DEVELOPMENTAL BIOLOGY/MORPHOGENESIS

Regeneration via somatic embryogenesis of the endangeredwild arum (Arum palaestinum)

Rida A. Shibli & Mahmud A. Duwayri &Jamal S. Sawwan & Mohamad A. Shatnawi &Tamara S. Al-Qudah

Received: 9 May 2011 /Accepted: 26 March 2012 /Published online: 3 May 2012 / Editor: Eric Bunn# The Society for In Vitro Biology 2012

Abstract Somatic embryogenesis was obtained from callusof wild arum (Arum palaestinum). Callus was induced fromsterilized corm bud sprouts cultured on basal medium contain-ing 4.4 μM 6-benzyladenine and 5.4 μM 1-naphthaleneaceticacid. Callus was maintained under dark conditions using basalmedium with 4.4 or 8.8 μM 6-benzyladenine and 5.4 or10.8 μM 1-naphthaleneacetic acid. The highest callus weightand most desirable callus phenotype were achieved usingbasal medium containing 8.8 μM 6-benzyladenine and5.4 μM 1-naphthaleneacetic acid. Friable calli were culturedin the dark on basal medium containing 4.5 μM 2, 4-dichlorophenoxyacetic acid, 0.46 μM 6-furfurylaminopurine,5.4 μM 1-naphthaleneacetic acid, and 1.7 mM proline toinduce embryogenesis before transfer to regeneration medium.Embryos that developed on regeneration medium were trans-ferred to medium minus plant growth regulators for germina-tion. Ninety percent of the germinating embryos developedinto rooted plantlets. Rooted plants were grown in the green-house and acclimatized successfully with a 95 % survival rate.This is the first report of successful somatic embryogenesis andplant regeneration in A. palaestinum.

Keywords Arum palaestinum . Callus . Embryogenesis .

Regeneration

Introduction

Arum palaestinum is a tuberous perennial plant belonging tothe Araceae family (Boyce 1993; Mayo et al. 1997; Al-Loziet al. 2008; Makhadmeh et al. 2010). In Jordan, A. palae-stinum is commonly known as “al-loof” and is widely usedas a spice, which is cooked like a leafy vegetable. A. palae-stinum is also used as a homeopathic treatment for cancer,circulatory system disorders, obesity, internal bacterial infec-tions, diabetic symptoms, and poisoning (Al-Eisawi 1982; Al-Lozi et al. 2008; Makhadmeh et al. 2010). The plant containsalkaloids, flavonoids, and other medicinal components (Wil-liams et al. 1981; El-Desouky et al. 2007). A. palaestinum isone of about 26 species of Arum, a genus native to Europe,northern Africa, and western Asia, with the highest speciesdiversity in the Mediterranean region. In Jordan, A. palae-stinum grows naturally in the hills and mountainous rockyplaces near water; and in the upper Jordan valley in manyregions, including Ajlun, Irbid, Jarash, Al Balqa’, WadiShua’ib, and Amman (Al-Eisawi 1982; Feinbrun-Dothan1986; Al-Lozi et al. 2008; Makhadmeh et al. 2010). Becauseof overexploitation through uprooting and continuous remov-al of the plants, natural habitat destruction, increasingdemands on the plant as a food or medicine, and rural devel-opment, A. palaestinum populations are in danger of extinc-tion in the wild. Thus, there is a need to establish a reliablestrategy for multiplying and conserving germplasm of thisplant (Oran 1994; Oran and Al-Eisawi 1998; Al-Lozi et al.2008; Makhadmeh et al. 2010). In vitro propagation, whichincludes the use of somatic embryogenesis, is one of the mosteffective methods for propagation and multiplication of

R. A. Shibli (*) :M. A. Duwayri : J. S. SawwanDepartment of Horticulture and Agronomy, Faculty of Agriculture,University of Jordan,Amman, Jordane-mail: r.shibli@ju.edu.jo

