Loading

JSM Cell and Developmental Biology

In vitro Selection and Hormonal Regulation in Cell Culture of Artemisia annua L. Plant

Research Article | Open Access Volume 3 | Issue 1 |

  • 1. Green Technology Department, Ipca Laboratories Ltd., India
  • 2. Department of Botany, Faculty of Science, University of Baroda, India
  • 3. Division of Gastroenterology and Liver Disease, Case Western Reserve University, USA
+ Show More - Show Less
Corresponding Authors
Mauji Ram, Green Technology Department, Ipca Laboratories Ltd., Sejavta-457002, Ratlam, Madhya Pradesh, India, Tel: +91-7412-278310; Fax: +91-7412-279083
Abstract

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. plant using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. Callus cultures were induced from leaf and stem explants of A. annua L., at different auxin and cytokinin concentrations. Moderate concentrations of plant growth regulators either in combination or in single in Murashige and Skoog (MS) medium produced friable, light green and non-embryogenic callus from both explants. These totipotent cells gave rise to shoots when transferred to same or different plant growth regulator containing medium as second subculture. Direct shoot induction was raised from leaf and stem explants. High percentages of direct regeneration were obtained from leaf and stem explants on medium supplemented of BAP/NAA (1.5/0.05mg/L). Complete rooting was achieved on full and half strength basal MS medium supplemented with different auxin concentrations. Synergetic effect of plant growth regulator plays an important role in callus induction and cell differentiation. This system exhibited a potential for a rapid propagation of shoots from the leaf and stem explants and makes it possible to develop a clonal selection and propagation of high artemisinin yielding A. annua L. plants.

Keywords

Artemisia annua; Plant growth regulators; Artemisinin; Clonal selection.

Citation

Dangash A, Ram M, Niranjan R, Bharillya A, Misra H, et al. (2015) In vitro Selection and Hormonal Regulation in Cell Culture of Artemisia annua L. Plant. JSM Cell Dev Biol 3(1): 1013.

ABBREVATIONS

2,4-D: 2,4-Dichloro Phenoxy Acetic Acid; NAA: Nephthalene Acetic Acid; IBA: Indol Butyric Acid; IAA: Indol Acetic Acid; BAP: Benzyl Amino Purine; Kin: Kinetin

INTRODUCTION

Artemisia annua, also known as Sweet Wormwood, Sweet Annie, Sweet Sagewort or Annual Wormwood, belonging to the family Asteraceae, is a common type of wormwood that grows throughout the world. It is a crop for the production of antimalarial and possibly antibacterial agents and natural pesticides. It was originally collected by the Chinese as an herbal medicine and is currently processed by pharmaceutical firms for the production of artemisinin for artemisinin-based combination therapies (ACTs) in the treatment of malaria. ACTs have been shown to have rapid resolution to fever and parasitaemia, low toxicity and are well tolerated. Artemisinin, a sesquiterpenelactone isolated from the aerial parts of Artemisia annua L. plants. Artemisinin has proved to be a dramatically effective anti-malarial against multi-drug resistant Plasmodium spp [1,2]. Besides being currently the best therapeutic agent against both drugresistant and cerebral malaria causing strains of Plasmodium sp., [3]. It is also effective against other infectious diseases such as Schistosomiasis, hepatitis B and Lishmmaniasis [4-7]. More recently, it has also been shown to be effective against a variety of cancer cell lines including breast cancer, human leukemia, colon cancer and small cell-lung carcinomas [8,9]. Due to its current use in artemisinin based-combination therapy (ACT), its global demand continuously is increasing. The relatively low yield of artemisinin in A. annua L. plant leaves (0.01-0.8%) however, a serious limitation to the commercialization of the drug [10- 12]. However, cross-pollination in A. annua L. plants is serious limitation to maintain the genetic fidelity and higher artemisinin yield throughout the population.

Micropropagation and organogenesis of different Artemisia species have been previously established by using several parts of plants, in order to obtain large number of plants, such as A. mutellina [13]; A. scorpia [14]; A. vulgaris [15]; A. absinthium [16]. An efficient in vitro method for multiple bud induction and regeneration has been developed in Artemisia annua, using leaf, stem, shoot tip, explants or by using young inflorescence segments [17-21].

