers have reported thatmutagens including chemical and physical mutagens caninteract with cellular molecules particularly water to pro-duce free radicals (H OH)These free radicals could combineto form toxic substances such as hydrogen peroxide (H

2O2)

which indirectly lead to the destruction of cells [4] Thisindirect effect is especially significant in vegetative cells asthe cytoplasm contains about 80 of water [14] Finallythe excessive radicals resulted in damage or modification ofimportant cell components and affected themorphology bio-chemistry and physiology of plants The degree of this injurydepends on the sensitivity of radiating material In this studygermination percentage of the irradiated seedlings decreasedsignificantly with a steady trend as the irradiation dosesincreased showing relative sensitivity to irradiation Thisreductionstimulation in seed germination where the hypo-thetic origins can accelerate cell division rates [15]might havebeen due to the effect of mutagens on meristematic tissuesof the seed [6] Our result agreed with researches reportedby [16ndash18] The decrease in germination at high doses ofthe mutagens may be attributed to disturbances at cellularlevel (caused at either physiological level or physical level)including chromosomal damage [6] And it was reported thathigher exposures are usually inhibitor on seed germination

of Gymnosperm and Angiosperm while lower exposures areinducer on seed germination [19ndash21]This could be the reasonwhy the abnormal performance of S davidii seed germinationoccurred at 20Kr and 140Kr (a slight rise occurred at 20Krdose treatment and 140Kr treatment)

Many researchers reported that gamma rays treatedplants can change the vegetative traits rhizome character-istics and flowering development and its maturity in eithera positive direction or a negative direction [4 6 22ndash24]In this study significant variations were observed in stemdiameter and number of branches per plant after gammairradiation But the variation was not proportional to thechange in dosages and was not in a definite pattern Highdoses of ionizing radiation have been reported to damagemacromolecular cellular components such as cell wallsmembranes and DNA [25] So we supposed these changesto happen because of the effects of excessive irradiationon cells However a small tendency towards a decrease inleaf size was observed in mutagenised seedlings though thedifferences were not significant The leaf length and leafwidth decreased as the radiation doses increased especiallyin irradiated plants treated with doses from 40Kr to 140KrSimilar decreases have been reported by Taheri et al [4]and Tangpong et al [23] Same trends were also detectedin seedling height and branch length Taheri et al [4] havereported similar decreases in seeding height of mutagenisedpopulations inCurcumaalismatifoliaGagnep Such effects areknown to arise due to chromosomal aberrations in additionto genetic mutations [4] It is a fact that the cells whichhave relativelymore chromosomal damage at high irradiationexposures are at a disadvantage due to diplontic section asthese cells cannot compete well with the normal cells and arethus prevented from making any further contribution [6]

That is this progressive reduction in S davidii mor-phological traits can be interpreted as the interference onnormal mitosis and frequent occurrence of mitotic aberra-tionsMoreover some researchers thought that the inhibitionof assimilation rate and consequent change in the nutrientlevel of plant and the inactivation of vital enzymes espe-cially those associated with respiration account for this Inaddition mutagenic effects such as auxin destruction inhi-bition of auxin synthesis failure of assimilatory mechanismand changes in the specific activity of enzymes can alsocause growth reductions [26] Since no major reduction ofparameters was found in our study we propose that this is


