American Journal of Plant Sciences, 2013, 4, 2206-2217
Published Online November 2013 (http://www.scirp.org/journal/ajps)
http://dx.doi.org/10.4236/ajps.2013.411274
Open Access AJPS
Efficient Somatic Embryogenesis and Organogenesis of
Self-Pollination Artemisia annua Progeny and Artemisinin
Formation in Regenerated Plants
Fatima Alejos-Gonzalez, Kelly Perkins, Malcolm Isaiah Winston, De-Yu Xie*
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, USA.
Received September 23rd, 2013; revised October 20th, 2013; accepted October 26th, 2013
Copyright © 2013 Fatima Alejos-Gonzalez et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua
plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin
formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On
agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation
of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon
embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryo-
genic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which
can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal
medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3 - 6
form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3 - 4 weeks. In all six posi-
tional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds.
Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both so-
matic embryogenesis and organogenesis complete self-pollination to produce seeds in 80 - 90 days of growth in growth
chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the
highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the
understanding of artemisinin biosynthesis and metabolic engineering.
Keywords: Artemisi a an n ua; Artemisinin; Biosynthesis; Self-Pollination; Somatic Embryogenesis; Organogenesis;
HPLC-MS
1. Introduction
To date, Artemisia annua is the only natural resource
producing artemisinin which is the main compound used in
the artemisinin-based combination therapy (ACT) fight-
ing against malarial diseases caused by parasites, such as
Plasmodium falciparum and P. viva [1-5]. As we know,
malaria is one of the most severe infectious diseases
causing life loss of approximately one million people
every year. Since 1970s when artemisinin was identified
to be an endoperoxide lactone sesquiterpene in A. annua
by Chinese scientists [6,7], its medicinal activity helped
Chinese people to effectively fight against and control
malarial disease in China. Later on, this medicine was
recommended to other epidemic countries and regions by
World Health Organization (WHO) [1,3,8]. Over the past
years, due to the low and variable content of artemisinin
in plants, its yield has never met the high demanding for
therapy. To fight against malaria, both institutional labo-
ratories and companies globally have started to investi-
gate A. annua and biosynthesis of artemisinin. Many
great efforts have made multiple progresses in the areas
of selection of ecotypes [9-11], genetic breeding
[2,5,11,12], tissue culture [2,13-15], genetic transforma-
tion [16-19], gene cloning and metabolic engineering
[20-23]. Particularly, the biochemical and transgenic
elucidation of biosynthetic steps from amorpha-4,
*Corresponding author.

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants 2207
11-diene to artemisinic acid [20,24,25] and dihyroar-
temisinic acid [25,26] has provided a high potential for
semi-synthesis of artemisinin. The introduction of these
steps into yeast has allowed the production of artemisinic
acid from fermentation [24]. This invention has devel-
oped a promising potential approach to synthesize ar-
temisinin.
Currently, plant growth in the field is still the main
approach to produce artemisinin for ACT of malaria.
Over the past many years, breeding efforts have largely
increased the yield of artemisinin [11,27] and enhanced
the understanding of artemisinin biosynthesis [5,12].
However, due to the feature of A. annua preferring to
cross pollination and hybridity of progenies [5,27,28],
the variation problem of artemisinin yield has remained
to be resolved. In addition, no success in mutagenesis has
been reported to use forward genetic tools to understand
artemisinin biosynthesis. To overcome this problem, we
have been endeavoring to breed self-pollination plants
[2]. To date, we obtained F6 progenies of plants, in
which no segregation occurs. Accordingly, this self-pol-
lination population allows us being able to investigate
genetics and regulation of artemisinin biosynthesis. Par-
ticularly, self-pollination plants will allow us continuing
to use forward and reverse genetics to dissect the bio-
synthetic pathway of artemisinin and to use metabolic
engineering approaches for high production.
As we know, a successful tissue culture system is the
basis for genetic transformation. Over the past approxi-
mately 30 years, numerous experiments have been per-
formed to use tissue culture to regenerate and propagate
A. annua clones for artemisinin production, as a good
result, basal medium and phytohormone combinations
have been optimized for different ecotypes [13-15,29-36].
