indirect regeneration and assessment of genetic...

Research ArticleIndirect Regeneration and Assessment of Genetic Fidelity ofAcclimated Plantlets by SCoT ISSR and RAPD Markers inRauwolfia tetraphylla L An Endangered Medicinal Plant

Gulab Khan Rohela 12 Phanikanth Jogam1 Prasad Bylla1 and Christopher Reuben3

1Department of Biotechnology Kakatiya University Warangal 506 009 Telangana India2Biotechnology Section Moriculture Division Central Sericultural Research amp Training Institute Central Silk BoardMinistry of Textiles Government of India Pampore 192 121 Jammu and Kashmir India3Department of Botany Kakatiya University Warangal 506 009 Telangana India

Correspondence should be addressed to Gulab Khan Rohela gulab biotechyahoocoin

Received 14 January 2019 Accepted 25 March 2019 Published 11 April 2019

Academic Editor Atanas Atanassov

Copyright copy 2019 Gulab Khan Rohela 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

Rauwolfia tetraphylla L is an important medicinal plant species which is well known for its pharmaceutically important alkaloidsIn the present study we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol ofindirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codontargeted (SCoT) inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) marker based analysisInitially friable callus was induced in maximum amounts (3787 3238 and 4128 in mg) from leaf root and stem explants onMurashige and Skoog (MS) media supplemented with 50mgL 30mgL of 24-dichlorophenoxyacetic acid (24-D) and 50mgLof naphthalene acetic acid (NAA) respectively Shoot regeneration with the maximum number of shoot buds (25 and 20) wasobtained from leaf and stem calluses on MS media supplemented with TDZ (025mgL) + BAP (2mgL) The regenerated shootswere rooted successfully with maximum rooting percentage of 980 on full strength MS media amended with IAA (10mgL) andIBA (10mgL) The regenerated plantlets were hardened using 21 ratio of sterile garden soil and sand followed by acclimatizationin field conditions with 86 of survival SCoT ISSR and RAPD primers based polymerase chain reaction (PCR) analysis wascarried out to check possible genetic variations in micro propagated plants in comparison with mother plant Among the ten SCoT(S) ISSR (R) and RAPD (OPA) primers used S2 R10 and OPA3 has given good amplification with scorable DNA bands Theresults revealed that the regenerated plants did not have any polymorphism with mother plant Hence the in vitro regenerated Rtetraphylla plantlets were confirmed as true-to-type

1 Introduction

In India Ayurveda is a traditional medicinal system whichhas been practiced for thousands of years and remains asone of the most ancient and yet living traditions across thecountry which has a sound philosophical and experimentalbasis [1 2] India is also the largest producer of medicinalherbs and hence appropriately called as the Botanical Gardenof the World [3] Some of these medicinal plants have beenfeatured on Indian postage stamps where the first set ofstamps came out in 1997 showing four different medicinal

plants Ghritkumari (Aloe barbadensis) Sarpagandha (Rau-wolfia serpentina) Tulsi (Ocimum sanctum) and Haridra(Curcuma longa) [4] followed by another set of fourmedicinalplants Brahmi (Bacopa monnieri) Guggulu (Commiphorawightii) Amla (Emblica officinallis) and Aswagandha (With-ania somnifera) as postal stamps were released [5]

Presently medicinal plants play a very important role inthe modern economy of developing countries In India themedicinal plant related trade is estimated to be approximatelyUS $ 1 billion per year [6] Recent trends have indicatedfurther increase in this trade with the herbal cosmetic and

HindawiBioMed Research InternationalVolume 2019 Article ID 3698742 14 pageshttpsdoiorg10115520193698742

2 BioMed Research International

pharmaceutical industry playing a major role in fulfilling thedemand for herbals worldwide In addition to the interna-tional trade there is a substantial volume of internal trade formedicinal plants in India which has created a lot of demandto these medicinal herbs and shrubs [7]

Among the different plant families Apocynaceae hasgained lot of medicinal importance as most of the plantspecies of this family produce milky sap which has phar-maceutical applications such as for gastrointestinal ailmentsmalaria pain and diabetes [8] Almost every part (rootsstem bark leaves and latex) of Apocynaceae family memberswas used for several treatments and disorders like leprosydysentery worms ulcers tumours skin and liver diseases[9]

Rauwolfia is an important genus in Apocynaceae familywhich has gained much popularity due to the presence ofpharmaceutically significant alkaloids with higher concen-trations in roots and stem bark Approximately 130 speciesof Rauwolfia have been identified and are located mostlyin tropical and semitropical regions Among the differentspecies R serpentina Benth ex Kurz is indigenous to India[10 11] and this species is on the verge of extinction [12]due to its overexploitation by ayurvedic practitioners andpharmaceutical industries [13] and Government of India hasalso restricted the exploitation of this endangered species[14] In this scenario the attention of industrialists andmedicinal practitioners was diverted to find an alternativeplant species as a source of pharmaceutical alkaloids

Among the other species of Rauwolfia the most impor-tant is Rauwolfia tetraphylla L which has gained lot ofimportance as an alternative source of reserpine and otheralkaloids [15] R tetraphylla is native of tropical America andWest Indies [16] but now it is naturalized [17] throughout thetropics which includes the moist deciduous forests of South-east Asia including Burma Bangladesh India Malaysiaand Srilanka [18] The common names are wild snake rootplant devil pepper be-still tree American serpent wooddevil root milk bush etc It is being cultivated widely asboth an ornamental and a source of pharmaceuticals Thisspecies produces different type of pharmaceutically impor-tant alkaloids in their stem bark and roots such as reserpinedeserpidine ajmalicine ajmaline canescine yohimbine andrecinnamine [19 20]

Due to presence of various commercial indole alkaloidsthis plant species was also exploited during the last threedecades and presently it is in endangered status [21] Prop-agation of R tetraphylla through vegetative cuttings and seedmaterial is limited (5-10) due to its poor rooting abilityand due to presence of phenolic compounds in seed [22]In view of the above facts about the mass exploitation ofthis plant from natural resources and due to its restrictedcultivation there is an urgent need to conserve this medici-nally important plant species by proper conservation strategyHence the present study is undertaken with the objective ofmass propagation of R tetraphylla through the standardizedindirect regeneration protocol by utilizing leaf and stembased callus and to confirm the genetic fidelity of acclimatedplants by using SCoT ISSR and RAPD primer based PCRanalysis

Even though there are some reports about its mul-tiplication through nodal shoot tip cultures [23] and invitro flowering [24] but there is scanty information aboutits regeneration through callus based indirect regenerationprotocols In our previous report we have used single typeof cytokinin (TDZ) in regenerating the low frequency ofmultiple shoots from leaf and stem based callus [25] Nowin this study we are reporting a protocol for its conservationthrough high frequency of plantlet production by usingcombination of cytokinins amended media

2 Materials and Methods

In this study most of materials and methods were followedas per the report on indirect regeneration studies in aMorusspecies [26]

21 Plant Material and Explant Source Two-year-old Rtetraphylla plants available in the medicinal arboretumDepartment of Biotechnology KakatiyaUniversity were usedas parent plant material in this in vitro propagation studiesFully expanded leaf of about 7 cm in length and 4 cm inbreadth stem (internodal segments) and root parts werecollected during the second week of June and were used asexplants for the study

22 Murashige and Skoog Media Preparation MS medium[27] was prepared by using sucrose (3) and appropriatequantities of vitamins Iron macronutrients and micronu-trients from stock solutions Before dispensing the preparedmedium to culture tubes required concentrations and com-binations of plant growth regulators were added to it from thehormonal stock solutionsThemediawas solidifiedwith 08Agar and pHwas adjusted to 56 with 1 or 2 drops of 01 NHClor NaOH solution before autoclaving at 121∘C for 15 minutes

23 Aseptic Techniques for Sterile Transfer All operationsincluding transfer and sterile filtration were made underlaminar airflow cabinet Prior to operation the laminarairflow bench was swabbed with 70 alcohol to maintainmaximum sterility The laminar airflow was switched on andkept on UV mode for 30 minutes before using it The scalpelforceps and spatula were sterilized by flaming with alcoholbefore using the laminar airflow cabinet