favorable for breeding attempts especially with 40Kr and80Kr irradiation doses

42 ISSR Marker Analysis in the Gamma Rays TreatedPlants Mutation breeding is based on induction ofmutationgenetic diversity evaluation and screening and molecularidentification of desired character During this processmolecular markers have made great contributions in thegenetic characterization screening of mutant plants and themolecular identification of specific agronomic trait Amongvarious molecular markers ISSR is easy to apply highlyinformative reliable repeatable and inexpensive [27 28] andis a practicable method for the assessment of genetic diversitystudies especially for plants with no or little specific primersreleased In present research amplifications were successfullyperformed for all the 20 ISSR primers assayed 183 scorablefragments in total were generated of which 94 (5137)werefound to be polymorphic (the average polymorphism per-centage per primer was 4569) It is indicated that gammarays irradiation is an effective method of mutant inductionwhich has been proven previously [4 29 30] and ISSR isof relatively high discriminative power of mutants or mutantloci (or the genetic diversity between them) corresponding tosimilar studies [4 29ndash32] The genetic similarity of seedlingsamong different doses treatments ranged from 06885 to1000 with an average genetic similarity of 07884 It isdirectly revealed that DNA changes had happened to theseseedlings and the dendrogram showing the formation of fivemain groups of mutants indicated that the effects of differentirradiation dosages on seedlings are far from each otherThisresult was in accordance with studies in lily [32] banana[31] Jatropha curcas L [6] and sugar beet [33] So it can beconcluded that gamma ray treatment was an effective wayfor mutation induction in S davidii and the mutants weresuccessfully identified through ISSR analysis

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was funded through projects of the National Nat-ural Science Foundation of China (31260572 and 71263012)the Science and Technology Innovation Talent Team Con-struction of Guizhou Province ([2016]5617) the Talent Teamof Guizhou Province (2016GZ13881) and the National KeyResearch Plan (2016YFC0502603)

References

[1] T Tanaka T Ito M Iinuma M Ohyama M Ichise andY Tateishi ldquoStilbene oligomers in roots of Sophora davidiirdquoPhytochemistry vol 53 no 8 pp 1009ndash1014 2000

[2] X Qi K Wang and C Zhang ldquoEffectiveness of ecologicalrestoration projects in a karst region of southwest Chinaassessed using vegetation successionmappingrdquo Ecological Engi-neering vol 54 pp 245ndash253 2013

[3] X Wu H Liu X Huang and T Zhou ldquoHuman driving forcesanalysis of rocky desertification in karst region in GuanlingCounty Guizhou Provincerdquo Chinese Geographical Science vol21 no 5 pp 600ndash608 2011

[4] S Taheri T L Abdullah Z Ahmad and N A P AbdullahldquoEffect of acute gamma irradiation on Curcuma alismatifoliavarieties and detection of DNA polymorphism through SSRmarkerrdquo BioMed Research International vol 2014 Article ID631813 pp 245ndash256 2014

[5] X M Luo N A Tinker Y Jiang P Xuan H Q Zhang and YH Zhou ldquoSuitable dose of 60Co 120574-ray formutation in Roegneriaseedsrdquo Journal of Radioanalytical and Nuclear Chemistry vol295 no 2 pp 1129ndash1134 2013

[6] D Dhakshanamoorthy R Selvaraj and A L A ChidambaramldquoInduced mutagenesis in Jatropha curcas L using gamma raysand detection of DNA polymorphism through RAPD markerrdquoComptes RendusmdashBiologies vol 334 no 1 pp 24ndash30 2011

[7] R Rustikawati E Suprijono A Romeida C Herison and S HSutjahjo ldquoIdentification of M4 gamma irradiated maize mutantbased on RAPD markersrdquo Agrivita vol 34 no 2 pp 161ndash1652012

[8] J Mudibu K K C NkongoloMMehes-Smith and A Kalonji-Mbuyi ldquoGenetic analysis of a soybean genetic pool usingISSR marker effect of gamma radiation on genetic variabilityrdquoInternational Journal of Plant Breeding amp Genetics vol 5 pp235ndash245 2011

[9] S Mejri Y Mabrouk M Voisin et al ldquoVariation in quantitativecharacters of faba bean after seed irradiation and associatedmolecular changesrdquoAfrican Journal of Biotechnology vol 11 no34 pp 8383ndash8390 2014

[10] H Afrasiab and J Iqbal ldquoGenetic analysis of somaclonalvariants and inducedmutants of Potato (Solanum tuberosum L)CV diamant using RAPDmarkersrdquo Pakistan Journal of Botanyvol 44 pp 215ndash220 2012

