chemical compositions, somatic embryogenesis, and...

Research ArticleChemical Compositions Somatic Embryogenesisand Somaclonal Variation in Cumin

Moslem Bahmankar1 Seyed MohammadMahdi Mortazavian1 Masoud Tohidfar2

Seyed Ahmad Sadat Noori1 Ali Izadi Darbandi1 Giandomenico Corrado3 and Rosa Rao3

1Department of Agronomy and Plant Breeding College of Aburaihan University of Tehran Tehran Iran2Department of Plant Biotechnology Faculty of Life Sciences and Biotechnology Shahid Beheshti University GC Tehran Iran3Facolta di Agraria Universita degli Studi di Napoli Federico II Portici Italy

Correspondence should be addressed to Seyed Mohammad Mahdi Mortazavian mortazavianutacir

Received 20 May 2017 Revised 16 August 2017 Accepted 9 October 2017 Published 6 November 2017

Academic Editor Hely M Haggman

Copyright copy 2017 Moslem Bahmankar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis ofthe content of catalase ascorbate peroxidase proline protein terpenic compounds alcoholphenols aldehydes and epoxides)and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan)Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 01mgL kinetin proved to be thebest primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet Results indicatedthat different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potentialThe high content of catalase ascorbate peroxidase proline and terpenic hydrocarbons and low content of alcoholic and phenoliccompositions had a stimulatory effect on somatic embryogenesis Band patterns of RAPD markers in regenerated plants weredifferent from those of the mother plants This may be related to somaclonal variations or pollination system of cumin Generallymeasurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cuminAlso somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs

1 Introduction

Cumin (Cuminum cyminum L) is one of the most importantspicy and medical plants in the world which belongs tothe Apiaceae family The Apiaceae family consists of 455genera and over 3500 species and is regarded as one of thelargest plant families [1 2] An effective and reproducible invitro regeneration system is required for studies on geneticmanipulation in each plant species [3] Somatic embryo-genesis is the developmental restructuring of somatic cellstoward the embryogenic pathway and forms the basis ofcellular totipotency in higher plants [4] Somatic embryo-genesis is one of the most effective techniques to be usedin biotechnological studies such as producing transgenicplants artificial seeds micropropagation and germplasmconservation [5 6] Somatic embryogenesis was initiallyreported in the Apiaceae family and in vitro culture of carrot

[7 8] Genotypic properties of plants physiological charac-teristics of the explant and cell status in plant tissues can bementioned as factors influencing somatic embryogenesis [9]Somatic embryogenesis is a model system for understandingthe physiological biochemical and molecular biologicalevents occurring during plant embryo development [4]Studies showed that various genotypes in a plant species havedifferent embryogenesis capabilities depending on geneticand biochemical properties ability level of key elementsin regeneration path and also endogenous phytohormonesmetabolism [10] Dave and Batra [11] conducted the firststudy of somatic embryogenesis in cumin using differentexplants including root hypocotyls and cotyledons Theystated that hypocotyl explants in MSmedium complementedwith 8mgL BA showed the best response to embryogenesisand emphasized the necessity of cytokinins hormone forproduction and development of cumin somatic embryos [11]

HindawiBioMed Research InternationalVolume 2017 Article ID 7283806 15 pageshttpsdoiorg10115520177283806

2 BioMed Research International

Table 1 Location coordinates and weather parameter of the studied cumin landraces

Location name Location coordinates Elevation (MSL) Mean annual rainfallNorth Khorasan 37∘0910158404210158401015840N 57∘031015840203010158401015840E 13566 130mmGolestan 37∘14101584017910158401015840N 55∘09101584034810158401015840E 375 363mm

It seems that auxin hormoneswere stimulators of intracellularbiochemical reactions and specific genes in preembryo cellswhich are required for producing globular embryos Theresearchers observed that elimination of auxin frommediumcan lead to the continuation of somatic embryogenesis[8 12 13] Measurement of chemical compositions suchas contents of proline protein and antioxidant enzymesincluding catalase ascorbate peroxidase and peroxidase hasbeen used as a marker to evaluate somatic embryogenesis indifferent studies [10 14 15] Studies suggest that antioxidantenzymes such as catalase and ascorbate peroxidase play animportant role in maturity and differentiation of plant tissuesand their high activity affects cellular processes such assomatic embryogenesis [10 16 17] Terpenic hydrocarbonsalcohols and phenols existing in the essential oil of plantsare among the genetic properties affecting somatic embryo-genesis Researchers stated that terpenic hydrocarbons havepositive effects on somatic embryogenesis process whilealcohols and phenols have negative effects on the process[18ndash20] Ebrahimie et al [3] used the cut seed embryo ofcumin as an explant in B5 medium and introduced 04mgIAA + 02mg NAA + 01mg BAP as the best hormonecombination Kahrizi and Soorni [21] used 24-D and Kinin cumin developed embryogenic calluses and reported thatan increase in the concentration of 24-D hormones has apositive effect on somatic embryogenesis but their studywas only confined to callus formation and they did notreport on regeneration and adaptation Although few studieswere conducted on somatic embryogenesis and regenerationin cumin already no report has been registered on therelationships between genetic properties like chemical com-positions existing in the plant and somatic embryogenesisprocess and evaluation of somaclonal variation of regeneratedplants Therefore the current study is aimed at measuringchemical compositions optimizing somatic embryogenesisand regeneration assessment of somaclonal variation inregenerated plants as compared to the mother plant andthen evaluating relationships of these properties in the twosuperior Iranian landraces of cumin

2 Materials and Methods

21 Plant Material Two Iranian superior cumin landracesseeds Golestan and North Khorasan were selected based onthe previous study [22] (Table 1 and Figure 1) In the presentstudy experiments were divided into two sets includingmeasurement of chemical characters and optimization ofsomatic embryogenesis regeneration rooting and evalua-tion of somaclonal variation of the landraces of cumin

22 Measurement of Chemical Characters In the first exper-iment the seeds of two landraces of cumin were planted in

N

16500000

0 30 60 90 120

150

(km)

Figure 1 Location of two superior Iranian cumin landraces on themap (modified from httpwwwenchantedlearningcom)

the experiment site of College of Aburaihan University ofTehran during two successive years (2014 and 2015) Eachlandrace was planted in 2m2 plots in a sandy-clay soil Thesoil was well drained with pH of 72 Recommended cropmanagement practices were implemented to raise the cropsto desirable growth stages Plots were kept weed- pest-and disease-free until harvesting time Chemical charac-ters including the content of catalase ascorbate peroxidaseprotein proline essential oil and its composition of twolandraces of cumin were measured It is worth noting thatcatalase and ascorbate peroxidase play an important role inthe maturity of plant tissues and their high activities affectsomatic embryogenesis [10 16 17] Each essential oil is madeup of numerous different organic molecules and some ofthese including terpenic hydrocarbons alcohols and phenolsexisting in the essential oil are among the genetic propertiesaffecting somatic embryogenesis [18ndash20] In order tomeasurechemical characteristics of two landraces of cumin leaftissue samples were immediately frozen in liquid nitrogenProtein and proline contents of the leaves were measuredaccording to Bradford and Bates et al [23 24] respectivelyThe activities of antioxidant enzymes including catalase andascorbate peroxidase were estimated as described by Beersand Sizer [25] and Nakano and Asada [26] The essentialoil of the ripened seeds was extracted by the Clevengerapparatus using the hydrodistillation method The driedpowdered seeds of cumin (40 g) were placed in a distillationapparatus with 400mL of distilled water and hydrodistilledfor three hours Then the essential oils were stored in glassvials at 5∘C until the essential oil compositionsrsquo analysisThe analysis of essential oil composition was performed byGC-MS analysis (a combined analytical method to identify

BioMed Research International 3

different substances within a sample) Varian CP-3800 GC(gas chromatography) coupled with Varian 4000 (ion trap)MS (mass spectrometry) equipped with a capillary VF-5fused silica column (30m times 025mm id film thickness025 120583m) Helium was used as the carrier gas at the constantflowof 10mlminminus1 with split ratio of 150Mass spectra weretaken at 70 Ev and Mass range was from 119898119911 35ndash400 amuThe oven temperature was held at 60∘C for 1min and thenprogrammed to 250∘C at a rate of 3∘C minminus1 and heldfor 10min The injector and detector (FID) temperatureswere kept at 250 and 280∘C respectively The essential oilcompositions were identified by calculation of their retentionindices under temperature-programmed conditions for n-alkanes (C6ndashC24) and the oil on a VF-5 column underthe same chromatographic conditions The compounds wereidentified by comparison of their mass spectra with thoseof the internal reference mass spectra library (Wiley 7) orwith authentic compounds and confirmed by comparison oftheir retention indices with authentic compounds or withthose reported in the literature For quantification purposesrelative area percentages obtained by FID were used withoutthe use of correction factors

221 Experimental Design and Data Analysis A randomizedcomplete block design with five replications was used Seedswere sown by hand with 10 cm distance in rows The datarelated to chemical characteristics were recorded during twoyears (2014 and 2015) and means of these were used foranalysis Five samples were recorded in each plot Data forthe experiment were subjected to paired samples 119879-test toevaluate the statistical significance and were expressed as themean plusmn SE SPSS 160 and Excel software were used for dataanalysis

