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Scientia Horticulturae 168 (2014) 33–37

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

hort communication

nhancement of regeneration efficiency in banana (Musa acuminatav. Berangan) by using proline and glutamine

urain Husina, Mahanom Jalil a,b, Rofina Yasmin Othmana, Norzulaani Khalida,∗

Centre of Biotechnology for Agriculture Research, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, MalaysiaCenter for Foundation Studies in Science, University Malaya, 50603 Kuala Lumpur, Malaysia

r t i c l e i n f o

rticle history:eceived 7 March 2013eceived in revised form 6 January 2014ccepted 11 January 2014

a b s t r a c t

Regeneration of Musa acuminata cv. Berangan (AAA) from embryogenic cell suspension was enhancedby using proline and glutamine. Sixteen embryo development media consisting of different concen-trations of proline and glutamine were tested. Glutamine was preferred as the source of organicnitrogen supplement compared to proline. The embryos developed in liquid media were shown to

eywords:ananaell suspensionmbryo developmentitrogen-supplemented regenerationomatic embryos

produce 10.6-fold higher number of plantlets in lesser duration (1.5 to 2.5 months) as compared tosolid media. Optimum somatic embryo development was observed in liquid media supplemented with400 mg/l glutamine (M4GL) with an average regeneration of 33,844 ± 402 plantlets per ml of settled cellvolume (SCV). Although both amino acids have proven to promote the embryo development, concen-trations of proline between 200 to 400 mg/l in liquid media caused abnormal embryo differentiation.

© 2014 Published by Elsevier B.V.

. Introduction

Embryogenic cell suspensions (ECSs) are comprised of rapidlyroliferating totipotent single cells. These totipotent single cellsake the embryogenic cells amenable for genetic manipulations

s well as an ideal system for mass propagation. Many publishedeports have described plant regeneration from somatic embryo-enesis in banana through callus intermediary derived from leafases and rhizome fragments (Kulkarni et al., 2006), highly prolif-rating scalps (Sipen et al., 2008), immature male flowers (Sidhat al., 2007, Wong et al., 2006 and Jalil et al., 2003,2008) and imma-ure female flowers (Grapin et al., 2000), and bract (Divakaran andair, 2011).

Based on the literature, to date, immature male flowers haveeen extensively used as the source of explants and mostly culturedn media established by Côte et al. (2006) to induce embryogenicallus. However, the current protocols for banana somatic embryo-enesis are constrained by low embryo germination concomitantly

eading to low plant regeneration rates and long cultural period.

Amino acids are source of organic nitrogen supplement duringulture. Previous studies in several species such as alfalfa, maize,

Abbreviations: 2,4-D, 2,4-Dichlorophenoxyacetic acid; BAP, benzylaminopurine;v, cultivar.∗ Corresponding author: Tel.: +60379677142; fax: +60379674178.

E-mail addresses: norzulaani@gmail.com, lani@um.edu.my (N. Khalid).

304-4238/$ – see front matter © 2014 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.scienta.2014.01.013

sorghum, pineapple, rice and other monocots have shown thatamino acid supplements could enhance somatic embryogenesisand in vitro plant regeneration (Claparols et al., 1993; Rao et al.,1995; Hamasaki et al., 2005; Grewel et al., 2006). It was due to theassociation of the organic nitrogen compared to inorganic sourceswith the ease of transportation of the former into the plant cells(Kim and Moon, 2007).

Although both amino acids have been widely used in manyplant species to improve somatic embryo maturation, study ofthe inclusion of amino acids into the culture media of bananafor embryo development and concomitant improvement in theregeneration was lacking. Therefore, this work was done tostudy the effect of L-proline and L-glutamine on somatic embryodevelopment of banana (cv. Berangan, AAA) and subsequentlydetermine the optimum concentration to improve the existingprotocol and develop the most efficient banana regenerationsystem.

2. Materials and Methods

2.1. Initiation and maintenance of ECS

Immature male flower clusters of Musa acuminata cv. Berangan

were cultured for six months to induce embryogenic calli, whichwere then used to initiate cell suspensions according to the methoddescribed by Jalil et al. (2003). ECSs were maintained in M2 propa-gation medium consisting of Murashige and Skoog (MS) (Murashige

34 N. Husin et al. / Scientia Horticulturae 168 (2014) 33–37

Table 1Composition of M3 development media tested. L denotes liquid M3 media and S denotessolidified M3 media.