These past endeavors greatly helped us save time and
labor to avoid testing all phytohormones. Therefore, in
our investigation, we only selected a few of combinations
of plants hormones to test regeneration capacity of
self-pollination plants and develop protocols. Young
seedlings were used as material resources. Leaves from
the first node to the sixth node of seedlings were used as
explants to compare their regeneration efficiency. Of
them, the sixth leaf showed 100% efficiency in both so-
matic embryogenesis and organogenesis. This high re-
generation efficiency allows us to further utilize self-
pollination plants for future genetic transformation and
knockout of genes to understand the biosynthetic path-
way and regulation of artemisinin in the future.
2. Materials and Methods
2.1. Chemicals
Indo-3-butyric acid (IBA), naphthaleneacetic acid (NAA),
6-benzylaminopurine (BAP or 6-BA), sucrose, and phy-
toagar as well as chemicals of macronutrients, micronu-
trients and organic nutrients used in basal MS medium
[37] were purchased from Plant Media (Dublin, OH,
USA). Thidiazuron (TDZ) was purchased from Sigma
(St. Louis, MO, USA).
2.2. Medium Preparation and Photoperiod
The basal MS medium was used in our experiments.
Phytohormones used were sterilized using a filtration
through a 0.2 µm membrane. All media used in experi-
ments were added 2% (W/V) sucrose and 0.45% (W/V)
phytoagar, adjusted to pH 5.7 with 1 N NaOH and then
autoclaved 35 min at 121˚C. After autoclaved, media
were cooled down to 50˚C - 60˚C, necessary phytohor-
mones were added to reach working concentrations used
in each medium described below. Twenty milliliters of
liquefied agar medium was poured into one petri dish (15
× 100 mm, height × diameter) and then solidified at room
temperature.
The photoperiod and temperature for callus induction
and regeneration were set up at light/dark (16/8 hrs) and
24˚C - 25˚C, respectively. The light intensity was set up
at 50 µmol·m−1·s−1.
2.3. Seed Germination and Selection of Explants
Seeds from self-pollination progeny plants (F5 and F6)
of A. annua grown in phytotron were used in this ex-
periment. Seeds were treated 1 min in 0.5 ml of 70%
ethanol contained in a sterile Eppendorf tube. During this
treatment, the tube was vortexed thoroughly. Ethanol was
then removed to a waste container. Seeds were subse-
quently washed four times using autoclaved deionized
H2O. These surface-sterilized seeds were then treated 5
min using 10% Clorox in a sterile Eppendorf tube, during
which the tube was vortexed 1 min thoroughly. After
Clorox was disposed into a waste container, seeds were
washed four times using autoclaved deionized H2O. Ster-
ilized seeds were placed on phytoagar-solidified MS me-
dium contained in petri dishes, which were then placed in
an incubator with necessary photoperiod and temperature
described above.
After three weeks of seed germination, seedlings (Fig-
ure 1(a)) developed the first two true simple leaves (#1
and #2) in addition to the two cotyledons. The size of
two leaves was approximately 0.8 - 1 cm in length. The
first and second leaves of these three-week old seedlings
were excised for explant materials. The 3rd, 4th, 5th and 6th
leaves (Figure 1(b)) of 35-old seedlings were excised as
explant materials.
For explant preparation, the first and second leaves
were wounded on both adaxial and abaxial surfaces with
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Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants
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Figure 1. Growth of seedlings and leaves used for explants. (a) Seedlings (three-week old) grown on agar-solidified ME1
(basal MS) medium in a petri dish; (b) Morphologies of the 1st and 2nd leaves from three-week old seedlings and the 3rd
through 6th leaves of seedlings from 35-day old seedlings.
2.6. Induction of Adventitious Roots to Obtain
Plantlets
a sterilized razor. The 3rd - 6th leaves were cut into ap-
proximately 0.8 × 1 cm size pieces. Wounded leaf pieces
were used as explants for induction of calli and adventi-
tious buds described below. Based on many optimized media for rooting of adventi-
tious buds/shoots reported previously (seeing discussion),
we selected one medium consisting of basal MS medium
supplemented with 0.05 mg/l NAA (Table 1). Agar-so-
lidified rooting medium was contained in 15 cm long
glass tubes.