23 Optimization of Somatic Embryogenesis and RegenerationIn second set of experiment the cumin seeds were surface-sterilized following the procedure previously described [22]To obtain plantlets the sterilized seeds were cultured inthe half-strength MS medium and stored in the growthchamber [21] One month after seed culturing when grownplantlets were about 5 cm in height about 05 cm of eachexplant including hypocotyl and cotyledonwas preparedTheprepared explants were cultured in MS medium containing3 (wv) sucrose and 08 (wv) agar and supplementedwith7 different concentrations of PGRs (24-D and Kin) (Table 2)The cultured explants were incubated in the growth chamberat 24∘C plusmn 2∘C 75 relative humidity and dark conditionduring the first month of culture After this step the formedcalli were subcultured into fresh mediums containing theprevious combination of PGRs and then were stored in thegrowth chamber at 24∘C plusmn 2∘C and 75 relative humidityunder 168 h photoperiod with 50 120583molminus2 sminus1 photosyntheticphoton flux density provided by cool white fluorescent lightData related to the percentage of callus formation wasrecorded five weeks after culture Seven weeks after culturecalli images were taken by a camera under the same situationsand then perimeter and area of calli were calculated byDigimizer an image analysis software (version 431MedCalcSoftware Belgium) In the eighth week the symptoms of

Table 2 The different combination of PGRs (24-D and Kin) usedin the present study

Medium code 24-D(mgL)

Kin(mgL)

A 05 01B 1 01C 2 01D 05 mdashE 1 mdashF 2 mdashG mdash 01

somatic embryogenesis in some of the studied mediumswere observed without transferring the calli to the PGRs-freemedium then different stages of the somatic embryogenesiswere recorded using a microscope

Thereafter the calli were transferred to the PGR-freemedium for excluding of auxin effect and further stimulationof somatic embryogenesis The percentage of induced calliwith embryogenic potential was calculated on the basisof a number of explants producing somatic embryogene-sisnumber of total cultured explants in each Petri dish aftertwo to three weeks Data related to the percentage of plantregeneration were recorded during 12 weeks after the firstculture The percentage of plant regeneration was calculatedon the basis of the number of regenerated explantsthenumber of induced calli with embryogenic potential in eachPetri dish

231 Experimental Design and Data Analysis The experi-ment was performed in a factorial form using a completelyrandomized design with three factors and three replications(as Petri dishes) Five explant segments were placed ineach Petri dish containing 25ml different medium Studiedfactors included two Iranian cumin landraces Golestan andNorth Khorasan two explants hypocotyl and cotyledonand 7 different concentrations of PGRs (Table 2) The datarelated to the percentage of callus formation perimeterand area of calli were subjected to ANOVA and DMRT toevaluate the statistical significance and were expressed asthe mean plusmn SE Since the distribution of the recorded datarelated to measured characters including the percentage ofinduced calli with embryogenic potential and the percentageof plant regeneration was not normal and no transformationwas able to induce normality the Kruskal-Wallis and theMannndashWhitney nonparametric tests were used and mediangrouping was made [27] SAS 92 SPSS 160 and Excelsoftware were used for data analysis

24 In Vitro Rooting and Acclimatization Since the bestresponse of plant regeneration was observed in the MSmedium containing 01mgL Kin the regenerated 3 to 4 cmlong plantlets were excised from mentioned medium andthen were cultured in pots containing half-strength MSmedium The following treatments were investigated formore rooting MS + PGR-free MS + IAA (035mgL)


GolestanNorth Khorasan

A

A

B B

0

1

2

3

4

5

6

Ascorbateperoxidase

Catalase

(Um

g)Co

nten

t

Studied characters

(a)


A

A

B

A

0

05

1

15

2

25

3

35

4

Proline Protein

(mg

g FW

)

Studied characters Co

nten

t

(b)


A

A

095

1

105

11

115

12

125

13

Essential oil

()

Cont

ent

Studied characters

(c)

Figure 2Means comparison of studied chemical characters ((a) (b) and (c)) in the studied cumin landraces based on paired-samples119879-testIn each character landraces with one common letter at least are not significantly different at the 5 level

MS + NAA (037mgL) and MS + IAA (035mgL) + NAA(037mgL) Pots were observed every 3-4 days regularly andthen some of the characters including days to production ofthe main rooting the percentage of produced main rootingand the percentage of produced lateral rooting were recordedto compare investigated treatments After 3 weeks suffi-ciently developed cumin plantlets containing rooting weretransplanted into plastic pots containing sterilized gardensoil Then the pots were covered with the plastic cover formaintaining of plantsmoisture and kept in the greenhouse fortwo weeks Thereafter they were acclimatized to greenhouseconditions by removing the covering plastic Percentage ofsurvival plantlets was recorded and then they were kept untilflowering and seed production

241 Experimental Design and Data Analysis The rootingexperiment was performed as a completely randomizeddesign with three replications (as pots) Ten regeneratedplantlets were placed in each pot containing 50ml of inves-tigated medium The data related to the rooting experimentwere subjected to ANOVA and DMRT to evaluate thestatistical significance and were expressed as the mean plusmn SE

25 Evaluation of Somaclonal Variation by RAPD MarkersSomaclonal variation is the variation seen in plants thathave been produced by plant tissue culture It is particularlycommon in plants regenerated from callus [28 29] Typ-ical somaclonal variations are the change in chromosomenumber chromosome structure and DNA sequence [29]In this experiment genetic homogeneity of the regeneratedplantlet as compared to mother plants was examined byRAPD markers Total genomic DNA was extracted fromyoung leaves of the in vitro regenerated cumin and motherplant following the CTAB procedure according to Murrayand Thompson [30] with minor modifications Quality andquantity of extracted genomic DNA were checked on 1agarose gel and spectrophotometer and the samples were

diluted to a final concentration of 50 ng120583l Ten RAPDprimers (OPU6OPU12 OPS17 OPJ18 OPJ19 OPJ21 OPC13OPC8 OPQ6 and OPF5) were used for DNA amplification[31] PCR amplification was performed in a 25120583l reactionvolume containing 95 120583l double-distilled water 125 120583l PCRamplification kit (10x) 1 120583l primer (10 pmol) and 2120583l DNA(50 ng120583l) The amplification program included initial 5mindenaturation at 95∘C followed by 30 cycles of 1min at 95∘C30 sec at 35∘C and 2min at 72∘Cwith a final extension at 72∘Cfor 8min The PCR products were electrophoresed on 2agarose gels stained with ethidium bromide and visualizedon a UV light transilluminator

3 Results and Discussion

31 Chemical Characters The antioxidant enzymes play arole in metabolizing reactive oxygen species and preventingdamage due to oxidative stresses auxin metabolism cross-links in the cell wall and response to environmental stresses[17 32] Results of the present study showed that level of cata-lase and ascorbate peroxidase enzymes in Golestan landracewas significantly much more than that in North Khorasanlandrace (Figure 2(a)) The presence of catalase in plant cellsplays an important role in increasing resistance to oxidativestresses In addition to ascorbate peroxidase catalyzing theH2O2 reduction reaction also this enzyme plays a role inmany cellular processes such as auxin metabolism cross-links in cellular wall and response to environmental stresses[10 16 32]

The results suggested that in terms of protein contentsthere was no significant difference between the two landracesof Golestan and North Khorasan (Figure 2(b)) The proteincontent of cells is regarded as one of the most important fac-tors of cell growth [33] Proteins play important physiologicalroles such as structural and enzymatic roles on the level ofcells and it is not possible for cells to continue growing inthe long run without these materials Any factors that inhibit


protein synthesis lead to cell growth failure [32 34] Proline isalso the key amino acid that develops stress tolerance in plantsthrough protection and sustainability of cell membranes [3234] The results showed that Golestan landrace had higherproline content than North Khorasan landrace (Figure 2(b))Proline in addition to its osmotic role is effective as a sourceof nitrogen for rapid growth of plant cells [33 35] Therewas no significant difference between the two landraces interms of the amount of essential oil (Figure 2(c)) In termsof chemical structure the essential oils are not homoge-neous and have different compositions Dubey et al [36]reported significant differences between the Indian landracesof cumin in terms of the amount of essential oil Table 3represents GC-MS profile of essential oil of two studiedcumin landraces The results showed that the identifiedcompounds in their essential oil may be classified into fiveorganic groups including terpenic hydrocarbons alcoholsand phenols aldehydes epoxides and other compositions(Table 3)