In vitro direct organogenesis of different parts of Artemisia annua was investigated, in this research, to obtain a large number of plants true to type. The ultimate goal was the multiplication of the selected clones with high levels of secondary metabolites and the utilization of the protocol in any future genetic transformation of this species.

Tissue culture uses standard protocols with shoot tips of mature field grown plants [22]. Shoot-tips and lateral buds of A. annua L. produced numerous shoots on MS medium and formed 100% roots on half strength Murashige and Skoog minimal organic medium. The medicinal use of A. annua in the tropics should be emphasized. There is therefore an urgent need for the conservation and rapid propagation of the seedlings using tissue culture techniques. This experiment was carried out to study growth response of A. annua L. plant shoot tip and stem explants in vitro, under different combinations of plant growth regulators, to develop a protocol for production of clean healthy plantlets for subsequent multiplication. In vitro selection has been proved very effective method for the selection of high artemisinin yielding genotypes of A. annua L. plants. Thus we have been able to maintain genetic fidelity and production of quality seed of A. annua L. plants

MATERIALS AND METHODS

Plant Materials and Chemicals

This experiment was carried out at Green Technology Department, Ipca Laboratory, Ratlam. Seeds of A. annua L. (CIM AROGYA) were purchased from CIMAP, Lucknow. These seeds were used for the preparation of nursery and 45 days old seedlings of A. annua L. plants were transplanted in the experimental field of Ipca Laboratories Ltd. The leaf and stem explants were selected from high artemisinin yielding A. annua L. plant grown in experimental field of Ipca Laboratory Ltd., Ratlam. Explants were washed under running tap water for 30 min and then surface sterilized with 5% teepol for 5-7 min. Further sterilization of explants was done in Laminar Air Flow; 1.0% bavistin (w/v) and 0.25% streptomycin (w/v) was used for 5 min. 5% solution of sodium hypochlorite (v/v) used for 4 min followed by disinfection with 70% ethyl alcohol for 30 seconds. Explants were then rinsed five times in sterile distilled water before inoculation on media. All chemicals and hormones were procured from Himedia Laboratories (India) and SigmaAldritch (USA). Sucrose and agar were procured from Himedia Laboratories, India. All the buffers and solutions were prepared by using autoclaved MilliQ water.

Media and Culture Conditions

Basal medium used was full strength [23]. The medium containing 3% (w/v) sucrose, B5 vitamins, 0.1 g Inositol, was augmented with different cytokinins and an auxin. For callogenic study, four auxins; 2, 4-D, IAA, NAA and IBA with different concentrations (0.25-1.5mg/L) in combination with two cytokinins; Kinetin and BAP (0.25-1.5mg/L) were used in basal MS. Different concentrations of BAP and Kin (0.25-2.0mg/L) in combination with amino acids (Glutamine-100mg/L; Cystine. HCl-5mg/L; Arginine-50mg/L; Asparagine-40mg/L) and two constant concentrations of NAA (0.05mg/L and 0.1mg/L) were tested for the shoot induction from callus and leaf. While different concentrations of IAA IBA and NAA (0.1-2.0mg/L) were used for root induction. Hormone free MS medium served as control. The pH of the medium was adjusted to 5.8 prior to the addition of 0.8%w/v agar. 20 ml aliquots were dispensed into jam bottles and autoclaved at 121°C at 15 lb pressure for 15 min. Surface sterilized explants were aseptically inoculated in jam bottles and the cultures were maintained at 25 ± 2°C using 16/8 light/dark period, under a light intensity of 3000 lux provided by cool-white fluorescent lamps and 50 to 55% relative humidity.

Statistical analysis

The experiments were entirely randomized with six replicates for each growth regulator(s) concentration(s). Statistical analyses were carried out by the ANOVA and Dunkens Multiple Test, at a 0.5% probability level.

RESULT AND DISCUSSION

Micro propagation is an advanced technique for producing a large number of genetically uniform and pathogen free plants in limited time and space [24]. In vitro clonal propagation of species through tissue culture has been frequently based on the successful adjustment of the type and combinations of plant growth hormones [25,26] .