24 Explant Surface Sterilization Leaf stem (internodal)and root explants of R tetraphylla were collected from themedicinal arboretum and initially washed under running tapwater for 2 minutes Then different protocols were followedfor the sterilization of leaf stem and root explants as leafexplants are more delicate and sensitive in nature to sterilantsand due to greater degree of fungal count on stem and rootexplants Leaf explants were treated with Tween-20 solutionfor 1 minute followed by 60 ethanol for 2 minutes andin 01 mercuric chloride (HgCl

2) solution for 2 minutes

Stem and root explants were treated initially with 05bavistin for 10 minutes followed by Tween-20 solution for2 minutes and with 01 mercuric chloride solution for 4

BioMed Research International 3

minutes After every treatment explants were rinsed in steriledistilled water for 2minutes to remove the adhering chemicalsterilants

25 Inoculation and Incubation Surface sterilized leaf stemand root explants were blotted dry on sterile tissue paperand inoculated onto MSmedium under aseptic conditions oflaminar air flow cabinet with the help of sterile forceps andculture containers were immediately closed During inocula-tion the test tubes and flasks containing solid medium wereheld horizontally this strongly reduces the contaminationsCulture tubes with inoculated explants were kept in cultureroom under controlled conditions of 168 hours of photope-riod with 3000 lux intensity light at 27∘C For each treatment25 replicates were used and each experiment was repeatedthrice

26 Callus Induction For callus induction fully expandedleaf stem (inter nodal) and root explants of R tetraphylla(two years old) were inoculated on to MS medium sup-plemented with 24-D or NAA or Kn (10-50mgL) Dataon callus induction from three replicates were recorded atthe end of 3 weeks of culture Each replicate consists of 25cultures

27 Determination of Fresh and Dry Weight of Callus Fordetermination of fresh and dry weight of callus 10 identicalculture tubes were used The mean value of each callus wasdetermined After determining the callus weight they weredried in an oven at 60∘C for 1 hour and average dry weight ofthe callus was determined The average fresh and dry weightof the callus was subtracted from the respective callus cultureto find out the average gain in fresh and dry weight of thecallus Fresh and dry weights of the callus were noted with agap of 22 days

28 Shoot Regeneration In vitro shoot regeneration wascarried from the friable callus by using individual andcombinations of cytokinins (BAP and TDZ) supplementedMS media While preparing combinational cytokinin mediaBAP of 2mgL was kept constant across all the combinationswith altering concentrations of TDZ in low amounts (01-10mgL) Data on number shoot bud regeneration fromfriable callus in three replicates was recorded at the end of2nd week of culture Each replicate of regeneration studiesconsists of 10 cultures

29 Rooting of the Regenerated Shoots The regeneratedshoots were separated from the culture tubes and trans-ferred onto MS medium containing various concentrations(05ndash20mgL) of individual (IAAor IBA) aswell as combina-tion (IAA+IBA) of auxins Data on root induction from shootlets of three replicates was recorded at the end of 2nd week ofculture Each replicate consists of 10 cultures

210 Hardening and Acclimatization of Rooted PlantletsRooted plantlets of R tetraphylla were separated carefullyfrom the culture tubes along with the media and the adhered

medium was removed gently by washing under running tapwater and then hardened in a plastic pot or plastic cup byusing 21 ratio of sterile garden soil and sand

211 DNA Isolation and Genetic Fidelity Analysis GenomicDNA was isolated from acclimated plantlets of R tetraphylla(seven plants were selected randomly four from leaf callusbased and three from stem callus based regenerants) fromfield along with the mother plant Fresh leaf tissue (500mg)of both mother plant and in vitro regenerated plants was usedas rawmaterial for DNA isolation by following modified cetyltrimethyl ammonium bromide (CTAB) method [28 29]Thequality parameters of DNA were checked by spectrometricand electrophoretic methods

For testing the genetic homogeneity of regeneratedplantlets with that of mother plant three types of primers(RAPD ISSR and SCoT) were used A total of 30 primerswere used ie SCoT (10 primers) ISSR (10 primers)and RAPD (10 primers) for genetic fidelity studies PCRamplifications were performed using a total volume of 50 120583lcontaining 100 ng DNA 1X PCR master mix (GCC biotech)and 10 p mole primer The amplification reaction was carriedout in a thermal cycler (Eppendorf Germany) for 30 cycleswith an initial denaturation at 94∘C for 5 minutes followedby denaturation at 94∘C for 30 seconds and annealing wascarried at 50∘C for 45 seconds for SCoT and ISSR and at 37∘Cfor 45 seconds for RAPD primer based analysis followedby extension at 72∘C for 2 minutes and final extension at72∘C for 5 minutes After completion of cycles amplifiedproducts were allowed to cool down at a holding temperatureof 4∘C PCR products were then separated on 1 agarose gelelectrophoresis by using TAE (Tris acetic acid EDTA) bufferThe size of amplicons was estimated using 1kb DNA ladder(Thermo Scientific) All amplification reactions with RAPDISSR and SCoT primers were repeated at least three times tocheck the reproducibility The gels were photographed usinggel documentation system and only clear and scorable DNAbands were considered for analysis

212 Statistical Analysis The data obtained in this researchstudy was statistically analyzed by using one-way ANOVA inSPSS Version 17 (SPSS Inc Chicago USA) and means werecompared using Tukeyrsquos tests at the 5 level of significanceAll means are presented with plusmn Standard Error (SE)

3 Results and Discussion

31 Callus Induction In this study leaf stem and rootexplants of R tetraphylla cultured on MS basal medium didnot produce any callus but when cultured on MS mediumsupplemented with various concentrations of plant growthregulators (24-D NAA and Kn) copious amounts of friablecallus were produced after 4 weeks of culture (Figures1(a)ndash1(d)) The produced callus is of different types such asfriable white friable pale brown and compact brown callusAt lower concentrations (1-2mgL) of 24-D NAA and Knall the explants have produced friable white callus (Figures1(a) and 1(b)) whereas friable green callus (Figure 1(c)) to


(a) (b)

(c) (d)

Figure 1 Friable callus induction from various explants of R tetraphylla (a) Friable white callus induced from leaf explants cultured on MS+ NAA (2mgL) (b) Friable white callus induced from stem explants cultured on MS +Kn (1mgL) (c) Friable green callus induced fromleaf explant cultured on MS+ Kn (3mgL) (d) Friable brown callus induced from root explant cultured on MS + 24-D (5mgL)

friable brown callus (Figure 1(d)) was produced from theoptimum (3mgL) and higher concentrations (4-5mgL)of plant growth regulators (24-D NAA and Kn) respec-tively

On 24-D supplemented MS media maximum amountof mean fresh and dry weight (mg) of callus (3787plusmn067192plusmn015) (2415plusmn034 103plusmn008) and (3238plusmn036155plusmn012) was observed in leaf stem and root explantscultured on 5 4 and 3mgL of concentrations respectivelyafter 3 weeks of culture (Table 1) On NAA supplementedmedia maximum amount of mean fresh and dry weight(mg) of callus (3135plusmn023 195plusmn027) (4128plusmn049 251plusmn015)and (2774plusmn014 161plusmn003) was observed in leaf stem androot explants cultured on 2 6 and 2mgL concentrationsrespectively after 3 weeks of culture (Table 2) On Knsupplemented media maximum amount of mean freshand dry weight (mg) of callus (3495plusmn046 228plusmn019) and(3432plusmn064 144plusmn026) was observed in leaf and stemexplants cultured on 5mgL concentration of Kn after 3weeks of culture (Table 3) Root explants have not respondedon Kn supplemented media even after 4 weeks of culture

Though the rate of callus growth was negligible in thefirst two weeks the entire explant was covered with healthycallus after 3 weeks Major portion of callus that developedfrom leaf stem and root was friable creamy white in colorafter 3 weeks of culture but became brown when culturedfor more than six weeks on the same medium due to therelease of phenolic compounds which is commonly observedin the callus cultures of shrub and tree species [30] As theprimarily induced callus of leaf and stem explants were friableand creamy white in color in order to make them friablegreen and organogenic the primary callus of leaf and stemexplants of R tetraphylla were subcultured for 3 passages oftime (2 months) on the same combination of plant growthregulators

Anitha and Kumari (2006a) [31] were first to report callusinduction from leaf explants of R tetraphylla on MS + 24-D (9 120583ML) and the fresh and dry weights of callus werereported as 03354 and 00826 grams respectively Callusinduction was also reported from petiole cotyledonary leafand hypocotyl explants of R tetraphylla cultured on MSmedium supplemented with 452120583M of 24-D [32] In our


