Gene 533 (2014) 313–321

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Methods paper

Genetic and phytochemical analysis of the in vitro regenerated Pilosocereus robinii byISSR, SDS–PAGE and HPLC

Salah Khattab a, Fadia El Sherif a, Hoda A. El-Garhy b, Safwat Ahmed c, Amany Ibrahim c,⁎a Department of Horticulture, Faculty of Agriculture, Suez Canal University, 41522, Ismailia, Egyptb Department of Genetics, Faculty of Agriculture, Benha University, Qalubia, Egyptc Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, 41522, Ismailia, Egypt

Abbreviations: SDS–PAGE, sodium dodecyl sulfate polsequence repeat; HPLC, high performance liquid chromaadenine; BA, benzyl adenine.⁎ Corresponding author. Tel.: +20 1092638387; fax: +

E-mail address: am_kamal66@yahoo.com (A. Ibrahim

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.gene.2013.09.026

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 9 September 2013Available online 14 September 2013

Keywords:Pilosocereus robiniiMicropropagationRegenerationGenetic fidelityProtein analysisPhytochemistry

Pilosocereus robinii is a rare specieswhich is experiencing sudden population collapse. Identifying and developingeffective conservation and management strategies to halt the forestall extinction of this species is crucial. Thepresent study was conducted to assess the best conditions for in vitro propagation of this plant in regard to itsmorphogenic, genetic as well as the chemical potentials. A successful in vitro propagation system of P. robiniihas been developed. MS hormone-free medium induced the best root morphogenic potential. The plants wereacclimatized in the greenhouse at 100% survival rate. Besides, the somaclonal variations between the in vitroraised plants were analyzed using PCR-ISSR markers and SDS–PAGE protein, where the regenerated explantsonMSmedium supplementedwith TDZwere thehighest in inducing new specificmarker bands. Sh6 ISSR primershowed thehighest polymorphismvalue, 81.8%with 33 total amplified fragments,while Sh3 ISSR primer showedthe lowest value with polymorphic percentage of 14.3%. Furthermore, SDS–PAGE protein analysis showed novariation in protein pattern of the studied treatments. On the other side, HPLC analysis of the in vitro plantlets ex-tracts has shown that 2iP based treatments were the highest in organic acids accumulation, while the phenolicconstituents' accumulation was found to reach its peak in the BA based treatments.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The Cactaceae is one of the most popular, easily recognizable andmorphologically distinct families of plants, and it includes approximate-ly 1,600 species (Nobel, 2002). Pilosocereus robinii (Lemaire), the keytree-cactus, has experienced local extirpations and is vulnerable toextinction as a result of habitat loss and other anthropogenic factors(Borhidi and Muñiz, 1983). It is native in Florida and Cuba (Avery,1982; Benson, 1982). Traditionally the propagation of this species ismade through cuttings and seeds. Seed propagation involves differentproblems such as low germination rate and slow initial growth(Quiala et al., 2009). Cacti seeds are frequently difficult to be obtained(Mauseth, 1977) and plantlets are reported to be susceptible todamping-off (Ault and Blackmon, 1987; Mauseth, 1979; Quiala et al.,2009). Many species of cacti have been shown to contain psychoactiveagents, chemical compounds that can cause changes in mood, percep-tion and cognition through their effects on the brain. Also cacti havebeen shown to have useful compounds for the treatment of ischemiaand oxidative stress-induced neurodegenerative disease (Huang et al.,

yacrylamide; ISSR, inter-simpletography; 2iP, N6-[2-isopentyl]

20 643230741.).

ights reserved.

2009). In addition,many plants from the Cactaceae family have demon-strated their value against diabetesmellitus and inmonitoring thebloodcholesterol levels (Gutierrez, 1998; Stintzing et al., 2000, 2001).

In vitro cultivation has relevance in the clonal propagation of valu-able or endangered plant germplasmand in theproduction of transgenicplants (Frederic et al., 2007; Johnson and Emino, 1979; Smith et al.,1991).

To date, micropropagation techniques are employed to produce alarge number of new and true-to-type plants in a relatively short periodof time. It has already proved to be successful for severalmembers of theCactaceae family (Cardarelli et al., 2010; Clayton et al., 1990; Escobaret al., 1986; Garcı´a-Rubio and Malda-Barrera, 2010; Garcıa-Saucedoet al., 2005; Johnson and Emino, 1979; Mauseth, 1979; Wyka et al.,2006). However, for P. robinii micropropagation, only the effect of BAwas tested (Quiala et al., 2009).

Somaclonal variations are usually associated with in vitro culturingwhich can be considered as a novel source of genetic variability forplant improvement (Larkin and Scowcroft, 1981). Genetic variationanalysis can be observed from morphological characters and othermarkers, as protein or DNAbasedmarkers (Paterson et al., 1991). Sever-al strategies are available for detecting these variations includingmolec-ular techniques like inter simple sequence repeat (ISSR)-PCR. ISSRshave been used to generate species with gene specific, trait specificmarkers and investigating variability at intraspecific level. Severalworkers have used ISSRs to monitor genetic variability in in vitro

314 S. Khattab et al. / Gene 533 (2014) 313–321

culture-derived plants (Gesteira et al., 2002; Lata et al., 2010; Lombardoet al., 2011; Qin et al., 2006; Reddy et al., 2002; Tawar et al., 2008; Yuand Pauls, 2008).

