Direct somatic embryo formation (without intervening callus) from garlic clove basal tissue was induced

in which the influence of plant growth regulators (PGRs) on various explants was examined. Medium

added with 2.0 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D)

were the most effective PGR combination for somatic embryo induction. It induced embryos directly in

85.5% of the basal clove explant. Callus induction was also obtained from other parts of explant and 2.0

mg/l 2,4-D induced callusing in 86.5% of the inoculated explants. Protein, amino acid and alliin content

were measured in callus and in embryos. Somatic embryos had more soluble protein and free amino acid

compared to callus. HPTLC analysis revealed that alliin was significantly high in somatic embryos com-

pared to undifferentiated callus tissue; the content was even more in older embryos. The present study of

Allium indicates that the event of morphogenetic development including in vitro embryogeny can effec-

tively be analysed by monitoring the changes of biochemical profiles.

Keywords: Allium sativum – alliin – callus – HPTLC – somatic embryo

INTRODUCTION

Garlic (Allium sativum) is an important culinary and medicinal plant used worldwide.

It shows several biological activities such as antibiotic, antitumour, antiatheroscle-

rotic [6, 7], cholesterol-lowering effect [46] and it also prevents cardiovascular dis-

orders [37]. Propagation of garlic is mainly accomplished by vegetative methods

Acta Biologica Hungarica 60 (4), pp. 441–454 (2009)

DOI: 10.1556/ABiol.60.2009.4.10

0236-5383/$ 20.00 © 2009 Akadémiai Kiadó, Budapest

IMPROVED ALLIIN YIELD IN SOMATIC EMBRYOS

OF ALLIUM SATIVUM L.

(CV. YAMUNA SAFED) AS ANALYZED BY HPTLC

S. A. NASIM, A. MUJIB,* K. RASHMI,

F. SAMAR, A. JUNAID and MAHMOODUZZAFAR

Department of Botany, Cellular Differentiation and Molecular Genetics Section, Hamdard University,

New Delhi 110 062, India

(Received: November 17, 2008; accepted: January 26, 2009)

* Corresponding author; e-mail: amujib3@yahoo.co.in

Abbrevations: 2,4-dichlorophenoxyacetic acid (2,4-D); naphthalene acetic acid (NAA); 6-benzy-

laminopurine (BAP); 6-furfuryl aminopurine (KIN); plant growth regulator (PGR); somatic embryogen-

esis (SE); Least Significant Difference (LSD), High Pressure Thin Layer Chromatography (HPTLC).

442 S. A. NASIM et al.

Acta Biologica Hungarica 60, 2009

which demonstrate a low coefficient of multiplication [32]; therefore it takes many

years to produce sufficient number of seed bulbs for practical cultivation of new elite

variety [31]. Similarly, the crop improvement by cross fertilization is limited as gar-

lic shows sexual incompatibility [27]. In Allium, callus culture and in vitro morpho-

genesis have been achieved from various plant parts [3, 30, 39, 42] but the rate of

multiplication and the number of plantlets regenerated per explant were not always

significantly high [38]. Plant regeneration through somatic embryogenesis is rare but

is not uncommon in Allium [42]. It has several advantages over organogenesis and

appears to be the most promising technique for fast propagation of plants [17]. The

developmental protocols to establish embryonic cultures with synchronous embryo

forming ability may able to eliminate many of the problems associated with zygotic

embryo development.

Garlic, like many other members of Alliums, contains high organic sulphur com-

pounds in the form of alkylcysteine sulphoxides and γ-glutamyl peptides. On tissue

damage and with alliinase enzyme’s activity, the alkyl cysteine sulphoxide releases

compounds that give unique Allium’s odour and flavour. In garlic, the major flavour

precursor is alliin while in onion it is isoalliin. Alliin has been proposed to be syn-

thesized by two pathways, in one, serine combines with allyl thiol source to produce

allyl cysteine which on oxidation produce alliin while in the other, glutathione

derived cysteine unites with an allyl source, forming an intermediate allyl glu-

tathione, later converts into allyl cysteine which finally give rise to allin [23].