[11] S Taheri T L Abdullah N A P Abdullah and Z AhmadldquoUse of intersimple sequence repeat assay for detection ofDNA polymorphism induced by gamma rays in Curcumaalismatifoliardquo HortScience vol 48 pp 1346ndash1351 2013

[12] J-H Lee and T-H Han ldquoSelection of mutants obtained bygamma ray irradiation and analysis of genetic variation usingRAPDmarkers inAcorus calamus LrdquoHorticulture Environmentand Biotechnology vol 55 no 3 pp 207ndash212 2014

[13] L Zhang Q-J Li H-T Li J Chen and D-Z Li ldquoGeneticdiversity and geographic differentiation in Tacca chantrieri(Taccaceae) an autonomous selfing plant with showy floraldisplayrdquo Annals of Botany vol 98 no 2 pp 449ndash457 2006

[14] E Kovacs and A Keresztes ldquoEffect of gamma and UV-BCradiation on plant cellsrdquoMicron vol 33 no 2 pp 199ndash210 2002

[15] R Zaka C Chenal and M T Misset ldquoEffects of low dosesof short-term gamma irradiation on growth and developmentthrough two generations of Pisum sativumrdquo Science of the TotalEnvironment vol 320 no 2-3 pp 121ndash129 2004

[16] S Gaur M Singh N Rathore P S Bhati and D Kumarldquoradiobiological responses of Cowpeardquo in Proceedings of theNational Symposium on Arid Legumes for Food NutritionSecurity and Promotion of Trade pp 75ndash78 Hisar India 2002

[17] C Toker B Uzun H Canci and F Oncu Ceylan ldquoEffectsof gamma irradiation on the shoot length of Cicer seedsrdquoRadiation Physics and Chemistry vol 73 no 6 pp 365ndash3672005


[18] M Melki and A Marouani ldquoEffects of gamma rays irradiationon seed germination and growth of hard wheatrdquo EnvironmentalChemistry Letters vol 8 no 4 pp 307ndash310 2010

[19] M R Saric R Curic I Ceric and D Hadzijev ldquoEffect ofgamma radiation of some varieties of wheat seed on themorphological characteristics of the seedlingsrdquo inProceedings ofthe Symposium on the Effects of Ionizing Radiations on Seeds andTheir Significance for Crop Improvement pp 103ndash116 KarlsruheGermany 1961

[20] C B Thapa ldquoEffect of acute exposure of gamma rays on seedgermination and seedling growth of Pinus kesiya gord and PwallichianaAB JacksrdquoOur Nature vol 2 no 1 pp 13ndash17 2004

[21] F G Taylor Jr ldquoSome effects of acute gamma radiation in giantSequoia seedlingsrdquo Radiation Botany vol 8 no 1 pp 67ndash701968

[22] T L Abdullah J Endan and B M Nazir ldquoChanges in flowerdevelopment chlorophyll mutation and alteration in plantmorphology of Curcuma alismatifolia by gamma irradiationrdquoAmerican Journal of Applied Sciences vol 6 no 7 pp 1436ndash14392009

[23] P Tangpong T Taychasinpitak C Jompuk and P JompukldquoEffects of acute and chronic gamma irradiations on in vitroculture ofAnubias congensisNE BrownrdquoKasetsart Journal vol43 pp 449ndash457 2009

[24] A R Ambavane S V Sawardekar S A Sawantdesai and N BGokhale ldquoStudies on mutagenic effectiveness and efficiency ofgamma rays and its effect on quantitative traits in finger millet(Eleusine coracana L Gaertn)rdquo Journal of Radiation Researchand Applied Sciences vol 8 no 1 pp 120ndash125 2015

[25] S G Wi B Y Chung J H Kim et al ldquoUltrastructural changesof cell organelles in Arabidopsis stems after gamma irradationrdquoJournal of Plant Biology vol 48 no 2 pp 195ndash200 2005

[26] R V Hegde Studies on Induced Mutagenesis and In VitroRegeneration in Turmeric (Curcuma longa L) University ofAgricultural Sciences Dharwad Dharwad India 2006