Table 1 Effect of 2 4-dichlorophenoxy acetic acid (2 4-D) on callus induction from leaf stem (internode) and root explants of Rauwolfiatetraphylla L

Explant MS + 24-D (mgL) Morphogenetic Response CallusMean fresh weight (mg) Mean dry weight (mg)

Leaf

10 Friable white callus 2344 plusmn 012i 082 plusmn 044k

20 Friable white callus 2559 plusmn 023g 096 plusmn 024j

30 Friable white callus 2878 plusmn 091f 114 plusmn 049h

40 Friable white callus 3306 plusmn 076b 167 plusmn 068b

50 Friable white callus 3787 plusmn 067a 192 plusmn 015a

Stem (Inter node)

10 Friable white callus 926 plusmn 019l 103 plusmn 011n

20 Friable white callus 1235 plusmn 028k 230 plusmn 067m

30 Friable white callus 1856 plusmn 036j 066 plusmn 023l

40 Friable white callus 2415 plusmn 034h 123 plusmn 008f

50 Compact brown callus 2322 plusmn 024i 094 plusmn 024j

Root

10 Friable white callus 1236 plusmn 034k 074 plusmn 055k

20 Friable white callus 2567 plusmn 012i 117 plusmn 023g

30 Friable brown callus 3238 plusmn 036c 155 plusmn 012c

40 dark brown callus 2943 plusmn 045d 146 plusmn 067d

50 dark brown callus 2883 plusmn 044e 138 plusmn 011e

Data represents average of three replicates Each replicate consists of 25 cultures Data scored at the end of 3 weeks of culture Mean plusmn standard error Meanfollowed by the same superscript in a column is not significantly different at P=005

Table 2 Effect of naphthalene acetic acid (NAA) on callus induction from leaf stem (internode) and root explants of Rauwolfia tetraphyllaL

Explant MS + NAA (mgL) Morphogenetic Response CallusMean fresh weight (mg) Mean dry weight (mg)

Leaf

10 Friable white callus 3117 plusmn 031c 191 plusmn 023c

20 Friable white callus 3135 plusmn 023d 195plusmn 067b

30 Friable pale brown callus with roots 3036 plusmn 055e 190 plusmn 054d

40 Friable pale brown callus with roots 2915 plusmn 018f 172plusmn 033e

50 Friable pale brown callus with roots 2424 plusmn 023j 156 plusmn 080g

Stem (Inter node)

10 Friable white callus 1533 plusmn 014l 22plusmn 043m

20 Friable white callus 2554 plusmn 040i 49 plusmn 045l

30 Friable white callus 3036 plusmn 047e 101 plusmn 013h

40 Pale brown callus 3431 plusmn 021b 158 plusmn 036f

50 Pale brown callus 4128 plusmn 049a 251 plusmn 015a

Root

10 White friable callus 2565 plusmn 038i 95 plusmn 019k

20 White friable callus 2774 plusmn 014g 161 plusmn 003f

30 Pale brown callus 2621 plusmn 021h 146 plusmn 043h

40 Brown callus 2584 plusmn 074i 139 plusmn 067i

50 Dark brown callus 2438 plusmn 036j 132 plusmn 031i


study we have induced much higher value of mean fresh anddry weight (mg) of friable callus (3787plusmn067 192plusmn015) at5mgL concentrations of 24-D from leaf explants They haveinduced and used the callus cultures for reserpine estimationstudies [31] (Anitha and Kumari 2006a) and studied the effectof NaCl amended media on callus growth [33]

From the above reports it was clear that callus inducedfrom various explants of R tetraphylla was used for dif-ferent studies by previous researchers but not for in vitroregeneration studies whereas in our study we have utilized

the induced callus for clonal multiplication of R tetraphyllathrough in vitro regeneration studies

32 Shoot Regeneration In our earlier studies we have usedsingle cytokinin (TDZ) for regeneration of shoot buds fromleaf and stem based callus and obtained maximum frequencyof shoot regeneration (120plusmn028) from stem based callusat 227 120583Ml concentration of TDZ [25] Now through thisresearch paper we are reporting a standardized protocol


Table 3 Effect of Kinetin (Kn) on callus induction from leaf stem (internode) and root explants of Rauwolfia tetraphylla L

Explant MS + Kn (mgL) Morphogenetic Response CallusMean fresh weight (mg) Mean dry weight (mg)

Leaf

10 No response -- --20 Friable white callus 2014 plusmn 011h 111 plusmn 014f

30 Friable green callus 2263 plusmn 041f 135 plusmn 022d

40 Pale brown callus 3346 plusmn 102c 185plusmn 068b


Stem (Inter node)

10 Friable white callus 2186 plusmn 025g 36 plusmn 019i

20 Friable White callus 2333 plusmn 023f 42 plusmn 017h

30 Friable White callus 2649 plusmn 091e 78 plusmn 023g

40 Pale brown callus 3094 plusmn 087d 115 plusmn 061e

50 Pale brown callus 3432 plusmn 064b 144 plusmn 026c

Root

10 Single shoot bud - -20 Two shoot buds - -30 Multiple shoot buds - -40 Multiple shoot buds - -50 Multiple shoot buds - -


02468

101214

010 025 050 075 100Concentration of TDZ (mgL)

Leaf CallusStem Callus

c c cd

e

c b b a a

Mea

n N

o o

f Sho

ot B

uds

Figure 2 Effect of TDZ (Thidiazuron) on mean number of shootbuds induced in leaf and stem calluses of R tetraphylla Barsrepresent meanplusmn SE and bars denoted by the same letter are notsignificantly different by Tukeyrsquos test used at P = 5 probability

with much higher frequency of shoot regeneration from thefriable callus of leaf and stem explants on MS media supple-mented with individual (Figures 2 and 3) and combination ofcytokinins (TDZ and BAP) (Figure 4)

In this study three times subcultured callus of leaf andstem explants of R tetraphylla has shown good response ofshoot regeneration with maximum number of shoot buds oncombinational media (TDZ+BAP) rather than individuallysupplemented media (TDZ or BAP) OnMSmedia amendedwith individual TDZ plant growth regulators regenerationof shoot buds with a frequency of 32plusmn01 and 122plusmn028 wasobtained at 025mgl and 050mgl concentration from leafand stem based callus respectively (Figure 5(a)) On BAPamendedmedia regeneration of shoot buds with a frequency

0

2

4

6

8

10

12

1 2 3 4 5


Concentration of BAP (mgL)

c

a

d

b

f

d

hg

ef

Mea

n N

o o

f Sho

ot B

uds

Figure 3 Effect of BAP (6-benzyl aminopurine) on number of shootbuds induced in leaf callus and stem callus of R tetraphylla Barsrepresent meanplusmn SE and bars denoted by the same letter are notsignificantly different by Tukeyrsquos test used at P = 5 probability

of 100plusmn042 and 90plusmn034 was obtained at 40mgl concen-tration from leaf and stem based callus respectively Overallthe maximum number of shoot buds with high frequency ofregeneration (25plusmn025 and 20plusmn076) was obtained from leafand stem calluses respectively when cultured on MS mediasupplemented with TDZ (025mgL) + BAP (2mgL) after 4weeks of culture (Table 4 and Figure 6(a))

Thidiazuron (TDZ) (N-phenyl-N1015840-123-thiadiazol-5-ylurea) is a thiadiazole-substituted phenylurea which wasreported to possess more cytokinin activity as compared tophenyl urea derivatives and other active adenine types ofcytokinins [34] Thidiazuron was also reported to possessactions of other plant growth regulators such as auxinsgibberellins and ethylene [35] Thidiazuron (TDZ) was used


Table 4 Effect of TDZ and BAP on shoot bud differentiation from 3 times subcultured leaf stem and root callusof Rauwolfia tetraphylla L

Three timessub-cultured callus

TDZ(mgL)