It should be mentioned that the two groups of ter-penic hydrocarbons and aldehydes were the highest existingcompositions in the essential oil of the studied cumin Ina study on the chemical compositions of the landracesof Indian cumin the main ingredients of essential oil ofterpenic hydrocarbons and aldehydes were reported [2] Inthe present study the extracted essential oil from Golestanlandrace contained 6227 terpenic hydrocarbons whilethe amount of these compositions was 5114 in NorthKhorasan landrace (Table 3) It would be worth noting thattwo compositions of alpha-thujene and gamma-terpinenewere themain constituent elements of terpenic hydrocarbonsand their expanding range varied from 691 to 248Alpha-thujene was significantly higher in North Khorasanlandrace while the number and amount of other identifiedterpenic hydrocarbons in Golestan landrace were highercompared to North Khorasan landrace (Table 3) In thepresent study a total of 45 compositions were identified byGC-MS based analysis of cumin essential oil representing9842 to 9915 of the total compositions (Table 3) In otherstudies some researchers reported that the total numbersof identified compositions in Egyptian landraces Indianlandraces Chinese landraces and Tunisian landraces were41 29 19 and 38 respectively [2] Discrepancies in theresults reported on chemical compositions of cumin essentialoil may be due to factors including the sensitivity of thesystem used or method adapted for analysis The chemicalcompositions of the essential oil of medicinal plants areaffected by numerous internal and external factors includingplant genotypes geographical location climatic conditionsgrowth season and essential oil extraction method [2 37]Analysis of essential oils of the two landraces showed thatamong the identified compositions in the essential oilscumin aldehyde has been the main constituent of cuminessential oil whose value in the Northern landrace has beensignificantly higher compared to Golestan landrace (Table 3)Cumin aldehyde smells good and is the main reason for thearomatic smell of cumin seeds and spice This substance iscommercially used in perfume and in hygienic and cosmeticindustries It is worth noting that in local and international

markets cumin seeds containing more cuminaldehyde arepreferred to cumin seeds containing terpinene [2]

32 Somatic Embryogenesis and Regeneration Two types ofembryogenic and nonembryogenic calluses were observed indifferent treatment combination (Figure 3)

The knotted and compact calluses were embryogenic cal-luses Nonembryogenic calluses were agglomerate and fragileand their size was much larger than embryogenic callusesSomatic embryos were observed in the outer layer of callusesabout 2-3weeks after the transfer of calluses to embryogenesisinductionmedium (PGRs-free) It is worth noting that differ-ent developmental stages of somatic embryos (from globularto cotyledonary stage) were simultaneously observed on acallus with embryogenic potential (Figure 4)

This observation suggests that development of somaticembryos is highly unsynchronized process in cumin Thelong-term culture of embryo increased the number ofembryos but all embryogenic callus sections were not able toproduce globular embryos It is worth noting that researchersfound that the embryogenic ability was not the same incalluses only the globular calluses with soft surfaces hadthe embryogenic ability and rough dry and brittle callusesdid not have embryogenic ability [38 39] Some researchersreported that differentiation of somatic embryos in cuminwas observed about twoweeks after calluses transfer to PGRs-free MS medium [21] Results of another study suggestedthat somatic embryos appeared one week after the callusestransfer to a PGRs-freemedium [8]The results of the presentstudy indicated that among the studied factors and theirinteractional effects on the percentage of callus formationonly explants effect was significant so that cotyledon explantshowed a superior response compared to hypocotyl explant(Table 4)

The sensitivity of cultured cells and tissues to externalhormones signaling can determine their embryogenic abil-ity Some researchers stated that somatic tissues of plantsvaried in terms of callus production and also cotyledonembryogenesis sensitivity was higher than hypocotyls [4041] Results showed that the interactional effect of mediumlandrace and explant on the percentage of callus formationwas not significant while these significantly influenced callusperimeter and area (Table 4) It is worth noting that mostperimeter and area of callus were observed in hypocotylexplant of North Khorasan on 05mgL 24-D + 01mgL Kinmedium (Table 4) In order to study the effect of hypocotyllength and placement in the medium on callus propertiessuch as callus perimeter area and density Mansouri et al[42] stated that the main and interactional effects of intendedfactors had no significant effect on the callus propertiesIn the present study the overall assessments suggested thatmost callus dimensions (callus perimeter and area) havebeen obtained in 0 5mgL 24-D + 01mgL Kin growthmedium and also hypocotyl explant was superior in terms ofthe callus properties (Table 4) Since no induced calli withembryogenic potential and regeneration were observed inmost treatment combinations their analysis was excludedDue to the fact that the data associated with the percentageof induced calli with embryogenic potential and regeneration


Table 3 Chemical composition of the essential oil of two studied Iranian superior cumin landraces

S number Compounds RI Golestan landrace RI North Khorasan landraceTerpenic compound1 Alpha-thujene 1453 1722 plusmn 026

b1090 248 plusmn 010a

2 Gamma-terpinene 998 1335 plusmn 021a 998 691 plusmn 011b

3 Alpha-pinene 948 045 plusmn 0005a 948 014 plusmn 0017b

4 Alpha-phellandrene 969 076 plusmn 001a 969 073 plusmn 0005a

5 Beta-farnesene 1440 058 plusmn 001a mdash mdash6 Beta-pinene 943 9 plusmn 022a 943 335 plusmn 009b

7 Beta-phellandrene 964 050 plusmn 001a 964 027 plusmn 002b

8 Bergamotene 1425 012 plusmn 0005a mdash mdash9 Di-epi-alpha-cedrene 1403 065 plusmn 0017a 1403 018 plusmn 0008b

10 Myrcene (b-pinene) mdash mdash 958 026 plusmn 0005a

11 Limonene mdash mdash 1013 042 plusmn 0008a

12 Beta-myrcene 958 056 plusmn 003a mdash mdash13 B-Cymene 1042 548 plusmn 008a 1042 258 plusmn 003b

14 Gamma-cadinene 1435 042 plusmn 001a mdash mdash15 120573-Bisabolene 1500 010 plusmn 0003a mdash mdash16 Beta-caryophyllene 1494 025 plusmn 0003a mdash mdash17 Cyclopropane 928 052 plusmn 004a mdash mdash18 Androstane mdash mdash 2145 007 plusmn 0003a

19 Origanene 902 014 plusmn 0008a 902 006 plusmn 0006b

20 15-Cyclodecadiene 1570 008 plusmn 000a mdash mdash21 Beta-caryophyllene oxide 1507 006 plusmn 0003a mdash mdash

22 Cholestane mdash mdash 2770 023 plusmn 0008a

3-Nonen-5-yne 4-ethyl- (Z) 1117 1122 plusmn 020a 1117 1114 plusmn 008a

23 Benzene ndashDimethoxymethyl 1080 027 plusmn 001a mdash mdash24 Silane 1115 056 plusmn 0008a mdash mdashTotal terpenic compound 6227 5114

Alcoholsphenols

p-Thymol 1262 014 plusmn 0003a mdash mdashIsoeugenol mdash mdash 1410 118 plusmn 003a

Phenol 3-(1-methylethyl) 1149 005 plusmn 0005b 1149 015 plusmn 0006a

p-Cymen-8-ol 1197 007 plusmn 0003a mdash mdashCarotol 1593 035 plusmn 0008a 1593 006 plusmn 0006b

cis-Sabinol 1085 007 plusmn 0003a 1085 006 plusmn 00a

120573-linalool 1082 013 plusmn 0005b 1082 024 plusmn 0006a

p-Mentha-14-dien-7-al 1240 012 plusmn 0003b 1240 044 plusmn 0031a

Phellandral 1175 438 plusmn 012b 1175 691 plusmn 008a

trans-Pinocarveol 1131 011 plusmn 0003b 1131 014 plusmn 0006a

18-Cineole 1247 023 plusmn 0005a mdash mdashHenicosan-1-ol 2351 009 plusmn 0001a mdash mdashtrans-Shisool mdash mdash 1250 020 plusmn 0005a

Terpinen-4-ol 1137 014 plusmn 0003b 1137 023 plusmn 001a

Total alcoholsphenols 588 961

Aldehydes

Cuminaldehyde 1230 304 plusmn 027b 1230 378 plusmn 024a

Carene 919 009 plusmn 0001a 919 0078 plusmn 00016b

Nonanal 1104 043 plusmn 0008a 1104 032 plusmn 0005b

Cyclopentene-1 carboxaldehyde mdash mdash 1176 020 plusmn 0006a

Total aldehydes 3091 384Epoxides Limonene epoxide 1031 005 plusmn 0003a mdash mdashTotal epoxides 005 0

Others Dehydro-neotigogenin benzoate mdash mdash 4336 070 plusmn 001a

Corylon 972 012 plusmn 0006a mdash mdashmdash not detected In each row values followed by the same letter indicate no significant differences at 5 (119879 test) Means of five replications plusmn SE RI retentionindex determined by gas chromatography-mass spectrometry (GC-MS)


(a) (b)

(c) (d)

Figure 3 Nonembryogenic ((a) and (b)) and embryogenic (c amp d) calli in cumin

percentageswere not normally distributed andwere notmadenormally distributed through data transformation methodsKruskal-Wallis nonparametric method was used to analyzethese The results showed that the highest percentage ofinduced calli with embryogenic potential and regenerationwas achieved in treatment combination of cotyledon explantof Golestan landrace in a medium containing 01mgL Kin(Figures 4 5 and 6) In this study it was observed thatcotyledon was superior to hypocotyl in terms of induced calliwith embryogenic potential and regeneration and hypocotylshowed no somatic embryogenesis response

