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Simple protocol for plant regeneration of Lilium ledebourii using transverse thin cell layer


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Simple protocol for plant regeneration of

Lilium ledebourii

using transverse thin

cell layer

Progress in Biological Sciences

Vol. 3, Number 2, Summer/Fall 2013/117-122

Received: May 1, 2013; Accepted: July 7, 2013

Masoud Mirmasoumi1; Pejman Azadi2*; Ali Sharafi3; Valentine Otang Ntui4 ; Masahiro Mii4

1- Department of Botany, School of Biology, Collage of Science, University of Tehran, Tehran, Iran; 2- Tissue Culture and Genetic Engineering Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran 3- Novin Giti Gene (NGG) Co., Bio-Incubator Center NIGEB, Tehran, Iran 4- Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba 271-8510, Japan



Transverse thin cell layer sections excised from in vitro scales of Lilium ledebourii were cultured on Murashige and Skoog (MS) medium supplemented with various plant growth regulators (PGRs) at different concentrations. Although, bulblets were produced on PGR-free MS medium during organogenesis, addition of 0.25 mg l-1 6-benzyladenine or 5.0 mg l-1 indole-3-acetic acid to the medium increased the organogenesis response and produced 4.4 bulblets per explants. Two months later, the bulblets were transferred to MS PGRs-free medium. Bulblets were successfully transplanted to the soil after a cold treatment of 8 weeks, with a survival rate of 85%.

Key Words:Lilium; Auxin; Organogenesis; Regeneration.

* Corresponding author: azadip22@gmail.com

Perspectives in taxonomy and phylogeny of the genus


(Fabaceae): a review Shahin Zarre*, Nasim Azani

Center of Excellence in Phylogeny of Living Organisms and Department of Plant Science, School of Biology, College of Science, University of Tehran, Tehran, Iran

Received: November 10, 2012; Accepted: November 24, 2012


The genus Astragalus L. (Fabaceae) is reviewed from both phyloenetic and taxonomic points of view. As the largest genus of flowering plants it has attracted many researchers, but much work remains to be done. A short taxonomic history with special focus on infrageneric classification of the genus, a list of phylogenetic studies including the applied markers and sampling strategies as well as a short discussion on evolution of morphological characters are presented.

Keywords:Astragalus, Fabaceae, Taxonomy, Phylogeny


With some 2900 species distributed in both Old- (1; ca. 2400 spp.) and New World (2; ca. 500 spp.),

Astragalus L. is by far the largest genus of flowering plants, following by Bulbophyllum

Thouars of Orchidaceae with approximately 2032 species and Psychotria L. of Rubiaceae comprising about 1951 species (1). The plants vary from short living annual herbs (ca. 80 spp.) to perennial rhizomatous or hemicryptophytic herbs (ca. 2500 spp.) and to cushion forming spiny shrubs (ca. 300 species) in habit (Fig. 1). Most species grow in semi-arid and arid areas throughout the world, but a few species prefer humid habitats (e.g.

A. glycyphyllos L.), or are known as weeds. The plants show the typical papilionaceous flowers and are characterized by any unique morphological synapomorphy. As a result of this fact, the delimitation of the genus is sometimes very difficult and the assignment of some species (such asA. annularis Forssk.) to this genus is doubtful and not supported by phylogenetic studies (2-4). According to recent investigations, the genus is currently placed in the well supported Astrgalean clade of tribe Galegeae s. l. close to genera

R.Br. and few small genera (5). The taxonomy and phylogeny of Astragalus are very challenging. In the present paper we will review the background of taxonomic and phylogenetic studies on Astragalus, the sources of complexity in the taxonomy and phylogeny of the genus, and future perspectives in studies on this genus.