Callogenic response

The callogenic response form leaf and stem explants was observed at different plant growth regulators concentration either singly or in combination (Table 1).

Table 1: Callogenic response from leaf and stem explant at different growth regulatorsZ.

Growth Regulator

Concentration (mg/l)

Leaves Explant

Stem Explant

Callus formation (%)X

Response

Callus formation (%)X

Response

2, 4-D

0.25

0.5

0.75

1.0

1.25

1.5

62.5

76.0

100.0

100.0

58.0

52.0

++

+++

++++

+++

++

+

59.5

76.0

100.0

86.0

42.5

32.5

++

+++

++++

+++

++

+

IAA

0.25

0.5

0.75

1.0

1.25

1.5

65.0

85.0

100.0

100.0

53.0

44.0

++

+++

++++

++++

++

+

82.5

92.0

100.0

100.0

57.0

41.0

++

+++

++++

+++

++

+

NAA

0.25

0.5

0.75

1.0

1.25

1.5

76.0

100.0

100.0

100.0

100.0

69.0

++

+++

++++

++++

+++

+

71.0

76.5

100.0

100.0

54.0

38.5

++

+++

++++

++++

+++

+

IBA

0.25

0.5

0.75

1.0

1.25

1.5

77.0

88.0

100.0

100.0

68.0

45.0

++

+++

++++

+++

++

+

68.0

79.0

100.0

100.0

53.5

51.0

++

+++

++++

+++

++

+

BAP

0.25

0.5

0.75

1.0

1.25

1.5

62.0

78.0

85.5

53.5

-

-

+

+++

++++

+++

+

-

55.0

72.0

80.0

36.5

-

-

+

++

+++

+++

+

-

Kin

0.25

0.5

0.75

1.0

1.25

1.5

51.0

64.0

74.0

22.0

-

-

++

+++

++++

++

+

-

47.0

61.0

72.5

14.0

-

-

++

+++

++++

++

-

-

BAP/IBA

0.5/0.5

0.5/1.0

0.5/1.5

81.5

100.0

100.0

++

++++

+++

77.0

100.0

100.0

++

++++

+++

BAP/NAA

0.5/0.5

0.5/1.0

0.5/1.5

100.0

100.0

100.0

++++

++++

++++

100.0

100.0

100.0

++++

++++

++++

Kin/NAA

0.5/0.5

0.5/1.0

0.5/1.5

100.0

100.0

100.0

+++

++++

++++

100.0

100.0

100.0

+++

++++

++++

BAP/2, 4-D

0.5/0.5

0.5/1.0

0.5/1.5

87.0

100.0

100.0

+++

++++

+++

82.0

100.0

100.0

+++

++++

+++

Kin/IBA

0.5/0.5

0.5/1.0

0.5/1.5

84.0

100.0

100.0

+++

++++

+++

78.0

100.0

100.0

+++

++++

+++

BAP/IBA/Kin

0.5/0.5/1.0

0.5/1.0/1.0

0.5/1.5/1.0

100

100

100

++++

++++

+++

100

100

100

++++

++++

+++

BAP/NAA/Kin

0.5/0.5/1.0

0.5/1.0/1.0

0.5/1.5/1.0

100

100

100

+++

++++

++++

100

100

100

+++

++++

+++

BAP/2, 4-D/Kin

0.5/0.5/1.0

0.5/1.0/1.0

0.5/1.5/1.0

100

100

100

+++

+++

++++

100

100

100

+++

++++

+++

Control

 

64.0

++

72.0

+++

X %age response of 6 replicates.

Z Rated after 30 days of culture: + = Low, ++ = good, +++ = very good, ++++ = excellent, - = nil.