Media Composition

MSOL MS basal medium, 20 g/l sucroseM1GL MS basal medium, 100 mg/l L-glutamine, 20 g/l sucroseM2GL MS basal medium, 200 mg/l L-glutamine, 20 g/l sucroseM3GL MS basal medium, 300 mg/l L-glutamine, 20 g/l sucroseM4GL MS basal medium, 400 mg/l L-glutamine, 20 g/l sucroseM1PL MS basal medium, 100 mg/l L-proline, 20 g/l sucroseM2PL MS basal medium, 200 mg/l L-proline, 20 g/l sucroseM3PL MS basal medium, 300 mg/l L-proline, 20 g/l sucroseM4PL MS basal medium, 400 mg/l L-proline, 20 g/l sucroseMSOS MS basal medium, 30 g/l sucrose, 2 g/l PhytagelTMM1GS MS basal medium, 100 mg/l L-glutamine, 30 g/l sucrose, 2 g/l PhytagelTMM2GS MS basal medium, 200 mg/l L-glutamine, 30 g/l sucrose, 2 g/l PhytagelTMM3GS MS basal medium, 300 mg/l L-glutamine, 30 g/l sucrose, 2 g/l PhytagelTMM4GS MS basal medium, 400 mg/l L-glutamine, 30 g/l sucrose, 2 g/l PhytagelTMM1PS MS basal medium, 100 mg/l L-proline, 30 g/l sucrose, 2 g/l PhytagelTM

crose,crose,crose,

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2

istapwm

M2PS MS basal medium, 200 mg/l L-proline, 30 g/l suM3PS MS basal medium, 300 mg/l L-proline, 30 g/l suM4PS MS basal medium, 400 mg/l L-proline, 30 g/l su

nd Skoog, 1962) basal medium supplemented with 1.1 mg/l 2,4-, 0.25 mg/l zeatin, 10 mg/l ascorbic acid and 20 g/l sucrose withH 5.7 prior to autoclaving. Observation on somatic embryo initia-ion was done for about 3–6 months culture. ECSs were subculturedvery 14 days and sieved through 425 �m mesh sieve every thirdubculture to maintain fine cells. Approximately 2 ml of settledell volume (SCV) was inoculated into 250 ml conical flask contain-ng 50 ml of M2 media. The cultures were maintained at 25 ± 1 ◦Cn orbital shaker at 80 rpm with a photoperiod of 16 h/day under1.4 �mol m-2s-1 light intensity provided by fluorescent lamps.xperiments were repeated twice with 3 replications done for eachreatment (3 flasks per treatment).

.2. Optimization of embryo development media (M3)

In this study, the influence of varying concentrations of L-prolinend L-glutamine in solid and liquid M3 embryo development mediaas compared. In liquid media, the solidifying agent was removed

Table 1). M3 media consists of MS basal supplemented with 30 g/lucrose solidified in 2 g/l phytagel (Duchefa, Netherland) and 20 g/lucrose in liquid media with varying concentrations of glutaminer proline. Glutamine or proline of concentrations at 0, 100, 200,00 and 400 mg/l were tested. All media were adjusted to pH 5.7rior to autoclaving.

ECSs in M2 media were sieved with 425 �m mesh sieve andllowed to settle in 50 ml Falcon tube. The SCV to M2 media wasdjusted to a ratio of 1:4. The cells were then suspended and 200 �las inoculated either in 20 ml of liquid M3 media or on 70 mm-iameter Whatman No. 1 filter paper placed on solid M3 media.ulture conditions for cells placed in M3 liquid medium were theame as the initiation stage, whereas cells in M3 solid media wereaintained in dark conditions until formation of somatic embryosas observed. Experiments were repeated twice with 3 replications

or each treatment (3 petri dishes containing cells per treatment).

.3. Shoot regeneration of somatic embryos

The mature embryos obtained from M3 media were culturedn M4 regeneration media containing MS basal, 6 mg/l BAP, 30 mg/lucrose and 2 g/l phytagel. After 30 days, the plantlets were subcul-ured onto M4 media with 3 mg/l BAP. Cultures were incubated in

photoperiod of 16 h/day under 31.4 �mol m-2s-1 light intensityrovided by fluorescent lamps for about 2 months. Experimentsere repeated twice with three replications done for each treat-ent (3 petri dishes containing somatic embryos per treatment).

2 g/l PhytagelTM 2 g/l PhytagelTM 2 g/l PhytagelTM

2.4. Rooting of shoots and plantlet acclimatization

Plantlets about 3 cm in height were cultured in M5 rootingmedia consisting of MS basal, 30 g/l sucrose, 1% (w/v) activatedcharcoal and 2.2 mg/l phytagel adjusted to pH 5.7. Cultures wereincubated for16 h/day under 31.4 �mol m-2s-1 light intensity pro-vided by fluorescent lamps. Plants were ready to be acclimatizedafter reaching about 8 cm minimum height. Acclimatization wasdone by transferring the plants into polybags containing soil mix-ture and placed in the netted nursery under natural condition (80%humidity, 28–30 ◦C, 2000–5000 lx, 12 h/day) for 1.5–2.0 monthsbefore transferring to the field.