Taxonomic background

Since its description in volume 2 of ‘Species Plantarum’ (6), Astraglus has been subjected to many taxonomic studies aiming mostly to achieve a natural subgeneric classification. Among these systems, Bunge’s (7) classification of the genus in 1868 into eight subgenera and 105 sections, has been widely used until recently (8, 9). Based on detailed morphological studies with emphasis on indumentum type (focusing on hair attachment: basifixed vs. medifixed), Podlech (10) explained the convergent nature of many of the morphological characters used in the delimitation of subgenera as proposed by Bunge (7) and reduced the number of recognized subgenera to three: subgen. Astragalus, subgen. Cercidothrix


Progress in Biological Sciences

Vol. 3, Number 2, Summer/ Fall 2013


1. Introduction

Lilium ledebourii (Baker) Boiss (Liliaceae) is a perennial and endangered rare species endemic to Iran. This plant is at the risk of rapid eradication because of irregular grazing and poaching. It is under surveillance of Iranian Regional Environmental Protection Agency(1). L. ledebourii has a high ornamental value because of its beautiful large white flowers that can be used as a commercial lily. However, the main problem for the commercialization of this species is its sensitivity to moderate or high temperature conditions and the inability of the bulbs to produce mature plants in greenhouse. Molecular breeding could possibly overcome these challenges. In our previous study, a successful micropropagation system using scale culture to conserve this species, , was reported (2). Using this method, we produced an enormous number of plants for breeding program, but our attempts to transform the scale explants via Agrobacterium-mediated transformation were abortive. The scale explants were not suitable for Agrobacterium -mediated transformation because of low surface contact with both Agrobacterium and selection medium. Therefore, we considered use of the transverse thin cell layer (tTCL) method as it is known to be an excellent source of material for genetic transformation(3, 4). The transverse thin cell layer (TCL) method provides a large number of cells that are exposed to Agrobacterium infection and maximizes the exposure of putative transformed tissue to the selective medium, reducing the probability of escapes often associated with large explants (3).

TCLs have been used to study lily differentiation with the successful manipulation of all morphogenic programs. The effect of

TCL explant source, sucrose concentration, explant position, activated charcoal (AC) and plant hormone regulators (PGRs) on the mass propagation of Lilium using tTCL explants were studied previously(4). Plant growth regulators are the most effective factors in tissue culture response using the tTCL method for lily plants(5, 3, 6–8, 4). In the present study, the effect of different PGRs on thin cell layer culture was considered for plant regeneration in L. ledebourii.

2. Materials and methods

Plant material, explant source and media culture

In vitro bulblets of L. ledebourii were obtained from the scales culture as described previously (2) and were used as explant source for the experiments. Transverse thin cell layers (tTCL) with 1.2 – 1.4 mm thickness were excised from the basal segment of scales of four-month-old bulblets. The tTCLs were cultured on MS medium (9) containing 30 g/l sucrose, 0.8% agar and supplemented with 6-benzyladenine (BA) alone at concentration of 0.0 and 0.25 mgl-1 or in combination with 0.0, 1.5, 3.0, 4.5,


119 Culture conditions

The pH of media was adjusted to 5.7 before autoclaving at 121ºC and 1.5 kg cm-1 pressure

for 20 minutes. The cultures were incubated at 25 ± 1ºC in the dark for the first month and then transferred to a 16 hours photoperiod at a light intensity of 45 μ mol m-2 s-1 emitted

by cool-white fluorescent tubes under the same temperature condition.

Cold treatment and transplantation After the scaly leaves had wilted, rooted plantlets were removed from jars, washed to remove all adhering gelling materials and then transferred to jars containing autoclaved peat. The jars were then closed slightly with lids and maintained for 8 weeks at 4ºC. After cold treatment, plantlets were transplanted into pots containing perlite, leaf-mold and peat moss (1:1:1 v/v/v) and placed in a greenhouse with 22 ± 2ºC mean temperature, 70 ± 5% relative humidity and natural light conditions.