Callogenic response from leaf and stem explants started at the margins or from injuries. Plant growth regulator (PGR)-free basal MS medium also induced callogenic response from both explants where leave explants showed 90% callogenic response while 73% stem explants produced calli. Nin et al. [27] reported no callogenic response from leaf explant on PGR-free medium and explants died after few days. 2, 4-D as callus inducing hormone produced light green, soft, friable and compact callus from leaf and stem explants. But at all concentration of 2, 4-D organogenic response was not observed within observation time. Nin et al. [27] stated that low concentration of 2, 4-D stimulated adventitious root development from 86% of all explants of A. annua L. At all concentrations of BAP and Kin, the callogenic response was poor. Very low callus was developed which was green, soft and compact. Small and few numbers of leaves also emerged, when the callus remained on the same medium for six weeks or the callus turned to hard and embryogenic. Callus produced at different concentrations of IAA and IBA was yellowish, soft and friable and callogenic response was 100% at lower concentration of both hormones. Nin et al. [27] reported that callogenesis occurred in 100% of explants, independent of the cytokinins/auxin ratio. But at different concentrations of NAA, light green, soft and friable callus was observed. At low concentrations of NAA, small shoots emerged while at higher concentrations callus turned hard and compact. The result shows that media supplemented with BAP either with Kin, NAA, 2, 4-D or IBA produced 100% callogenic response from both explants. 0.5 mg/l BAP and 0.5-1.5 mg/l NAA in combination produced green, soft and friable callus from both explants (Figure 1 & 2).

Figure 1: Callus induction from leaf explant of Artemisia annua L. plant on MS medium (BAP 0.5mg/l + NAA 1.5mg/l).

Figure 2: Callus induction from stem explant of Artemisia annua L. plant on MS medium (BAP 0.5mg/l + NAA 1.5mg/l).

Ganesan and Paulsamy [19] observed 98.66% callogenic response from leaf discs with NAA at 0.9mg/l in A. annua L., while Nin et al. [27] reported best callogenic response with BAP and NAA in the medium for A. absinthium whereas Benjamin et al. [28] observed callus induction from shoot buds using BAP plus IAA for Artemisia pallens. 2, 4-D at varying concentration (0.05 - 0.25 mg/l) in combination with BAP (0.5 mg/l) also produced light green and soft callus when supplemented in MS medium. When IBA and NAA were combined with Kin, the callogenic response was also low and callus was not good in texture. Xu and Jia [29] observed best callus result in the presence of 2, 4-D with Kin for Artemisia sphaerocephala.

Shoot Induction and Regeneration

Shoots were induced from callus and leaf explants by supplementing various combinations of BAP, Kin and NAA. All combinations of these plant growth regulators in callus and leaf explant culture could not induce shoot induction (Table 2 & 3). The period of shoot induction was 4 weeks and it varies in different species of Artemisia [13-16,30].

Shoot induction from callus was observed at different concentration of BAP and Kin, alone and in combination with NAA and amino acids (Glutamine-100mg/L; Cystine.HCl-5mg/L; Arginine-50mg/L; Asparagine-40mg/L) (Table 2).

Table 2: Effect of growth regulators on in vitro shoot induction of Artemisia annua from callus on MS mediumZ.

Growth Regulator

Conc.

 (mg/l)

Response (%)Y

Average No. of of shootsXT

General description

BAP

0.25

0.5

0.75

1.0

1.5

2.0

00.0

28.0

46.0

69.2

79.0

15.0

-

-

0.83 ± 0.234d

1.66 ± 0.412cd

2.16 ± 0.614ab

-

No response

No response

1-2 shoots with small green leaves

1-3 shoots with small green leaves

2-3 shoots with small green leaves

No response but embryogenic callus

Kin

0.25

0.5

0.75

1.0

1.5

2.0

00.0

21.0

36.0

56.2

41.0

00.0

-

-

0.83 ± 0.352d

1.5 ± 0.335cd

-

-

No response but embryogenic callus

No response but embryogenic callus

1-2 shoots with small green leaves

1-3 shoots with small green leaves

No response but embryogenic callus

No response but embryogenic callus

NAA

0.1

0.5

00.0

00.0

-

-

No response but embryogenic callus

No response but embryogenic callus

BAP/NAA

1.5/0.05

1.5/0.1

83.6

26.0

2.83 ± 0.234a

2.16 ± 0.271bbc

3-4 shoots with green leaves

1-2 shoots with green leaves

Kin/NAA

1.5/0.05

1.5/0.1

65.0

16.5

2.33 ± 0.724cd

1.16 ± 0.121d

2-3 shoots with green leaves

1-2 shoots with green leaves

LSD

 

 

1.366

 

XMean ± standard error

Interval of confidence 95%.

YData are mean of 6 replicates.