In this study,we aimed to establish an applicable and stable regener-ation system to save the endangered P. robinii, detection of any proba-bility of somaclonal variations in the in vitro regenerated plants andthe chemical profile of the plantlets in terms of their phenolic content,as well as assessing the organic acids content.

2. Materials and methods

2.1. Seed germination

Seeds of P. robinii were purchased from (Flora-Fey Solingen)Germany. The seeds were sterilized by soaking in 70% ethanol solutionfor 30 s and then were rinsed in 5% (v/v) sodium hypochlorite solutionfor 5 min and washed three times with sterile tap water under laminarair-flow hood. Sterilized seeds were cultured into 40 mL capacity jarscontaining 10 mL half strength MS (Murashige and Skoog, 1962) basicsalts and vitamins, supplemented with 2% (w/v) sucrose, 7 g/L agarand the pH was adjusted to be 5.7 before autoclaving at 121 °C and1.2–1.3 kg/cm2 pressure for 20 min. One hundred seeds were cultured,one seed per jar. The cultures were incubated under growth roomconditions (22 ± 2 °C, at dark). After germination, the seedlings weretransferred into 16 h photoperiod with a light intensity of 4000 luxprovided by florescent lamps (Phillips TLM 40W/33RS). The rate ofgermination was determined after 8 weeks from seed culture.

2.2. Effects of BA on shoot induction

After 8 weeks the roots of the germinated seeds were isolated, in-duced stem cuttings (1.0 cm height) was excised from base alongwith some portion of mother explants and placed on MS medium con-taining 3% (w/v) sucrose, 7 g/L agar, and benzyl adenine (BA) concen-trations (0.0, 0.5, 1.0, 2.0 and 4.0 mg/L). Cultures were established in40 mL culture vessels. After 3 months the shoot tip explants fromthese regenerated shootswere transferred to 40 mL culture vessels con-tainingMSmedium for studying the effect of different concentrations ofBA, Kin and 2iP and the following data were recorded: number of shoot,plant height and plant weight. Some multiplied shoots were collectedand dried under room temperature before extraction for HPLC analysisand determination of total phenolic constituents.

2.3. Proliferation of shoots from axillary buds

The proliferation was carried out by means of axillary buds activa-tion, the six-month-old stem cutting was cut transversely with theheight of (1.0 cm) and cultured onMSmedium containing 3% (w/v) su-crose, 7.0 g/L agar and different concentrations of benzyl adenine (BA),kinetin (Kin), 2-isopentenyladenine (2iP) (0.0, 1.0, 2.0 and 4.0 mg/L),with pH 5.7 and autoclaving at 121 °C and 1.2–1.3 kg/cm2 pressurefor 20 min. Every jar contained three explants and each treatment hadten replicates, i.e., ten jars. The cultureswere grown for 3 months beforedatawere recorded in terms of the number of shoots, plant height, plantweight and number of roots. Some explants were sub-cultured in thesame MS medium supplemented with kin, 2iP and BA for ISSR andSDS–PAGE analysis and some multiplied shoots were collected anddried under room temperature before extraction for HPLC analysis anddetermination of total phenolic constituents.

2.4. Effects of TDZ on shoot induction

Fully developed shoot and root were excised from the six-month-old in vitro cultured explants to be used for further experimental succes-sion. Shoot tip, stem cutting of about 1.0 cm, root segment of about1.0 cm2 in size and a developed stem (1.0 g) were cut to very small

pieces (as small as possible) under sterile conditions. These explantswere distributed separately on Petri dishes containing MS media(Murashige and Skoog, 1962) basic salts and vitamins and supplementedwith 3% (w/v) sucrose, 7.0 g/L agarwith pH 5.7. Different concentrationsof thidiazuron (TDZ) (0.0, 0.005, 0.01, 0.02 and 0.04 μM) were added tothe medium after being autoclaved and cooled to 47 °C (after filtration-aided sterilization as required). The explants were placed on Petri disheswith ad-axial surface in contact with the medium. Every Petri dish wasinoculated with five root explant discs; each treatment was run in tenreplicates (Petri dishes). After 3 months the number of explants was cal-culated and the regeneration explants were transferred to the rootingmedium. Regenerated explants from small pieces of stem were culturedon MS hormone free medium for ISSR and SDS–PAGE analysis.

2.5. Rooting and acclimation

For rooting, nine-month-old shoots (0.5–1 cm in length) formedin vitro were excised and transferred to MS medium supplementedwith different concentrations of IAA, IBA or NAA (0.0, 0.1, 0.2 and0.4 mg/L) and placed under same growth room conditions for rootformation. After 4 weeks of culture, data were recorded in terms ofnumber of roots, root length, plant fresh weight and plant height. Therooted plantlets were transferred to the greenhouse for acclimatizationin pots with a moist mixture of (1:1) sand and perlite. The plants weremaintained inside a plant growth chamber and irrigatedwith a finemistof water for 3 weeks. The percentage of the survived plants was deter-mined after 4 weeks.