BAP(mgL) No of shoot buds

Leaf

010 200 4 plusmn 012f

025 200 25 plusmn 025a

050 200 10 plusmn 017d

075 200 8 plusmn 011e

100 200 2 plusmn 015g

Stem

010 200 4 plusmn 043f

025 200 20 plusmn 076b

050 200 14 plusmn 013c

075 200 4 plusmn 028f

100 200 2 plusmn 026g


0

5

10

15

20

25

30

Mea

n N

o o

f Sho

ot B

uds

01+20 025+20 05+20 075+20 10+20Concentration of TDZ + BAP (mgL)


g

f

de

c

ba a

bb

de

cc

bba a

b

Figure 4 Effect of combination of TDZ and BAP on number ofshoot buds induced from leaf callus and stem callus of R tetraphyllaBars representmeanplusmn SE and bars denoted by the same letter are notsignificantly different by Tukeyrsquos test used at P = 5 probability

by several researchers in regeneration studies of medicinalplants with higher frequency of shoot let production fromfriable callus [36 37] In our earlier studies we have inducedlow frequency of multiple shoots from stem callus of Rtetraphylla on TDZ amended media [25]

It was reported that thidiazuron in combination withother plant growth regulators induced callus [38] somaticembryos [39] and shoot regeneration from friable callus [40]Hence in this study we have used TDZ in combination withBAP for the indirection regeneration of R tetraphylla Inour study combination of TDZ (025mgL) + BAP (2mgL)has resulted in inducing maximum number of shoot buds(25plusmn025 and 20plusmn076) from leaf and stem calluses respec-tively which is much higher compared to our earlier reportsTDZ+BAP combinations have been reported for successfulmultiple and rapid shoot regeneration of various specieseg Ancistrocladus heyneanus [41] Luffa cylindrical L [42]Rauwolfia tetraphylla L [25] Oryza sativa [43] Mirabilisjalapa L [44] Achyranthes aspera [40] and Morus Sps cvPPR-1 [26]

Table 5 Effect of auxins (IAA and IBA) on root induction from invitro shoot lets of Rauwolfia tetraphylla L

IAA (mgL) IBA (mgL) Rooting () No of rootsshoot05 - 724 plusmn 56e 354 plusmn 062c

10 - 521 plusmn 64c 312 plusmn 024c

15 - 483 plusmn 38b 218 plusmn 028b

20 - 327 plusmn 22a 208 plusmn 013b

- 05 822 plusmn 57f 516 plusmn 044e

- 10 906 plusmn 62g 740 plusmn 072g

- 15 603 plusmn 34d 322 plusmn 084c

- 20 448 plusmn 28b 486 plusmn 032d

05 05 846 plusmn 46f 604 plusmn 054f

05 10 924 plusmn 58g 630 plusmn 028f

10 05 698 plusmn 42d 144 plusmn 012a

10 10 980 plusmn 12h 832 plusmn 084h

Data represents average of three replicates Each replicate consists of 10cultures Data scored at the end of 2 weeks of culture Mean plusmn standarderror Mean followed by the same superscript in a column is not significantlydifferent at P=005

As the regenerated shoots areminute in the form ofmicroshoots they were transferred onto MS basal media (Figures5(b) and 6(b)) and cultured for 2 weeks for elongation ofmicro shoots into shoot lets (Figures 5(c) 5(d) 5(e) and6(c))

33 Rooting of In Vitro Regenerated Shoots The regeneratedshoots of R tetraphylla L were rooted successfully withmaximum rooting frequency of 980plusmn12 on combinationalmedia of auxinswith IAA (10 mgL) and IBA (10mgL) after2 weeks of culture and well developed plantlet with extensiverooting was observed after 4 weeks of culture (Figures5(f) 6(d) and 6(e)) On individual auxins supplementedmedia rooting was induced with a frequency of 724plusmn56and 906plusmn62 from IAA (05mgL) and IBA (10mgL)respectively (Table 5 and Figure 7) Rapid growth of plant lets


(a) (b)

(c) (d)

(e) (f) (g)

Figure 5 In vitro plant regeneration from leaf callus (3 times subcultured) inR tetraphylla (a) Shoot bud induction (representedwith arrows)in leaf callus cultured on MS + TDZ (05mgL) (b) Transfer of individual micro shoots on MS basal media (c) Single shoot elongation onMS basal medium (d) Increase in shoot length up to 1 cm onMS basal medium after 1 week of culture (e) Increase in shoot length up to 2 cmonMS basal medium after 2 weeks of culture (f) Root induction in single shoot cultured onMS + IAA (1mgL) + IBA (1mgL) (g) Completeplantlet transferred to plastic pot containing sterile soil for hardening


(a) (b)

(c) (d) (e)

(f) (g) (h)

Figure 6 In vitro plant regeneration from stem callus (3 times sub cultured) in R tetraphylla (a) Shoot bud induction from stem calluscultured on MS + TDZ (025mgL) + BAP (2mgL) (b) Multiple shoot buds transferred onto MS basal media (c) Elongation of multipleshoot buds into small shoot lets on MS basal media (d) Root induction in single shoot cultured on MS + IAA (1mgL) + IBA (1mgL) (e)Root induction in single shoot cultured on MS + IAA (1mgL) + IBA (1mgL) (f) Complete plantlet transferred to plastic cup containingsterile soil for hardening (g) Field transfer (2 months old) (h) Acclimatized plant (24 months old)

was observed once the primary roots were initiated from theends of shoot lets

34 Plant Acclimatization to Ex Vitro Conditions In vitroraised plantlets ofR tetraphyllawere separated carefully fromthe media and hardened in a plastic pot (Figures 5(g) and6(f)) using 21 ratio of sterile garden soil and sand Initially

the hardened plantlets were kept in hardened pots and cupsfor 4-6 weeks under controlled conditions by irrigating withbroth solution of half strength MS salts and then transferredand acclimatized to field conditions (Figures 6(g) and 6(h))with 86 survival With garden soil and sand similar typesof results were obtained in acclimatizing the in vitro raisedMorus Sps [45 46]


0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10

Concentration of Auxins (mgL)

ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd

Figure 7 Effect of Auxins (IAA amp IBA) on root induction from in vitro shoot lets of Rauwolfia tetraphylla L

Table 6 List of primers their sequence number and size of DNA bands generated by SCoT

Primer code Primer Sequence Number of Bands Band Length (in bp)1 S1 CAACAATGGCTACCACCA 2 900-12002 S2 CAACAATGGCTACCACCC 2 1000-15003 S3 CAACAATGGCTACCACCG 3 1000-14004 S4 CAACAATGGCTACCACCT 4 500-20005 S5 CAACAATGGCTACCACGA 4 500-20006 S6 CAACAATGGCTACCACGC 4 500-20007 S7 CAACAATGGCTACCACGG 3 500-20008 S8 ACGACATGGCGACCAACG 3 400-21009 S9 ACCATGGCTACCACCGAC 2 500-120010 S10 CCATGGCTACCACCGCAG 3 400-1200

Table 7 List of primers their sequence number and size of DNA bands generated by ISSR

Primer code Primer Sequence Number of Bands Band Length (in bp)1 R1 AGAGAGAGAGAGAGAGG 5 500-20002 R2 AGAGAGAGAGAGAGAGYT 5 750-25003 R3 AGAGAGAGAGAGAGAGTC 4 700-17004 R4 CTCTCTCTCTCTCTCTRA 4 500-20005 R5 GAGAGAGAGAGAGAGAT 6 400-20006 R6 GAGAGAGAGAGAGAGAC 4 500-2007 R7 ACACACACACACACACT 3 750-15008 R8 CTCCTCCTCCTCCTCCTC 3 600-11009 R9 GAGAGAGAGAGAGAGAYT 6 300-200010 R10 GAGAGAGAGAGAGAGAYG 6 600-2500

35 Assessment of Genetic Stability Using SCoT ISSR andRAPD Markers Genetic fidelity of micropropagated andacclimated plantlets of R tetraphylla was assessed by usingthree generations of markers ie RAPD ISSR and SCoTFor better analysis of genetic homogeneity and for discardingthe possibility of somaclonal variants it was always recom-mended to use more than one marker while carrying out thegenetic stability studies [47]

Analysis with SCoT primers (10 primers) has produced atotal of 30 monomorphic bands and 3 as the average numberof bands per primer with band size (in bp) ranging from 400

to 2000 (Table 6) Among the 10 SCoTprimers three primersnamely S4 S5 and S6 have produced the highest number ofbands (4 bands) and three primers namely S1 S2 and S9have produced the least number of bands (2 bands) whichwere monomorphic to the mother plant Amplification with10 ISSR primers has yielded 45 bands in total with 4 as theaverage number of bands per primer and band size (in bp)ranging from 300 to 2500 (Table 7) ISSR primer R10 hasgiven good amplification and produced 6 DNA bands whichare clear distinct and scorable in the range (in bp) of 600-2500 Similarly with 10RAPDprimers a total of 42 bandswith


