Scientia Horticulturae 121 (2009) 223–227

Multiple shoot regeneration in seed-derived immature leaflet explantsof peanut (Arachis hypogaea L.)

Siddharth Tiwari, Rakesh Tuli *

Plant Molecular Biology and Genetic Engineering Division, National Botanical Research Institute,

Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow 226001, U.P., India

A R T I C L E I N F O

Article history:

Received 23 April 2008

Received in revised form 9 January 2009

Accepted 21 January 2009

Keywords:

Organogenesis

Peanut

Phytohormone

Plant regeneration

A B S T R A C T

A protocol was developed for organogenesis from immature leaflet explants derived from mature

seeds of peanut. Immature leaflets pre-incubated on MS medium supplemented with 13.32 mM

BAP + 4.95 mM NAA for 7 days, turned green and enlarged. The enlarged green leaflets produced multiple

shoot buds after 1–2 cycles of sub-culture on MS medium supplemented with 13.32 mM BAP. Three

cycles of shoot buds on the elongation medium (13.32 mM BAP) produced 6.17 � 0.47 elongated shoots

per explant. The shoot bud formation was genotype independent. All elongated shoots rooted on the medium

containing 4.95 mM NAA. The complete protocol gave efficient (>81%) direct organogenesis, leading to the

development of plantlets within 4 months.

� 2009 Elsevier B.V. All rights reserved.

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1. Introduction

Peanut or groundnut (Arachis hypogaea L.) has captured theattention of several researchers due to its highly nutritive value asa source of protein and oil. The crop suffers from many biotic andabiotic stresses which cause major losses in terms of quality andquantity. Conventional breeding has contributed towards theimprovement of peanut (Reddy et al., 1996; Garcia et al., 2006) butstrong interspecific barriers, low recovery of hybrids and linkage ofundesirable traits limit the introgression of variability from wildspecies (Halward et al., 1993; Tallury et al., 2005). Genetictransformation overcomes these limitations and allows introduc-tion of agronomically important genes across taxa (Li et al., 1997;Singsit et al., 1997; Yang et al., 1998; Magbanua et al., 2000;Sharma and Anjaiah, 2000; Livingstone et al., 2005; Tiwari et al.,2008). The availability of an efficient regeneration system ishowever an essential prerequisite for utilizing this approach.

Immature leaflets from young seedlings of peanut had beenutilized to achieve organogenesis (Mroginski et al., 1981; McKentlyet al., 1991; Cheng et al., 1992; Sukumar and Sree Rangasamy, 1984;Narasimhulu and Reddy, 1983; Akasaka et al., 2000; Chengalrayanet al., 2001). However, among those reports some had shown lowregeneration efficiency and excessive time required for the

* Corresponding author. Tel.: +91 522 2205848; fax: +91 522 2205839.

E-mail addresses: rakeshtuli23@rediffmail.com, rakeshtuli@hotmail.com

(R. Tuli).

Abbreviations: BAP, 6-benzylaminopurine; MS, Murashige and Skoog (1962)

medium; NAA, a-naphthalene acetic acid.

0304-4238/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2009.01.029

development of the regenerants. For instance, Mroginski et al.(1981) and Cheng et al. (1992) reported bud primordia developmentthat failed to regenerate normal plants. Sukumar and SreeRangasamy (1984) reported calli development in seven Arachis

spp. but none of them formed shoots. Narasimhulu and Reddy(1983) reported callus mediated plant development in only 19 % ofthe explants. Akasaka et al. (2000) reported various abnormalities inshoot development and low conversion rate (34.7%) from shoot budsto shoots. Mature zygotic embryo derived leaflet explants have beendeployed by Chengalrayan et al. (2001) to examine the effect ofphytohormones on organogenesis and somatic embryogenesis.

The present paper reports in vitro direct organogenesis fromimmature leaflet explants derived from zygotic embryos of matureseeds. Mature dry seeds guarantee year-round availability ofexplants for continuous research. The proposed protocol couldimprove peanut micropropagation with the potential of enhancingexogenous gene transfer.

2. Materials and methods

2.1. Plant materials and explant preparation

The pods of four peanut cultivars extensively cultivated insouthern states of India viz., JL-24 (Maharastra and Karnataka),TMV-2 (Tamil Nadu and Karnataka), TAG-24 (Maharastra, Karna-taka and Tamil Nadu) and Dh-3-30 (Karnataka) were obtainedfrom the University of Agricultural Sciences, Dharwad (Karnataka,India). Mature dry seeds were surface-sterilized (Tiwari and Tuli,2008) in 0.1% aqueous mercuric chloride for 10 min, rinsed 6–7times with sterile water and left soaked overnight in sterile water.