Data collection and statistical analysis: A completely randomized design was used with 3 replicates per treatment. All collected data were subjected to the analysis of variance, and mean separation was performed using Duncan’s multiple range tests. All percent data were subjected to arc sine (√x) transformation before statistical analysis.

3. Results

All media containing auxin (picloram, 2,4-D, IAA or dicamba) alone or in combination with 0.25 mg l -1BA showed callus induction except media containing high concentrations of 2,4-D. Increasing the 2,4-D concentration dramatically decreased callus production, and only the medium containing 1.5 mg l-1

2,4-D and 0.25 mg l-1 BA produced calli,

with a low number of bulblets (Table 1; data for dicamba not shown ). The highest rate of callus induction 44.4% and 40.0% was occurred in medium containing 1.5 mg l-1 or


Figure 1.


Table 1. Effect of plant growth regulators on callus formation and regeneration of Lilium ٢٣٢

ledebourii transverse thin cell layer.


Plant growth regulators (mg l-1)* No. of explants

producing calli**


Number of bulblets/

explant** BA Picloram 2,4-D IAA

0.0 0.0 0.0 0.0 30.0±4.2 b 1.1±0.2d

0.0 1.5 0.0 0.0 44.4±5.1a 0.0e

0.0 3.0 0.0 0.0 37.3±2.4ab 0.0e

0.0 4.5 0.0 0.0 38.1±3.2ab 0.0e

Figure 1. Plant regeneration using transverse thin cell layers of Lilium ledebourii. Early stages of shoot formation (arrows) after 25 (a) and 35 days (b) of culture (bar = 1 mm) on MS medium supplemented with 0.25 mg l-1 BA in combination with 2 mg l-1 IAA and (c,d) regenerated bulblets after 60 days

on Petri dishes containing PGRs-free medium(bar = 3mm). (e) Enlargement of bulblets after three weeks of culture in jars containing MS PGRs-free medium (bar = 1 cm). (f) Adaption of plantlets in the greenhouse (bar = 4 cm).


Progress in Biological Sciences

Vol. 3, Number 2, Summer/ Fall 2013



Table 1. Effect of plant growth regulators on callus formation and regeneration of Lilium


ledebourii transverse thin cell layer.