2.4. Treatment of Explants with TDZ and IBA
Combinations
Three different concentrations of TDZ and 0.1 mg/l IBA
were selected to form three combinations (Table 1).
Meanwhile, MS basal medium was used as a control.
Thirty explants from the 1st and 2nd leaves (Figure 1(b))
were inoculated onto agar-solidified medium contained
in one petri dish (100 × 15 mm, diameter × height, in
size). The other petri dish was performed as a technical
repeat for each medium. Petri dishes were sealed with
parafilm and placed under the culture condition described
above. Explants were examined every day and taken
pictures at different days (e.g. 2, 7, 17 and 30 days) after
inoculation. The dates of callus and adventitious bud
formation were recorded in detail. This experiment was
repeated 4 times. In addition, this experiment was tested
with both F5 and F6 progeny plants, respectively.
Adventitious buds (0.3 - 0.5 cm in length) from so-
matic embryos induced by TDZ and IBA combinations
were excised from calli for root induction. Adventitious
buds (0.5 - 1 cm in length with 2 - 3 leaves) induced by
BAP and NAA were separated from explants or calli for
root induction. Adventitious buds were inoculated on
rooting medium (ME5, Table 1) contained in glass tubes.
All tubes were sealed with parafilm and placed under the
culture condition described above.
2.7. Plant Growth in Soil, Self-Pollination and
Seed Germination
After one month of root induction, plantlets were trans-
planted to small pots (15 × 15 cm, diameter by height)
filled with premier Pro-Mix-PGX (fine granulated) soil.
One pot was planted with one plantlet and then was
placed on a nursery bed facilitated with a photoperiod of
12/12 (light/dark) at 25˚C in the Phytotron. The light
intensity was set at 50 µE/m2/sec. During the period in
the nursery bed, plants were misted with tap water one
time per 3 sec during the light cycle and one time per 3
min during the dark cycle. After two weeks of growth,
each regenerated plant was transplanted into a 10 cm pot
filled with premier Pro-Mix-PGX (fine granulated) soil.
All plants were then placed in a growth chamber facili-
tated with a photoperiod of 9/15 hrs (light/dark). A tem-
perature cycle was set at 26˚C/22˚C (light/dark) as re-
ported previously [2]. Plants were watered every other
day with nutrients and alternate days with tap water.
2.5. Test of Regeneration Capacity among
Leaves #1 through #6
In this study, we selected two combinations of plant
hormones to test regeneration capacity of explants from
different positional leaves. One was 0.6 mg/L TDZ and
0.1 mg/L IBA (ME4, Table 1) and the other was 1 mg/l
BAP and 0.05 mg/l NAA.
Explants were obtained from 1st, 2nd, 3rd, 4th, 5th and 6th
leaves (Figure 1(b)) respectively, of which explants
from leaves #1 and 2 were considered as one group,
while each of others was as an individual group, respec-
tively. Fifteen explants from each group were inoculated
onto agar-solidified medium contained in one petri dish.
Two petri dishes were tested as a technical repeat for
each group of explants. Inoculation, observation and
taking picture were as described above. This experiment
was repeated 4 times and tested using both F5 and F6
progenies of plant, respectively.
To test self-pollination, each plant was covered using a
plastic bag with an opening of the top and management
of flowering and seed harvest were the same as reported
previously [2].

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants 2209
Seeds harvested from regenerated plants were tested
for germination on ME1 medium as described above.
Seeds germinated on medium were recorded to evaluate
the capacity of germination rate.