It should be noted that the level of endogenous hormonesis one of the required factors affecting the responding abilityof explants Quantity and quality of endogenous hormonesvary across explants so they affect their responding poten-tiality [43 44] Some researchers believe that although thetotipotency property is a very important trait in plants cellsonly gain this ability under certain conditions Even whenit is thought that all cells are equally totipotent the numberof embryos as compared to the number of cells in cellularmass is insignificant Thus only a limited portion of the cellmass has the ability to produce somatic embryos and thisprocess is confined to the particular tissue of a genotype[4 39 45] Experiences gained through tissue culture indicatethat there is a type of slope between different parts of theplant in response to embryogenesis so that embryogenicability has been high in embryo-derived tissues and low inhypocotyl and root [41] Results of the present study suggestthat somatic embryogenesis is highly dependent on the typeof explant genotype and culture medium Other researchershave also reported that the type and concentration of growthregulators and physiological state of explants are the most

important factors which play a role in somatic embryoge-nesis [8 39] In the present study the level of percentageof induced calli with embryogenic potential and regenera-tion under the treatment of 01mgL Kin was significantlyhigher than other treatments which suggest the role ofcytokinin hormones in somatic embryogenesis (Figures 5 and6)

It is worth noting that in the first embryogenesis experi-ment conducted on cumin the role of cytokinin hormonesfor the occurrence of this process has been emphasized[11] According to the results of the present study althoughinduced calli with embryogenic potential have been observedin other media containing hormone combination of auxinand cytokinin (05mgL 24-D + 01mgL Kin) or auxin alone(05mgL 24-D) the rate of regeneration in these was signif-icantly lower than that in the medium containing cytokinin(Figures 5 and 6) It would be worth noting that inducedcalli with embryogenic potential were also observed in sometreatment combinations containing 24-D in the presentstudy (Figure 5)This process can be attributed to the key roleof auxin in the induction of somatic embryogenesiswhich candirectly or indirectly regulate embryogenesis process throughaffecting the internal metabolism of other phytohormones[39 46] It should be mentioned that in the presence of 24-D embryos were not able to pass through the globular stageto heart-shaped stage and then complete embryogenesis Theinhibitory effect of 24-D hormone was also reported in otherplants belonging to Apiaceae family including carrot andcelery [47] It is worth noting that percentage of inducedcalli with embryogenic potential and the rate of regenerationhave decreased in treatment combinations containing higherauxin concentration compared to treatment combinations


(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)

Figure 4 Different developmental stages of somatic embryogenesis and plant regeneration in cotyledon of cumin (Cuminum cyminum L)Golestan landrace onto MS medium containing 01mgL Kin ((a) (b) and (c)) Globular embryos developing from cotyledon explants ((d)(e) and (f)) Simultaneous induction stages of the somatic embryo from cotyledon explants ((g) (h) and (i)) Regenerated and elongatedshoots from induced somatic embryo

containing lower auxin concentration (Figures 5 and 6)Higher concentration of auxin hormones in the culturemedium resulted in the biosynthesis of hydrocarbons such asethylene which results in cells and plant organs getting oldIn fact auxin acts as an effective inducer of somatic embryo-genesis whose reduction and omission from the culturemedium resulted in developing somatic embryogenesis [48]Therefore researchers have recommended prevention fromcontinued exposure to high levels of auxin during the somaticembryogenesis process [8 13] Some researchers also believethat sometimes auxin in the culture medium is decomposedthrough photooxidation and leads to undesirable effectson the effectiveness of somatic embryogenesis through theproduction of unwanted compositions [49 50] It should benoted that plants in Apiaceae family such as cumin contain

high levels of secondary metabolites and endogenous growthregulators particularly auxin and cytokinins Therefore itis possible that high levels of endogenous regulators in theplant have caused the external use of them to be unnecessary[51] Valizadeh et al [52] have mentioned the occurrence ofregeneration in some kinetin-free treatments as reasons forthis claim The researchers believe that the most importantfactor involved in differences between embryogenic andnonembryogenic cultures is their difference in terms ofthe level of endogenous hormones because these regulateexplants differentiation processes in the media [38 53] Ingeneral factors such as plant genetic properties type ofexplants the explant growth phase and cells position in plantcultured tissue are effective for their somatic embryogenicability


Table 4 Mean comparisons of medium times landrace times explant effect in the induction of callus production and related characters in Iraniansuperior cumin landraces

Medium code Landrace Explant callus production Callus area (cm) Callus perimeter (cm)A Golestan Cotyledon 934 plusmn 68a 093 plusmn 009cd 499 plusmn 016bc

A Golestan Hypocotyl 934 plusmn 68a 113 plusmn 012bc 491 plusmn 034bc

A North Khorasan Cotyledon 867 plusmn 137ab 163 plusmn 007a 600 plusmn 037a

A North Khorasan Hypocotyl 867 plusmn 68ab 101 plusmn 015bcd 453 plusmn 031bcde

B Golestan Cotyledon 100 plusmn 0a 124 plusmn 024b 502 plusmn 042b

B Golestan Hypocotyl 934 plusmn 68a 086 plusmn 02d 386 plusmn 027efgh

B North Khorasan Cotyledon 934 plusmn 68a 079 plusmn 004de 417 plusmn 004defg

B North Khorasan Hypocotyl 867 plusmn 68ab 083 plusmn 017de 399 plusmn 043efgh

C Golestan Cotyledon 100 plusmn 0a 087 plusmn 001d 471 plusmn 017bcd

C Golestan Hypocotyl 867 plusmn 68ab 066 plusmn 00e 362 plusmn 005fgh

C North Khorasan Cotyledon 100 plusmn 0a 079 plusmn 009de 358 plusmn 003fgh

C North Khorasan Hypocotyl 80 plusmn 117ab 068 plusmn 001e 353 plusmn 0006gh

D Golestan Cotyledon 100 plusmn 0a 080 plusmn 05de 408 plusmn 05defg

D Golestan Hypocotyl 80 plusmn 117ab 058 plusmn 006 337 plusmn 019h

D North Khorasan Cotyledon 934 plusmn 68a 084 plusmn 002de 371 plusmn 007efgh

D North Khorasan Hypocotyl 867 plusmn 68ab 073 plusmn 003e 358 plusmn 004fgh

E Golestan Cotyledon 934 plusmn 68a 072 plusmn 004e 367 plusmn 008efgh

E Golestan Hypocotyl 80 plusmn 117ab 061 plusmn 004e 336 plusmn 012h

E North Khorasan Cotyledon 934 plusmn 68a 073 plusmn 003e 359 plusmn 007fgh

E North Khorasan Hypocotyl 667 plusmn 18b 063 plusmn 004e 338 plusmn 013h

F Golestan Cotyledon 100 plusmn 0a 098 plusmn 015cd 433 plusmn 050cde

F Golestan Hypocotyl 667 plusmn 18b 077 plusmn 002de 353 plusmn 012gh

F North Khorasan Cotyledon 100 plusmn 0a 082 plusmn 002de 402 plusmn 02efgh

F North Khorasan Hypocotyl 934 plusmn 68a 083 plusmn 03de 374 plusmn 01efgh

G Golestan Cotyledon 944 plusmn 577a 090 plusmn 003cd 422 plusmn 011def

G Golestan Hypocotyl 867 plusmn 68ab 098 plusmn 005cd 431 plusmn 021de

G North Khorasan Cotyledon 100 plusmn 0a 087 plusmn 003d 401 plusmn 009defgh

G North Khorasan Hypocotyl 80 plusmn 117ab 096 plusmn 005cd 413 plusmn 015defg

Mean values in each column followed by different letters indicate significant differences according to the DMRT test at 119875 lt 005

B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n

Figure 5 Rank comparisons of percentage of induced calli withembryogenic potential in the studied treatments Values in eachcolumn followed by the same letters are not significantly differentaccording to the Kruskal-Wallis nonparametric test

B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n

Figure 6 Rank comparisons of plant regeneration in the studiedtreatments Values in each column followed by the same letters arenot significantly different according to the Kruskal-Wallis nonpara-metric test


Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)

Figure 7 Induction of rooting and developed plantlets in cumin (Cuminum cyminum L) Golestan landrace ((a) and (b)) Main and lateralrooting inMSmedium containing IAA (035mgL) + NAA (037mgL) ((c) and (d)) Regenerated cumin planted (e) Pot covered by a plasticcover (F1) Developed cumin plantlets and (F2) mature cumin plant

33 In Vitro Rooting and Acclimatization The results showedthat the hormone treatment IAA (035mgL) + NAA(037mgL) as compared to other treatments was superiorin terms of rooting rate the percentage of lateral root pro-duction and survival rate of regenerated seedlings (Figure 7)According to these results it can be said that IAA andNAA auxin have synergistic effects on properties related torooting and act supplementally (Table 5) Ginger also wasobserved amongmedical plants regenerated organs of whichhave produced roots under the application of IAA and NAAhormones [54] Rooting in plants is usually induced inmediacontaining auxin hormones [54] In the present study in themedium free of auxin limited rooting was also observedwhich may be associated with plant genotypes and endoge-nous hormone levels (Table 5) It is worth noting that someresearchers also reported that cumin regenerated seedlingshave produced roots in the auxin-free medium but rootsproduced in the growth medium containing IBA auxin weremuch more and stronger [55] Other researchers reportedthat rooting in calluses obtained fromhypocotyl of cumin hasbeen significantly increasedwith an increase in concentrationof NAA hormone [52] Researchers found that the usage ofIBA hormone to induce rooting in cumin is effective andreported that although the usage of PEG in the medium hasled to decreased rooting in cumin it has increased survivalability of regenerated plants [55] The researchers believethat PEGhas inducedmorphological and anatomical changes

associated with increased viability and adaptability of plants[56 57] Since the survival rate of plants regenerated in thepresent study was lower than the rate observed by Tawfic andNoga [55] perhaps in addition to differences in the type ofgenotype lack of PEG can bementioned as another reason forit One of the most important problems of micropropagationis the survival rate of the seedling during transferring to exvitro conditions to acclimatization to the new climate in thegreenhouse or fieldThe type of environment and pot soil arealso factors affecting the establishment and the survival rateof regenerated seedlings [55]