2.6. Genomic DNA extraction

DNAwas extracted from four fresh explants of in vitro derived plant-lets (one-year old) which produced in multiplication and regenerationstages on BA, Kin, 2iP and TDZ aswell as control plants, using i-genomicplant DNA Extraction Mini Kit (iNtron Biotechnology, Inc., cat. no.17371). Purified concentrated products were obtained at high qualityand high yield and were used directly in a variety of downstreamapplications.

2.7. ISSR-PCR reaction and thermocycling profile

ISSR-PCR was performed in 25 μL reaction mix containing 1× PCRbuffer, 2 mM MgC12, 0.2 mM of each dNTPs, 1 μM oligonucleotideprimer, 25 ng genomicDNA and 1 unit of hot start taqDNA polymerase.A high stringency touchdown and hot start thermocycling profile wasperformed as follows: an initial hot start and denaturation step at94 °C for 15 min followed by one cycle at 94 °C for 1 min; 55 °C for1 min and 72 °C for 2 min. The annealing temperature was lowered ineach cycle by 1 °C during nine cycles. This was followed by thirty cyclesat 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min and an extensioncycle at 72 °C for 7 min then 4 °C (infinitive). The PCR products wereseparated on 1.3% agarose gel, stained with 0.5 μg/mL ethidium bro-mide, and visualized under ultraviolet light, then photographed. DNAfragment sizes were determined by comparisons with the 1 kb DNAladder marker.

The obtained datawere entered in a computer file as binarymatriceswhere “0” stands for the absence of a band and “1” stands for thepresence of a band in each individual sample. Similarity coefficientswere calculated according to dice matrix (Nei and Li, 1979; Rolf, 1993).

2.8. Protein extraction

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was used to determine protein electrophoretic pattern. Proteinextraction was performed as follows: fresh explants (1 g) were excisedfrom four in vitro derived plantlets of one-year old were used. The usedplants were produced in the multiplication and the regeneration stages

Table 2The effect of different BA concentrations onmultiple shoot induction of Pilosocereus robinii.

BA concentration(mg/L)

No. of shootsper explant

Length ofthe longestshoot (cm)

Explant freshweight (g)

Callus %

0.0 1.0⁎b 2.0a 1.35a 25.0ab

0.5 3.0b 2.5a 2.4a 54.2a

1.0 10.2a 2.6a 3.6a 41.7ab

2.0 7.0ab 2.8a 4.4a 40.0ab

4.0 8.6ab 2.0a 1.7a 0.0b

⁎ Means followed by the same letter within a column are not significantly different at0.05 level of probability according to LSD test.

Table 3The effects of BA, Kin and 2iP concentrations on multiple shoot induction of Pilosocereusrobinii.

Growth regulator(mg/L)

No. of shootsper explant

Length of thelongest shoot, (cm)

Explant freshweight (mg)

BA 2iP Kin

315S. Khattab et al. / Gene 533 (2014) 313–321

using BA, Kin, 2iP and TDZ as well as control plants. The explants werehomogenized with a mortar and pestle using 2 mL of ice-cold 20 mMTris–HCl and the pH was adjusted to 7.5, before being centrifuged at10,000 ×g for 20 min to remove cell debris. The supernatant was fil-tered through four layers of Miracloth. Ten micrograms of proteinswere separated by12% sodiumdodecyl sulfate–polyacrylamide gel elec-trophoresis with a mini PROTEIN II apparatus (Bio-Rad, Hercule, CA)and stained with a solution containing 0.002% Coomassie Blue-R-250,and then destained with a mixture of glacial acetic acid, methanol andwater. A prestained marker protein of known molecular weights of(250, 130, 95, 72, 55, 36, and 28 kDa) was run on the correspondinggel and used for characterization and determination of the molecularmass.

2.9. Assessment of the phytochemical profile

2.9.1. HPLC method for determination of malic acid

2.9.1.1. Instrumentation. Chromatographic experiments were performedonHPLC device (Bischoff, Germany), the instrumentwas equippedwithmodel series 2250 LC pump, Rheodyne 7125 injector with a 20 μL loopand an LC lambda 1010 variable wavelength spectrophotometric detec-tor (Bischoff). The sample was injected with 25 μL Hamilton analyticalsyringe. Data acquisition was performed on a model McDAcq dataacquisition system (version 1.3×) and Hewlett Packard LaserJet 6Lprinter. The detector was set at λ 210 nm.

2.9.1.2. Materials and reagents.Authentic standard ofmalic acidwas sup-plied from Oxford Lab. Inc., India. The standard was certified to contain99.8%. The used acetonitrile was HPLC grade (Fisher, Leis LE 11 5 RGUK). Water was HPLC grade (Scharlau, Spain). Phosphoric acid was ofanalytical grade (Sigma-Aldrich, USA).