2.8. Scanning Electron Microscope Observation
of Somatic Embryogenesis
After inoculation of 10, 12, 14, 16 and 18 days respec-
tively, calli induced from explants on medium ME4 (Ta-
ble 1) were collected for SEM observation. Calli were
immersed in 3.0% glutaraldehyde dissolved in 0.05 M
potassium phosphate buffer (pH 6.6) at 4˚C. After two
weeks, calli were washed with 0.05 M potassium phos-
phate buffer (pH 6.6), one change of buffer per 20 min
for three changes at 4˚C. Washed calli were successively
treated with 30%, 50%, 70%, 95% and 100% ethanol on
ice, 2.5 hrs per treatment, to remove water from calli.
Dehydrated calli were warmed to room temperature. To
obtain complete dehydration, calli were treated additional
twice in 100% ethanol at room temperature, each 2 hrs.
Dehydrated calli were summited to a critical point dry for
15 minutes at critical point using liquid carbon dioxide
(Tousimis Samdri-795, Tousimis Research Corporation,
Rockville, MD) and then were mounted on stubs with
double-stick tape. Finally, mounted calli were sputter
coated with approximately 50Å gold-palladium (Hum-
mer 6.2 sputtering system, Anatech USA; Union City CA)
and stored in a vacuum desiccator. Coated calli were
scanned at 20 kV using a JEOL JSM-5900LV (JEOL
USA; Peabody, MA).
2.9. Extraction of Artemisinin and LC-MS
Analysis
To understand if regenerated plants produce artemisinin,
rosette leaves were collected from seedlings that were
grown for 30 days in the photoperiod of 15/9 hrs
(light/dark). One hundred milligrams of fresh leaves was
used to extract artemisinin using LC-MS grade methanol.
Identification of artemisinin was carried out using
LC-MS analysis on a 2010EV Shimadzu LC-PDA-ESI-
Table 1. Media tested for regeneration of self-pollinated A.
annua progeny.
Medium Components
ME1 Basal MS medium solidified
with 0.45% phytoagar
ME2 (regeneration) MS2 + 0.1 mg/L IBA + 0.2 mg/L TDZ
ME3 (regeneration) MS2 + 0.1 mg/L IBA + 0.1 mg/L TDZ
ME4 (regeneration) MS2 + 0.1 mg /L IBA + 0.6 mg/L TDZ
ME5 (rooting) 0.05 mg/L NAA
MS instrumentation. Extraction and LC-MS analysis
protocols were as described previously [2].
3. Results
3.1. Induction of Embryogenic Calli, Somatic
Embryos and Development of Plantlets
In our experiments, before we tested other leaves, we
firstly focused to use leaves #1 and 2 (Figure 1(b)) to
investigate effects of selected media on induction of cal-
lus and adventitious bud from explants. The reason was
that experiments could be started after seed germination
of three weeks. This method saved time. As described in
methods, we investigated 4 media, ME1-ME4 (Table 1).
The number of explants forming calli on each medium
was recorded. In comparison, explants on ME4 showed
the rapidest responses. In the first week, explants on this
medium started to obviously expand to form calli from
wounded sites. Approximately 3 weeks of inoculation,
explants formed obvious friable yellow-greenish calli
(Figure 2(a)) and developed a certain number of adven-
Figure 2. Regeneration from explants cultured on agar-
solidified MS medium supplemented with TDZ (0.6 mg/l)
and IBA (0.1 mg/l) (ME4, Table 1). Petri dish pictures (a)-(e)
were taken after inoculation of explants for 17 days. (a)
Explants from leaves #1 and 2, in which “a-i” is an inserted
image showing adventitious bud formation from calli at day
24 after inoculation of explants; (b)-(e) Explants from leaf
#3 (b), leaf #4 (c), leaf #5 (d) and leaf #6 (e) show their dif-
ferential responses; (f) Formation of adventitious buds from
embryogenic calli induced from explants of leaf #6 at day 24
after inoculation; (g) Plantlets obtained from rooting me-
dium (ME5, Table 1).
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Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants
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titious buds, which continuously developed leaf struc-
tures in the following 4th and 5th weeks of culture (Figure
2(a-i)). In addition, although unlike the rapid responses
on ME4, yellow-greenish calli and adventitious buds
were induced from explants on ME2 and ME3, respec-
tively. In contrast, explants neither formed calli nor ad-
ventitious buds on ME1, the basal MS medium.