Table 8 List of primers their sequence number and size of DNA bands generated by RAPD

Primer code Primer Sequence Number of Bands Band Length (in bp)1 OPA1 CAGGCCCTTC 3 300-15002 OPA2 TGCCGAGCTG 5 300-10003 OPA3 AGTCAGCCAC 6 300-22004 OPA4 AATCGGGCTG 3 400-12005 OPA5 AGGGGTCTTG 4 500-16006 OPA6 TGGGCGTCAA 6 400-20007 OPA7 GGCATGACCT 5 300-18008 OPA8 TGGGCGTCAA 4 400-19009 OPA9 CCAGCAGCTT 3 600-110010 OPA10 GACTGCACAC 3 600-1200

10000 bp5000 bp

2500 bp

500 bp

1000 bp

Figure 8 SCoT profiles of mother plant (Lane 2) and tissue culture raised plantlets (Lanes 3-9) of R tetraphylla using S2-SCoT primer Lanes1 and 10 DNA ladder of 1Kbp size Lane 2 DNA banding pattern of mother plant Lanes 3-6 DNA banding pattern of acclimated plants whichwere raised from leaf explants Lanes 7-9 DNA banding pattern of acclimated plants which were raised from stem explants

4 as the average number of bands per primer and band size (inbp) ranging from 300 to 2000 (Table 8) were obtained RAPDprimer OPA3 has given good amplification and produced6 DNA bands which are clear distinct and scorable in therange (in bp) of 300-2200

Even though we have carried amplification and producedmonomorphic bands with RAPD primers as we cannotensure the reproducibility of results with RAPD primershence in our study along with RAPD primers we havealso used other advanced primers ie ISSR and SCoTwhich are reliable and reproducible type in assessing thegenetic stability of regenerants compared to RAPD [48] Allamplification reactions with RAPD ISSR and SCoT primerswere repeated three times to check the reproducibility andresults were ie reproducible type with all the markers (R10-ISSR S2-SCoT and OPA3-RAPD)

Among the various markers nowadays SCoTmarkers aregaining much attention due to their superiority and betterresolvability over other markers [49 50] SCoT markers can

be used effectively for both genetic diversity and similaritystudies [51 52] SCoT markers were already reported forassessing the genetic stability of several plant species such asCleome gynandra [53] Pittosporum eriocarpum [48] Albiziajulibrissin [54] and Simmondsia chinensis [55] SimilarlyISSR primer based assessment of regenerans was reported inTylophora indica [56] Simmondsia chinensis [57] RauwolfiatetraphyllaL [58] Spilanthes calva [59]CucumismeloL [60]Cornus alba [61] and Zea mays [62]

In our study DNA bands produced by amplification withRAPD ISSR and SCoT markers were monomorphic acrossthe micro propagated plantlets and the mother plant (Figures8 9 and 10) which confirms the true-to-type and geneticallystable nature of acclimated plantlets of R tetraphylla RAPDand ISSR primers were also used by earlier researchersfor confirming the genetic homogeneity of regenerants inR tetraphylla [25 63 64] But this is the first report ofusing SCoT primers for the assessment of genetic stability ofregenerants in R tetraphylla


10000 bp5000 bp

2500 bp

500 bp

1000 bp

Figure 9 ISSR profiles of mother plant (Lane 2) and tissue culture raised plantlets (Lanes 3-9) of R tetraphylla using R10-ISSR primer Lanes1 and 10 DNA ladder of 1Kbp size Lane 2 DNA banding pattern of mother plant Lanes 3-6 DNA banding pattern of acclimated plants whichwere raised from leaf explants Lanes 7-9 DNA banding pattern of acclimated plants which were raised from stem explants

10000 bp5000 bp

2500 bp

500 bp

1000 bp

Figure 10 RAPD profiles of mother plant (Lane 2) and tissue culture raised plantlets (Lanes 3-9) of R tetraphylla using OPA3-RAPDprimerLanes 1 and 10 DNA ladder of 1Kbp size Lane 2 DNA banding pattern of mother plant Lanes 3-6 DNA banding pattern of acclimated plantswhich were raised from leaf explants Lanes 7-9 DNA banding pattern of acclimated plants which were raised from stem explants

4 Conclusion

An efficient and repeatable protocol was developed forconservation of economically important plant species ie Rtetraphylla L by high frequency of indirect shoot regener-ation from leaf and stem based callus Further the genetichomogeneity of in vitro regenerated plantlets was confirmedby using SCoT ISSR and RAPD markers This standardizedprotocol could be utilized for the mass propagation of true-to-type genotype of this endangered medicinal plant

Data Availability

The data used to support the findings of this study areincluded within the article and can be freely available toauthors with proper citation in their research work

Conflicts of Interest

The authors declare that there are no conflicts of interest inthis research study

Acknowledgments

The first author gratefully acknowledges University GrantsCommission (UGC) NewDelhi for providing financial sup-port under Maulana Azad National Fellowship ProgrammeThe authors thank Prof VS Raju for authentication of Rtetraphylla L and providing an accession number (No 1866)for the specimen preserved in his research lab Departmentof Botany Kakatiya University Warangal

References

[1] S Dahanukar and U Thatte Ayurveda Revisited PopularPrakashan Mumbai 3rd edition 2000

[2] A Chopra and V V Doiphode ldquoAyurvedic medicine coreconcept therapeutic principles and current relevancerdquoMedicalClinics of North America vol 86 no 1 pp 75ndash89 2002

[3] M Ahmedulla and M P Nayar Red data book of Indian Plantsvol 4 Botanical survey of India Calcutta India 1999

[4] A N Sahu S Hemalatha and K Sairam ldquoPhyto-Pharmac-ological review of Mesua ferrea Linnrdquo InternationalJournal ofPhytology vol 5 no 1 pp 6ndash14 2014


[5] P Lingaraj ldquoMedicinal Plants of IndiaWith special reference toOdishardquo International Journal of Advanced Research In Ideas inEducation vol 2 no 5 pp 121ndash135 2016

[6] K Joshi P Chavan D Warude and B Patwardhan ldquoMolecularmarkers in herbal drug technologyrdquoCurrent Science vol 87 no2 pp 159ndash165 2004

[7] K M Singh K Abhay R K P Singh and K Ujjwal ldquoMedic-inal and aromatic plants for enhancing farm income thecase of biharrdquo Munich Personal Repec Archive 2013 httpsmpraubuni-muenchende505711MPRA paper 50571pdf

[8] C Wiart Medicinal Plants of Asia and the Pacific CRC PressTaylor and Francis Boca Raton Fla USA 2006

[9] K M Rajkarnikar H K Sainju and B D Bhatta ldquoIn vitro cul-ture of Rauwolfia serpentina L benth ex Kurzrdquo in Proceedingsof Nepal Japan Joint Symposium pp 232ndash234 2002

[10] R J VAKIL ldquoA clinical trial of Rauwolfia serpentina in essentialhypertensionrdquo British Heart Journal vol 9 no 4 pp 350ndash3551949

[11] G K Rohela P Bylla S Pendli K Rajender B Dharavath andC R Thammidala ldquoISSR marker based DNA profiling studiesin Rauwolfia speciesrdquo Annals of Plant Sciences vol 7 no 5 p2289 2018

[12] A Chevaliere Encyclopedia of Medicinal Plants DK Publish-ing Inc New York NY USA 1st edition 1996

[13] P Singh A Singh A K Shukla L Singh V Pande and TK Nailwal ldquoSomatic embryogenesis and in vitro regenerationof an endangered medicinal plant sarpgandha (rauvolfia ser-pentina l)rdquo Life Science Journal vol 6 no 2 pp 57ndash62 2009

[14] M Faisal N Ahmad and M Anis ldquoShoot multiplication inRauvolfia tetraphylla L using thidiazuronrdquo Plant Cell Tissueand Organ Culture vol 80 no 2 pp 187ndash190 2005

[15] G K Rohela B Prasad C H Ravi et al ldquoIn vitro clonalpropagation of Rauwolfia tetraphylla a relative of indian snakeroot plantrdquo Research Journal of Biotechnology vol 10 pp 23ndash312015