[27] M P Reddy N Sarla and E A Siddiq ldquoInter simple sequencerepeat (ISSR) polymorphism and its application in plant breed-ingrdquo Euphytica vol 128 no 1 pp 9ndash17 2002

[28] K Semagn B Rnstad and M N Ndjiondjop ldquoAn overviewof molecular marker methods for plantsrdquo African Journal ofBiotechnology vol 5 pp 2540ndash2568 2006

[29] R K N Pestanana E P Amorim C F Ferreira et al ldquoAgro-nomic and molecular characterization of gamma ray inducedbanana (Musa sp) mutants using a multivariate statisticalalgorithmrdquo Euphytica vol 178 no 2 pp 151ndash158 2011

[30] P Orazem N Stajner and B Bohanec ldquoEffect of X-rayirradiation on olive shoot culture evaluated by morphologicalmeasurements nuclear DNA content and SSR and AFLPmarkersrdquoTreesmdashStructure and Function vol 27 no 6 pp 1587ndash1595 2013

[31] A Khatri S Bibi M U Dahot I A Khan and G S NizamanildquoIn vitro mutagenesis in banana and variant screening throughISSRrdquo Pakistan Journal of Botany vol 43 pp 2427ndash2431 2011

[32] M Xi L Sun S Qiu J Liu J Xu and J Shi ldquoIn vitromutagenesis and identification of mutants via ISSR in lily(Lilium longiflorum)rdquo Plant Cell Reports vol 31 no 6 pp 1043ndash1051 2012

[33] A Sen and S Alikamanoglu ldquoAnalysis of drought-tolerantsugar beet (Beta vulgaris L) mutants induced with gammaradiation using SDS-PAGE and ISSR markersrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 738-739 no 1 pp 38ndash44 2012

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Effects of gamma radiation on seed germination percentage and morphological traits of S davidii at seedling stage

Dose SG () LL (cm) LW (cm) SH (cm) BL (cm) SD (mm) NBControl 4000 plusmn 100b 148 plusmn 036a 069 plusmn 017a 2711 plusmn 331a 1156 plusmn 189a 288 plusmn 032c 967 plusmn 153ab

20Kr 4567 plusmn 321a 148 plusmn 032a 067 plusmn 018a 2707 plusmn 416a 1042 plusmn 255a 311 plusmn 032c 933 plusmn 208ab

40Kr 3967 plusmn 252b 149 plusmn 025a 070 plusmn 012a 2543 plusmn 589a 1041 plusmn 233a 333 plusmn 028bc 1033 plusmn 152ab

60Kr 3367 plusmn 115c 142 plusmn 029a 061 plusmn 019a 2310 plusmn 439a 1009 plusmn 402a 386 plusmn 029ab 967 plusmn 115ab

80Kr 3133 plusmn 115c 136 plusmn 034a 055 plusmn 021a 2321 plusmn 463a 946 plusmn 252a 393 plusmn 047a 1200 plusmn 265a

100Kr 2267 plusmn 231d 131 plusmn 030a 051 plusmn 020a 2285 plusmn 448a 852 plusmn 281a 323 plusmn 021c 967 plusmn 115ab



Each value was expressed as mean plusmn standard deviation SG seed germination LL leaf length LW leaf width SH seedling height BL branch length SD stemdiameter NB number of branches per plant Within columns means plusmn standard deviation followed by the same letter are not significantly different at 119875 =005

DNApolymorphism among themutants through intersimplesequence repeat (ISSR) marker analysis

2 Materials and Methods

21 Plant Source Irradiation and Morphology Study Theseeds of Sophora davidii (Franch) Kom ex Pavol werecollected in October 2013 from a karst mountain region(105∘381015840N 25∘551015840E) in Guanling Town Guizhou China Allseeds were dried under sunshine and then stored in a cooldry location Prior to radiation the moisture content of allseeds was equilibrated by placing them inside paper bagsstored in a calcium oxide-dried container

Then the seeds were irradiated with different doses (2040 60 80 100 120 and 140Kr) of 60Co-120574 in October 2013 atthe Institute of Biological and Nuclear Technology GuizhouProvince Academy of Agricultural Sciences China

