callogenesis and organogenesis from inflorescence segments of curcuma alismatifolia and curcuma...

*Corresponding author (A. Jala). Tel/Fax: +66-2-5644440-59 Ext. 2450. E-mail address: [email protected]. 2013. American Transactions on Engineering & Applied Sciences. Volume 2 No. 3 ISSN 2229-1652 eISSN 2229-1660 Online Available at http://TuEngr.com/ATEAS/V02/213-222.pdf

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American Transactions on Engineering & Applied Sciences

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Callogenesis and Organogenesis from Inflorescence Segments of Curcuma Alismatifolia and Curcuma Hybrid ‘Laddawan’

Anchalee Jala a*

a Department of Biotechnology, Faculty of Science and Technology, Thammasat University, 12120 THAILAND A R T I C L E I N F O

A B S T R A C T

Article history: Received February 21, 2013 Received in revised form April 22, 2013 Accepted May 03, 2013 Available online May 09, 2011 Keywords: Curcuma sp.; Callogenesis; Shoot organogenesis; 2,4-Dichlorophenoxy acetic acid.

Callogenesis of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ were proliferated from young inflorescences when cultured them on Murashige and Skoog (MS) medium supplemented with various concentration (8, 10, 12, and 14 mg/l) 2,4-dichlorophenoxyacetic acid (2,4-D) at dark period for 4 weeks to form somatic embryo. The highest percentage growth rate is 90 percent for somatic embryo proliferated on MS medium supplemented with 14 mg/l 2,4-D. When induced these somatic embryo to form new shoot organogenesis by culturing them on MS medium supplemented with various types ( glucose, sucrose and maltose ) of sugar, the highest percentage of new shoots were proliferated from Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ with 0.25 g/l maltose which were 50.00 and 46.67, respectively.

2013 Am. Trans. Eng. Appl. Sci.

1. Introduction The genus Curcuma (Zingiberaceae) comprises more than 80 species of rhizomatous perennial

2013 American Transactions on Engineering & Applied Sciences.

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herbs and is widespread in tropical Asia, extending to Africa and Australia (Purseglove et al.

1981). Within the genus Curcuma, Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ is a

highly valued ornamental, which is indigenous to Burma and Thailand (Apavatjrut et al. 1996;

Schaffer et al. 2011). It is considered as a novel ornamental plant in the floricultural market and

grows well after having been introduced to Guangzhou, China. It is usually used in tropical and

subtropical landscape design as a potted, foliage, and flower plant. It has a long flowering period

(April to November) and vase cut flowers can last 20 days. For these reasons it has seen an

increasing demand in the world ornamental market. However, seeds obtained are limited and

can’t germinate during cold season due to their dormancy, as traditional multiplication involves

propagation by rhizome. Since it is difficult to keep pace with the demands created by market

exploitation, it is thus essential to establish an efficient propagation and regeneration system,

preferably in vitro, to ensure the clonal propagation of desired genotypes.

In this study, anthers were used as explants to induce somatic embryo, which was then used to

form shoots organogesis. This study reports on an efficient callus and plant regeneration for

Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’.

2. Materiasls and Method Young inflorescence of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ were used as

explants and cleaned surface with liquid detergent and washed with tap water for 2 min., immersed

in 70% alcohol for 5 min. and sterilized with 20% sodium hypochlorite for 20 min. then with 5%

sodium hypochlorite for 10 min and immersed in sterile distilled water 3 times for 2 min each.

Petal and Sepal and the end of inflorescence were removed from the sterilized inflorescence.

Sterilized inflorescence about 2.5–3.5 cm long sections were inoculated on Murashige and Skoog

(MS.1962) supplemented with 2mg/l BA. Explants were incubated on agarified MS medium.

All medium contained 30 g m/l sucrose and pH was adjusted to 5.8 with 1.0 N HCl or 1.0 N NaOH

before adding 2.5 gram Phytagel (Sigma, USA) and autoclaving at 121°C for 20 min and incubated

at culture room at 27 ± 2°C exposed to 60 µmol m-2 s-1 fluorescent light (TLD 36W/84 3350lm

Philips Thailand) in a 16-hour photoperiod. All cultures were subcultured every two weeks, for

three times to multiply callus.