Generally decreased survival rate of plants regeneratedin tissue culture experiments can be attributed to differentfactors including low photosynthetic capacity resulting fromthe presence of sugar in the culture medium low lightinadequate amount of CO2 and high relative humidity in themedium which eventually undesirably affect the efficiency ofthe plantrsquos photosynthesis and its survival rate [58]

34 Evaluation of the Relationship between Chemical Com-position and Somatic Embryogenesis The processes of celldifferentiation and somatic embryogenesis are highly depen-dent on the genetic and chemical composition of plantsMeasurement of chemical parameters can be used as amarkerto assess somatic embryogenesis [14 15] Studies suggest thatantioxidant enzymes such as catalase and ascorbate peroxi-dase play an important role in the growth and differentiation


Table 5 Mean comparisons of the effect of different mediums on the rooting and survival of regenerated plantlets in Golestan landrace

Medium Main rootinginduction

Days to initiation ofmain rootinginduction

Lateral rootinginduction Ex vitro survival

PGR-free 013 plusmn 005c 123 plusmn 088a 013 plusmn 003b 0b

IAA (035mgL) 027 plusmn 021c 11 plusmn 115ab 023 plusmn 013b 01 plusmn 003b

NAA (037mgL) 043 plusmn 015b 103 plusmn 09ab 027 plusmn 01b 017 plusmn 009b

IAA (035mgL) + NAA(037mgL) 067 plusmn 006a 933 plusmn 067b 063 plusmn 007a 058 plusmn 008a


of plant cells and tissues and their high activities are associ-ated with cell differentiation process [10 16 17] The resultsof the present study showed that the contents of catalaseand ascorbate peroxidase and the percentage of induced calliwith embryogenic potential in Golestan landrace were signif-icantly higher compared to North Khorasan landrace Sincesomatic embryogenesis is heavily dependent on the genotypeone of the factors which are effective in somatic embryogen-esis differences among the studied cumin landraces may besignificant differences in their chemical compositions [5 5960] Studies have shown that ascorbate peroxidase is requiredto maintain size and shape of cells during embryogenesisIt also plays an important role in the formation of ligninand protects embryo cell walls [10 61] Researchers have alsostated that variations in the content of antioxidant enzymescorrelate with the regeneration of plants [10 16 32] It isworth noting that catalase and ascorbate peroxidase havebeen used as markers to study the developmental stagesof somatic embryos and their highest activity level wasobserved in torpedo stage of the somatic embryogenesis[10] In the present study Golestan landrace in addition toantioxidant enzymes had much more proline and proteinThese compositions may be considered as other geneticproperties of this landrace which lead to increased somaticembryogenesis in it It should be mentioned that increasedsomatic embryogenesis through proteins and amino acidsis due to the fact that the compositions easily supply thesource of nitrogen for the cell growth [35] Proline plays animportant role in somatic embryogenesis and stress condi-tions in plants [62] For example studies have shown that theproline-rich protein is required in the cell wall of Arabidopsisfor correctly placing cell plate during cytokinesis processfor embryos development The carrot cell wall protein hasbeen secreted into the growth medium and regulates somaticembryogenesis development [63] Researchers observed thatprotein content of cells in preembryogenesis calluses washigher than that in embryogenic calluses In fact as thecallus enters somatic embryogenesis stage its protein leveldecreases which indicates protein consumption for somaticembryogenesis development Studies have shown that cellsuse their protein content to initiate embryogenesis so it canbe said that the cellular protein content is among geneticproperties that lead to increased somatic embryogenesis [1034] As somatic embryogenesis initiates and develops anincrease in protein consumption has also been reported inother studies [9 10] The cultured cells are naturally able to

synthesize all their necessary amino acids however embryo-genesis and regeneration levels in cumin can be increased byadding proline and other amino acids to the culture mediumand this ability can be applied in gene transfer programs[35 59] Results of the essential oil analysis showed thatphytochemical profiles of the two landraces of Golestan andNorth Khorasan have significant differences with each otherwhich is another confirmation of the existence of geneticdifferences between them For example the number ofterpenic hydrocarbons was higher in essential oil of Golestanlandrace than that in North Khorasan landrace (Table 3)Researchers have reported that the genetic characteristicsare regarded as terpenic hydrocarbons which could have apositive effect on somatic embryogenesis in plants [20] Theresults of this study suggest that one of themain factors affect-ing the occurrence of somatic embryogenesis in Golestanlandrace is the phytochemical properties especially the highterpenic hydrocarbons content It should be noted that NorthKhorasan landrace has higher alcoholic and phenolic com-positions and these compositions along with other factorsmay lead to decreased somatic embryogenesis in it Howphenolic compositions affect the induction and developmentof somatic embryo is not clear yet but these compositionsmay be involved in the metabolism of endogenous hormonessuch as auxin [18 19] Phenolic compositions prevent thegrowth and development of somatic embryogenesis fromgoing beyond the globular stages Embryos formed in thepresence of phenolic compositions lack sufficient amounts ofsucrose to carry out the metabolic activity and thus stop inthe globular stage It should be noted that the inhibitory effectof phenolic compositions due to their high concentrationin cells leads to protein deposition enzyme deactivation ormembrane damage [18 64] However in some studies ithas been reported that phenols usually have a reinforcingeffect on somatic embryogenesis but it is possible thatphenols interaction with alcohols and other phytochemicalcompositions of essential oil inhibits their reinforcing effecton embryogenesis Results of the present study showed thatessential oil percentage of North Khorasan was higher thanGolestan landrace however the difference was not signifi-cant Also it should be noted that cuminaldehyde contentthe most primarily essential oil composition of cumin wassignificantly higher in North Khorasan landrace than inGolestan landrace A high percentage of essential oil andmore cuminaldehyde content in North Khorasan landracemay be regarded as two of the factors affecting the lack of its


somatic embryogenesis As no study has been reported on theeffect of cuminaldehyde on somatic embryogenesis in cuminthe final decision on this issue requires further studiesResults of the present study showed that variation in thegenetic and chemical structure of cumin landraces is amongthe important factors determining somatic embryogenesisprocess and can be used as a marker for assessment ofoccurrence of this process

35 Evaluation of Somaclonal Variation in the RegeneratedPlants in Comparison with the Mother Plants Somaclonalvariation is a common process in plants cell and tissue cultureand was firstly introduced by Larkin and Scowcroft [28]This process has led to changes in nuclear and cytoplasmicgenomes and is of genetic and epigenetic nature [28 29]Ten primers were initially used 7 of which generated infor-mative and reproducible bands (6ndash10 bands per primer)ranging from approximately 200 to more than 1000 bp insize The 7 primers generated a total of 52 scorable bandswith an average of 74 per primer which were polymorphicacross all the analyzed in vitro regenerated plantlets origi-nating from cotyledons of the mother plant (Figure 8) Inthe present study RAPD markers suggested polymorphismbands between regenerated plants of Golestan landrace andseed plants (Figure 8) Itmay be said that the variation is asso-ciated with the heterozygous nature of the cumin landraceand its pollination system Some studies suggest somaticvariations in plants regenerated from tissue culture and someothers suggest genetic stability [65ndash67] Bakhtiar et al [67]in their study on the genetic diversity ofThymus regeneratedseedlings and seedlings frommother plant found that RAPDmarker suggests the presence of polymorphism bands amongthem

They believed that the observed variation may be relatedto Thyme pollination system They also stated that theoptimized protocol for Thyme regeneration is a useful toolto protect germplasm and it does not seem that interferenceshave been made in the genetic uniformity of the regeneratedplants [67] Analysis of Arabidopsis regenerated using RAPDand RFLP markers indicated the lack of diversity in theDNAprofile of regenerated plants [68] Evaluation of somaticvariation frequency is valuable for any regeneration systembut the variation has been studied in few regenerated plants[43] Although molecular markers have been used as amolecular tool to evaluate somaclonal variation with regardto the fact that morphological and cytogenetic diversitycannot be detected at DNA level there is a little doubt abouttheir effectiveness [29] In regenerated plants changes inDNA methylation levels are considered as another sourceof somaclonal variation It is believed that the occurrenceof somaclonal variation resulting from DNA methylationespecially when auxin is consumed is more than somaticvariation due to changes in the DNA sequence [69 70] Itis also well known that increasing numbers of subculturesincreases the likelihood of somaclonal variation [70] Geneticuniformity of produced plants through tissue culture dependson several factors the most important ones are the propaga-tion method and genotype [51 67] As compared to classicalmethods of vegetative propagation the regenerated plants

M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)

Figure 8 RAPD marker analysis generated by the primers OPC8(a) and OPU12 (b) Lane M corresponds to 100 bp DNA ladderLanes 1ndash11 correspond to DNA from randomly selected regeneratedplantlets Lane S corresponds to seed plantlet Lane NC correspondsto negative control

from calluses depending on the species origin and cultureage may show varying amounts of structural or physiologicaldisorders Although these disorders seem inappropriate atfirst since a limited number of the above-mentioned plantshave desirable characteristics missing in the main plantsbreeders will use this genetic variation in breeding programs[51]