Plant growth regulators (mg l-1)* No. of explants producing calli**


Number of bulblets/ explant** BA Picloram 2,4-D IAA

0.0 0.0 0.0 0.0 30.0±4.2 b 1.1±0.2d

0.0 1.5 0.0 0.0 44.4±5.1a 0.0e

0.0 3.0 0.0 0.0 37.3±2.4ab 0.0e

0.0 4.5 0.0 0.0 38.1±3.2ab 0.0e

0.0 6.0 0.0 0.0 40.0±7.5a 0.0e

0.0 7.5 0.0 0.0 33.3±6.4b 0.0e

0.0 0.0 1.5 0.0 20.8±3.5c 0.0e

0.0 0.0 3.0 0.0 25.0±6.8 bc 0.0e

0.0 0.0 4.5 0.0 0.0 f 0.0e

0.0 0.0 6.0 0.0 0.0 f 0.0e

0.0 0.0 7.5 0.0 0.0 f 0.0e

0.0 0.0 0.0 1.0 25.0±6.0 bc 1.0±0.3d

0.0 0.0 0.0 2.0 25.0±6.0bc 1.5±0.3c

0.0 0.0 0.0 3.0 22.0±2.5c 1.4±0.2c

0.0 0.0 0.0 4.0 25.0±3.0 bc 2.1±0.5bc

0.0 0.0 0.0 5.0 25.0±5.0bc 4.4±0.3a

0.25 0.0 0.0 0.0 35.4±7.4bc 4.4±0.7a

0.25 1.5 0.0 0.0 25.9±4.5bc 1.0±0.2d

0.25 3.0 0.0 0.0 29.5±5.5bc 0.0e

0.25 4.5 0.0 0.0 33.3±2.8b 0.0e

0.25 6.0 0.0 0.0 33.3±2.8b 0.0e

0.25 7.5 0.0 0.0 30.0±4.0b 0.0e

0.25 0.0 1.5 0.0 12.5±4.4d 0.7±0.4d

0.25 0.0 3.0 0.0 8.3±2.5e 0.0e

0.25 0.0 4.5 0.0 12.5±3.1e 0.0e

0.25 0.0 6.0 0.0 0.0 f 0.0e

0.25 0.0 7.5 0.0 0.0f 0.0e

0.25 0.0 0.0 1 10.0±2.0e 3.2±1.0b

0.25 0.0 0.0 2.0 25.0±3.8bc 4.2±0.6a

0.25 0.0 0.0 3.0 16.6±2.5d 1.8±0.3c

0.25 0.0 0.0 4.0 23.3±3.4c 1.9±0.3c

0.25 0.0 0.0 5.0 8.3±1.8e 2.0±0.2c

*Mean values followed by the same letters are not significantly different at the 5% level (Duncan’s multiple range test);**The values represent


the mean ± S.D (standard deviation) of 54 explants.



Table 1. Effect of plant growth regulators on callus formation and regeneration of Lilium ledebourii transverse thin cell layer.

6 mg l-1 picloram respectively; no bulblets

were produced under these conditions. The media containing 0.25 mg l-1 BA alone or in

combination with 2 mg l-1 IAA produced the

highest number (4.4 and 4.2 respectively) of bulblets per explant (Fig. 1a-d). The tTCL explants on MS free hormone (1 per explant)

or containing 0.25 mg l-1 BA alone (4.4 per

explant) produced bulblets (Table 1).



mg l-1) in combination with 0.25 mg l-1 BA. In

this study, all media containing dicamba alone or in combination with BA produced calli, but only medium with 1.5 mg l -1 dicamba and 0.25

mg l-1 BA showed low regeneration frequency

(data not shown).

The produced bulblets were cultured in jars containing PGRs-free MS medium to enlarge the bulblets (Fig. 1e). After cold treatment for eight weeks, the bulblets were successfully transplanted to soil with a survival rate of 85%. The plantlets were uniform with emerging new leaves and no significant differences were observed between plantlets (Fig. 1f).

4. Discussion

In plants, the composition of plant growth regulators in the culture medium directs the callus morphotype(10, 11). In this study, effect of different combinations of plant growth regulators on plant regeneration of L. ledebourii using tTCL as explants was studied. Interestingly, in MS medium lacking any plant growth regulators (PGRs), 30.0% of explants formed calli with 1.1 bulblets per explants via organogenesis. Production of calli and a high number of bulblets were observed on the medium containing 0.25 mg l-1 BA

alone. In contrast, Nhut et al. (2001b) reported necrosis when tTCLs of Lilium longiflorum

were cultured on media lacking PGRs, and enlargement of explants when they were cultured on MS medium supplemented with BA alone in. Production of calli and bulblets on PGRs-free medium suggests a high level of endogenous hormones in the scales of bulblet of L. ledebourii .

Callus formation has been reported to occur using picloram in L. formolongi(12, 13). There is no report considering the effect of the above mentioned auxins in regeneration of Lilium using tCTL explants. In our study, the media supplemented with picloram alone or in combination with BA showed higher percentage of callus production compared to media supplemented with the other auxins. However, it failed to regenerate plantlets except at low concentration in combination with BA (Table 1). Media containing dicamba could not produce any bulblets, probably because of the toxicity of dicamba on the bulb scales(14).

The effectiveness of IAA on regeneration of Chrysanthemum, using tTCLs has been shown(4). Our results showed that the highest number of bulblets was achieved in media supplemented with IAA. This suggests that IAA could be a suitable PGR for plant regeneration using tTCL as an explant in L. ledebourii. The media supplemented with high concentration of 2,4-D could not produce bulblets. Nhut et al. (2001b), also reported a significant decrease in bulblet regeneration from tTCL using high concentration of 2,4-D

in L. longiflorum.