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2.1 Somatic Embryo Induction Proliferated callus was transferred to MS medium supplemented with various concentration

(2, 4, 6, 8, 10, 12 and 14 mg/l ) 2,4-D , 3% sucrose and pH was adjusted to 5.8 with 1.0 N HCl or 1.0

N NaOH before adding 2.5 gram Phytagel (Sigma, USA) and autoclaving at 121°C for 20 min and

incubated at culture room at 27±2°C in dark period for four weeks to induce somatic

embryogenesis. Percentage number of somatic embryos is recorded.

2.2 Shoot Organogenesis Callus from Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ which cultured on MS

medium supplemented with 14 mg/l 2,4-D and 0.25 gram per liter of different types of sugar

(glucose, sucrose, maltose ) and pH was adjusted to 5.8 with 1.0 N HCl or 1.0 N NaOH, before

adding 2.5 gram Phytagel (Sigma, USA) and autoclaving at 121°C for 20 min and incubated at

culture room at 27 ± 2°C exposed to 60 µmol m-2 s-1 fluorescent light (TLD 36W/84 3350lm

Philips Thailand) in a 16-hour photoperiod. All cultures were subcultured every three weeks for

four times. Shoot proliferation was investigated after culturing for 45 days.

3. Data Analysis Somatic embryo experiment was used Randomized Complete Block Design with 8 treatments

three replicates and each replication was 10 explants. Shoot Induction was used Randomized

Complete Block Design with three treatments three replicates and each replication was 10 explants.

Means were analyzed by Analysis of Variance and significant differences between means were

compared by the Duncan’s New Multiple Range Test (DMRT) using SPSS v. 13.0. For all

comparisons, statistical significance was considered at p<0.01.

4. Result Somatic embryos from Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ were

proliferated after cultured on MS medium supplemented with different concentration of 2,4-D in

dark period for 4 weeks. It was found that number of somatic embryos proliferated on MS

medium supplemented with 8, 10, 12 and 14 mg/l 2,4 – D. When compared average percentage

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Table 1 Effect of 2,4-D which induced somatic embryo from young inflorescence of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ after cultured for 4 weeks. 2,4-D (mg/l) Percentage of somatic embryo proliferated from young

inflorescence of Curcuma (%) C. alismatifolia** C. hybrid ‘Laddawan’ .**

0 0.00 e 0.00 d 2 0.00 e 0.00 d 4 0.00 e 0.00 d 6 0.00 e 0.00 d 8 36.67 d 60.00 c

10 46.67 c 66.67 bc 12 80.00 b 76.67 b 14 90.00 a 90.00 a

** - highly Significant difference p ≤ 0.01 abc - in the same row was not significant difference when compared their mean with DMRT at p ≤ 0.01

Figure 1 Young inflorescence Curcuma alismatifolia and Curcuma hybrid ‘Laddawan cultured on

MS Medium supplemented with 12 and 14 mg/l 2,4-D after cultured for 4 weeks 1a.Compact callus ( C. alismatifolia) proliferated on MS medium supplemented with 12 mg/l 2,4-D 1b.Friable callus(C. alismatifolia) proliferated on MS medium supplemented with 14 mg/l 2,4-D 1c.Compact callus(C. hybrid ‘Laddawan’) proliferated on MS medium supplemented with 12 mg/l 2,4-D 1d. Friable callus (C. hybrid ‘Laddawan) proliferated on MS medium supplemented with 14 mg/l 2,4-D.

number of somatic embryos in statistic. They were highly significant difference (p<0.01) and in

MS medium supplemented with 14 mg/l 2,4-D gave the highest average percentage number of

somatic embryos in both Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ which are 90

as shown in Table 1. When compared type of callus in 12 and 14 mg/l 2,4-D in each Curcuma

alismatifolia and Curcuma hybrid ‘Laddawan,’ they showed that characteristic of callus which

proliferated in 12 mg/l 2,4-D their cells are smaller and more compact than from 14 mg/l 2,4-D as

shown in Figure 1.


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The callus originated from the young inflorescence surface and not from inner tissue in

Curcuma alismatifolia (Figure 1a) and Curcuma hybrid ‘Laddawan (Figure 1c). The callus

which proliferated from MS medium supplemented with 14mg/l 2,4-D are friable , their cells are

bigger and uninucleated which ready to form somatic embryo in both Curcuma alismatifolia

(Figure 1.b) and Curcuma hybrid ‘Laddawan (Figure 1d).