Among three combinations of TDZ and IBA (Table 1,
ME2-ME3), the percentage of explants forming calli was
similar on ME2, ME3 and ME4 after nearly 3 weeks of
inoculation (Figure 3(a)). In contrast, the percentage of
adventitious bud formation from calli was significantly
lower on ME2 than on ME3 and ME4, between which
the average value on ME4 was higher (Figure 3(a)). As a
result, we used ME4 to compare regeneration capacities
of leaves #1 through 6. In contrast, neither calli nor ad-
ventitious buds were formed from explants on ME1
(Figure 3(a)).
To understand the features of calli, we collected callus
samples induced on ME4 at different dates and then im-
mediately fixed them for SEM observation. Under SEM,
different stages of somatic embryo structures were ob-
served, including globular, torpedo, heart and early coty-
ledon embryos (Figures 4(a)-(d)). These results demon-
strated that TDZ and IBA tested induced embryogenic
calli. The formation of adventitious buds on ME2-ME4
was via a procedure of somatic embryogenesis.
On ME4, somatic embryos could continuously develop
into vegetative structures, such as buds and leaves (Fig-
ures 2(a-i) and (f)). Many adventitious buds with leaves
were formed from calli after three weeks of induction. As
culture continued, multiple independent adventitious
shoots (elongated adventitious buds) became highly ob-
vious. This result showed that on this medium, somatic
embryos could further develop to form shoot apical mer-
istems and leaves. Furthermore, this observation was
highly obvious on explants from leaves #3 - 6 described
below. However, no plantlets were obtained on ME4.
Neither was a plantlet formed on ME2 and ME3.
To obtain plantlets, adventurous shoots were inocu-
lated onto a rooting medium (ME5, Table 1), which was
supplemented with 0.1 mg/l NAA. On this medium,
nearly 100% of shoots developed roots to form plantlets
(Figure 2(g)). Therefore, the use of ME4 and ME5 were
effective to induce regeneration via somatic embryo-
genesis.
3.2. Comparison of Leaves #1 through #6
Responding to TDZ and IBA
To understand regeneration capacity of different leaves
from seedlings, we compared six positional leaves, in-
cluding the 1st and 2nd leaves (leaves #1 and 2) from
three-week old seedlings and the 3rd - 6th leaves (leaves
#3 - 6) from five-week old seedlings. For this compari-
son, we tested explants on ME4. Responses of explants
were recorded in detail at different dates. After inocula-
tion of 17 days, almost all explants from different leaves
formed embryogenic calli (Figures 2(a)-(e)); although
the average percentage value of induction from leaves #1
and 2 was slightly lower (Figure 3(b)).
The formation of adventitious buds (from somatic em-
bryogenesis) was also obvious at day 17 after explant
inoculation (Figures 2(a)-(e)). After three weeks of in-
duction, multiple adventitious buds developed from so-
matic embryos were characterized with one-two leaves
(Figures 2(a-i) and (f)) but without roots. In the six posi-
tional leaves, explants from the 6th leaf exhibited the
highest average percentage value (Figure 3(b)). As cul-
ture was extended to 3 - 4 weeks, embryogenic calli in-
duced from all explants of the 6th leaf formed multiple
adventitious buds (Figure 2(f)).
Somatic embryos induced from different leaf explants
could not form roots on ME4. For root induction, adven-
titious buds were cultured onto ME5, on which, nearly
100% of them formed roots to develop into complete
plantlets (Figure 2(g)).
3.3. Regeneration on Medium Supplemented
with BAP and NAA
Over the past 30 years, many concentration combinations
of BAP and NAA were tested to induce organogenesis of
A. annua plants. Multiple combinations of different phy-
tohormones such as BAP, NAA, IAA, KT and IBA have
been optimized for different ecotypes [13-15,34,35,
38-43]. Based on these previous reports, we only chose
one combination consisting of 1 mg/l BAP and 0.05 mg/l
NAA to test regeneration capacity of leaves.