An early biochemical marker(s) for the identification of embryonic potency would

be of immense value in studying efficient plant regeneration. It has earlier been noted

that the morphogenetic pattern is associated with specific biochemical changes that

can be used as markers [45]. Several quantitative and qualitative studies were con-

ducted and their developmental pattern were analysed during somatic embryogene-

sis and organogenesis events [21, 24]. Dave and Batra [10] studied the metabolic fac-

tors which promote to induce somatic embryos in culture and the embryogenic tis-

sues were compared with non-embryogenic callus. Earlier, biochemical differences

between embryonic and non-embryonic callus cultures with respect to polypeptide

pattern were also investigated in rice [8]. But, there are only a few biochemical

reports that discussed events during differentiation in medicinal plants [18]. In this

communication, a simple high frequency direct somatic embryogenic system is

reported from basal part of clove in Allium sativum cv. Yamuna Safed in which we

investigated the role of BAP and 2,4-D in embryogenesis. Attention has also been

paid to identify the biochemical differences that existed between callus and embryo-

genic tissues/embryos in Allium sativum during plant regeneration.

MATERIALS AND METHODS

Plant material

Local Indian garlic (Allium sativum L. c.v Yamuna safed, voucher specimen IC-

375117, deposited at NBPGR, New Delhi) a moderate maturing variety, was used as

experimental material. It was procured from National Horticultural Research and

Development Foundation, Nasik (Maharashtra). Bulbs were harvested from 4–5

months old plants and stored at 15 °C, and individual clove of bulb was used as

explant.

Explant preparation

Healthy garlic cloves were selected and washed under running water for 1 h, surface

sterilized with 0.1% HgCl2 for 15 min and 70% ethanol for 30 sec and washed sev-

eral times with sterilized distilled water. The clove was divided into basal, middle

and tip part; and inoculated in MS [29] medium. Nearly 3–5 mm cloves were trans-

ferred to culture tubes containing 15 ml of solidified basal medium supplemented

with various concentrations of BAP and 2,4-D.

Medium and culture conditions

The MS medium was used that was added with various plant growth regulators,

PGRs such as 2,4-D, BAP and KIN (6-furfuryl amino purine). The medium pH was

adjusted to 5.6 prior to autoclaving at 121 °C for 20 min. All the cultures were incu-

bated at 25 ± 2 °C in a culture room under 16-h photoperiod provided by cool white

fluorescent light at intensity of 40 Wm–2.

The frequency of callusing, i.e. the number of explants that produced callus was

counted after six weeks of incubation. Similarly, the frequency of globular somatic

embryos and the mean number of embryos per explant were recorded after two

months of incubation. Each treatment had at least 10 explants with 5 replicates.

Values were represented as mean ± standard error at 5% significance level.

Biochemical analyses

Estimation of protein

Bradford method [5] was used to estimate the protein content, 0.5 g tissue was

ground in prechilled mortar and pestle with 1.0 ml (0.1M) phosphate buffer (pH 7.0)

on ice and centrifuged at 5,000 rpm for 10 min at 4 °C. TCA (0.5 ml) was added and

the sample was again centrifuged at 5,000 rpm for 10 min. The supernatant was dis-

Plant growth regulators and somatic embryogenesis 443

Acta Biologica Hungarica 60, 2009

444 S. A. NASIM et al.

Acta Biologica Hungarica 60, 2009

carded and the pellet was dissolved in 1.0 ml of 0.1N NaOH after washing with dou-

ble-distilled water. After adding 5.0 ml of Bradford reagent, the optical density was

measured at 595 nm.