2.9.1.3. HPLC conditions. The HPLC separation and quantitation weremade on a 250 × 4.6 mm (i.d.) 5 μmODS column (Luna, Phenomenex,USA). The mobile phase was prepared by mixing acetonitrile andc(H2PO4) = 6.1−3 mol/L (pH 2.1) in a ratio 5:95 v/v. The flow ratewas 1 mL min−1. All determinations were performed at ambienttemperature (at 25 °C). The mobile phase was filtered using 0.45 μmmembrane filter (Millipore, Milford, MA) and degassed by vacuumprior to use. The samples were also filtered using 0.45 μm disposablefilters.

2.9.1.4. Standard solutions and calibration. Stock standard solutions wereprepared by dissolving 5 mg of malic acid in 5 mL of mobile phase. Thestandard solutionswere prepared by dilution of the stock standard solu-tions with the mobile phase to reach concentration ranges of 20–100.Triplicate 20 μL injectionsweremade for each compound concentrationand chromatographed under the specified conditions described previ-ously. The peak area valueswere plotted against corresponding concen-trations to obtain the calibration graph.

2.9.1.5. Sample preparation. The extracts were prepared from the driedsamples by ultrasonic-aided extractionwithmethanol. Different identi-fiedweights of the dried samples were soaked in the extractionmixture

Table 1Name and sequence of the primers used in ISSR detection.

Primer code Sequence Primer code Sequence

Sh 1 (AG)8CTC Sh 6 CGC(GATA)4

Sh 2 (AG)8CTG Sh 7 GAC(GATA)4

Sh 3 (AC)8CTT Sh 8 (AGAC)4GCSh 4 (Ac)8CTG Sh 9 (GATA)4GCSh 5 (GT)8CTG Issr3 TTT(TCC)5

Issr2 CAC(TCC)5 Issr10 (TCC)5n AC

and the solvent was replaced every day for five consequent days toinsure complete extraction before drying under vacuum. Complete ex-traction was confirmed by thin layer chromatography and high perfor-mance liquid chromatography. For the HPLC analysis, 10 mg of eachsample was dissolved in 10 mL of the mobile phase in a volumetricflask producing a final concentration of 100 mg%. The content of eachflask was shaken vigorously for 10 min, sonicated for 15 min before fil-tration through 0.45 μm disposable filters. A sample of 20 μL was theninjected using the general procedures described under calibration andthe concentrations of malic acid was calculated.

2.9.2. Determination of total phenolic constituents

2.9.2.1. Instrumentation. A double-beam Shimadzu (Japan) UV–visiblespectrophotometer, model UV-1601 PC, connected to an IBM compati-ble computer was used. The bundled software was UVPC personal spec-troscopy software version 3.7 (Shimadzu). The spectral bandwidth was2 nm and the wavelength scanning speed was 2800 nm min−1.

2.9.2.2. Methodology. Total phenolic constituents determination wasperformed in reference to the previously reported method involvingFolin–Ciocalteu reagent with gallic acid as a standard (Sengul et al.,2009). The plant samples were extracted in the sameway as previouslymentioned under Sample preparation in the HPLC analysis for malicacid. Extract solution for each treatment (0.1 mL) containing 1 mL ex-tract was taken in a volumetric flask, 45 mL distilled water and 1 mLFolin–Ciocalteu reagentwere added and theflaskwas shaken thorough-ly. After 3 min, 3 mL of Na2CO3 (2%) was added and the mixture wasallowed to stand for 2 h with intermittent shaking. The absorbancewas measured at 760 nm. The same procedure was repeated for allstandard gallic acid solutions (0–1000 mg, 0.1 mL−1) and the standardcurve was obtained.

0.0 0.0 0.0 1.3⁎e 2.2abc 2.4bc

1.0 0.0 0.0 10.4cd 1.6cd 7.9ab

2.0 0.0 0.0 19.3b 1.7cd 2.1c

4.0 0.0 0.0 2.25de 1.0d 0.97c

0.0 1.0 0.0 7.0cde 2.7ab 10.7a

0.0 2.0 0.0 8.9cde 1.8cd 8.4ab

0.0 4.0 0.0 9.2cde 1.8bcd 2.7bc

0.0 0.0 1.0 4.25de 2.8a 2.4bc

0.0 0.0 2.0 30.0a 2.8a 9.1ab

0.0 0.0 4.0 13.6c 3.0a 7.9ab

⁎ Means followed by the same letter within a column are not significantly different at0.05 level of probability according to LSD test.

(A) (C)

(D) (E) (F)

(B)

Fig. 1. (A) Seed germination of Pilosocereus robinii, (B) shoot regeneration on MS medium containing (2.0 mg/L Kin), (C) root formation on MS medium, (D, E and F) adventitious shootregeneration from small pieces of stem on MS medium containing (0.005 μM TDZ) 4, 8 and 12 weeks after subculture (explant showing hyperhydration is arrowed).