M. A. ShatnawiDepartment of Agricultural Biotechnology, Faculty of Agriculture,Al-Balqaa Applied University,Salt, Jordan

T. S. Al-QudahHamdi Mango Center for Scientific Research (HMCSR),University of Jordan,Amman, Jordan

In Vitro Cell.Dev.Biol.—Plant (2012) 48:335–340DOI 10.1007/s11627-012-9438-z

medicinal plants (Mostafa et al. 2010; Al-Qudah et al. 2011;Shatnawi et al. 2011). Somatic embryogenesis allows theproduction of large numbers of genetically uniform plantswhich can have the same growth characteristics as seed-derived plants (Shibli and Ajlouni 2000; Shibli et al. 2001;Duquenne et al. 2006; Abdul Karim and Ahmed 2010). In thisstudy, a protocol for plant regeneration via somatic embryo-genesis was developed because A. palaestinum plant estab-lishment from seeds has been difficult in vitro.

Somatic embryogenesis from a number of different tis-sues has been reported for several genera in the Araceae.Leaf tissues were used as explants for somatic embryogen-esis in Anthurium scherzerianum and Anthurium andraea-num (Matsumoto et al. 1996; Hamidah et al. 1997), whereasseeds were used for Pinellia pedatisecta (Wu et al. 1996),and stem apices were used for Xanthosoma sagittifolium(Gomez et al. 1992). In Spathiphyllum, somatic embryogen-esis was induced from anther filaments (Werbrouck et al.2000) and from etiolated nodes of a single genotype (Chenand Kuehnle 1999). Propagation by somatic embryogenesishas the advantages of scale-up for propagation using bio-reactors and production of synthetic seeds (Rani and Raina2000). Additionally, direct somatic embryogenesis has alower probability of introducing genetic variation than otherpropagation methods (Merkle 1997).

Until now, no reports exist in the literature on the in vitroestablishment or regeneration of A. palaestinum. Develop-ing a protocol for callus establishment and somatic embryo-genesis is the first step toward the in vitro conservation andcryopreservation of this valuable, threatened genetic re-source. Hence, this study was conducted to develop a pro-cedure for in vitro callus establishment, rapid multiplicationvia somatic embryogenesis of wild arum (A. palaestinum),and production of disease-free propagules for Arum.

Materials and Methods

Plant material collection and sterilization. Corms of A.palaestinum L. were collected from the wild in Januaryand February 2010 from Wadi shua’ib, Salt, Jordan (long31°57′34″ E, lat 35°43′04″ N). Corms were surface steril-ized by washing thoroughly under running tap water for30 min with a few drops of mild detergent until all soiland other debris were removed. The corms were then cutinto sections containing sprouts, washed thoroughly underrunning tap water for 15 min with antibacterial soap 5 ml l−1

Devomycin (Norbrook Laboratories Ltd, Newry, Ireland)and then washed thoroughly with a solution containing1.0 %w/v Benomyl (Changzhou Good-Job BiochemicalCo., Ltd, Jiangsu, China). The corm sections were trans-ferred to a laminar air-flow cabinet and placed in an anti-septic solution of 5.5 %v/v sodium hypochlorite for 7 min,

followed by three rinses with sterile distilled water (15 minper rinse). The corm sections were transferred into 70 %v/vethanol for 30 s, and then rinsed 3× with sterile distilledwater (15 min per rinse). The corm sections were then usedfor callus induction.

In all experiments, the basal medium (BM) containedMurashige and Skoog (1962) mineral nutrients and vitaminswith the following: 3 %w/v sucrose (except in the experi-ments that used different concentrations of carbohydratesource; sucrose, fructose, or glucose for embryogenesis);and 0.8 %w/v Difco Bacto agar (Voigt Global DistributionINC, Kansas, USA). The pH was adjusted to 5.8. Flasks andtest tubes containing BM were closed and autoclaved at121°C and 1.5 kg/cm2 pressure for 20 min. All growthregulators were autoclaved in the medium, except gibberel-lic acid (GA3), which was filter sterilized and added to thesterilized medium. The medium also contained 0.03 %w/vpolyvinyl pyrrolidone to avoid browning caused by pheno-lic exudations.