Estimation of free amino acids

Free amino acids content was estimated by the method of Lee and Takahashi [24]. In

brief, 0.1 g tissue was incubated overnight in 70% ethanol followed by washing with

double-distilled water. Then, 1.5 ml of 55% glycerol and 0.5 ml ninhydrin solution

were added, boiled at 100 °C for 20 min and cooled down. The final volume was

made up to 6 ml with double-distilled water and the optical density was measured at

570 nm.

Quantification of alliin

Extraction of garlic material

Arnault et al. [1] method was used for the extraction of garlic. One gram of garlic in

the form of dry powder was extracted at room temperature using 10 ml methanol –

water (80 : 20, v/v) plus 0.05% formic acid at pH < 3.

Quantification of alliin by HPTLC

The content of alliin in in vitro cultures was estimated using alliin (Sigma Chemical,

MI, USA) as standard.

Standard Alliin: One mg/ml solution of standard alliin as prepared in methanol

and 1 ml of this solution was diluted to 10 ml with methanol to get 100 µg/ml solu-

tion, these solutions were used for application on TLC plate for preparation of stan-

dard plot. The UV spectrum of standard alliin solution in methanol (50 µg/ml) was

recorded using UV spectrophotometer (UV-1601, Shimadzu Japan) for authentica-

tion. The λmax was matched with standard alliin.

HPTLC instrument and conditions

The samples were spotted in the form of bands (width 4 mm) with Camag microlitre

syringe on precoated silica gel aluminium plate 60F-254 (20 cm × 10 cm × 0.2 mm

thickness, E. Merck, Germany) using a Camag Linomat V (Switzerland). A constant

application rate of 80 nl/s was employed and space between two bands was 5.2 mm.

The slit dimension was kept a 4 mm × 0.1 mm; and a 20 mm/sec scanning speed was

employed. The mobile phase consisted of n-butanol : methanol : ammonia (8 : 1 : 1.5,

v/v). Linear ascending development was carried out in twin through glass chamber,

saturated with mobile phase. The optimum chamber saturation time for mobile phase

was 30 min at room temperature. The length of chromatogram run was 65 mm. TLC

plates were dried with air current provided by an air dryer. Densitometric scanning

was performed on Camag TLC scanner IV (absorbance mode 510 nm) after spraying

with anisaldehyde sulphuric acid and dried at 100C. Deuterium and tungsten lamps

were used as radiation sources.

Calibration curve of alliin

Different volumes of standard solution (0.5 – 6 µl) were spotted in triplicate on TLC

plate to obtain 50–1000 ng per spot of alliin. The data of peak area vs. alliin con-

centration were treated by linear least square regression and the regression equation

thus obtained from standard curve, was used to estimate alliin in different samples.

Quantification of alliin

Sample (5 µl) of in vitro cultures (calli and embryos) was applied in triplicate on

TLC plate. The alliin yield was quantified by using regression equation of calibration

curve.

Statistical analysis

All data are expressed as mean ± standard error. Mean number of somatic embryos

per explant and mean number of explant showing callus were determined using

ANOVA followed by LSD test at P= 0.05.

RESULTS AND DISCUSSION

Explant type and effect of PGRs on callusing and embryogenesis

Various sections of garlic clove (basal, middle and tip part) were inoculated in MS

added with 2,4-D and BAP. Of the several implanted explants, the tip and the middle

part of the clove developed callus (Fig. 1a, b) but did not produce embryos in cul-

ture. High frequency somatic embryos were only observed at the basal part of the

clove (Fig. 1c, d) which proliferated and produced more number of embryos on sub-

sequent passages.

