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.

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

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.

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

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

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.

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.

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.

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.

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.

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.

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

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