4.1 Shoot Induction from Somatic Embryo The callus originated from the young inflorescence surface and not from inner tissue in

Curcuma alismatifolia and Curcuma hybrid ‘Laddawan and formed somatic embryo .When culture

these somatic embryo on MS medium supplemented with 0.25g/l different types of sugar (glucose

sucrose and maltose) for 12 weeks. It was found that somatic embryo responded to different types

of sugar. They proliferated to young shoot in MS medium supplemented with different type of

sugar. When compared the result in statistic, induction shoot from MS medium supplemented with

0.25g/l maltose was highly significant difference (p ≤ 0.01) from glucose and sucrose as shown in

Table 2. The highest average percentage of shoot induction in Curcuma alismatifolia and

Curcuma hybrid ‘Laddawan were 50.00 and 46.67, respectively.

Table 2 Shoot Induction of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan from somatic embryo which cultured on MS medium supplemented with different types of sugar after cultured

for 12 weeks. Types of sugar

(0.25 g/l) Percentage of shoot induction (%)

C. alismatifolia ** C. hybrid ‘Laddawan’ ** No sugar 0.00 c 0.00 c glucose 23.33 b 13.33 c sucrose 23.33 b 30.00 b maltose 50.00 a 46.67 a

** - highly Significant difference p ≤ 0.01 abc - in the same row was not significant difference when compared their mean with DMRT at p ≤0.01

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Figure 2 Shoot induction from somatic embryo on MS medium supplemented with 0.25g/l maltose

on Curcuma alismatifolia and Curcuma hybrid ‘Laddawan after culturing 12 weeks: (a) Somatic embryo proliferation in C. alismatifolia (b) Differentiation of Somatic embryo in C. alismatifolia (c) Shoot proliferation in C. alismatifolia (d) Somatic embryo proliferation. In

C. hybrid ‘Laddawan’ (e) Differentiation of Somatic embryo in C. hybrid ‘Laddawan’ (f) Shoot proliferation in C. hybrid ‘Laddawan’

5. Discussion Young inflorescence of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan cultured on

MS medium supplemented with different concentration of 2,4-D in dark period for 4 weeks

showed different responses. Callus proliferated on MS medium supplemented with 8, 10, 12 and

14 mg/l 2,4-Dand MS medium supplemented with 14mg/l 2,4-D gave the highest average

percentage of friable callus which were 90 in both curcuma. As worked of Yaping et al. (2013)

showed that 2,4-D in the induction medium could induce a high percentage of callus (56.2 %). This

callus was compact and friable. In C. alismatifolia, regeneration has been reported from young

inflorescences (Wannakrairoj, 1997). Also, Toppoonyanont et al. ( 2005) had reported that C.

alismatifolia inflorescences were used as explants and, when inoculated on MS basal medium

containing 44.0M BA and 0.57M NAA for 1 month they developed and reverted to vegetative

shoots directly from flower organs (or floral buds) and not via callus. Mohanty et al. (2008)

reported that plant regeneration from callus cultures of C. aromatica was possible with 2,4-D

(9.1M) and 2.3M KT. Callus could be induced from the base of adventitious shoots of C.

kwangsiensis on MS medium containing 1.4M TDZ, 4.4M BA and 2.3M 2,4-D (Zhang et al.

2011). Plant regeneration from the culture of immature of C. longa inflorescences was possible

by direct shoot development on MS basal medium supplemented with BA (22.0 or 44.0M) in

combination with 0.9M 2,4-D or 0.54M NAA and 4.5 or 9.0M TDZ in combination with 0.57M


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IAA (Salvi et al. 2000). Moreover, they located at the same positions and were arranged spirally

within the bracteole, similarly to ex vitro shoots (Udomdee et al. 2003). All of these reports

showed that callus were induced from different explants by using of 2,4-D single or NAA in

combination with BA, KT or TDZ. The callus doubled in size in less than two weeks when

subcultured in a fresh MS medium supplemented with 1 mg/l 2,4-D.

In this treatment, the concentration was maintained at 1 mg/l due to its response in producing

favorable rapid results. In plant tissue culture, sucrose is the most commonly used carbohydrate

source because of the wide spread of this disaccharide as a transporter molecule, and its high

solubility in water. Many in vitro studies have proven that sucrose supports near optimum rates of

growth and also plays multiple roles in the provision of carbon and energy that promotes cell

growth and division (Balachandran et al. 1990). Viu et al. (2009) reported that Callus was

suspended in a liquid medium to enhance proliferation. For initiation, only friable callus was used.