4 Conclusion

Results of this study identified effective factors in somaticembryogenic ability such as plant genetic properties typeof explant and type and concentration of PGRs and thecotyledon of Golestan landrace in MS + 01mgL Kinmedium was the best treatment The results also suggestedthat different developmental stages of somatic embryoswere simultaneously observed on a callus with embryogenicpotential This observation suggests that development ofsomatic embryos is a highly unsynchronized process incumin The high content of catalase ascorbate peroxidaseproline and terpenic hydrocarbons and low contents ofalcoholic and phenolic compositions had a positive effecton somatic embryogenesis Therefore measurement of thesecharacters may be useful as a marker to predict somaticembryogenesis in cumin Results suggested that althoughthe cultured cells are naturally able to synthesize all theirnecessary chemical parameters modulation of them throughmanipulation of culture mediummay be an effective method


to improve somatic embryogenesis in cumin and it can beapplied in gene transformation Band patterns of RAPDmarkers in regenerated plants were different from those ofthe seed plants It may be related to somaclonal variations orimpure nature and pollination system of cumin It should benoted that plant breeders can apply the observed variation ofregenerated plants from tissue culture in breeding programs

Abbreviations

PGRs Plant growth regulatorsIAA Indole-3-acetic acidIBA Indole-3-butyric acidNAA 1-Naphthaleneacetic acidBAP 6-Benzylaminopurine24-D 24-Dichlorophenoxyacetic acidKin KinetinPEG Polyethylene glycolMS Murashige and SkoogB5 Gamborgrsquos B5CTAB Cetyl trimethylammonium bromideRAPD Random Amplification of Polymorphic DNARFLP Restriction Fragment Length PolymorphismPCR Polymerase chain reactionGC-MS Gas chromatography-mass spectrometryANOVA Analysis of VarianceSE Standard errorDMRT Duncanrsquos Multiple Range Test

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

The authors gratefully acknowledge the support provided forthis survey by the Biotechnology Development Council ofthe Islamic Republic of Iran (Grant no 950503) The authorsalso would like to thank Dr Pasquale Chiaiese Lab Specialistof the Department of Agricultural Sciences University ofNaples Federico II andMs Maryam Salehi Ph D student ofPlant Breeding Department Faculty of Agriculture TarbiatModares University Tehran for their helpful assistance andcomments during the experiment

References

[1] R Baranski ldquoGenetic transformation of carrot (Daucus carota)and other apiaceae speciesrdquo Trans Plant Journal vol 2 no 1 pp18ndash38 2008 httpwwwglobalsciencebooksinfoJournalsSupimages0806

[2] P N Dubey S N Saxena B K Mishra R K Solanki M KVishal and B Singh B ldquoPreponderance of cumin ( Cuminumcyminum L ) essential oil constituents across cumin growingAgro-Ecological Sub Regions Indiardquo Industrial Crops Productsvol 95 pp 50ndash95 2017

[3] E Ebrahimie A A Habashy M Mohammadie-DehcheshmehM R Ghannadha B Ghareyazie and B Yazdi-Amadi ldquoDirectshoot regeneration from mature embryo as a rapid and

genotype-independent pathway in tissue culture of heteroge-neous diverse sets of cumin (Cuminum cyminum L) geno-typesrdquo In Vitro Cellular amp Developmental Biology - Plant vol42 no 5 pp 455ndash460 2006

[4] O Karami B Aghavaisi and A Mahmoudi Pour ldquoMolecularaspects of somatic-to-embryogenic transition in plantsrdquo Journalof Chemical Biology vol 2 no 4 pp 177ndash190 2009

[5] Y Guan S Li X Fan and Z Su ldquoApplication of SomaticEmbryogenesis in Woody Plantsrdquo Frontiers in Plant Sciencevol 07 2016

[6] S Von Arnold I Sabala P Bozhkov J Dyachok and LFilonova ldquoDevelopmental pathways of somatic embryogenesisrdquoPlant Cell Tissue and Organ Culture vol 69 no 3 pp 233ndash2492002

[7] F C Steward M O Mapes and K Mears ldquoGrowth andOrganized Development of Cultured Cells II Organizationin Cultures Grown from Freely Suspended Cellsrdquo AmericanJournal of Botany vol 45 no 10 p 705 1958

[8] A A Tawfik and G Noga ldquoCumin regeneration from seedlingderived embryogenic callus in response to amended kinetinrdquoPlant Cell Tissue and Organ Culture vol 69 no 1 pp 35ndash402002

[9] G I Nic-Can R M Galaz-Avalos C De-la-Pena A Alcazar-Magana KWrobel andVM Loyola-Vargas ldquoSomatic embryo-genesis Identified factors that lead to embryogenic repressionA case of species of the same genusrdquo PLoS ONE vol 10 no 6Article ID e0126414 2015

[10] M Jariteh H Ebrahimzadeh V Niknam M Mirmasoumiand K Vahdati ldquoDevelopmental changes of protein prolineand some antioxidant enzymes activities in somatic and zygoticembryos of Persian walnut (Juglans regia L)rdquo Plant Cell Tissueand Organ Culture vol 122 no 1 pp 101ndash115 2015

[11] A Dave and A Batra ldquoRole of protein metabolism constituentsin somatic embryo formation in cuminrdquo Indian Journal of PlantPhysiology vol 12 no 1 pp 25ndash27 1995

[12] J L Zimmerman ldquoSomatic embryogenesis A model for earlydevelopment in higher plantsrdquo The Plant Cell vol 5 no 10Article ID 160372 pp 1411ndash1423 pp 1411-14231993 httpwwwpubmedcentralnihgovarticlerenderfcgiartid=160372ampamptool=pmcentrezampamprendertype=abstract

[13] Y Tokuji and K Kuriyama ldquoInvolvement of gibberellin andcytokinin in the formation of embryogenic cell clumps in carrot(Daucus carota)rdquo Journal of Plant Physiology vol 160 no 2 pp133ndash141 2003

[14] B H Abbasi H Ali B Yucesan S Saeed K Rehman and MA Khan ldquoEvaluation of biochemical markers during somaticembryogenesis in Silybummarianum Lrdquo 3 Biotech vol 6 no 1article no 71 2016

[15] M Jeyaseelan and M V Rao ldquoBiochemical studies of embryo-genic and non-embryogenic callus of Cardiospermum halica-cabum Lrdquo Indian Journal of Experimental Biology (IJEB) vol43 no 6 pp 555ndash560 2005

[16] J Thakar and S Bhargava ldquoSeasonal variation in antioxidantenzymes and the sprouting response of Gmelina arborea Roxbnodal sectors cultured in vitrordquo Plant Cell Tissue and OrganCulture vol 59 no 3 pp 181ndash187 1999

[17] A N Molassiotis K Dimassi G Diamantidis and I TheriosldquoChanges in peroxidases and catalase activity during in vitrorootingrdquo Biologia Plantarum vol 48 no 1 pp 1ndash5 2004

[18] E Reis M T Batista and J M Canhoto ldquoEffect and analysis ofphenolic compounds during somatic embryogenesis induction


in Feijoa sellowiana Bergrdquo Protoplasma vol 232 no 3-4 pp193ndash202 2008

[19] G G Gross C Janse and E F Elstner ldquoInvolvement of malatemonophenols and the superoxide radical in hydrogen peroxideformation by isolated cell walls from horseradish (Armoracialapathifolia Gilib)rdquo Planta vol 136 no 3 pp 271ndash276 1977

[20] G Alonzo F Saiano N Tusa and S Fatta Del Bosco ldquoAnalysisof volatile compounds released from embryogenic cultures andsomatic embryos of sweet oranges by head space SPMErdquo PlantCell Tissue and Organ Culture vol 66 no 1 pp 31ndash34 2001

[21] D Kahrizi and J Soorni ldquoStudy on shoot regeneration andsomatic embryogenesis in cumin (Cuminum cyminum L)landracesrdquo Biharean Biologist vol 7 no 1 Article ID 131107 pp37ndash41 2013

[22] M Bahmankar M SMMortazavian S A Sadat Noori and AIzadi Darbandi ldquoIzadi Darbandi Determination of ThresholdConcentration of Kanamycin to Transfer Gene in CuminrdquoElectronic Journal of Biology vol 11 no 4 pp 161ndash164 2015httpejbioimedpubcomdetermination-of-threshold-con-centration-of-kanamycin-to-transfer-gene-in-cuminphpaid=7829

[23] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquo Analytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[24] L S Bates R PWaldren and I D Teare ldquoRapid determinationof free proline for water-stress studiesrdquo Plant and Soil vol 39no 1 pp 205ndash207 1973

[25] R F Beers and I W Sizer ldquoA spectrophotometric method formeasuring the breakdown of hydrogen peroxide by catalaserdquoThe Journal of Biological Chemistry vol 195 no 1 pp 133ndash1401952

[26] Y Nakano and K Asada ldquoPurification of ascorbate peroxidasein spinach chloroplasts its inactivation in ascorbate-depletedmedium and reactivation by monodehydroascorbate radicalrdquoPlant amp Cell Physiology (PCP) vol 28 no 1 pp 131ndash140 1987