Callus induction. Several different explants (leaf, stem,corm sections with bud sprouts, and other corm sections)were evaluated and sterilized by a method similar to thatused for the corm sections with sprouts. Sterilized explantswere cultured on BM supplemented with 4.4 μM 6-benzyladenine (BA) and 5.4 μM 1-naphthaleneacetic acid(NAA). However, the only explants that developed calluswere the sections of corm with bud sprouts. Calli weresubcultured every 3 wk and maintained in the dark. Allcallus culture experiments in this study were carried out in9-cm Petri dishes containing 12 ml medium.

Callus multiplication and maintenance. Conditions for cal-lus multiplication and maintenance were tested by transferring0.5 g friable callus onto BM supplemented with differentconcentrations of 4.4 or 8.8 μM BA and 5.4 or 10.8 μMNAA. In a second experiment, we tested different concentra-tions of 4.5, 9.0, or 13.5 μM 2, 4-dichlorophenoxyacetic acid(2, 4-D) and thidiazuron (TDZ) in combination with 5.4 μMNAA. Data on callus weight and texture were collectedafter 5 wk.

Induction of embryogenesis. Friable callus was transferredto embryogenesis induction medium containing 4.5 μM 2,2, 4-D, 0.46 μM 6-furfurylaminopurine, 5.4 μM NAA, and1.7 mM proline and incubated in the dark in the growthroom (24±1°C). Callus fresh weight (FW) was recordedat the time of transfer to embryogenesis-inducing BM.After 4 wk, callus was transferred to regeneration mediumcontaining 0, 4.4, 8.8, or 13.2 μM BA, and 0, 4.9, 9.8,14.7 μM 2-isopentenyl adenine or 0, 4.6, 9.2, 13.8 μM 6-furfurylaminopurine in combination with 0.49 μM indole-3-butyric acid (IBA), 0.45 μM 2, 4-D, and 1.7 mM

336 SHIBLI ET AL.

proline. Callus was then incubated in a growth room(16-h light/8-h dark, photosynthetic photon flux density(PPFD)040–45 μmol m−2 s−1, 24±1°C).

In another experiment, callus fragments were also sub-cultured onto embryogenesis induction medium containing4.5 μM 2,4-D, 0.46 μM 6-furfurylaminopurine, 5.4 μMNAA, and 1.7 mM proline; with increasing concentrations(1, 3, and 5 %, w/v) of sucrose, fructose, or glucose. Afterculture in the dark for 4 wk, the callus pieces were trans-ferred to regeneration medium containing 4.4 μM BA,0.45 μM 2, 4-D, 0.49 μM IBA, and 1.7 mM proline. Inboth experiments, after 4 wk, data were collected on thenumbers of somatic embryos and shoots developed on re-generation medium. Somatic embryo and shoot numbers pergram of callus subcultured onto regeneration medium wererecorded.

In vitro rooting. The somatic embryos produced on callusfrom the different experiments were transferred to BM (minusplant growth regulators) in 250-mL Erlenmeyer flasks con-taining 50 mL solid BM to allow embryo development and toproduce rooted plantlets. Callus fragments were divided intopieces of approximately 0.4–0.5 cm in diameter to improveembryo rooting. Data were collected after 4 wk on the numberof rooted plantlets and the number of embryos developingsecondary embryos.

Ex vitro acclimatization. Rooted plantlets from the differentexperiments were transferred to acclimatization BM in 250-mL Erlenmeyer flasks containing 2.9 μM GA3, 1.3 μM BA,and 0.3 μM indole-3-acetic acid (IAA) to enhance shoot androot development in the growth room (16-h light/8-h dark,[PPFD]040–45 μmol m−2 s−1, 24±1°C).

After 4 wk, plantlets with well-developed roots wereremoved from the culture medium. The roots were washedgently under running tap water to remove the adheringmedium, and the plantlets were transferred to plastic cups(10-cm diameter) containing growing medium (1:1 peat/perlite). Each cup was covered with a perforated plasticbag to reduce evaporation. The plants were irrigated withdistilled water every 2 d for 3 wk, then by tap water every2 d for another 2 wk. The potted plantlets were maintainedinside the culture room (16-h light/8-h dark, PPFD040–45 μmol m−2 s−1; 24±1°C) to acclimatize for 3 wk and thentransferred to the greenhouse conditions (24±2°C day/20±2°C night) with overhead irrigation for 3 wk (once a day).