Leaves #1 through #6 of seedlings were used for ex-
plants to compare their responses to BAP and NAA. Re-
sultant data showed differences in induction of both cal-
lus and adventitious bud among explants (Figure 3(c);
Figures 5(a)-(e)). After inoculation of 17 days, all ex-
plants from leaves #4, 5 and 6 formed greenish compact
calli. The average induction rate of calli from leaf #1 and
2 was approximately 63%, significantly lower than those
values from other leaves (Figure 3(c)). As culture con-
tinued, all explants from different leaves produced calli.
Under microscope, calli induced by BAP and NAA were
relatively compact and different from embryogenic calli
induced by TDZ and IBA described above.
In addition, of 6 positional leaves tested, explants from
leaf #6 gave the highest induction rate of adventitious
bud formation at day 17, the average percentage value of
which was significantly higher than those values from
leaves #1, 2, 3 and 4 (Figure 3(c)). Approximately 92%
of explants from leaf #6 produced adventitious buds.
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Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
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Figure 3. Effec ts of media and le af positions on callus induction and adventitious bud for m ation. (a) Data show percentages of
induction of embryogenic calli and formation of adventitious buds from le af #1 and 2 explants on ME1-ME4 (Table 1) after
cultured 21 days; percentage values are mean values from 4 independent experiments, each of which was performed with 30
explants and technically repeated once; error bars are calculated from standard deviation. Columns labeled with different
letters such as “A” and “B” indicate significant differences evaluated by Student’s T-test, P < 0.05, while labeled with the
same letter such as “B” and “C” indicate insignificant differences. (b) Data show percentage values of embryogenic calli in-
duction and formation of adventitious buds from explants of leaves #1 - 6 after inocul ation of 17 days on medium ME4; per-
centage values are mean values of 4 independent experiments, each of which was performed with 15 explants and technically
repeated once; error bars were calculated from standard deviation. Columns labeled with the same capitalized letters indi-
cate insignificant differences (P values > 0.05), while columns labeled with different capitalized letters indicates significant
differences (P < 0.05) evaluated by Student’s T-test. (c) Data show percentage values of induction of calli and formation of
adventitious buds from explants of leaves #1 - 6 after cultur ed 17 days on MS medium supplemented with BAP (1 mg/l) and
NAA (0.1 mg/l); percentage values are mean values of 4 independent experiments, each of which was performed with 15 ex-
plants and technically repeated once; standard error bars were calculated from standard deviation; columns labeled with the
same capitalized letter(s) indicate insignificant differences (P > 0.05), while columns labeled with different capitalized letters
indicates significant differences (P < 0.05) evaluated by Student’s T-test.

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants
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Figure 4. Images of scanning electron microscopy showing different stages of somatic embryo structures formed on embryo-
genic calli induced from explants of leaves #1 and 2 on ME4. SEM images were taken from embryogenic calli induced from
explants after inoculation of 10 (a), 12 - 14 (b), 16 (c) and 18 (d) days, respectively. ge: global embryos, te: torpedo embryos,
he: heart embryos; ce: coty ledon embryos.
Furthermore, as culture continued, all explants of leaf #6
produced multiple adventitious buds.
On this medium, adventitious buds could not develop
roots. To induce roots, we cultured adventitious shoots
on ME5. After three weeks of induction, approximately
100% of adventitious shoots formed roots to obtain
plantlets (Figure 5(f)).
3.4. Growth of Plantlets in Pot Soil,
Self-Pollination and Seed Germination
By following the growth protocol of seedlings, flowering
induction and self-pollination that we developed previ-
ously [2], we grew regenerated plants in growth chamber
to induce flowers and self-pollination. After plantlets
were transplanted to small pots (15 × 15 cm) filled with
premier Pro-Mix-PGX (fine granulated) soil, most of
them grew to develop new leaves and elongated stems in
the photoperiod of 15/9 hrs (light/dark) in phytotron
(Figure 6(a)). After nearly 5-week’s growth in long pho-
toperiod, regenerated plants were transferred into a
growth chamber with a short photoperiod (9/15 hrs,
light/dark), in which plants started to develop flowers
after additional two weeks of growth (Figure 6(b)) and
then covered with sleeve-like plastic bag for self-polli-
nation. All regenerated plants grew 25 - 35 centers tall to
start to bloom and set seeds (Figure 6(b)), as seedling
growth reported previously.