TMean separation by LSD.

ZRated after 30 days of culture.

Values with the different letters on the same column are significantly different.

At 1.5 mg/l BAP, 2.16 ± 0.614 shoots emerged while at 0.5 mg/l no shoot induction was observed. Nin et al. and Zia et al. [16,27] also did not observe any shoot induction at low concentrations of BAP in A. absinthium. However Le (2001) reported that new axillary shoots development was promoted in Artemisia annua by addition of BAP in MS medium. The best shoot induction 83.6% (2.83 ± 0.234) was observed on BAP (1.5 mg/l) in combination with NAA (0.05 mg/l) (Figure 3).

Figure 3: Shoot induction from callus of Artemisia annua L. plant on MS medium (BAP 1.5mg/l + NAA 0.05mg/l + Glutamine-100mg/L; Cystine.HCl-5mg/L; Arginine-50mg/L; Asparagine-40mg/L).

At different concentrations of Kin alone or in combination with NAA response of shoot induction was observed low. At 1.0 mg/l Kin, shoot induction was 56.2% (1.5 ± 0.335) and at higher concentrations response was absent while the callus turned hard, compact and embryogenic. In the present study, direct shoot induction from leaf explant was also carried out. The best shoot induction 85.7% (3.16 ± 0.434) was observed on BAP (1.5mg/l) in combination with NAA (0.05mg/l) (Figure 4).

Figure 4: Direct shoot induction from leaf explant of Artemisia annua L. plant on MS medium (BAP 1.5mg/l + NAA 0.05mg/l).

Geng et al. [31] observed shoot cluster in A. annua L. on MS medium supplemented with BAP and NAA. Mackay and Kitto [32] and Nam-cheol et al. [33] also reported shoot induction on MS medium supplemented with BAP and NAA in different Artemisia species. Shoot induction was very low or absent at different concentrations of Kn alone or in combination with NAA. At 1.5mg/l Kn along with 0.05mg/l NAA, shoot induction was 65% (2.0 ± 0.724) and at higher concentrations, response was absent (Table 3).

Table 3: Effect of growth regulators on in vitro direct shoot induction of Artemisia annua from leaf explants on MS mediumZ.

Growth Regulator

Conc.

 (mg/l)

Response (%)Y

Average No. of of shootsXT

General description

BAP

0.25

0.5

0.75

1.0

1.5

2.0

00.0

28.0

46.0

69.2

79.0

15.0

-

-

0.833 ± 0.324d

1.5 ± 0.622cd

2.83 ± 0.615ab

-

No response

No response

1-2 shoots with green leaves

1-3 shoots with green leaves

2-3 shoots with green leaves

No response but embryogenic callus

Kin

0.25

0.5

0.75

1.0

1.5

2.0

00.0

21.0

36.0

56.2

41.0

00.0

-

-

0.833 ± 0.352d

2.16 ± 0.335cd

-

-

No response but embryogenic callus

No response but embryogenic callus

1-2 shoots with green leaves

1-3 shoots with green leaves

No response but embryogenic callus

No response but embryogenic callus

NAA

0.1

0.5

00.0

00.0

-

-

No response but embryogenic callus

No response but embryogenic callus

BAP/NAA

1.5/0.05

1.5/0.1

85.7

26.0

3.16 ± 0.434a

1.83 ± 0.271bbc

4-5 shoots with green leaves

2-3 shoots with green leaves

Kin/NAA

1.5/0.05

1.5/0.1

65.0

16.5

2.0 ± 0.724cd

1.66 ± 0.121d

2-3 shoots with green leaves

1-2 shoots with green leaves

LSD

 

 

1.233

 

XMean ± standard error

Interval of confidence 95%

YData are mean of 6 replicates

TMean separation by LSD

ZRated after 30 days of culture.

Values with the different letters on the same column are significantly different.