Experimental design and statistical analysis. Treatmentswere arranged in a completely randomized design. Eachtreatment was replicated 5×, and each experiment was per-formed twice. Each Petri dish or flask was considered asingle replicate. The data were statically analyzed by usingthe Statistical Package for the Social Sciences analysis

system. Analysis of variance was used to determine the results.Means were compared by using Tukey’s Honestly SignificantDifference (HSD) test with a probability level of 0.05.

Results and Discussion

Callus induction and maintenance. Callus cultures of A.palaestinum were induced from bud sprouts of corms within4 wk. The induction rate was about 90 % on BM containing4.4 μM BA and 5.4 μM NAA. Other explant sources andmedia compositions did not give any callus induction (datanot shown). In Typhonium flagelliforme, the highest numberof shoots per tuber explant was obtained using 22.0 μM BAwith 5.4 μM NAA (Nobakht et al. 2009). Yang et al. (2008)found that Leonurus heterophyllus formed loose, yellowishcallus when subcultured on BM containing 8.8 μM BA and2.7 μM NAA.

For callus multiplication and maintenance, the highestcallus weight (4.7 g) and most desirable phenotype wasobtained using a medium containing 8.8 μM BA with5.4 μM NAA (Fig. 1C). Callus weight was significantlylower when both NAA and BA were used at 10.8 and8.8 μM; respectively (Fig. 1D). BA and NAAwere the onlygrowth regulators that were successful for callus inductionand maintenance. Callus failed to multiply when either TDZor 2, 4-D were used in combination with NAA (data notshown). In Punica granatum, the highest frequency of callusestablishment was obtained from cotyledon explants on BMcontaining 13.5 μM NAA and 13.2 μM BA (Deepika andKanwar 2010).

Figure 1. Effect of different concentrations of BA and NAA (A) 4.4 μMBAwith 5.4 μMNAA, (B) 4.4 μM BAwith 10.8 μMNAA, (C) 8.8 μMBAwith 5.4 μM NAA, and (D) 8.8 μM BAwith 10.8 μM NAA, on thecallus multiplication of A. palaestinum. Error bars represent the standarderror.Means with the same letter are not significantly different accordingto Tukey’s HSD test at probability level of ≤0.05.

REGENERATION IN WILD ARUM 337

Induction of embryogenesis. Friable callus with embryonicstructures were classified as embryogenic callus. Embryodevelopment from embryogenic callus started 3–4 wk aftertransfer to regeneration medium. Embryos turned green afterexposure to light, and some embryos started to germinate onthe surface of the callus. regeneration medium containing8.8 μM BA gave the highest numbers of embryos andshoots (Table 1). In this study, embryogenesis in the controltreatment (regeneration medium without BA, Kinetin, or 2-isopentenyl adenine (2ip)) was 25× less than for 8.8 μM BA(Table 1). Shoots from all treatments were regenerated onthe regeneration medium before transfer to BM (minus plantgrowth regulators). The regenerated shoots were green andmorphologically normal. Deepika and Kanwar (2010)reported regeneration of P. granatum on BM supplementedwith 13.5 μM NAA and 13.2 μM BA. In Zantedeschiahybrids, somatic embryogenesis was achieved only fromtuber explants on BM containing 2.6 or 8.8 μM BA incombination with 10.8 μM NAA (Duquenne et al. 2006).Chaurhury and Qu (2000) found that inclusion of 0.04 μMBA in Bermudagrass (Cynodon dactylon) callus inductionmedium-induced formation of a compact, nodular embryogenic

structure on approximately 20% of the calli; plants regeneratedfrom these calli were green and morphologically normal(Fig. 2).

Among the regeneration medium with 2iP, the mediumwith 4.9 μM 2iP resulted in the highest number of embryos(524.9 embryos/g callus FW) and shoots (201.8 shoots/gcallus FW; Table 1). In “Nabali” olive (Olea europea L), thehighest rate of embryogenesis (1,680 embryos/g callus FW)was obtained using a medium containing 9.8 μM 2iP (Shibliet al. 2001), whereas in Iris nigricans, a medium containing4.9 μM 2iP resulted in 2,686 embryos/g callus FW (Shibliand Ajlouni 2000). Somatic embryos of Zantedeschiahybrids developed into plantlets on BM supplemented with4.9 μM 2iP (Duquenne et al. 2006).