Here, we successfully developed a simple plant regeneration system using tTCL as explants. In our previous studies, a high efficiency transformation protocol for Lilium using Agrobacterium-mediated system was developed(15–17). Experiments in which these two methods are combined are in progress for L. ledebourii.


Progress in Biological Sciences

Vol. 3, Number 2, Summer/ Fall 2013




1. Jalili,A. and Jamzad,Z. (1999) Red Data Book of Iran Research Institute of Forest and Rangelands, Tehran. 2. Azadi,P. and Khosh-Khui,M. (2007) Micropropagation of Lilium ledebourii (Baker) Boiss as affected

by plant growth regulators, sucrose concentrations, harvesting season and cold treatments. Electron J Biotechn, 10.

3. Nhut,D., Le,B., Da Silva,J. and Aswath,C. (2001) Thin cell layer culture system in Lilium: regeneration and transformation perspectives. Vitr. Cell Dev B, 37, 516–523.

4. Teixeira da Silva,J.A. and Dobránszki,J. (2013) Plant Thin Cell Layers: A 40-Year Celebration. J PlantGrowthRegul, 10.1007/s00344-013-9336-6.

5. Bui,V.L., Nhut,D.T. and Tran Thanh Van,K. (1999) Plant production via shoot regeneration from thin

cell layer pseudo-bulblet explants of Lilium longiflorum in vitro. C. R. Acad. Sci. Paris, 322, 303–310.

6. Nhut,D., Le,B. and Van,K. (2001) Manipulation of the morphogenetic pathways of Lilium longiflorum

transverse thin cell layer explants by auxin and cytokinin. Vitr. CellDevB, 37, 44–49.

7. Nhut,D., Bui,V., Minh,N., Teixeira da Silva,J., Fukai,S., Tanaka,M. and ran Thanh Van,K. (2002)

Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum.

PlantGrowthRegul, 37, 193–198.

8. Bakhshaie,M., Babalar,M., Mirmasoumi,M. and Khalighi,A. (2010) Somatic embryogenesis and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species. PlantCellTissOrg, 102, 229–235.

9. Murashige,T. and Skoog,F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. PhysiolPlant., 15, 473–497.

10. Gaspar,T., Kevers,C., Penel,C., Greppin,H., Reid,D. and Thorpe,T. (1996) Plant hormones and plant growth regulators in plant tissue culture. Vitr. CellDev B, 32, 272–289.

11. Lin,H.-S., van der Toorn,C., Raemakers,K.J.J.M., Visser,R.G.F., De Jeu,M.J. and Jacobsen,E. (2000) Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria. PlantCellRep., 19, 529–534.

12. Mii,M., Yuzawa,Y., Suetomi,H., Motegi,T. and Godo,T. (1994) Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium ×formolongi by utilizing meristematic nodular cell clumps. PlantSci, 100, 221–226.

13. Godo,T., Matsui,K., Kida,T. and Mii,M. (1996) Effect of sugar type on the efficiency of plant

regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium×formolongi hort. PlantCellRep, 15, 401–404.

14. Famelaer,I., Ennik,E., van Tuyl,J., Meijer,H. and Creemers-Molenaar,C. (1996) The establishment of suspension and meristem cultures for the development of a protoplasts regeneration and fusion in lily. ActaHort, 414, 161–168.

15. Azadi,P., Chin,D., Kuroda,K., Khan,R. and Mii,M. (2010) Macro elements in inoculation and

co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in

Lilium. Plant Cell Tiss Org, 10.1007/s11240-010-9677-9.

16. Azadi,P., Ntui,V., Chin,D., Nakamura,I., Fujisawa,M., Harada,H., Misawa,N. and Mii,M. (2010) Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway. PlantBiotechRep, 4, 269–280.


Table 1. Effect of plant growth regulators on callus formation and regeneration of Lilium


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