316 S. Khattab et al. / Gene 533 (2014) 313–321

2.9.3. Statistical analysisExperiments were set up in completely randomized design. Data

were statistically analyzed using ANOVA/MANOVA of Statistica 6 soft-ware (StatSoft, Inc., 2001). The significance of differences amongmeans was carried out using the least significant test (LSD) at p =0.05. For statistical analysis of HPLC results and total phenolic constitu-ents, data were computed using Microsoft Excel program.

3. Results and discussion

3.1. Shoot multiplication

At the end of 2 months, seeds cultured on half-strengthMSmediumshowed 65% germination. In the first experiment when the usedMS medium was supplemented with different concentrations of BA

Table 4The effects of TDZ concentrations on shoot regeneration induction of Pilosocereus robinii.

TDZ (μM) Explants

Shoot tip Stem cutting

No. ofshoots perexplant

Length of thelongest shoot(cm)

No. ofroots perexplant

Length of thelongest root(cm)

No. ofshoots perexplant

Length oflongest sh(cm)

0.0 1.5d 1.0⁎b 1.0e 2.0ab 1.5d 0.5cd

0.005 2.0d 0.5cd 0.5e 2.5ab 6.5d 0.6c

0.01 1.0d 0.5cd 4.5cde 4.5a 4.0d 0.5cd

0.02 1.0d 0.5cd 2.0cde 0.5b 2.0d 0.5cd

⁎ Means followed by the same letter within a column are not significantly different at 0.05 l

(Table 2), these levels of BAwere able to positively affect themultiplica-tion of the explants without affecting the plant growth. The controltreatments without BA were found to be unable to induce shoot prolif-eration. All the used concentrations of BA, Kin and 2iP (Table 3) werefound to facilitate bud and shoot differentiation. After 3 months of cul-ture Kinwas found to be themost efficient in terms of number of shootsper explant. Kin at a concentration (2.0 mg/L) gave the highest numberof shoots per explant (30 shoots per explant) with an average length of(2.8 cm) and lower percentage of hyperhydration symptoms. Based onthese findings, Kin is supposed to be themost efficient growth regulatortested for the optimal multiplication of this plant material. On the otherhand, the total number of shoots had shown an increase with theincrease of 2iP concentration. While, shoot length decreased with theincrease of BA and 2iP concentration, on the contrary, the shoot lengthincreased with the increase of Kin concentration (Table 3 and Fig. 1B).

Small pieces of leaves (1.0 g)

theoot

No. ofroots perexplant

Length of thelongest root(cm)

No. ofshoots perexplant

Length of thelongest shoot(cm)

No. ofroots perexplant

Length of thelongest root(cm)

0.5e 1.0b 8.3cd 3.0a 15.0a 3.7ab

0.3e 1.3b 14.9bc 3.0a 6.0bcd 2.8ab

0.5e 1.3b 18.7b 2.8° 6.7bc 3.5ab

4.3cde 1.0b 29.a 3.0a 9.7b 4.5a

evel of probability according to LSD test.

Table 5The effects of concentrations of IAA, IBA and NAA on root induction of Pilosocereus robinii.

Growth regulator(mg/L)

No of rootsper explant

Length of theroot (cm)

Plant freshweight (g)

Plant height(cm)

IAA IBA NAA

0.0 0.0 0.0 4.6⁎a 2.0a 0.7ab 1.5a

0.0 0.0 0.1 3.86ab 1.86a 0.69ab 0.86b

0.0 0.0 0.2 2.0cde 1.5ab 0.24b 1.1ab

0.0 0.0 0.4 2.3bcde 1.5ab 0.68ab 0.83b

0.0 0.1 0.0 3.0abcd 1.5ab 0.52b 1.0ab

0.0 0.2 0.0 3.67abc 1.33ab 0.41b 0.67b

0.0 0.4 0.0 1.5de 1.08ab 0.82ab 1.0b

0.1 0.0 0.0 0.83e 0.75b 0.76ab 1.25ab

0.2 0.0 0.0 2.3cde 1.25ab 0.42b 0.83b

0.4 0.0 0.0 0.67e 0.27b 1.3a 1.33ab

⁎ Means followed by the same letter within a column are not significantly different at0.05 level of probability according to LSD test.

317S. Khattab et al. / Gene 533 (2014) 313–321

Hyperhydration of the tissues is a serious problem for in vitro cultureof cacti. This physiological disorder is due to the physical and chemicalconditions of in vitro culture, i.e., high humidity, excess of carbohydratesandminerals, high levels of plant growth regulators and low light inten-sity (Elias-Rocha et al., 1998; Pérez-Molphe-Balch et al., 1998; Quiala

1 2 3 4 5 5 4 3 2 1 5 4 3 2 1

1 2 3 4 5 5 4 3 2 1 1 2 3 4

1 2 3 4 5 1 2 3 4 5

Fig. 2. ISSR-PCR pattern of Pilosocereus robinii with 12 primers, M refers to the DNA marker, (1respectively.

et al., 2009). In this work, hyperhydration was presented in allkinds of the used cytokinins only in case of the high concentrations,i.e., 4.0 mg/L (data not shown).