Fig. 1. Stages in the preparation of immature leaflet explants. (A) Seed coat peeled off from pre-soaked sterilized seed. (B) Embryonated cotyledon. (C) Embryo axis excised by

removing the cotyledon (arrow). (D) Immature leaflet lamina used as explant (arrow).

S. Tiwari, R. Tuli / Scientia Horticulturae 121 (2009) 223–227224

The embryo axes were excised by removing the cotyledonsaseptically from the sterilized seeds. The radical portion of embryoaxis was cut out and the immature leaflet lamina was used asexplant for formation of multiple shoots (Fig. 1).

2.2. Regeneration via organogenesis

Immature leaflets were cultured in petridish (94 mm diameter),on semi-solid medium containing MS salts (Murashige and Skoog,1962), B5 vitamins (Gamborg et al., 1968), 100 mg/l myo-inositoland 30 g/l sucrose supplemented with 6-benzylaminopurine (BAP)(13.32 mM) and a-naphthalene acetic acid (NAA) (4.95, 9.90 and14.85 mM). Twenty to twenty five leaflets were cultured for pre-incubation in each petridish. Three petridishes, containing a totalof 60–75 explants were cultured and incubated for 7, 15 and 30days. There were three replications in each experiment. Percentageof explants showing shoot development response was noted. Afterpre-incubation, the explants in the three lots were transferred toshoot formation and elongation medium. The shoot formation andelongation medium contained MS medium supplemented with13.32 mM BAP. Shoot buds appeared on the explants within threeweeks and increased in number to make clusters. Percent response,mean and standard deviation in each experiment were calculatedfrom data generated at this stage. Subsequently, shoot clusterswere sub-cultured on similar medium every three weeks forenhancing shoot elongation. After 3–4 cycles, the number ofelongated shoots was recorded. Shoots (3–4 cm) derived from theshoot clusters were excised and rooted on MS medium supple-mented with NAA (4.95 mM).

2.3. Genotypic response

Immature leaflet explants prepared from four peanut cultivarswere cultured for 7 days on pre-incubation medium andtransferred on shoot formation and elongation medium(13.32 mM BAP) for 3 cycles of 3 weeks each. The multiple shootbud formation (percent � SD) and shoot elongation (mean numberof elongated shoots per explant � SD) efficiency were compared.

2.4. Culture conditions and hardening

Throughout the study, the cultures were incubated at 25 � 2 8Cin 80 mmol photon m�2 s�1 light intensity with a photoperiod of 16/8 h. All the media were solidified with 0.8% agar. The pH was adjustedto 5.8 before autoclaving at 121 8C for 20 min. The hardening of theregenerated plantlets was carried out with irrigation in plastic potscontaining Soilrite mix (Keltech Energies Ltd., Bangalore, India). Potswere initially kept under plexiglass acclimatization hoods (Basco Pvt.Ltd., India) with 85% relative humidity for 15 days. For the next 15days, the hoods were raised gradually, decreasing the humidity. After

4 weeks, the plants were planted in sandy loam soil and kept in aglass-house till maturity.

All biochemicals and media constituents, unless stated other-wise were molecular biology/cell culture grade from SigmaChemical Company (St. Louis, MO, USA).

3. Results and discussion

The present report describes a rapid, reproducible and efficientprotocol for in vitro propagation of peanut. Earlier reports on in

vitro organogenesis in peanut showed strong influence of genotype(Mroginski et al., 1981; Seitz et al., 1987; McKently et al., 1990;Cheng et al., 1992; Banerjee et al., 2007; Matand and Prakash,2007) and culture conditions (Chengalrayan et al., 1995; Akasakaet al., 2000; Palanivel and Jayabalan, 2002; Vasanth et al., 2006;Tiwari and Tuli, 2008) on the response of explants.