[27] K Pruski T Astatkie and J Nowak ldquoTissue culture propa-gation of Mongolian cherry (Prunus fruticosa) and Nankingcherry (Prunus tomentosa)rdquo Plant Cell Tissue and OrganCulture vol 82 no 2 pp 207ndash211 2005

[28] P J Larkin andW R Scowcroft ldquoSomaclonal variation - a novelsource of variability from cell cultures for plant improvementrdquoTheoretical and Applied Genetics vol 60 no 4 pp 197ndash214 1981

[29] A Nwauzoma and E Jaja ldquoA review of somaclonal variation inplantain (Musa spp) mechanisms and applicationsrdquo Journal ofApplied Biosciences vol 67 no 0 p 5252 2013

[30] M G Murray and W F Thompson ldquoRapid isolation of highmolecular weight plant DNArdquoNucleic Acids Research vol 8 no19 pp 4321ndash4326 1980

[31] A Bahraminejad G Mohammadi-Nejad M A Kadir andM R B Yusop ldquoMolecular diversity of Cumin (Cuminumcyminum L) Using RAPDmarkersrdquo Australian Journal of CropScience vol 6 no 2 pp 194ndash199 2012

[32] S Pandey M K Patel A Mishra and B Jha ldquoPhysio-bio-chemical composition and untargeted metabolomics of cumin(CuminumcyminumL)make it promising functional food andhelp in mitigating salinity stressrdquo PLoS ONE vol 10 no 12Article ID e0144469 2015

[33] M Hasanuzzaman K Nahar M M Alam R Roychowdhuryand M Fujita ldquoPhysiological biochemical and molecularmechanisms of heat stress tolerance in plantsrdquo InternationalJournal ofMolecular Sciences vol 14 no 5 pp 9643ndash9684 2013

[34] B Adamczyk A Smolander V Kitunen and M GodlewskildquoProteins as nitrogen source for plants a short story aboutexudation of proteases by plant rootsrdquo Plant Signaling andBehavior vol 5 no 7 pp 817ndash819 2010

[35] B Pawar P Kale J Bahurupe A Jadhav A Kale and S PawarldquoProline and Glutamine Improve in vitro Callus Induction andSubsequent Shooting in Ricerdquo Rice Science vol 22 no 6 pp283ndash289 2015

[36] P N Dubey S N Saxena B KMishra et al ldquoPreponderance ofcumin (Cuminum cyminumL) essential oil constituents acrosscumin growing Agro-Ecological Sub Regions Indiardquo IndustrialCrops and Products vol 95 pp 50ndash59 2017

[37] I Bettaieb S Bourgou W A Wannes I Hamrouni F Limamand B Marzouk ldquoEssential oils phenolics and antioxidantactivities of different parts of cumin (Cuminum cyminum L)rdquoJournal of Agricultural and Food Chemistry vol 58 no 19 pp10410ndash10418 2010

[38] V M Jimenez and F Bangerth ldquoEndogenous hormone levels inexplants and in embryogenic and non-embryogenic cultures ofcarrotrdquo Physiologia Plantarum vol 111 no 3 pp 389ndash395 2001

[39] F Quiroz-Figueroa C Fuentes-Cerda R Rojas-Herrera andV Loyola-Vargas ldquoHistological studies on the developmentalstages and differentiation of two different somatic embryoge-nesis systems of Coffea arabicardquo Plant Cell Reports vol 20 no12 pp 1141ndash1149 2002

[40] M Klems M Truksa I Machackova J Eder and S Pro-chazka ldquoUptake transport and metabolism of 14C-24-di-chlorophenoxyacetic acid ( 14C-24-d) in cucumber (Cucumissativus L) explantsrdquo Plant Growth Regulation vol 26 no 3 pp195ndash202 1998

[41] A Mujib and J Samaj Somatic Embryogenesis vol 2 Springer-Verlag BerlinHeidelberg 2006 85ndash101

[42] A Mansouri A Fadavi and S M M Mortazavian ldquoEffectsof length and position of hypocotyl explants on Cuminumcyminum L callogensis by image processing analysisrdquo PlantCell Tissue and Organ Culture vol 121 no 3 pp 657ndash666 2015

[43] M D Gaj ldquoFactors influencing somatic embryogenesis induc-tion and plant regeneration with particular reference to Ara-bidopsis thaliana (L) Heynhrdquo Plant Growth Regulation vol 43no 1 pp 27ndash47 2004

[44] C Simoes N Albarello C H Callado T C de Castro and EMansur ldquoSomatic embryogenesis and pant regeneration fromcallus cultures of Cleome rosea Vahlrdquo Brazilian Archives ofBiology and Technology vol 53 no 3 pp 679ndash686 2010

[45] MA J Toonen THendriks E D L SchmidtH AVerhoevenA van Kammen and S C de Vries ldquoDescription of somatic-embryo-forming single cells in carrot suspension culturesemploying video cell trackingrdquo Planta vol 194 no 4 pp 565ndash572 1994

[46] A Feher T P Pasternak and D Dudits ldquoTransition of somaticplant cells to an embryogenic staterdquo Plant Cell Tissue andOrganCulture vol 74 no 3 pp 201ndash228 2003

[47] P V Ammirato and F C Steward ldquoSome effects of environmenton the development of embryos from cultured free cellsrdquoBotanical Gazette vol 132 no 2 pp 149ndash158 1971

[48] Z X Lim A Pick K Ling S Hussein K Lumpur and B Divi-sion ldquoCallus Induction of Ocimum sanctum and Estimationof Its Total Flavonoids Contentrdquo Asian Journal of AgriculturalSciences vol 1 no 2 pp 55ndash61 2009

[49] B L Lad S Jayasankar F Pliego-Alfaro P A Moon and RE Litz ldquoTemporal effect of 24-D on induction of embryo-genic nuclellar cultures and somatic embryo development of


lsquoCarabaorsquo mangordquo In Vitro Cellular amp Developmental Biology -Plant vol 33 no 4 pp 253ndash257 1997

[50] S Manish T A Khair S A Ghaya et al ldquoNucellar embryogen-esis and plantlet regeneration in monoembryonic and polyem-bryonic mango (Mangifera indica L) cultivarsrdquoAfrican Journalof Biotechnology vol 15 no 52 pp 2814ndash2823 2016

[51] G R Rout S Samantaray and P Das ldquoIn vitro manipulationand propagation of medicinal plantsrdquo Biotechnology Advancesvol 18 no 2 pp 91ndash120 2000

[52] M Valizadeh A Safarnejad G H Nematzadeh and S KKazemitabae ldquoRegeneration of plantlets from embryo explantsof bunium persicum (Boiss) B fedtschrdquo Research Journal ofMedicinal Plant vol 1 no 2 pp 42ndash47 2007

[53] B Grieb F Schafer J Imani K Nezamabadi Mashayekhi BArnholdt-Schmitt and K H Neumann ldquoChanges in solubleproteins and phytohormone concentrations of cultured carrotpetiole explants during induction of somatic embryogenesis(Daucus carota L)rdquo Journal of Applied Botany and FoodQualityvol 71 no 3-4 pp 94ndash103 1997

[54] R T Faria and R D Illg ldquoMicropropagation of Zingiberspectabile Griffrdquo Scientia Horticulturae vol 62 no 1-2 pp 135ndash137 1995

[55] A A Tawfik and G Noga ldquoAdventitious shoot proliferationfrom hypocotyl and internodal stem explants of cuminrdquo PlantCell Tissue and Organ Culture vol 66 no 2 pp 141ndash147 2001

[56] M N Nas and P E Read ldquoImproved rooting and acclimatiza-tion of micropropagated hazelnut shootsrdquo HortScience vol 39no 7 pp 1688ndash1690 2004

[57] I Dami andH G Hughes ldquoEffects of PEG-induced water stresson in vitro hardening of rsquovaliantrsquo graperdquo Plant Cell Tissue andOrgan Culture vol 47 no 2 pp 97ndash101 1996

[58] B N Hazarika ldquoMorpho-physiological disorders in in vitroculture of plantsrdquo Scientia Horticulturae vol 108 no 2 pp 105ndash120 2006

[59] X Ge Z Chu Y Lin and S Wang ldquoA tissue culture system fordifferent germplasms of indica ricerdquo Plant Cell Reports vol 25no 5 pp 392ndash402 2006

[60] L M Guan and J G Scandalios ldquoCatalase transcript accumu-lation in response to dehydration and osmotic stress in leaves ofmaize viviparous mutantsrdquo Redox Report vol 5 no 6 pp 377ndash383 2000

[61] N Fernandez-Garcıa M Carvajal and E Olmos ldquoGraft unionformation in tomato plants Peroxidase and catalase involve-mentrdquo Annals of Botany vol 93 no 1 pp 53ndash60 2004

[62] S K Ghanti K G Sujata S Rao M Udayakumar and PB K Kishor ldquoRole of enzymes and identification of stage-specific proteins in developing somatic embryos of chickpea(Cicer arietinum L)rdquo In Vitro Cellular ampDevelopmental Biology- Plant vol 45 no 6 pp 667ndash672 2009

[63] Q Hall and M C Cannon ldquoThe cell wall hydroxyproline-richglycoprotein RSH is essential for normal embryo developmentin ArabidopsisrdquoThePlant Cell vol 14 no 5 pp 1161ndash1172 2002