Vergauwe et al. [34] reported the shoot regeneration from leaf explants of A. annua L. on MS medium with 0.05mg/l NAA and 0.05mg/l BAP after 5 weeks of culture. In the present study, a result of shoot induction rate is in agreement with the report of Banyai et al. [35] who considered 1mg/L BAP with 0.1mg/L NAA as the best supplemented medium for leafexplants-derived shoot regeneration. Almaarri and Yu Xie [20] reported 100 and 66.6% shoot induction in different genotypes of A. annua on MS fortified with TDZ (1 mg/l) and BAP (1 mg/l), respectively. Similar results have also been reported by Sujata and Kumari, Sharma et al. Gonzalez et al. Tahir et al. and Hailu et al. [18,21,36-38].

However, we established an improved protocol for direct shoot regeneration of A. annua L. using leaf explants on MS medium supplemented with BAP and NAA resulting in a rapid and high number of shoots per explant in this study. Therefore, this regeneration system might be a useful method for high regeneration efficiency and has commercial advantage due to the shoot regeneration period over a combination of several plant growth regulators. The regeneration system developed in this study will be useful for plant improvement through micropropagation and genetic engineering of A. annua L. Moreover, this system can be available for the clonal propagation in order to obtain the strain containing a constant concentration of artemisinin in A. annua L.

Root Induction

Regenerated shoots were sub-cultured on same medium and multiplied on BAP (1.5mg/l) in combination with NAA (0.05mg/l) (Figure 5).

Figure 5: Multiplication of regenerated shoots of Artemisia annua L. plant on MS medium (BAP 1.5mg/l + NNA 0.05mg/l).

Individual shoots were isolated and transferred to elongation medium (half strength MS) for 30 days. Elongated shoots were transferred on rooting medium. Rooting was found very well in the present study (Table 4).

Table 4: Effect of growth regulators on in vitro rooting of Artemisia annuaZ.

Growth Regulator

Concentration (mg/l)

Full MS

½ MS

Response (%)Y

Average No. of rootsX

Response (%)Y

Average No. of rootsX

IAA

0.1

0.25

0.5

1.0

1.5

2.0

-

36.0

55.8

23.5

-

-

-

1.16 ± 0.5

2.33 ± 0.6

1.33 ± 0.4

-

-

-

-

45.8

-

-

-

-

-

0.83 ± 0.25

-

-

-

IBA

0.1

0.25

0.5

1.0

1.5

2.0

-

23.5

59.6

-

-

-

-

0.83 ± 0.3

2.0 ± 0.9

-

-

-

-

21.9

55.4

-

-

-

-

0.66 ± 0.2

1.66 ± 0.7

-

-

-

NAA

0.1

0.25

0.5

1.0

1.5

2.0

-

50.6

85.8

43.5

18.7

-

-

1.83 ± 0.6

2.5 ± 0.9

1.33 ± 0.5

1.16 ± 0.3

-

-

20.2

72.3

20.5

-

-

-

0.83 ± 0.3

1.83 ± 0.9

0.66 ± 0.2

-

-

XMean ± standard error

YData are mean of 6 replicates

ZRated after 30 days of culture

Six concentrations of auxins (IAA, IBA, and NAA) were tested in full and half strength MS medium. Highest response 85.8% with 0.5 mg/l of NAA in full MS followed by IBA (59.6%) and IAA (55.8%) was observed from shoots. At 0.5 mg/l NAA at full MS and ½ MS, 2-3 roots were observed. When these plants were transferred on the same medium, they produced further roots. Similar findings were observed by Zia et al. and Mohammad et al. [16,25]. Plants that produced roots were transferred to pots filled with soil and peat moss (3:1) under high humid condition till maturation of leaves, and then transferred to green house.

ACKNOWLEDGEMENT

Alka Dangash thankful to Ipca Laboratories Ltd., Ratlam for providing lab facility to carry out the present study

REFERENCES

1. Brown GD. Secondary metabolism in tissue culture of Artemisia annua. J Nat Prod. 1995; 57: 975-977.

2. Duke SO, Vaughn KC, Croom EM, and Elsohly HN. Artemisinin, a constituent of annual wormwood (Artemisia annua), is a selective phytotoxin. Weed Sci. 1987; 35: 499-505.

3. Newton P, White N. Malaria: new developments in treatment and prevention. Annu Rev Med. 1999; 50: 179-192.

4. Borrmann S, Szlezák N, Faucher JF, Matsiegui PB, Neubauer R, Binder RK, et al. Artesunate and praziquantel for the treatment of Schistosoma haematobium infections: a double-blind, randomized, placebo-controlled study. J Infect Dis. 2001; 184: 1363-1366.