Immature leaflet explants of peanut were reported torespond significantly in organogenesis (Mroginski et al., 1981;Cheng et al., 1992; Sukumar and Sree Rangasamy, 1984;Narasimhulu and Reddy, 1983; Seitz et al., 1987; Akasakaet al., 2000; Chengalrayan et al., 1995, 2001). Mroginski et al.(1981) used leaflets at different developmental stages andexposed them to MS medium supplemented with 12 combina-tions of NAA and BAP. The results showed that immature leafletexplants were most responsive and gave highest bud formationin 1 mg/l each of NAA and BAP containing medium. Seitz et al.(1987) reported that immature leaves induced up to 30% shootformation on the MS medium containing NAA (1 mg/l) and BAP(1 mg/l). Cheng et al. (1992) and Akasaka et al. (2000) reportedthat MS medium containing NAA and BAP was the mostpromising combination for shoot bud formation from immatureleaflet explants. In the present study, the zygotic embryoderived immature leaflet explants pre-incubated for 15 and 30days on MS medium supplemented with combinations of BAP(13.32 mM) and NAA (4.95 and 9.90 mM) showed very lowfrequency of shoot bud formation. The 13.32 mM BAP and14.85 mM NAA combination gave no shoot buds (Table 1).Seven-day pre-incubated leaflet explants formed no shoot budsin any combination, though they turned green and enlarged insize (Fig. 2A). When the cultures pre-incubated for differentdays were transferred to shoot formation and elongationmedium containing BAP (13.32 mM), the frequency of shootbud formation was substantially increased (Table 2). Seven daypre-incubated leaflets showed highest percent of multiple shootbud proliferation on the shoot formation and elongationmedium. Shoot bud clusters sub-cultured on the similarmedium resulted in enhanced shoot elongation. Therefore, thehighest shoot bud formation (81.5 %) and shoot elongation (6.17shoots/explant) were obtained when the explants pre-incubatedfor 7 days on medium containing 13.32 mM BAP and 4.95 mM

Table 1Interactive effect of phytohormone combination treatments and pre-incubation periods on multiple shoot bud formation in immature leaflets of JL-24 cultivar of peanut.

Treatment Phytohormone (mM) combination Shoot bud formation frequency (% � SD) during pre-incubation periods

BAP NAA 7 days 15 days 30 days

1 13.32 4.95 0 � 00 5.1 � 0.58 5.9 � 0.58

2 13.32 9.90 0 � 00 3.7 � 1.15 3.7 � 1.15

3 13.32 14.85 0 � 00 0 � 00 0 � 00

S. Tiwari, R. Tuli / Scientia Horticulturae 121 (2009) 223–227 225

NAA were transferred to the shoot formation and elongationmedium (Table 2). The results suggest that the physiologicalstatus of the explant, growth regulator combination, itsconcentration and exposure time determined response of theexplant. Initially the meristematic shoot buds were observed atthe base of lamina rachis (Fig. 2B). However, gradually the wholelamina turned into shoot clusters (Fig. 2C, D). These shootclusters proliferated and elongated on the shoot formation andelongation medium (Fig. 2D, E). In every cycle, loose watery calli

Fig. 2. Multiple shoot developmental steps from immature leaflet explants. (A) Green

containing 13.32 mM BAP and 4.95 mM NAA. (B) Shoot bud formation on leaflet in MS m

shoot buds after 1–2 cycles on MS medium containing 13.32 mM BAP. (D) Shoot cluster p

shoot on MS medium containing 13.32 mM BAP. (F) Rooted shoot on MS medium contain

the references to color in this figure legend, the reader is referred to the web version o

were excised out. After 3 cycles, the highest number(6.17 � 0.47) of elongated shoots per explant was recovered onthe shoot formation and elongation medium (Table 2). Multipleshoot buds cultured on the same medium proliferated new shootbuds on every sub-culture. This culture became a continuoussource of normal elongated shoots for 15–18 months. However,after this long period, the efficiency of normal shoot proliferationdeclined, morphologically abnormal somatic clones appeared andgave abnormal shoots and buds (data not shown). Normal

and enlarged immature leaflet as observed during pre-incubation on MS medium

edium containing 13.32 mM BAP after 3 weeks. (C) Leaflet turned into a cluster of

roliferated and maintained on MS medium containing 13.32 mM BAP. (E) Elongated

ing 4.95 mM NAA. (G) Tissue culture raised acclimatized plant. (For interpretation of

f the article.)

Table 3Genotypic response of multiple shoot bud formation from immature leaflet

explants cultured on BAP (13.32 mM).

Genotype Shoot bud formation

frequency (% � SD)

Average elongated

shoots/explant

JL-24 81.5 � 10.20 6.17 � 0.47

TMV-2 85.1 � 5.59 6.20 � 1.69

TAG-24 80.72 � 12.62 5.80 � 2.23

Dh-3-30 77.76 � 6.65 6.4 � 0.87

Table 2Effect of post-pre-incubation on multiple shoot development in explants of JL-24 cultivar sub-cultured from the three treatments in Table 1.