[64] A Ptak A El Tahchy E Skrzypek T Wojtowicz and DLaurain-Mattar ldquoInfluence of auxins on somatic embryogenesisand alkaloid accumulation in Leucojum aestivum callusrdquo Cen-tral European Journal of Biology vol 8 no 6 pp 591ndash599 2013

[65] C Gimenez E De Garcia N X De Enrech and I BlancaldquoSomaclonal variation in banana Cytogenetic and molecularcharacterization of the somaclonal variant CIEN BTA-03rdquo InVitro Cellular Developmental Biology-Plant vol 37 no 2 pp217ndash222 2001

[66] P Eshraghi R Zarghami and H Ofoghi ldquoRAPD analysis ofmicropropagated plantlets in date palmrdquo Pakistan Journal ofBiological Sciences vol 9 no 1 pp 111ndash114 2006

[67] Z Bakhtiar M Mirjalili A Sonboli M Farimani and MAyyari ldquoIn vitro propagation genetic and phytochemicalassessment of Thymus persicus mdash a medicinally importantsource of pentacyclic triterpenoidsrdquo Biologia vol 69 no 5 pp594ndash603 2014

[68] M D Gaj ldquoSomatic Embryogenesis and Plant Regenerationin the Culture of Arabidopsis thaliana (L) Heynh ImmatureZygotic Embryosrdquo in Plant Embryo Culture vol 710 ofMethodsin Molecular Biology pp 257ndash265 Humana Press Totowa NJ2011

[69] R Li A H Bruneau and R Qu ldquoTissue culture-induced mor-phological somaclonal variation in St Augustinegrass [Steno-taphrum secundatum (Walt) Kuntze]rdquo Plant Breeding vol 129no 1 pp 96ndash99 2010

[70] S M Kaeppler and R L Phillips ldquoTissue culture-induced DNAmethylation variation in maizerdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 90 no19 pp 8773ndash8776 1993

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 Location coordinates and weather parameter of the studied cumin landraces

Location name Location coordinates Elevation (MSL) Mean annual rainfallNorth Khorasan 37∘0910158404210158401015840N 57∘031015840203010158401015840E 13566 130mmGolestan 37∘14101584017910158401015840N 55∘09101584034810158401015840E 375 363mm

It seems that auxin hormoneswere stimulators of intracellularbiochemical reactions and specific genes in preembryo cellswhich are required for producing globular embryos Theresearchers observed that elimination of auxin frommediumcan lead to the continuation of somatic embryogenesis[8 12 13] Measurement of chemical compositions suchas contents of proline protein and antioxidant enzymesincluding catalase ascorbate peroxidase and peroxidase hasbeen used as a marker to evaluate somatic embryogenesis indifferent studies [10 14 15] Studies suggest that antioxidantenzymes such as catalase and ascorbate peroxidase play animportant role in maturity and differentiation of plant tissuesand their high activity affects cellular processes such assomatic embryogenesis [10 16 17] Terpenic hydrocarbonsalcohols and phenols existing in the essential oil of plantsare among the genetic properties affecting somatic embryo-genesis Researchers stated that terpenic hydrocarbons havepositive effects on somatic embryogenesis process whilealcohols and phenols have negative effects on the process[18ndash20] Ebrahimie et al [3] used the cut seed embryo ofcumin as an explant in B5 medium and introduced 04mgIAA + 02mg NAA + 01mg BAP as the best hormonecombination Kahrizi and Soorni [21] used 24-D and Kinin cumin developed embryogenic calluses and reported thatan increase in the concentration of 24-D hormones has apositive effect on somatic embryogenesis but their studywas only confined to callus formation and they did notreport on regeneration and adaptation Although few studieswere conducted on somatic embryogenesis and regenerationin cumin already no report has been registered on therelationships between genetic properties like chemical com-positions existing in the plant and somatic embryogenesisprocess and evaluation of somaclonal variation of regeneratedplants Therefore the current study is aimed at measuringchemical compositions optimizing somatic embryogenesisand regeneration assessment of somaclonal variation inregenerated plants as compared to the mother plant andthen evaluating relationships of these properties in the twosuperior Iranian landraces of cumin

2 Materials and Methods

21 Plant Material Two Iranian superior cumin landracesseeds Golestan and North Khorasan were selected based onthe previous study [22] (Table 1 and Figure 1) In the presentstudy experiments were divided into two sets includingmeasurement of chemical characters and optimization ofsomatic embryogenesis regeneration rooting and evalua-tion of somaclonal variation of the landraces of cumin

22 Measurement of Chemical Characters In the first exper-iment the seeds of two landraces of cumin were planted in

N

16500000

0 30 60 90 120

150

(km)

Figure 1 Location of two superior Iranian cumin landraces on themap (modified from httpwwwenchantedlearningcom)

the experiment site of College of Aburaihan University ofTehran during two successive years (2014 and 2015) Eachlandrace was planted in 2m2 plots in a sandy-clay soil Thesoil was well drained with pH of 72 Recommended cropmanagement practices were implemented to raise the cropsto desirable growth stages Plots were kept weed- pest-and disease-free until harvesting time Chemical charac-ters including the content of catalase ascorbate peroxidaseprotein proline essential oil and its composition of twolandraces of cumin were measured It is worth noting thatcatalase and ascorbate peroxidase play an important role inthe maturity of plant tissues and their high activities affectsomatic embryogenesis [10 16 17] Each essential oil is madeup of numerous different organic molecules and some ofthese including terpenic hydrocarbons alcohols and phenolsexisting in the essential oil are among the genetic propertiesaffecting somatic embryogenesis [18ndash20] In order tomeasurechemical characteristics of two landraces of cumin leaftissue samples were immediately frozen in liquid nitrogenProtein and proline contents of the leaves were measuredaccording to Bradford and Bates et al [23 24] respectivelyThe activities of antioxidant enzymes including catalase andascorbate peroxidase were estimated as described by Beersand Sizer [25] and Nakano and Asada [26] The essentialoil of the ripened seeds was extracted by the Clevengerapparatus using the hydrodistillation method The driedpowdered seeds of cumin (40 g) were placed in a distillationapparatus with 400mL of distilled water and hydrodistilledfor three hours Then the essential oils were stored in glassvials at 5∘C until the essential oil compositionsrsquo analysisThe analysis of essential oil composition was performed byGC-MS analysis (a combined analytical method to identify







Kin(mgL)








A

A

B B

0

1

2

3

4

5

6

Ascorbateperoxidase

Catalase

(Um

g)Co

nten

t

Studied characters

(a)


A

A

B

A

0

05

1

15

2

25

3

35

4

Proline Protein

(mg

g FW

)


nten

t

(b)


A

A

095

1

105

11

115

12

125

13

Essential oil

()

Cont

ent

Studied characters

(c)




















b1090 248 plusmn 010a
















Alcoholsphenols












Aldehydes









(a) (b)

(c) (d)







(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)



































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Kin(mgL)








A

A

B B

0

1

2

3

4

5

6

Ascorbateperoxidase

Catalase

(Um

g)Co

nten

t

Studied characters

(a)


A

A

B

A

0

05

1

15

2

25

3

35

4

Proline Protein

(mg

g FW

)


nten

t

(b)


A

A

095

1

105

11

115

12

125

13

Essential oil

()

Cont

ent

Studied characters

(c)




















b1090 248 plusmn 010a
















Alcoholsphenols












Aldehydes









(a) (b)

(c) (d)







(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)



































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



A

A

B B

0

1

2

3

4

5

6

Ascorbateperoxidase

Catalase

(Um

g)Co

nten

t

Studied characters

(a)


A

A

B

A

0

05

1

15

2

25

3

35

4

Proline Protein

(mg

g FW

)


nten

t

(b)


A

A

095

1

105

11

115

12

125

13

Essential oil

()

Cont

ent

Studied characters

(c)




















b1090 248 plusmn 010a
















Alcoholsphenols












Aldehydes









(a) (b)

(c) (d)







(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)



































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












b1090 248 plusmn 010a
















Alcoholsphenols












Aldehydes









(a) (b)

(c) (d)







(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)



































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)







(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)



































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

Heart-shaped

Torpedo-shaped

(d)

Heart-shaped

Torpedo-shaped

Cotyledonary

Globular

(e) (f)

(g) (h) (i)



































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































B B

AB

B

A

0102030405060708090

100

Studied treatments

o

f ind

uced

calli

with

embr

yoge

nic p

oten

tial

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

lowastN

orth

Kho

rasa

n

lowastG

oles

tan

Alowast

coty

ledo

n

Clowast

cote

ldon

Dlowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n


B B

A

Studied treatments

0102030405060708090

100

Rege

nera

tion

()

lowastG

oles

tan

lowastG

oles

tan

lowastG

oles

tan

Alowast

coty

ledo

n

Dlowast

coty

ledo

n

Glowast

coty

ledo

n



Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Main rooting

Lateral rooting

(a) (b) (c)

(d) (e)

F1

F2

(f)






















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















M S 1 2 3 4 5 6 7 8 NC 9 10 11

1000 bp

500 bp

(a)

M 1 2 NC 3 4 5 6 7 8 9 10 11 S

1000 bp

500 bp

(b)



4 Conclusion




Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Abbreviations




Acknowledgments


References



















































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

chemical compositions, somatic embryogenesis, and...

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