5. Utzinger J, Xiao S, N’Goran EK, Bergquist R, Tanner M. The potential of artemether for the control of schistosomiasis. Int J Parasitol. 2001; 31: 1549-1562.

6. Romero MR, Efferth T, Serrano MA, Castano B, Macias R, Briz O, Marin JJ. Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an ‘in vitro’ system. Antiviral Res. 2005; 68:75–83.

7. Sen R, Bandyopadhyay S, Dutta A, Mandal G, Ganguly S, Saha P, et al. Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes. J Med Microbiol. 2007; 56: 1213- 1218.

8. Efferth T, Dunstan H, Sauerbrey A, Miyachi H, Chitambar CR. The antimalarial artesunate is also active against cancer. Int J Oncol. 2001; 18: 767-773.

9. Singh NP, Lai H. Selective toxicity of dihydroartemisinin and holotransferrin toward human breast cancer cells. Life Sci. 2001; 70: 49-56.

10. van Agtmael MA, Eggelte TA, van Boxtel CJ. Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication. Trends Pharmacol Sci. 1999; 20: 199-205.

11. Laughlin JC. Agricultural production of artemisinin--a review. Trans R Soc Trop Med Hyg. 1994; 88 Suppl 1: S21-22.

12. Abdin MZ, Israr M, Rehman RU, Jain SK. Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production. Planta Med. 2003; 69: 289-299.

13. Mazzetti C, Donata M. Micropropagation of Artemisia mutellina, ISHS Acta Horticulturae 457; Symposium on Plant Biotechnology as a tool for the exploitation of Mountain Lands., Abst. 1998

14. Aslam N, Zia M, Chaudhary MF. Callogenesis and Direct Organogenesis of Artemisia scoparia. Pakistan J Biological Science. 2006; 9: 1783- 1783.

15. Govindaraj S, Kumari BD, Cioni PL, Flamini G. Mass propagation and essential oil analysis of Artemisia vulgaris. J Biosci Bioeng. 2008; 105: 176-183.

16. Zia M, Rehman R, Chaudhary MF. Hormonal regulation for callogenesis and organgenesis of Artemisia absinthium L. African J Biotechnol. 2007; 6: 1874-1878.

17. Lualon W, De-Eknamkul W, Tanaka H, Shoyama Y, Putalun W. Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron. Z Naturforsch C. 2008; 63: 96-100.

18. Sharma A, Yadav AS, Bajaj A, Rai A. Cost effective in vitro micropropagation protocol for conservation of plant resources with special reference to important medicinal plants. J Env Res Dev. 2008; 2: 357-364.

19. Ganesan CM, Paulsamy S. Standardized protocol for the in vitro culture of Artemisia annua L.–A medicinal plant at high altitudes of Nilgiris, the Western Ghats. J Res Biol. 2011; 1: 173-178.

20. Almaarri K and Yu Xie. In vitro direct organogenesis and micropropagation of Artemisia annua. J Biotechnologie Vegetale. 2010; 26: 327-337.

21. Gonzalez FA, Perkins K, Winston MI, Xie D. Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua Progeny and Artemisinin Formation in Regenerated Plants. American J Plant Sci. 2013; 4: 2206-2217.

22. Simon JE. Charles D, Cebert E, Grant L, Janick J, Whipkey A. Artemisia annua L.: A promising aromatic and medicinal. In Advances in new crops. Janick J, Simon JE, editors. Portland: Timber Press; 1990; 522- 526.

23. Saxena G, Banerjee S, Rahman L, Mallavarapu GR, Sharma S, Kumar S. An efficient in vitro procedure for micropropagation and generation of somaclones of rose scented Pelargonium. Plant Sci. 2000; 155: 133- 140.

24. Zobayed SMA, Saxena PK. In vitro grown roots, a superior explant for prolific shoot regeneration of St.John’s wort (Hypericum perforatum L. New stem) in a temporary immersion bioreactor. Plant Sci. 2003; 165: 463-470.

25. Murashige T. Plant propagation by tissue culture: a practice with unrealized potential. Handbook of plant cell culture. Ornamental species. 1990; 5: 3-9.