The preliminary experiment using entire leaf, shredded leaf and rootsegments cultured on hormone-free MS basal medium revealed that allexplants did not regenerate shoots effectively and became pale and ne-crotic after a period of 6–8 weeks. On MS medium supplemented withTDZ, shoot regeneration was not observed in case of using root derivedexplants. On the other hand, most of the shoot tips, stem cuttings andthe finely-cut stem had shown good shoot regeneration using thesame previous medium. The number of adventitious shoots varieddepending on the explant type and TDZ concentrations (Table 4,Figs. 1D, E and F). TDZ at higher concentration (0.02 μM) proved to bethe best treatment for direct shoot regeneration from finely-cut stem(29 shoots per culture) (Table 4). Hyperhydration was present only inthe regenerated plants from finely-cut stem explants (Fig. 1F).

3.2. Rooting and acclimatization

Rooting efficiency was found to be 100% in case of the three testedauxins. MS hormone-free media induced the highest number of roots,root length and explant height (4.6 root per explant, 2.0 cm and

1 2 3 4 5 5 4 3 2 1

5 1 2 3 4 5 1 2 3 4 5

) is the control plant, (2, 3, 4 and 5) refer to regenerated plants on BA, Kin, 2iP and TDZ,

Table 6Number of total amplified fragments, number of polymorphic bands and percentage of polymorphic bands of five Pilosocereus robinii regenerants, one-year old, based on ISSR-PCR analysiswith 12 primers.

Treatment ISSR primers

Total Sh1 Sh2 Issr2 Issr3 Issr10 Sh3 Sh4 Sh5 Sh6 Sh7 Sh8 Sh9

TAF 494 38.0 42.0 43.0 27.0 54.0 34.0 24.0 45.0 33.0 68.0 52.0 34.0

MB 36 4.0 0.0 5.0 3.0 2.0 3.0 1.0 5.0 1.0 5.0 4.0 3.0

PB 458 34.0 42.0 38.0 24.0 52.0 31.0 23.0 40.0 32.0 63.0 48.0 31.0

PB% 54.2% 55.56% 63.6% 75% 57.14% 61.5% 14.3% 80% 46.2% 81.8% 44.5% 33.33% 37.5%

Control AF 8.0 5.0 11.0 6.0 8.0 6.0 7.0 9.0 6.0 13.0 8.0 6.0SM 0.0 0.0 0.0 0.0 0.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0

BA AF 4.0 10.0 5.0 6.0 13.0 7.0 6.0 9.0 5.0 13.0 11.0 6.0SM 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0

Kin AF 9.0 8.0 8.0 6.0 13.0 7.0 3.0 12.0 7.0 13.0 11.0 6.0SM 0.0 1.0 0.0 0.0 0.0 0.0 1.0 2.0 0.0 0.0 0.0 0.0

2iP AF 8.0 9.0 10.0 3.0 7.0 7.0 3.0 8.0 10.0 14.0 11.0 8.0SM 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

TDZ AF 9.0 10.0 9.0 6.0 13.0 7.0 5.0 9.0 5.0 15.0 11.0 8.0SM 0.0 2.0 1.0 3.0 0.0 0.0 1.0 0.0 1.0 1.0 0.0 1.0

Total AF 38.0 39.0 42.0 24.0 54.0 33.0 21.0 45.0 32.0 66.0 52.0 33.0SM 0.0 3.0 1.0 3.0 0.0 1.0 3.0 2.0 1.0 2.0 0.0 1.0

TAF = total amplified fragment, MB = monomorphic bands, PB = polymorphic bands, AF = amplified fragments, SM = specific marker.

318 S. Khattab et al. / Gene 533 (2014) 313–321

1.5 cm, respectively) (Table 5 and Fig. 1C). The lowest root number(0.67 root per explant) and root length (0.27 cm) were recognized incase of using a medium containing 0.4 mg/L NAA. The use of MS medi-um for rooting of the in vitro induced shoots has already been reportedfor P. robinii (Quiala et al., 2009). Many cacti were found to produce anexcess of auxin under in vitro culture conditions, hence facilitating thein vitro rooting in hormone-free MS media (Clayton et al., 1990).

The success of any in vitro regeneration protocol largely depends onthe survival and growth performance of the propagated plantletsex vitro (Joshi and Dhar, 2003). In the present study, the applied accli-matization procedures were successful, where the in vitro regeneratedplantlets had shown a 100% survival rate when transferred to soil andshowed a vigorous growth.