The role of different concentrations of 2,4-D, BAP and KIN in producing callus

and to induce embryos was presented in Tables 1 and 2. It was observed from the

tables that 2,4-D at 2.0 mg/l was the most effective concentration for callus forma-

tion and percent callus induction was least on BAP added medium (Table 1). Medium

Plant growth regulators and somatic embryogenesis 445

Acta Biologica Hungarica 60, 2009

446 S. A. NASIM et al.

Acta Biologica Hungarica 60, 2009

supplemented with KIN (0.5 mg/l) and 2, 4-D (2.0 mg/l) was also very effective in

initiating callus when compared to the medium, added with BAP (0.5 mg/l) and 2,4-

D (2.0 mg/l). There was no significant difference in embryo induction ability in

medium that supplemented either with 2,4-D or BAP particularly at lower concen-

tration (Table 2). The embryo inductive potential, i.e. the mean number of embryos

per explant was more in 2,4-D added medium than that of BAP. The embryo induc-

tion frequency was significantly high in medium when amended with 0.25–1.0 mg/l

2,4-D and 2.0 mg/l BAP (Table 2). The best growth regulator combination identified

was 0.5 mg/l 2,4-D + 2.0 mg/l BAP, which showed maximum embryogenic frequen-

cy with highest number of embryos. It was also noted that 2,4-D at concentration

0.50 mg/l or more reduced embryogenesis percentage and mean numbers of embryos

when combined with BAP (2.0 mg/l). In this Allium cultivar, secondary or adventive

somatic embryo formation which is very common in plant embryogenesis research

was never noted on primary somatic embryo on various tested PGRs levels. The indi-

vidual application of auxins like 2,4-D invariably produced secondary embryos par-

ticularly in long-term culture, was found to be inefficient in producing secondary

embryos in Allium. Similarly, in medium amended with BAP (2.0 mg/l) and 2,4-D

Fig. 1. Callusing and direct somatic embryo induction in MS medium in Allium sativum. (a, b): Non-

embryogenic callus induced in medium added with 2.0 mg/l 2,4-D, (c, d): Somatic embryos (arrow

heads) induced directly in 0.5 mg/l 2,4-D + 2.0 mg/l BAP added medium, and (e) regenerated plantlet

from somatic embryo. Bars: a – d, 1 mm; e, 1 cm

(0.25–1.0 mg/l), where primary somatic embryos occurred more frequently, no sec-

ondary embryo was formed in this very medium in Allium.In this present investigation, different parts of the explant (basal, middle and tip of

clove) were used in which callus was induced from middle and tip part of the clove

while basal clove directly formed embryos; we also noted variable callus yield in dif-

ferent parts of the clove explant (data not shown). The PGRs such as 2,4-D, BAP and

KIN were active but the callusing responses varied from one PGR to other. Similar

observation of differential requirement was previously observed in other groups of

plants and this may be due to different physiological gradient, photosynthetic reserve

differences, endogenous PGRs level etc. that existed in parts (proximal and distal) of

the same clove explant [20]. In the present investigation of Allium, we observed that

the calli from two or more sources were different not only morphologically; their

morphogenetic response was also not the same. In callus developed from the basal

part, cells acquired embryogenic competence, embryos were induced, later germi-

nated into plantlets, this embryogeny is very similar to zygotic embryo development,

while in callus induced from middle or tip part of the clove this embryogenic signal

is altogether absent. The type and concentration of auxins employed is critical for the

induction and development of somatic embryos [14]. The presence of 2,4-D in par-

ticular has been found to be necessary for the induction of embryo (perhaps up to

Plant growth regulators and somatic embryogenesis 447

Acta Biologica Hungarica 60, 2009

Table 1

Effect of various concentrations of PGRs on callusing

PGR (mg/l)

% Callus induction

2,4-D BAP KIN

0.00 0.00 0.25 45.00 ± 15.00c

0.00 0.00 0.50 51.10 ± 12.50bc

0.00 0.25 0.00 19.00 ± 5.10d

0.00 0.50 0.00 21.50 ± 8.00d

0.25 0.00 0.00 42.80 ± 11.50c

0.50 0.00 0.00 61.00 ± 11.10b

0.50 0.50 0.50 23.20 ± 15.50cd

1.00 0.00 0.00 70.50 ± 15.30ab

2.00 0.00 0.00 86.50 ± 9.50a

3.00 1.00 0.00 59.20 ± 12.20b

2.00 0.50 0.00 48.20 ± 8.50c

2.00 0.00 0.50 75.40 ± 10.50ab

ANOVA

F 9.13

P 0.002*

Data are expressed as mean ± standard error of at least 5 replicates.