[16] B Jyothi G Penchala Pratap G Sudharshan et al ldquoEthnob-otanical investigation of undergroundplant parts fromChittoorDistrict Andhra Pradesh Indiardquo Life Science Leaf Lets vol 18pp 695ndash699 2011

[17] S K Panda D Debajoyti N Bichitra et al ldquoPhyto-pharmacognostical studies and quantitative determinationof reserpine in different parts of Rauwolfia(Spp) of EasternOdisha by UV Spectrocsopy Methodrdquo Asian Journal of PlantSciences and Research vol 2 no 2 pp 151ndash162 2012

[18] S K Bhattacharjee ldquoHandbook of Medicinal Plants PointerPublicationsrdquo India pp 293-294 1998

[19] G Brahmachari L C Mandal D Gorai A Mondal S Sarkarand S Majhi ldquoA new labdane diterpene from Rauvolfia tetra-phylla Linn (Apocynaceae)rdquo Journal of Chemical Research vol35 no 12 pp 678ndash680 2011

[20] K C Ghosh and N Banerjee ldquoInfluence of plant growthregulators on in vitro micropropagation of Rauwolfia tetra-phylla Lrdquo Phytomorphology An International Journal of PlantMorphology vol 53 no 1 pp 11ndash19 2003

[21] M Faisal andM Anis ldquoRapid in vitro propagation of Rauvolfiatetraphylla L - An endangered medicinal plantrdquo Physiology andMolecular Biology of Plants vol 8 no 2 pp 295ndash299 2005

[22] G C Mitra ldquoStudies on the formation of viable and non-viable seeds in Rauwolfia serpentinabenthrdquo Indian Journal ofExperimental Biology vol 14 no 1 pp 54ndash56 1976

[23] RHarisharanraj K Suresh andS Saravanababu ldquoRapidClonalPropagation Rauvolfia tetraphylla Lrdquo Academic Journal of PlantSciences vol 2 no 3 pp 195ndash198 2009

[24] D Sarma S Sarma and A Baruah ldquoMicropropagation and invitro flowering of Rauvolfia tetraphylla a potent source of anti-hypertension drugsrdquo Planta Medica vol 65 no 3 pp 277-2781999

[25] G K Rohela P Bylla S Kota S Abbagani R Chithakariand T C Reuben ldquoIn vitro plantlet regeneration from leafand stem calluses of Rauwolfia tetraphylla (Rcanescens) andconfirmation of genetic fidelity of plantlets using the ISSR PCRmethodrdquo Journal of Herbs Spices ampMedicinal Plants vol 19 no1 pp 66ndash75 2013

[26] G K Rohela P Jogam A A Shabnam P Shukla S Abbaganiand M K Ghosh ldquoIn vitro regeneration and assessment ofgenetic fidelity of acclimated plantlets by using ISSR markers inPPR-1 (Morus sp) An economically important plantrdquo ScientiaHorticulturae vol 241 pp 313ndash321 2018

[27] T Murashige and F Skoog ldquoA revised medium for rapidgrowth and bio assays with tobacco tissue culturesrdquo PhysiologiaPlantarum vol 15 no 3 pp 473ndash497 1962

[28] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990

[29] P Bylla K R Gulab T Radha et al ldquoDNA profiling ofcommercial chilli pepper (Capsicum annuum L) varietiesusing random amplified polymorphic DNA (RAPD) markersrdquoAfrican Journal of Biotechnology vol 12 no 30 pp 4730ndash47352013

[30] G K Rohela P Bylla R Korra et al ldquoPhytochemical screeningand antimicrobial activity of leaf stem root and their callusextracts in Rauwolfia tetraphyllardquo International Journal of Agri-culture amp Biology vol 18 pp 521ndash528 2016

[31] S Anitha and B D Ranjitha Kumari ldquoIn vitro flowering inRauvolfia tetraphylla Lrdquo Pakistan Journal of Biological Sciencesvol 9 no 3 pp 422ndash424 2006

[32] S Anitha and D R Kumari ldquoIn vitro callus culture in Rauvolfiatetraphylla L Indole alkaloid productionrdquo Asian Journal ofPlant Sciences vol 12 no 1 pp 1ndash4 2012

[33] S Anitha and B D Ranjitha Kumari ldquoReserplne accumulationinNaCI treated calli of Rauvolfia tetraphylla Lrdquo ScienceAsia vol32 no 4 pp 417ndash419 2006

[34] C A Huetteman and J E Preece ldquoThidiazuron a potentcytokinin for woody plant tissue culturerdquo Plant Cell Tissue andOrgan Culture vol 33 no 2 pp 105ndash119 1993

[35] S R Pai and N S Desai ldquoEffect of TDZ on various plantculturesrdquo in idiazuron From urea derivative to plant growthregulator Ahmad N and M Faisal Eds Springer Singapore2018

[36] C H Ravi K S Avinash K R Gulab et al ldquoHigh frequencyin vitro clonal propagation of Solanum surattense burm FrdquoInternational Journal of Pharma and Biological Sciences vol 3pp 147ndash151 2012

[37] R Korra P Bylla and G K Rohela ldquoIn vitromicro propagationand confirmation of genetic fidelity using RAPD marker inethno medicinal plant Stachytarpheta jamaicensis L VahlrdquoInternational Journal of Advanced Research vol 5 pp 1494ndash1502 2017

[38] H Azeez K Ibrahim R Pop D Pamfil M Harta andO Bobis ldquoChanges induced by gamma ray irradiation onbiomass production and secondary metabolites accumulationin Hypericum triquetrifolium Turra callus culturesrdquo IndustrialCrops and Products vol 108 pp 183ndash189 2017


[39] P Baskaran and J Van Staden ldquoUltrastructure of somaticembryo development and plant propagation for Lachenaliamontanardquo South African Journal of Botany vol 109 pp 269ndash274 2017

[40] S R Pai V Upadhya H V Hedge R K Joshi and S DKholkute ldquoIn vitro rapid multiplication and determination oftriterpenoids in callus cultures of Achyranthes aspera LinnrdquoIndian Journal of Biochemistry and Biophysics vol 17 no 1 pp151ndash159 2018

[41] S R Pai M S Nimbalkar N V Pawar V V Kedage andG B Dixit ldquoIn vitro embryo culture and Ex situ regenerationstudies in Ancistrocladus heyneanus Wall Ex Grahrdquo Plant CellBiotechnology and Molecular Biology vol 9 no 3-4 pp 1ndash62008

[42] D Sujatha C H Ravi L Raghuvardhanet et al ldquoIn vitroplantlet regeneration and genetic transformation of spongegourd (Luffa cylindrica L)rdquo African Journal of Plant Sciencesvol 7 no 6 pp 244ndash252 2013

[43] A RMohdDin F Iliyas Ahmad AWagiran A Abd Samad ZRahmat and M R Sarmidi ldquoImprovement of efficient in vitroregenerationpotential ofmature callus induced fromMalaysianupland rice seed (Oryza sativa cv Panderas)rdquo Saudi Journal ofBiological Sciences vol 23 no 1 pp S69ndashS77 2016

[44] G Khan Rohela S Damera P Bylla R Korra S Pendli andC Thammidala ldquoSomatic embryogenesis and indirect regen-eration in Mirabilis jalapa LinnrdquoMaterials Today Proceedingsvol 3 no 10 pp 3882ndash3891 2016

[45] G K Rohela A S Aftab and S Pawan ldquoIn vitro clonalpropagation of PPR-1 a superior temperate mulberry varietyrdquoIndian Journal of Biotechnology vol 17 pp 619ndash625 2018

[46] G K Rohela A A Shabnam P Shukla A N Kamili andM K Ghosh ldquoRapid one step protocol for the in vitro micropropagation of morus multicaulis var goshoerami an elitemulberry variety of temperate regionrdquo Journal of ExperimentalBiology and Agricultural Sciences vol 6 no 6 pp 936ndash9462018

[47] V Lakshmanan S R Venkataramareddy and B NeelwarneldquoMolecular analysis of genetic stability in long-term micro-propagated shoots of banana using RAPD and ISSR markersrdquoElectronic Journal of Biotechnology vol 10 no 1 p 8 2007

[48] J Thakur M D Dwivedi P Sourabh P L Uniyal and A KPandey ldquoGenetic homogeneity revealed using SCoT ISSR andRAPD markers in micropropagated Pittosporum eriocarpumRoyle- an endemic and endangered medicinal plantrdquo PLoSONE vol 11 no 7 Article ID e0159050 2016