In the present study, kinetin was the least effective cyto-kinin for production of embryogenic callus, but it still gave410.8 embryos/g of callus and 146.2 shoots/g of callus at4.6 μM (Table 1). Somatic embryos were also producedfrom golden pothos (Epipremnum aureum) leaf and petioleexplants cultured on BM containing 9.3 μM kinetin and2.3 μM 2,4-D (Zhang et al. 2005). Embryogenic callus incumin (Cuminum cyminum L.) developed within 2 wk after

Table 1 Influence of the growthregulators in “regenerationmedium” on embryogenesisfrom callus of wild Arum (A.palaestinum) and the develop-ment of embryos on “BM(minus plant growth regulators)”

xOnly one control treatment wasconducted in each PGR treat-ment serieszMeans within columns (for eachgrowth regulator) havingdifferent letters are significantlydifferent according toTukey HSD at P≤0.05

Growth regulator(μM)

Regeneration medium BM (minus plant growth regulators)

No. embryos/gcallus

No. shoots/gcallus

No. rootedplantlets/g callus

No. embryos with secondaryembryogenesis/g callus

Kinetin

0.0x 20.0±1.9 dz 9.0±2.9 d 7.0±2.3 d 4.0±1.9 d

4.6 410.8±5.7 a 146.2±1.5 a 130.6±1.4 a 163.6±1.8 a

9.2 269.6±1.3 b 122.6±2.6 b 59.0±1.9 b 109.4±1.9 b

13.8 106.2±2.9 c 78.8±2.2 c 41.4±1.0 c 45.0±1.2c

BA

0.0 20.0±1.9 d 9.0±2.9 d 7.0±2.3 d 4.0±1.9 c

4.4 554.0±13.2 b 218.8±10.8 b 101.8±7.3 b 96.6±4.3 b

8.8 724.0±23.2 a 353.0±14.2 a 195.8±5.1 a 199.4±17.4 a

13.2 340.0±7.0 c 112.8±7.4 c 75.8±7.4 c 75.8±2.5b

2ip

0.0 20.0±1.9 d 9.0±2.9 d 7.0±2.3 d 4.0±1.9 d

4.9 524.89±7.1 a 201.8±1.2 a 105.0±1.7 a 128.2±1.8 a

9.8 305.2±4.3 c 89.6±3.7 c 58.6±1.3c 53.0±1.6 c

14.7 429.8±4.0b 116.0±15.8 b 89.4±2.0 b 89.8±2.3 b

A B CFigure 2. (A) shootregeneration of A. palaestinumon BM containing 8.8 μM BA,(B) shoot regeneration of A.palaestinum on BM containing4.9 μM 2iP, and (C) shootregeneration of A. palaestinumon BM containing 4.6 μMKinetin. Scale bar01.0 cm.

338 SHIBLI ET AL.

transferring the callus to BM (minus plant growth regula-tors); the presence of 2.3–4.6 μM kinetin in the callusinduction medium with 2,4-D enhanced both callus prolif-eration and subsequent differentiation of the embryos onhormone-free medium (Tawfik and Noga 2002).

Among the carbohydrate sources (sucrose, fructose, andglucose) and concentrations in embryogenesis inductionmedia tested here, 785.2 embryos/g callus developed aftertransfer of callus to an embryogenesis induction mediumcontaining 3 %w/v sucrose (Table 2). A similar result wasseen in I. nigricans, in which the maximum number ofembryos developed on BM containing 3.4 % sucrose (Shibliand Ajlouni 2000). In O. europea, increasing the sucrose,glucose, or fructose concentrations in the BM increasedembryogenesis, and maximum embryogenesis was obtainedwith 6.8 % sucrose (Shibli et al. 2001).

In this study, the most effective concentrations of fruc-tose and glucose were the lowest concentrations tested(1 %), whereas in studies of O. europea and I. nigricans,fructose and glucose gave maximum embryogenesis at3.6 % (Shibli et al. 2001; Shibli and Ajlouni 2000). In

carnation (Dianthus caryophyllus L.), development ofsomatic embryos was enhanced by increasing the sucroseconcentration from 1.5–12 %, whereas embryo developmentwas reduced at higher concentrations (15–18 %) (Karamiet al. 2006).