3.3. ISSR analysis

Tissue culture is a mutagenic technique that causes cytogenetic,genetic and epigenetic variation collectively referred to as tissue cultureinduced or somaclonal variation. Genetic diversity is one of the mostimportant factors for crop improvement. (Petolescu et al., 2011). Plantregeneration from P. robinii was used for detection and exploitationof DNA polymorphism using both protein SDS–PAGE and ISSR-PCRmarkers. DNA from four in vitro regenerated plants aswell as the controlplant (one-year old) of P. robinii was analyzed using 12 ISSR primers(Table 1 and Fig. 2). The number of total bands, the polymorphism,the percentage of polymorphism and specific marker bands werepresented in Table 6. The total number of amplified amplicons was494 fragments and the number of amplified DNA fragments by eachprimer ranged from 24 to 68 fragments. Sh7 ISSR primer amplifiedthe highest number of fragments (68 bands), while sh4 ISSR primer

Table 7Specific ISSR markers linked to tissue culture conditions for five Pilosocereus robinii regenerant

Treatment Molecular length (ML) for specific marker bands bas

Total Sh2 Issr2 Issr3 Sh3

Control 2 0 0 0 9835BA 1 0 0 0 0Kin 1 0 0 0 02iP 0 0 0 0 0TDZ 13 8592.5 bp 9112.6 bp 11224.5 bp

6386.9 bp 8533.6 bp8078.2 bp

produced the lowest number of amplicons (24 bands). All the primersused were found to produce polymorphic bands (Fig. 2 and Table 6),ranging in number from 23 to 63 bands. The percent of polymorphismrevealed by the different primers ranged from 14.3% for sh3 ISSR primerto 81.8% for sh6 ISSR primer. Nine primers were found to produce spe-cific markers for the effect of the used tissue culture phytohormoneswith different molecular length. Data revealed that the regenerated ex-plants on TDZ were the highest in inducing new specific marker bands,as there were 13 new bands that appeared with eight ISSR primers(Table 7 and Fig. 2). In vitro culture is usually associated withsomaclonal variation; it is necessary to establish a micropropagationsystem that produces genetically identical and stable plants, especiallyin the case of commercial plants, and to perform ISSR technique to eval-uate genetic variation which is more effective and reproducible forquick assessment of the genetic polymorphism of plants. The resultsobtained proved relatively genetic polymorphism related to tissue cul-ture conditions which had a significant effect on inducing an individualvariability or somaclonal variation. These results showed good discrim-inating power of the ISSR techniques for detecting the genetic varianceamong the studied samples (Abu-Qaoud et al., 2010; Frederic et al.,2007; Gaowka et al., 2007; Guo et al., 2007; Noor et al., 2012; Petolescuet al., 2011; Semagn et al., 2006).

3.4. SDS–PAGE analysis

For detecting changes in gene expression on four random in vitroderived plantlets as well as control plants, SDS–PAGE analysis of totalcellular proteins was used. The results of protein marker were showedin Fig. 3 and Table 8. Data revealed a total of seven monomorphicbands; the obtained bands were equal in number but some of them

s, one-year old, based on ISSR-PCR analysis with 12 primers.

ed on ISSR primers

Sh4 Sh5 Sh6 Sh7 Sh9

.7 bp 10475.7 bp 0 0 0 00 0 0 4554.1 bp 04072 bp 0 0 0 00 0 0 0 07058.1 6309.4 bp 5867.1 bp 6749.4 bp 6315.7 bp

5733.6 bp4924.022 bp

250 130 95 72 55

36

28

KDa

4 3 2 1 C M

Fig. 3. Electrophoretic patterns of Pilosocereus robinii based on SDS–PAGE of total plantproteins. M is the molecular weight marker, © is the control plant, (1, 2, 3 and 4) referto regenerated plants on BA, Kin, 2iP and TDZ, respectively.

0 1 2 3 4 5 6 7 8 9 10

Minutes

Det

ecto

r re

spon

se

Malic Acid

Fig. 4. HPLC chromatogram of organic extract of (2iP) Pilosocereus robinii showing malicacid.

319S. Khattab et al. / Gene 533 (2014) 313–321

had shown a difference in density (Fig. 3). The previous data showed novariation in protein pattern of the studied treatments. We concludedthat the genetic changes obtained in the studied propagated plantletswill be of impact on the protein in the following generations when theobtained DNA polymorphisms make an effect in genes expression.SDS–PAGE has been used widely in many studies to identify variationin protein pattern of many species (Kamikouchi et al., 2004; Khattaband El Sherif, 2011; Lortal et al., 1997).

3.5. Malic acid — HPLC method and total phenolic content

Crassulacean acid metabolism (CAM photosynthesis) is the mecha-nism which cacti use in order to synthesize their nutritional reservoirof carbohydrates through fixing CO2 in the form of organic acidsmainlymalic acid during night before releasing the CO2 back at day to theCalvin cycle in order to synthesize sugars. This mechanism allows thestomata to remain shut during day to reduce evapotranspiration(Kelly, 1982). The total organic acids content in cacti is used to beexpressed as malic acid (Nobel, 2002).