Pre-incubation

treatment

Multiple shoot development in post-pre-incubation period

7 days 15 days 30 days

Shoot bud formation

frequency (% � SD)

Average elongated

shoots/explant

Shoot bud formation

frequency (% � SD)

Average elongated

shoots/explant

Shoot bud formation

frequency (% � SD)

Average elongated

shoots/explant

1 81.5 � 10.20 6.17 � 0.47 36.6 � 9.23 2.40 � 0.59 13.3 � 3.13 1.53 � 0.51

2 28.1 � 6.23 2.30 � 0.67 30 � 7.12 1.12 � 0.64 16.6 � 6.34 1.10 � 0.55

3 5.9 � 4.26 2.08 � 0.50 30 � 6.14 1.90 � 0.56 16.6 � 7.13 1.95 � 0.49

In all cases the explants from medium in Table 1 were sub-cultured on shoot bud formation and elongation medium containing 13.32 mM BAP for 3 cycles of 3 weeks each.

S. Tiwari, R. Tuli / Scientia Horticulturae 121 (2009) 223–227226

elongated shoots were transferred to the rooting medium. Healthyroots were formed at the bottom of all shoots after three weeksincubation (Fig. 2F). All rooted plantlets survived when transferredto glass-house. No difference was observed in the phenotypes ofthese regenerated plants as compared to the seed-derived controlplants under glass-house conditions. The plants grew normally(Fig. 2G) and produced flowers and pods within 3–4 months, albeitthe number of pods developed from F0 tissue culture raised plantswas low as compared to that from seed-derived control plants(data not shown). In subsequent generation, the in vitro derivedplants performed as well as the seed-derived plants.

The present results on shoot bud formation from immatureleaflet explants are in agreement with the earlier reports wheresignificant response was observed on MS medium containingNAA and BAP. A plausible explanation for this response may bethat the plasticity of immature leaflet tissue lent it to enhancedcell division and resulted in the initiation of shoot buds. Matandand Prakash (2007) have also discussed that organogeneticplasticity and rejuvenating nature of the tissue taken from thebasal part of plant such as hypocotyl, cotyledonary node andimmature leaflet tissues were more responsive than the matureones. Unlike most of the earlier studies on peanut immatureleaflet organogenesis that had resulted in extended periodrequired for the development of normal plantlets (Mroginskiet al., 1981; McKently et al., 1991; Akasaka et al., 2000), thepresent protocol results in the development of rooted healthyplants within 4 months.

The proposed protocol was used to assess shoot regenerationpotential of immature leaflet tissue of four peanut cultivars(Table 3). The cultivars JL-24, TMV-2, TAG-24 and Dh-3-30 gave81.5 � 10.20, 85.1 � 5.59, 80.72 � 12.62, 77.76 � 6.65% shootformation, and an average of 6.17 � 0.47, 6.20 � 1.69,5.80 � 2.23, 6.4 � 0.87 elongated shoots/explants, respectively.Morphogenic developmental pattern of multiple shoot budformation and shoot elongation was similar to that described forJL-24 cultivar. Therefore, all the genotypes evaluated in the presentstudy responded equally well to conditions of multiple shoot budformation and shoot elongation. However, most of the earlierstudies on peanut organogenesis have reported a strong genotypicinfluence (Mroginski et al., 1981; Seitz et al., 1987; Banerjee et al.,2007; Matand and Prakash, 2007). Matand and Prakash (2007)

evaluated twenty-five peanut cultivars representing four markettypes from different countries. They reported that various explantsbelonging to different genotypic backgrounds cultured on MSshoot formation medium supplemented with thidiazuron (TDZ)responded differently. Most efficient response was observed inhypocotyl of Spanish and Valencia market types. Banerjee et al.(2007) evaluated organogenesis potential of tikka disease suscep-tible (four varieties) and resistant (one variety) Indian peanutcultivars. They found that different concentrations of BAP wererequired for efficient response of different varieties. In contrast Liet al. (1994), Sharma and Anjaiah (2000) and Tiwari and Tuli (2008)did not observe significant organogenic variations among thecultivated peanut varieties. The differences may be due to theextent of diversity among the genotypes selected in differentstudies.

In conclusion, the present protocol gives the advantage ofefficient shoot bud formation, shoot elongation and culturemaintenance on a single MS medium containing 13.32 mM BAP.The protocol allows continuous shoot formation through 15–18months and shows good response by a number of elite cultivars.Such cyclic regeneration system is ideally suited for genetictransformation research due to the ease of imposing antibioticselection on transformed tissues to eliminate transgenicchimeras. The present protocol is therefore a substantialimprovement over the immature leaflet explant derivedregeneration protocols reported earlier for this economicallyvaluable crop.

Acknowledgements

The authors express their gratitude to K.G. Parmeshwarappa,University of Agricultural Sciences, Dharwad for providing thepeanut germplasm, Council of Scientific and Industrial Research,New Delhi for funding the study to Siddharth Tiwari and toDepartment of Science and Technology, Government of India forJ.C. Bose Fellowship to Rakesh Tuli.

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