Within each column, values followed by same superscript letters are not signifi-

cantly different at p = 0.05 level according to LSD test.

F test significant at: *p < 0.05.

448 S. A. NASIM et al.

Acta Biologica Hungarica 60, 2009

globular stage). NAA, Picloram, Dicamba etc. are also used to a lesser extent [19, 36]

and in most instances the auxins are used alone or in combination with cytokinins

[13]. The continuous exposition of 2,4-D however, inhibit normal embryo develop-

ment; tend to produce secondary embryos and induce aberrant embryos with a very

low frequency of plant regeneration. The development/maturity of somatic embryos

is achieved by reducing or removing the auxins from the culture medium as the

exogenous auxins interact with polar auxin gradient and prevents apical-basal

embryo patterning during later stages of embryogenesis [25, 44]. In this Allium cul-

tivar, though 2,4-D induced prolific callus, it did not have much role in embryogen-

esis (absolutely no effect on secondary embryogenesis) when used alone, the syner-

gistic effect of 2,4-D and BAP was however, very active to promote somatic embryo

number which later germinated into plantlets (Fig. 1e). Even in some limited cases,

somatic embryos were formed in cytokinin amended medium when used alone as

sole source of PGR, with no requirement of 2,4-D [16, 41]. Compared to BAP, KIN

on the other hand was observed to be less potent cytokinin for embryogenesis as it

only induced non-embryogenic callus. In some earlier studies, Ferol et al. [11, 12]

reported embryogenesis on 2,4-D and KIN added medium (solid and liquid); histo-

Table 2

The influence of various PGRs (2,4-D, BAP and KIN) in inducing embryos

PGR (mg/l)Frequency No. of globular embryos

of globular embryos per explant2,4-D BAP KIN

0.00 0.00 0.25 04.00 ± 0.56d 03.11 ± 0.20d

0.00 0.00 0.50 05.12 ± 0.99d 04.03 ± 0.92d

0.00 0.25 0.00 07.50 ± 1.11d 04.51 ± 1.25d

0.00 0.50 0.00 08.55 ± 1.93d 05.12 ± 1.50d

0.25 0.00 0.00 08.21 ± 1.32d 05.52 ± 155d

0.50 0.00 0.00 09.11 ± 1.11d 06.05 ± 1.77d

0.50 0.00 0.50 08.16 ± 1.51d 06.44 ± 2.01d

0.50 0.50 0.50 38.50 ± 1.63c 14.90 ± 2.10c

0.25 1.00 0.00 60.50 ± 1.33b 27.34 ± 2.22b

0.25 2.00 0.00 74.25 ± 1.45ab 41.76 ± 2.44ab

0.50 2.00 0.00 85.50 ± 1.71a 51.41 ± 3.11a

1.00 2.00 0.00 75.00 ± 1.56ab 44.87 ± 2.76ab

2.00 2.00 0.00 67.50 ± 1.44ab 38.65 ± 2.54ab

2.00 1.00 0.00 55.55 ± 1.6b 30.98 ± 1.99b

ANOVA

F 8.19 3.43

P 0.001* 0.007*

Data are expressed as mean ± standard error of at least 5 replicates.

Within each column, values followed by same superscript letters are not significantly different at p = 0.05 level accord-

ing to LSD test.

F test significant at: * p < 0.05.

logical analysis was also conducted to confirm the somatic embryogenesis based

plant regeneration in Allium sativum.