26. Uranbey S, Sevimay CS, Ozcan S. Development of high frequency multiple shoot formation in Persian clover (Trifolium resupinatum L.). Plant Cell Tissue Organ Cult. 2005; 80: 229-232.

27. Nin S, Morosi E, Schiff S, Bennici A. Callus culture of Atremisia absinthium L. initiation, growth optimization and organogenesis. Plant Cell Tissue Organ Cult. 1996; 45: 67-72.

28. Benjamin BD, Sipahimalani AT, Heble MR. Tissue culture of Artemisia pallens: organogenesis, terpenoid production. Plant Cell Tissue Organ Cult. 1991; 21: 159-164.

29. Xu ZQ, Jia JF. Callus formation from protoplasts of Artemisia sphaerocephala Krasch and some factors influencing protoplast division. Plant Cell Tissue Org Cult. 1996; 44: 129-134.

30. Liu CZ, Murch SJ, EL-Demerdash M, Saxena PK. Regeneration of the Egyptian medicinal plant Artemisia judaica L. Plant Cell Rep. 2003; 21: 525-530.

31. Geng S, Chun YH, Gufeng L, Mi M, Chong K, Geng S, et al. Flowering of Artemisia annua L. test tube plantlets and Artemisinin production with hoot cluster induced from flower organs explants. Chinese J App Environ Biol. 2001; 7: 201-206.

32. Mackay WA, Kito SI. Factors affecting in vitro shoot proliferation of French Tarragon. Hortic Sci. 1988; 113: 282-287.

33. Nam-cheol K, kim JG, Lim JH, Hahn TR. Production of secondary metabolites by tissue culture of Artemisia annua L. J Korean Agri Chem Soc. 1992; 35: 99-105.

34. Vergauwe A, Cammaert R, Vandenberghe D, Genetello C, Inze D, Van Montagu M, et al. Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants. Plant Cell Rep. 1996; 15: 929-933.

35. Banyai W, Nakamura I, Mii M, Supaibulwatana K. High regeneration frequency of transgenic plants in Artemisia annua L. by Agrobacterium tumefaciens mediated gene transformation. Proceedings of the 10th international congress of SABRAO. 22–24 August 2005. The University of Tsukuba, Japan 2005.

36. Sujata G, Kumari BD. Effect of phytohormones on micropropagation of Artemisia vulgaris L. Acta Physiol Plant. 2007; 29: 189-195.

37. Tahir SM, Usmas IS, Katung MD, Ishiyaku MF. Micropropagation of Wormwood (Artemisia annua L.) using leaf primordia. Sci World J. 2013; 8: 1-7.

38. Hailu T, Abera B, Mariam EG. In vitro mass propagation of Artemisia (Artemisia annua L.) CV: Anamed. Plant Tissue Cul & Biotech. 2013; 23: 165-176.

39. Mohammad A, Alam P, Ahmad MM, Ali A, Ahmad J, Abdin MZ. Impact of plant growth regulators (PGRs) on callogenesis and artemisinin content in Artemisia annua L. plants. Indian J Biotechnol. 2014; 13: 26-33.

40. Le CL. In vitro propagation of Artemisia annua L. As a meaningful tool for the selection and domestication of high Artemisinin yielding clones. In: quality enhancement of plant production through tissue culture. Working group 2, Advanced propagation technique. 2nd meeting in the Saloniki, Greece. 2001; 22-25

Dangash A, Ram M, Niranjan R, Bharillya A, Misra H, et al. (2015) In vitro Selection and Hormonal Regulation in Cell Culture of Artemisia annua L. Plant. JSM Cell Dev Biol 3(1): 1013.

Received : 20 Dec 2014
Accepted : 18 Jan 2015
Published : 21 Jan 2015
Journals
Annals of Otolaryngology and Rhinology
ISSN : 2379-948X
Launched : 2014
JSM Schizophrenia
Launched : 2016
Journal of Nausea
Launched : 2020
JSM Internal Medicine
Launched : 2016
JSM Hepatitis
Launched : 2016
JSM Oro Facial Surgeries
ISSN : 2578-3211
Launched : 2016
Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
Author Information X