A new single, isocratic, selective reversed phase liquid chromato-graphic method has been developed for quantification of malic acid –

the main organic acid in the extracts – at different multiplication stagesof the in vitro cultures of P. robinii. Themethod allowed good separationand quantification of malic acid within 10 min. The average retentiontime ± standard deviation for malic acid was found to be 5.75 ±0.03 min. The selectivity of the RP-HPLC is illustrated in (Fig. 4),where there was a good separation of malic acid from the other compo-nents of the extract at different manipulations of the experiment. TheHPLC method was selective for malic acid. It was able to detect malicacid in the complex natural extract with minimal interference withother compounds in the extract. Peak purity was confirmed by compar-ing the response of malic acid at 2 different wavelengths over time[210 nm (UV maximum) and 235 nm] (Table 9). Only one sharp peak

Table 8Effect of different tissue culture phytohormones on protein electrophoretic patternCommassie Blue-R-250 stained protein gel of five Pilosocereus robinii regenerants explant.

M.W. values (kD) Lane 1Control

Lane 2BA

Lane 3Kin

Lane 42iP

Lane 5TDZ

Lane 6Marker

Band 1 120.033 118.069 118.069 120.033 122.010 250.000Band 2 98.133 99.770 101.450 104.929 104.929 130.000Band 3 82.191 85.586 86.740 89.074 89.074 95.000Band 4 49.706 49.706 49.706 51.498 51.498 72.000Band 5 36.780 36.978 37.385 37.385 37.178 55.000Band 6 34.490 34.305 33.754 33.937 34.120 36.000Band 7 26.578 26.044 27.111 26.756 26.756 28.000

for each component appeared on both wavelengths. The absorbanceratio in which the slice area is divided at wavelength 210 by the slicearea of wavelength 235 was found to be constant throughout all corre-sponding slices of the peaks (Drouen et al., 1984; Law and Das, 1987).Malic acid concentrations in correlation to different external phytohor-mone concentrations were listed in Table 9. Malic acid concentration inthe in vitro regenerated plantlets as well as the control plantlets at theage of 5 and 12 monthswas determined.Malic acid showed a peak con-centration of 0.299% dry weight in the micropropagated plants on MSmedium supplemented with 2iP (12 months), while the lowest malicacid content of 0.0003% dry weight was observed in MS hormone-freemedia (Table 9). The age of the plantlets was a crucial element for or-ganic acids accumulation as appears in Table 9 for the control treatmentof 5 months old and that of 1 year old where the concentration wasshifted up in the latter compared to the former one. At the same timethe treatment raised on BA-enriched mediumwas found to accumulateabout seven times more organic acids at the age of 1 year compared tothat of 5 months old and raised at the same conditions. The effect ofculture time on the accumulation of the chemical constituents withinthe tissues of in vitro cultured plants was previously reported (Ibrahimet al., 2009).

It is well-known that phenolic compounds contribute to qualityand nutritional value in terms of modifying color, taste, aroma, flavorand also in providing health beneficial effects (Vanisree et al., 2004).

Table 9The effect of different medium modifications on the % dry weightaccumulation of malic acid content in the cultures of Pilosocereusrobinii.

Treatments Malic acid % dry wt.

2iP 0.2994 ± 0.0006BA 0.1146 ± 0.0005Kin 0.0009 ± 0.00002Control 0.0003 ± 0.00007Controla 0.0140 ± 0.00009BAa 0.0150 ± 0.00005

a Micropropagated plants of 5 months old.

Table 10The effect of different medium modifications on the accumulation of phenolicconstituents in the cultures of Pilosocereus robinii.

Treatments Phenolic content mg GAEa/g dry wt.

Controla 13.18BAa 13.18Kin 12.272iP 0Control 11.8BA 11.8

GAE: gallic acid equivalent.a Micropropagated plants of 5 months old.

320 S. Khattab et al. / Gene 533 (2014) 313–321

They also serve in plant defense mechanisms to counteract reactive ox-ygen species (ROS) in order to survive and prevent molecular damageand damage by microorganisms, insects and herbivores (Sengulet al., 2009). The phenolic compounds were found in allmicropropagated plants except the micropropagated plants on MSmedium supplemented with 2iP (Table 10). The total phenolic con-tents of the investigated in vitro treatments are shown in Table 10.

4. Conclusion

Here we report the first study to be oriented towards micro-propagation alongside with in vitro regeneration of the endangeredkey tree-cactus P. robinii from stem cuttings, shoot tips and the finely-cut shoot explants. Assessment of the genetic stability in the cultivatedplants is also reported. ISSR-PCR technique proved relatively geneticpolymorphism related to tissue culture conditions and showed thatregenerated explant on MS media supplemented with TDZ was thehighest one in inducing new specific marker bands. Protein analysisshowed no variance in the studied generation since we expectedchanges in the following generation when the obtained genetic poly-morphism expressed. A new single, isocratic, selective reversed phaseliquid chromatographic method has been developed for quantificationof malic acid as a representative of the organic acids in this plant; onthe other hand the total phenolic content was determined in order toassess the secondary metabolites profile of these in vitro propagatedplants. The phytochemical analysis of P. robinii was meant to expandits nutraceutical and pharmaceutical utilization.

Conflict of interest

There is no conflict of interest.

Acknowledgments

All thanks to Tissue Culture Laboratories, Department of Horticultureand Department of Pharmacognosy at Faculties of Agriculture andPharmacy, Suez Canal University, Ismailia, Egypt.

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