Prior to any new treatment, 1 g of the friable callus was proliferated in a liquid medium with same

concentration as callus induction medium (MS medium supplemented with 1mg l-1 2,4-D and 30g

/l sucrose).

The highest average percentage of shoot proliferated from somatic embryo of Curcuma

alismatifolia and Curcuma hybrid ‘Laddawan, when cultured on MS medium supplemented with

0.25g per liter of maltose which are 50.00 and 46.67, respectively. But Wannakrairoj(1997) and

Jala, (2012) reported that plant regeneration from the culture of immature of C. longa

inflorescences was possible by direct shoot development on MS basal medium supplemented with

BA (22.0 or 440 M) in combination with 0.9 M 2,4-D in combination with 30g/l sucrose. And the

same as Zhang et al (2011) report that callus could be induced from the base of adventitious shoots

of C. kwangsiensis on MS medium containing 1.4M TDZ, 4.4 M BA and 2.3M 2,4-D. Normally

they used sucrose for carbon source. Sucrose is the most commonly used carbohydrate in plant

tissue or cell culture (Vu et al., 1993). Many factors affecting the growth of excised plant organs,

tissues and cells in vitro have been studied extensively over the last 30 years, including plant

growth regulators, light, temperature, medium pH, humidity, gas exchange and the presence of

harmful microorganisms (Leifert et al., 1995). On the other hand, plant carbohydrate

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requirements in vitro have rarely been studied in detail and still are poorly understood (Leifert et

al., 1995; Swedlund & Locy, 1993). It has also been well documented that certain plant tissues

may contain and/or utilize different carbohydrates at the same time. It is then not surprising that

carbon sources other than sucrose might be effective in promoting in vitro tissue specific growth

responses in a given species (Swedlund & Locy, 1993). Furthermore, several reports have pointed

out these carbon sources as inducers of cell differentiation in some plant species (Lemos & Blake,

1996; Karhu, 1997; Tamil et al. 2012). Galactose, glycerol and sorbitol are examples of

alternative carbon sources that have been tested in vitro for different purposes in a wide range of

plant species (Swedlund et al.1993).

6. Conclusion Young inflorescence of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan could

proliferated to somatic embryo when cultured on MS medium supplemented with 8, 10, 12, and 14

mg/l 2,4-D in dark condition for 4 weeks and got the highest average percentage at 14mg/l 2,4-D

which are 90 percent. Somatic embryo proliferated to shoot organogenesis from friable callus that

cultured on MS medium supplemented with 0.25g/l maltose. The highest average percentage of

shoot proliferated in Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’ were 50.00 and

46.67 , respectively.

7. References Apavatjrut P, T. Sirisawad, P. Sirirugsa, P. Voraurai,C. Suwanthada. (1996) Studies on

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Karhu, S.T. (1997) Sugar use in relation to shoot induction by sorbitol and cytokinin in apple. Journal of the American Society for Horticultural Science. 122: 476-480.

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cell and tissue cultures. Critical Reviews in Plant Science.14: 83-109.

Lemos, E.E.P., J. Blake. (1996) Micropropagation of juvenile and mature Annona muricata L. Journal of Horticultural Science.71: 395-403.

Mohanty S, M.K. Panda, E. Subudhi, S. Nayak. (2008) Plant regeneration from callus culture of Curcuma aromatica and in vitro detection of somaclonal variation through cytophotometric analysis. Biol Plant 52:783–786

Murashige, T. and F. Skoog. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 15: 473-479.

Purseglove JW, E.G. Brown, C.L. Green, S.R. Robbins. (1981) Spices. Longman, London, New York.

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Yaping Kou; Guohua Ma, A. Jaime, Teixeira da Silva, Nian Liu. (2013). Callus induction and shoot organogenesis from anther cultures of Curcuma attenuata Wall. Plant Cell, Tissue and Organ Culture (PCTOC).112 : 1-7.

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Dr.Anchalee JALA is an Associate Professor in Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Campus, Pathumtani , THAILAND. Her teaching is in the areas of botany and plant tissue culture. She is also very active in plant tissue culture research.

Peer Review: This article has been internationally peer-reviewed and accepted for

publication according to the guidelines given at the journal’s website.

callogenesis and organogenesis from inflorescence segments of curcuma alismatifolia and curcuma...

Technology

genus curcuma zingiberaceae

skoog ms medium supplemented

agarified ms medium

n naoh

sterilized inflorescence

somatic embryogenesis

gram phytagel sigma

efficient callus