Immediately after radiation 200 treated seeds were sownat a depth of 1 cm with a mixed medium of river sandred soil and farm yard manure at the ratio of 3 2 1 inplastic pots (23 times 27 cm 6 cm in height) After a month ofgermination the number of germinated seeds was countedand expressed as percentage of the total number of seedsplanted Ten seedlings from each treatmentwere transplantedto experimental field Morphological traits including leaflength leaf width plant height branch length stem diameterand number of bunches per plant were recorded twomonthslater after transplantation

22 DNA Extraction and ISSR-PCR Amplification The freshleaf material was harvested from the three-month-old plantstreated with gamma rays DNA was extracted using a mod-ified CTAB method [13] and dissolved in 1x TE butter forsubsequent use ISSR-PCR amplifications were performedin a GeneAmp PCR System 9700 DNA Thermal Cycler(PerkinElmer USA) with cycling profiles 4min at 94∘Cfollowed by 35 cycles of 30 s at 94∘C 30 s at 50∘C and 1min at72∘C and endingwith 10min at 72∘C 50 primers (Biotechnol-ogy Laboratory InnerMongolia AgricultureUniversity) werescreened initially to identify well-amplified polymorphicbands among the populations Of the 50 primers tested 20produced bright clear and reproducible bands

PCR was carried out in a total volume of 20 120583L whichincluded 50 ng of template DNA 2 120583L 10x PCR buffer (Mg2+Plus) 2 120583LMgCl

2(25mM) 10 120583L dNTPs mixture (25mM)

10U of Taq polymerase (TaKaRa Biotechnology (Dalian)Co Ltd Dalian China) and 04120583M primers (ShanghaiSangon Biological Engineering Technology and Service CoLtd Shanghai China) Amplification products along with aGeneRuler100 ladder (Fermentas UAB Inc) were separatedvia electrophoresis on 15 (wv) agarose gels with 05x TBEbuffer at 120V for 3-4 h stained with ethidium bromide(01mg120583L) They were then photographed with an Epsondigital still AF camera Negative controls which lackedtemplate DNA were also included in each PCR set to test forpossible contamination

23 Data Statistical Analysis Data of seed germination andmorphological traits were analyzed using one-way ANOVAwith SPSS software package (170) Data of ISSR markeranalysis were scored as discrete variables using ldquo1rdquo forpresence and ldquo0rdquo for absence of bands for each of the primerpairs The faint and unclear bands were not consideredfor data scoring The binary data generated were used toestimate levels of polymorphismby dividing the polymorphicbands by the total number of scored bands A dendrogrambased on Jaccardrsquos similarity coefficients was constructed byusingUnweighted Pair GroupMethodwith ArithmeticMean(UPGMA) with the SHANmodule of NTSYS-PC 20 to showphenetic representation of genetic relationships as revealedby the similarity coefficient

3 Results

31 Seed Germination and Seedling Morphological Traits inGamma Rays Treated Plants In this study we measured theeffect of different doses (control 20 40 60 80 100 120and 140Kr) of gamma irradiation on seed germination andseedling morphological traits The results (mean plusmn standarddeviation) are given in Table 1 Repeated measures ANOVArevealed a significant positive influence of the gamma irra-diation on seed germination stem diameter and number ofbranches per plant (119875 lt 005)




Total 183 94 91375Mean 915 47 4569











075 077 079 080 082 084 086

Control

120Kr

140Kr

80Kr

100Kr

40Kr

60Kr20Kr



4 Discussion


2O2)










Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Total 183 94 91375Mean 915 47 4569











075 077 079 080 082 084 086

Control

120Kr

140Kr

80Kr

100Kr

40Kr

60Kr20Kr



4 Discussion


2O2)










Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




075 077 079 080 082 084 086

Control

120Kr

140Kr

80Kr

100Kr

40Kr

60Kr20Kr



4 Discussion


2O2)










Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

effect of gamma rays on sophora davidii and detection of dna...

Documents