2.5. Preparation for histological sections

Specimens selected for histological studies were fixed for 24 hin formaldehyde- absolute ethanol-acetic acid (FAA) solution con-sisting of 5% (v/v) formaldehyde, 45% (v/v) absolute ethanol and 5%(v/v) glacial acetic acid. The samples were then dehydrated in anethanol series; 30% for 30 min, 50% for 45 min, 70% for 45 min, 80%for 60 min and 100% for 120 min. Specimen were then embedded inbasic resin (Technovit 7100) and sectioned at 3.5 �m. Sections weredouble-stained with naphtol blue black (Sigma, USA) [1 g naphtholblue black in 100 ml 7% (v/v) acetic acid] (Fisher, 1968).

2.6. Statistical analysis

The data collected were analysed by ANOVA and the mean val-ues were tested to evaluate the significance at 1% confidence levelusing Tukey’s HSD test.

3. Results and Discussion

Development of Musa acuminata cv. Berangan somatic embryoswas affected by both media composition and phase (liquid andsolid). The embryos developed in liquid media were shown to pro-duce more plantlets in a shorter time compared to solid media(Table 2). The maximum number of plantlets obtained in liquid cul-ture medium (M4GL) was 10.6-fold higher than on solid medium(M4GS). This result was in agreement with previous study by Wonget al. (2006) on Musa acuminata cv. Mas (AA). This could be due toease of nutrient uptake into the cells where media and cells were

in close proximity.

Different amino acid supplements resulted in different effects ondevelopment and regeneration of Berangan somatic embryos. Glu-tamine was the preferred source of organic nitrogen compared to

N. Husin et al. / Scientia Horticulturae 168 (2014) 33–37 35

Table 2Effects of liquid and solid development media with different concentrations of L-glutamine or L-proline on the production and culture duration of banana cv. Beranganplantlets.

Media phase Embryo development media M3 Plantlets per ml SCV* Culture duration (months) Abnormality (%)

Liquid MSOL 9,902 ± 350f 2.5–3 0M1GL 17,341 ± 442d 1.5–2.5 0M2GL 20,961 ± 325c 1.5–2.5 0M3GL 25,602 ± 721b 1.5–2.5 0M4GL 33,844 ± 402a 1.5–2.5 0M1PL 15,430 ± 360e 2–2.5 0M2PL 16,336 ± 494e 2–2.5 0.8M3PL 17,775 ± 452d 2–2.5 3.2M4PL 16,400 ± 187d,e 2–2.5 5.7

Solid MSOS 186 ± 17j 3.5–5.5 0M1GS 1,352 ± 88h,i,j 2.5–3.5 0M2GS 1,803 ± 73g,h,i 2.5–3.5 0M3GS 2,917 ± 192g,h 2.5–3.5 0M4GS 3,181 ± 199g 2.5–3.5 0M1PS 539 ± 38i,j 2.5–4 0M2PS 1,098 ± 53i,j 2.5–4 0M3PS 1,850 ± 67g,h,i 2.5–4 0

21g,h

* rence

poaeisalebt

Fw(m

M4PS 2,797 ± 1

Values are the means ± SE of 3 replicates; different letters indicate significant diffe

roline. The highest plantlet number was obtained in liquid devel-pment medium supplemented with 400 mg/l glutamine, yieldingn average 33,844 of regenerated plantlets per ml SCV. The pres-nce of both glutamine and proline promoted embryo developmentn Berangan. Media without amino acid supplementation (MSOolid and liquid) not only produced lower plantlet numbers (186nd 9,902 plantlets) per ml SCV respectively, but also required aonger time to achieve maturity than those cultured in the pres-

nce of amino acids. However, the embryos developed in glutamineased media turned opaque white within 1.5–2 months comparedo those on medium with proline (2–2.5 months). Embryos in

igure 1. Comparison on embryo development on different M3 media. (A–C): Embryos afere still at earlier stage of development. B) Some of the embryos in M4PS matured int

D–F): Embryos 1.5 months >after being cultured in liquid M3 media; (D) MSOL, (E), M4Paturity. Bar (A–F) = 1 mm.