Amino acid, protein and alliin content in callusand in somatic embryos

We analysed the amino acid and protein contents in callus and in embryos. In

embryos, the content of protein and amino acid were more (8.54 ± 1.21, 6.13 ± 1.54

mg/g fresh weight, respectively) compared to callus (6.58 ± 1.11, 4.45 ± 1.32 mg/g

fresh weight, respectively), which was maximum in 6 to 8 weeks old cultures (Figs

2, 3). This is in accordance with similar observation [18] where the levels of free

amino acid were increased in embryogenic system as compared to callus. In contrast,

Plant growth regulators and somatic embryogenesis 449

Acta Biologica Hungarica 60, 2009

Fig. 3. Free amino acid content in callus and in embryos, data scored after 4, 6 and 8 weeks of culture.

Means with common letter are not significantly different at p = 0.05 level according to LSD

Fig. 2. Total soluble protein content in callus and in embryos, data scored after 4, 6 and 8 weeks of

culture. Means with common letter are not significantly different at p = 0.05 level according to LSD

450 S. A. NASIM et al.

Acta Biologica Hungarica 60, 2009

Fig. 4 – a, b. HPTLC traces comparing the profile of the extract from garlic (a) callus, (b) embryos.

Both the cultures were 8 weeks old

a

b

Dave and Batra [10], Chanprame et al. [7] reported reduced level of protein in somat-

ic embryos, indicating thereby the utilization of protein at the site of embryoid for-

mation. Similarly, earlier, various physiological, biochemical and molecular markers

including proteins, amino acids, isoenzymes etc. have been studied and used to dif-

ferentiate embryogenic tissue from non-embryogenic one in a wide range of plants

[2, 9]. Quantitative analysis of alliin in somatic embryos and in callus of garlic was

examined by using HPTLC. We noted that the alliin content was significantly high

in embryos (3.01 ± 0.19 mg/g dry weight) as compared to callus (1.58 ± 0.14 mg/g

dry weight) and in field grown clove (1.12 ± 0.41 mg/g dry weight); the content

improved further as the cultures became comparatively old (Figs 4a, b, 5). This result

corroborated with earlier study where a significant increase in alkaloid content was

reported in Narcissus confusus somatic embryos [43]. Earlier, the biosynthesis of

flavour precursors has been attempted in garlic and in onion involving cultured tis-

sues like callus, cell suspensions and differentiating tissues [22, 33]. Recently,

Hughes et al. [15] reported significant amounts of allin in garlic callus following

incubation with allyl cysteine and allyl thiol. The role of allyl cysteine and allyl thiol

in improving alliin is not known clearly, but these may be used as intermediate sub-

strate in proposed pathways of alliin synthesis. How in vitro raised tissues like

embryo produce enhanced level of alkaloid was not investigated in this present study.

The enriched medium components with added carbon source on which tissues are

cultivated, optimized photoperiod, the cellular stresses etc. may enhance in vitro syn-

thesis of alkaloids [4, 40]. The plant growth regulators also influence synthesis by

acting as signaling molecule. In this investigation, we observed that the alliin pro-

duction was less in callus and was more in embryos, thus synthesis seems to be tis-

sue specific. This enhanced level may be attributed by the fact that certain degree of

differentiation of individual cell or tissues is required, which is present in embryos,

influence secondary metabolite synthesis [26, 34].

Plant growth regulators and somatic embryogenesis 451

Acta Biologica Hungarica 60, 2009

Fig. 5. Alliin content in callus and in embryos. Means with common letter are not significantly different

at p = 0.05 level according to LSD

452 S. A. NASIM et al.

Acta Biologica Hungarica 60, 2009

CONCLUSIONS

In conclusion, the present study demonstrates that in vitro cultures provide a unique

tool to study the biochemical difference(s) associated with morphological develop-

ment. It also indicates that in embryos, the content of primary compounds like solu-

ble amino acid and protein are high, so is alliin, which could both be used as mark-

ers to distinguish callus from comparatively complex tissues like developing

embryos.

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