[49] B C Y Collard and D J Mackill ldquoStart Codon Targeted(SCoT) polymorphism a simple novel dna marker techniquefor generating gene-targetedmarkers in plantsrdquo PlantMolecularBiology Reporter vol 27 no 1 pp 86ndash93 2009

[50] A M Gorji P Poczai Z Polgar and J Taller ldquoEfficiencyof arbitrarily amplified dominant markers (SCOT ISSR andRAPD) for diagnostic fingerprinting in tetraploid potatordquoAmerican Journal of Potato Research vol 88 no 3 pp 226ndash2372011

[51] S Cabo L Ferreira A Carvalho P Martins-Lopes A Martınand J E Lima-Brito ldquoPotential of Start Codon Targeted(SCoT) markers for DNA fingerprinting of newly synthesizedtritordeums and their respective parentsrdquo Journal of AppliedGenetics vol 55 no 3 pp 307ndash312 2014

[52] C Fang-Yong and L Ji-Hong ldquoGermplasm genetic diversity ofMyrica rubra in Zhejiang Province studied using inter-primer

binding site and start codon-targeted polymorphism markersrdquoScientia Horticulturae vol 170 pp 169ndash175 2014

[53] N S Rathore M K Rai M Phulwaria N Rathore and NS Shekhawat ldquoGenetic stability in micropropagated Cleomegynandra revealed by SCoT analysisrdquo Acta Physiologiae Plan-tarum vol 36 no 2 pp 555ndash559 2014

[54] M Rahmani P M Pijut N Shabanian and M Nasri ldquoGeneticfidelity assessment of in vitro-regenerated plants of Albiziajulibrissin using SCoT and IRAP fingerprintingrdquo In VitroCellularamp Developmental Biology - Plant vol 51 no 4 pp 407ndash419 2015

[55] S A Bekheet A M M Gabr A A Reda and M K El-Bahr ldquoMicropropagation and assessment of genetic stabilityof In Vitro raised jojoba (Simmondsia chinensis Link) plantsusing SCoT and ISSR markersrdquo Plant Tissue Culture andBiotechnology vol 25 no 2 pp 165ndash179 2015

[56] M M Sharma R N Verma A Singh and A Batra ldquoAssess-ment of clonal fidelity of Tylophora indica (Burm f) Merrillldquoin vitrordquo plantlets by ISSR molecular markersrdquo Springer Plusvol 3 pp 1ndash9 2014

[57] S KumarMMangal A K Dhawan andN Singh ldquoAssessmentof genetic fidelity of micropropagated plants of Simmondsiachinensis (Link) Schneider using RAPD and ISSR markersrdquoActa Physiologiae Plantarum vol 33 no 6 pp 2541ndash2545 2011

[58] M Faisal A A Alatar N Ahmad M Anis and A K HegazyldquoAn efficient and reproducible method for in vitro clonal mul-tiplication of Rauvolfia tetraphylla L and evaluation of geneticstability using DNA-based markersrdquo Applied Biochemistry andBiotechnology vol 168 no 7 pp 1739ndash1752 2012

[59] M Razaq M Heikrujam S K Chetri and V Agrawal ldquoIn vitroclonal propagation and genetic fidelity of the regenerants ofSpilanthes calva DC using RAPD and ISSR markerrdquo PhysiologyandMolecular Biology of Plants vol 19 no 2 pp 251ndash260 2013

[60] M R Raji M Lotfi M Tohidfar et al ldquoSomatic embryogenesisof muskmelon (Cucumis melo L) and genetic stability assess-ment of regenerants using flow cytometry and ISSR markersrdquoProtoplasma vol 255 no 3 pp 873ndash883 2018

[61] A Ilczuk and E Jacygrad ldquoIn vitro propagation and assessmentof genetic stability of acclimated plantlets of Cornus alba Lusing RAPD and ISSR markersrdquo In Vitro Cellular amp Develop-mental Biology - Plant vol 52 no 4 pp 379ndash390 2016

[62] M Ramakrishnan S A Ceasar V Duraipandiyan and SIgnacimuthu ldquoEfficient plant regeneration from shoot apexexplants of maize (Zea mays) and analysis of genetic fidelityof regenerated plants by ISSR markersrdquo Plant Cell Tissue andOrgan Culture vol 119 no 1 pp 183ndash196 2014

[63] A Alatar andM Faisal ldquoEncapsulation of Rauvolfia tetraphyllamicroshoots as artificial seeds and evaluation of genetic fidelityusing RAPD and ISSR markersrdquo Journal of Medicinal PlantsResearch vol 6 no 7 pp 1367ndash1374 2012

[64] M Faisal A A Alatar and K H Ahmad ldquoMolecular andbiochemical characterization in rauvolfia tetraphylla plantletsgrown from synthetic seeds following in vitro cold stor-agerdquo Applied Biochemistry and Biotechnology Article ID101007s12010-012-9977-0 2012

Hindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom


pharmaceutical industry playing a major role in fulfilling thedemand for herbals worldwide In addition to the interna-tional trade there is a substantial volume of internal trade formedicinal plants in India which has created a lot of demandto these medicinal herbs and shrubs [7]

Among the different plant families Apocynaceae hasgained lot of medicinal importance as most of the plantspecies of this family produce milky sap which has phar-maceutical applications such as for gastrointestinal ailmentsmalaria pain and diabetes [8] Almost every part (rootsstem bark leaves and latex) of Apocynaceae family memberswas used for several treatments and disorders like leprosydysentery worms ulcers tumours skin and liver diseases[9]

Rauwolfia is an important genus in Apocynaceae familywhich has gained much popularity due to the presence ofpharmaceutically significant alkaloids with higher concen-trations in roots and stem bark Approximately 130 speciesof Rauwolfia have been identified and are located mostlyin tropical and semitropical regions Among the differentspecies R serpentina Benth ex Kurz is indigenous to India[10 11] and this species is on the verge of extinction [12]due to its overexploitation by ayurvedic practitioners andpharmaceutical industries [13] and Government of India hasalso restricted the exploitation of this endangered species[14] In this scenario the attention of industrialists andmedicinal practitioners was diverted to find an alternativeplant species as a source of pharmaceutical alkaloids

Among the other species of Rauwolfia the most impor-tant is Rauwolfia tetraphylla L which has gained lot ofimportance as an alternative source of reserpine and otheralkaloids [15] R tetraphylla is native of tropical America andWest Indies [16] but now it is naturalized [17] throughout thetropics which includes the moist deciduous forests of South-east Asia including Burma Bangladesh India Malaysiaand Srilanka [18] The common names are wild snake rootplant devil pepper be-still tree American serpent wooddevil root milk bush etc It is being cultivated widely asboth an ornamental and a source of pharmaceuticals Thisspecies produces different type of pharmaceutically impor-tant alkaloids in their stem bark and roots such as reserpinedeserpidine ajmalicine ajmaline canescine yohimbine andrecinnamine [19 20]

Due to presence of various commercial indole alkaloidsthis plant species was also exploited during the last threedecades and presently it is in endangered status [21] Prop-agation of R tetraphylla through vegetative cuttings and seedmaterial is limited (5-10) due to its poor rooting abilityand due to presence of phenolic compounds in seed [22]In view of the above facts about the mass exploitation ofthis plant from natural resources and due to its restrictedcultivation there is an urgent need to conserve this medici-nally important plant species by proper conservation strategyHence the present study is undertaken with the objective ofmass propagation of R tetraphylla through the standardizedindirect regeneration protocol by utilizing leaf and stembased callus and to confirm the genetic fidelity of acclimatedplants by using SCoT ISSR and RAPD primer based PCRanalysis

Even though there are some reports about its mul-tiplication through nodal shoot tip cultures [23] and invitro flowering [24] but there is scanty information aboutits regeneration through callus based indirect regenerationprotocols In our previous report we have used single typeof cytokinin (TDZ) in regenerating the low frequency ofmultiple shoots from leaf and stem based callus [25] Nowin this study we are reporting a protocol for its conservationthrough high frequency of plantlet production by usingcombination of cytokinins amended media

2 Materials and Methods

In this study most of materials and methods were followedas per the report on indirect regeneration studies in aMorusspecies [26]