In vitro rooting. Splitting the embryogenic callus into twoto three pieces (0.4–0.5 cm) and subculturing onto BM(minus plant growth regulators) stimulated rooting andgermination of embryos. The highest number of roots onBM (minus plant growth regulators) was achieved fromthose shoots transferred from regeneration medium con-taining 8.8 μM BA (Table 1, Fig. 3A) or 3 %w/v sucrose(Table 2, Fig. 3B). Most of the plantlets produced onregeneration medium were rooted successfully in BM(minus plant growth regulators). Transferring the rootedplantlets to medium containing 2.9 μM GA3, 1.3 μM BA,and 0.3 μM IAA for 4 wk enhanced shoot and rootdevelopment prior to the acclimatization period in thegrowth chamber. In E. aureum, shoots and roots devel-oped on MS medium with no growth regulators; approx-imately 30–100 plantlets were regenerated from eachexplant (Zhang et al. 2005). In Syngonium podophyllum,up to 85 % of somatic embryos were able to germinateafter transferring to BM containing 8.8 μM BA and1.1 μM NAA, and approximately 50–150 plantlets wereregenerated from each petiole explant (Zhang et al. 2006).

Ex vitro acclimatization. Rooted plantlets were successfullyacclimatized in the growth chamber and had a 95 % survivalrate. The acclimatized plantlets were green and well devel-oped when transferred to the greenhouse. The plantlets pro-duced were phenotypically similar to each other and showednormal growth.

Table 2 Influence of sugarsource and concentration onembryogenesis from callus ofwild Arum (A. palaestinum) andthe development of embryos on“BM (minus plant growthregulators)”

zMeans within columns (for eachsugar source) having differentletters are significantly differentaccording to Tukey HSD atP≤0.05

Sugar sources(% w/v)

Regeneration medium BM (minus plant growth regulators)

No. embryos/gcallus

No. shoots/gcallus

No. rootedplantlets/g callus

No. embryos with secondaryembryogenesis/g callus

Sucrose

1 % 511.2±5.2 bz 234.6±2.0 b 112.6±1.2 c 97.6±0.8 b

3 % 785.2±9.5 a 400.4±2.6 a 214.4±3.4 a 178.8±0.7 a

5 % 335.8±4.8 c 104.8±1.9 c 161.4±1.8 b 65.4±1.2 c

Glucose

1 % 434.2±3.6 a 203.0±0.7 a 116.6±1.6 a 116.0±1.8 a

3 % 306.0±3.7 b 138.6±1.5 b 90.2±2.2 b 91.0±1.6 b

5 % 200.8±1.5 c 86.2±1.2 c 57.8±0.9 c 60.0±1.0 c

Fructose

1 % 399.4±0.8 a 169.4±0.74 a 127.0±6.8 a 193.8±2.1 a

3 % 325.6±1.6 b 146.2±1.5 b 97.2±0.8 a 99.0±0.7 b

5 % 215.0±2.0 c 94.6±1.5 c 45.0±1.1 c 53.6±2.2 c

A B

Figure 3. (A) rooted plantlets of A. palaestinum obtained on BMcontaining 8.8 μM BA, (B) rooted plantlets of A. palaestinum obtainedon BM containing 3 %w/v sucrose. Scale bar01.0 cm.

REGENERATION IN WILD ARUM 339

Conclusions

Efficient callus establishment and somatic embryogenesis fromcallus was possible for the first time in the endangered speciesA. palaestinum. The procedure described here can be used formass propagation and production of disease-free plants withoutaffecting wild populations. In addition, this procedure will beuseful for in vitro conservation or cryopreservation of thisvaluable genetic resource. The procedures developed in thisstudy could also be adapted for in vitro production of second-ary metabolites from Arum for medicinal purposes.

Acknowledgments The authors would like to thank the Deanship ofScientific Research at the University of Jordan, Amman, Jordan forfunding this research (project no. 1191).

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