2.5–4 0

s between media at P < 0.01.

media supplemented with glutamine produced normal plantlets.This indicated that exogenous amino acid serves as nitrogen sourcefor the synthesis of proteins which is important to embryo differ-entiation and maturation. Similar result was obtained in chestnut(Robichaud et al., 2004), eastern white pine (Garin et al., 2000),white spruce (Ashihara et al., 2001) and hybrid tea rose (Marchantet al., 1996).

The beneficial role of glutamine in the histodifferentiation of

embryos has been observed in many species such as date palm(Zouine and El-Hadrami, et al., 2007), bitter melon (Thiruvengadamet al., 2006) and horse gram (Varisai et al., 2004). It was reported that

ter 3 months in culture on M3 solid media. A) Translucent embryos in MSOS mediao solid white structure. (C) Most of the embryos in M4GS had turned solid white.L and (F) M4GL, a: embryo at early stage of development, b: solid white embryos at

36 N. Husin et al. / Scientia Horticulturae 168 (2014) 33–37

Figure 2. (A–G): Morpho-histodifferentiation stages of banana (cv. Berangan) somatic embryos. (A) Morphology of aggregate embryos. Bar 1 mm. (B) Histology sectionings yos ins pplemi the fi

toet2s

lsbisa(dtrb

iitpetat(Aep(I

howing globular embryos with protoderm (pd). Bar = 50 �m. (C) Regenerating embrhowing procambial strands (ps). Bar = 100 �m. (E) Abnormal embryos in proline-sun M5 media. Bar = 1 cm. (G) Acclimatized Berangan plantlets ready to be planted in

he frequency of somatic embryos increased with the incorporationf glutamine in the media (Thiruvengadam et al., 2006 and Varisait al., 2004). Exogenous supply of glutamine was also reportedo increase soluble storage protein (Zouine and El-Hadrami, et al.,007), which enhanced protein synthesis and allowed rapid divi-ion of embryogenic cells (Stasolla et al., 2001).

From this study, glutamine was shown to be superior to pro-ine as a supplement in the development media of Beranganomatic embryos in both solid and liquid media. Proline liquidased media also showed negative effects in the embryos, wherein

t caused abnormalities such as fused root-like structure, exten-ive browning cells and retardation (Figure 2). The percentage ofbnormalities increased with the increasing proline concentrationTable 2). All abnormalities appeared after 1–2 months of culture inevelopment media. Abnormal embryos with pseudo-radicle struc-ure and/or browning became necrotic after 1–3 weeks, whereasetarded embryos maintained its structure for as long as 6 monthsefore becoming necrotic.

According to Caligo et al. (1985), proline has an inhibitory effectf added during the embryo development stage of carrot, in whicht could produce embryos either with no polarity, or if they do,hey were lacking root meristem. Trovato et al. (2008) stated thatlant cells were capable of accumulating and quickly degradingxcess proline when needed. Proline usually accumulates or is syn-hesized at high levels during osmotic stress condition (Delauneynd Verma, 1993; Hu et al., 1992 and Kiyosue et al., 1996), and even-ually, the proline concentration will drop when stress is relievedKiyosue et al.,1996; Peng et al., 1996 and Nakashima et al., 1998).ccording to Hellmann et al. (2000), this adaptation is important as

xcess amount of proline is toxic to the cell, due to its conversionroduct P5C that causes the production of reactive oxygen speciesROS) which induces apoptosis and programmed cell death (PCD).n this study, the high concentration of proline may cause toxicity

M4 media prior to exposure to light. Bar = 1 mm. (D) Histology sectioning of embryoented liquid M3 media. Bar = 1 mm (→ abnormal root). (F) Rooted Berangan plantleteld. Bar =10 cm.

to cells which later causes abnormalities and necrosis of the devel-oping embryos. The toxicity level might also become more severein liquid culture as proline was supplemented every 14 days duringsubculture.

Embryo during the early stage of development in both mediaappeared as translucent globular structure, which turned solidwhite as it matured (Figure 1). Histological sections of the embryosshowed normal embryogenesis structure as described by Jalil et al.(2008). The embryos were later regenerated into morphologicallynormal plants (Figure 2) and are currently being acclimatized to betested in the field for clonal fidelity study.

4. Conclusion

From this study, it can be concluded that 400 mg/l glutamine insolid and liquid development media was superior to all the mediatested, in terms of duration and number of regenerated plantletsobtained. Proline supplementation to the media was inferior toglutamine in enhancing development in banana somatic embryosand also induced abnormality to the developing embryos at highconcentration in liquid media.

Acknowledgement

We would like to acknowledge University Malaya for the grantUMRG (RG0130-09BIO) (RP005A-13BIO) and Ministry of Science,Technology and Innovation (10-01-03-PB004) for the financialsupport.

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