21 Plant Material and Explant Source Two-year-old Rtetraphylla plants available in the medicinal arboretumDepartment of Biotechnology KakatiyaUniversity were usedas parent plant material in this in vitro propagation studiesFully expanded leaf of about 7 cm in length and 4 cm inbreadth stem (internodal segments) and root parts werecollected during the second week of June and were used asexplants for the study

22 Murashige and Skoog Media Preparation MS medium[27] was prepared by using sucrose (3) and appropriatequantities of vitamins Iron macronutrients and micronu-trients from stock solutions Before dispensing the preparedmedium to culture tubes required concentrations and com-binations of plant growth regulators were added to it from thehormonal stock solutionsThemediawas solidifiedwith 08Agar and pHwas adjusted to 56 with 1 or 2 drops of 01 NHClor NaOH solution before autoclaving at 121∘C for 15 minutes

23 Aseptic Techniques for Sterile Transfer All operationsincluding transfer and sterile filtration were made underlaminar airflow cabinet Prior to operation the laminarairflow bench was swabbed with 70 alcohol to maintainmaximum sterility The laminar airflow was switched on andkept on UV mode for 30 minutes before using it The scalpelforceps and spatula were sterilized by flaming with alcoholbefore using the laminar airflow cabinet

24 Explant Surface Sterilization Leaf stem (internodal)and root explants of R tetraphylla were collected from themedicinal arboretum and initially washed under running tapwater for 2 minutes Then different protocols were followedfor the sterilization of leaf stem and root explants as leafexplants are more delicate and sensitive in nature to sterilantsand due to greater degree of fungal count on stem and rootexplants Leaf explants were treated with Tween-20 solutionfor 1 minute followed by 60 ethanol for 2 minutes andin 01 mercuric chloride (HgCl

2) solution for 2 minutes

Stem and root explants were treated initially with 05bavistin for 10 minutes followed by Tween-20 solution for2 minutes and with 01 mercuric chloride solution for 4
















(a) (b)

(c) (d)









Leaf






Stem (Inter node)






Root









Leaf






Stem (Inter node)






Root














Leaf





Stem (Inter node)






Root



02468

101214



c c cd

e

c b b a a

Mea

n N

o o

f Sho

ot B

uds




0

2

4

6

8

10

12

1 2 3 4 5



c

a

d

b

f

d

hg

ef

Mea

n N

o o

f Sho

ot B

uds







TDZ(mgL)


Leaf

010 200 4 plusmn 012f

025 200 25 plusmn 025a

050 200 10 plusmn 017d

075 200 8 plusmn 011e

100 200 2 plusmn 015g

Stem

010 200 4 plusmn 043f

025 200 20 plusmn 076b

050 200 14 plusmn 013c

075 200 4 plusmn 028f

100 200 2 plusmn 026g


0

5

10

15

20

25

30

Mea

n N

o o

f Sho

ot B

uds



g

f

de

c

ba a

bb

de

cc

bba a

b













05 05 846 plusmn 46f 604 plusmn 054f

05 10 924 plusmn 58g 630 plusmn 028f

10 05 698 plusmn 42d 144 plusmn 012a

10 10 980 plusmn 12h 832 plusmn 084h





(a) (b)

(c) (d)

(e) (f) (g)



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

















(a) (b)

(c) (d)









Leaf






Stem (Inter node)






Root









Leaf






Stem (Inter node)






Root














Leaf





Stem (Inter node)






Root



02468

101214



c c cd

e

c b b a a

Mea

n N

o o

f Sho

ot B

uds




0

2

4

6

8

10

12

1 2 3 4 5



c

a

d

b

f

d

hg

ef

Mea

n N

o o

f Sho

ot B

uds







TDZ(mgL)


Leaf

010 200 4 plusmn 012f

025 200 25 plusmn 025a

050 200 10 plusmn 017d

075 200 8 plusmn 011e

100 200 2 plusmn 015g

Stem

010 200 4 plusmn 043f

025 200 20 plusmn 076b

050 200 14 plusmn 013c

075 200 4 plusmn 028f

100 200 2 plusmn 026g


0

5

10

15

20

25

30

Mea

n N

o o

f Sho

ot B

uds



g

f

de

c

ba a

bb

de

cc

bba a

b













05 05 846 plusmn 46f 604 plusmn 054f

05 10 924 plusmn 58g 630 plusmn 028f

10 05 698 plusmn 42d 144 plusmn 012a

10 10 980 plusmn 12h 832 plusmn 084h





(a) (b)

(c) (d)

(e) (f) (g)



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





Leaf






Stem (Inter node)






Root









Leaf






Stem (Inter node)






Root














Leaf





Stem (Inter node)






Root



02468

101214



c c cd

e

c b b a a

Mea

n N

o o

f Sho

ot B

uds




0

2

4

6

8

10

12

1 2 3 4 5



c

a

d

b

f

d

hg

ef

Mea

n N

o o

f Sho

ot B

uds







TDZ(mgL)


Leaf

010 200 4 plusmn 012f

025 200 25 plusmn 025a

050 200 10 plusmn 017d

075 200 8 plusmn 011e

100 200 2 plusmn 015g

Stem

010 200 4 plusmn 043f

025 200 20 plusmn 076b

050 200 14 plusmn 013c

075 200 4 plusmn 028f

100 200 2 plusmn 026g


0

5

10

15

20

25

30

Mea

n N

o o

f Sho

ot B

uds



g

f

de

c

ba a

bb

de

cc

bba a

b













05 05 846 plusmn 46f 604 plusmn 054f

05 10 924 plusmn 58g 630 plusmn 028f

10 05 698 plusmn 42d 144 plusmn 012a

10 10 980 plusmn 12h 832 plusmn 084h





(a) (b)

(c) (d)

(e) (f) (g)



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





Leaf





Stem (Inter node)






Root



02468

101214



c c cd

e

c b b a a

Mea

n N

o o

f Sho

ot B

uds




0

2

4

6

8

10

12

1 2 3 4 5



c

a

d

b

f

d

hg

ef

Mea

n N

o o

f Sho

ot B

uds







TDZ(mgL)


Leaf

010 200 4 plusmn 012f

025 200 25 plusmn 025a

050 200 10 plusmn 017d

075 200 8 plusmn 011e

100 200 2 plusmn 015g

Stem

010 200 4 plusmn 043f

025 200 20 plusmn 076b

050 200 14 plusmn 013c

075 200 4 plusmn 028f

100 200 2 plusmn 026g


0

5

10

15

20

25

30

Mea

n N

o o

f Sho

ot B

uds



g

f

de

c

ba a

bb

de

cc

bba a

b













05 05 846 plusmn 46f 604 plusmn 054f

05 10 924 plusmn 58g 630 plusmn 028f

10 05 698 plusmn 42d 144 plusmn 012a

10 10 980 plusmn 12h 832 plusmn 084h





(a) (b)

(c) (d)

(e) (f) (g)



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





TDZ(mgL)


Leaf

010 200 4 plusmn 012f

025 200 25 plusmn 025a

050 200 10 plusmn 017d

075 200 8 plusmn 011e

100 200 2 plusmn 015g

Stem

010 200 4 plusmn 043f

025 200 20 plusmn 076b

050 200 14 plusmn 013c

075 200 4 plusmn 028f

100 200 2 plusmn 026g


0

5

10

15

20

25

30

Mea

n N

o o

f Sho

ot B

uds



g

f

de

c

ba a

bb

de

cc

bba a

b













05 05 846 plusmn 46f 604 plusmn 054f

05 10 924 plusmn 58g 630 plusmn 028f

10 05 698 plusmn 42d 144 plusmn 012a

10 10 980 plusmn 12h 832 plusmn 084h





(a) (b)

(c) (d)

(e) (f) (g)



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(a) (b)

(c) (d)

(e) (f) (g)



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(a) (b)

(c) (d) (e)

(f) (g) (h)






0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

20

40

60

80

100

120

IAA IBA IAA+IBA

o

f Roo

ting

05+05 05+10 10+05 10+10


ab

cc d

ef

gf

g

d

h

05 10 15 20 05 10 15 20

aab

ccc d

ef f

dd












10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





10000 bp5000 bp

2500 bp

500 bp

1000 bp








10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



10000 bp5000 bp

2500 bp

500 bp

1000 bp


10000 bp5000 bp

2500 bp

500 bp

1000 bp


4 Conclusion


Data Availability




Acknowledgments


References






































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


indirect regeneration and assessment of genetic...

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