After nearly 80 days of transplanting, plants were
ready for seed harvest. Each pot containing one plant
with numerous dry inflorescence heads was moved to a
dry room, in which plants were not watered and naturally
dried for one additional week at room temperature. Then,
seeds (Figure 6(c)) were harvested from each individual
plant and were used for germination test on ME1 me-
dium. All mature seeds germinated to develop new seed-
lings in petri dishes. In addition, all seeds germinated in
soil.
3.5. LC-ESI-MS Analysis of Artemisinin
The establishment of regeneration protocols was to in-
vestigate artemisinin biosynthesis in and accelerate meta-
bolic engineering using self-pollination plants. In our ex-
periments, artemisinin formation was investigated using
LC-ESI-MS analysis. As reported previously to show
artemisinin biosynthesis in self-pollinated plants, positive
ionization mode was used to add one proton to artemisi-
nin [2]. In this condition, artemisinin standard was cre-
ated one main mass fragment, 341 [m/z]+ = [artemisinin
+ Na + Cl]+. In addition, another main mass fragment
was created to be 305 [m/z]+ = [artemisinin + Na]+. In
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Figure 5. Regeneration from leaf explants on agar-solidified
MS basal medium supplemented with BAP (1 mg/l) and
NAA (0.1 mg/l). (a)-(e) Explants from leaves #1 and 2 (a),
leaf #3 (b), leaf #4 (c), leaf #5 (d) and leaf #6 (e), which were
cultured 17 days, show their differential responses to this
induction medium; (f) Plantlets obtained from rooting me-
dium (ME5, Table 1).
our analysis, we used 341 [m/z]+ to create selected ion
chromatographs to detect artemisinin in extraction of leaf
samples. As artemisinin standard showed, a high abun-
dant peak of artemisinin was detected in leaf extracts at
the same retention time (Figure 7(a)). However, this
peak was not detected in root extracts (Figure 7(b)). This
result demonstrated that regenerated plants produced
artemisinin.
4. Discussion
In this study, our goal was to test regeneration capacity
of seedling leaves and then develop an efficient regen-
eration protocol for self-pollination A. annua plants. We
understood that in plant tissue culture, testing multiple
combinations of plant hormones was essential to develop
protocols, but, we did not follow this regular experimen-
tal logic in our experiments. The reason was that since
middle 1980s, numerous experiments have been per-
formed to use tissue culture to regenerate and propagate
A. annua clones for artemisinin production [13-15,29-36].
Although there were many challenges in optimizing con-
Figure 6. Growth, blooming and seeds of regenerated plants
in growth chamber. (a) Examples of regenerated plants
from somatic embryogenesis; (b) Blooming of a regenerated
plant; (c) Seeds from self-pollination of a regenerated plant.
ditions for regeneration of different ecotypes of A. annua,
numerous solid results regarding the use of basal medium
and combinations of plant hormones such as 2, 4-D, BAP,
NAA and IBA were obtained from those investigations.
These useful data helped us save time and labor to avoid
testing all of phytohormones. In comparison, TDZ was
seldom used for regeneration of A. annua. Accordingly,
we selectively tested 3 combinations consisting of TDZ
and IBA (Table 1) and one combination of BAP and
NAA to induce regeneration of self-pollination progenies
and compare regeneration capacities of different leaves.
The rationale to choosing TDZ was that this synthetic
cytokinin has been reported to be able to induce somatic
embryogenesis of many plants, such as Acacia mangium,
Catharanthus roseus and Bambusa edulis [44-46]. In our
experiments, results showed that 0.6 mg/l TDZ and 0.1
mg/l IBA highly efficiently induced somatic embryos
from explants, particularly from leaf #6 of seedlings
(Figures 3(b) and 4), nearly 100% of which produced
somatic embryos further forming adventitious buds. To
our best knowledge, this is the first report to induce so-
matic embryogenesis from leaf explants of self-pollina-
tion plants of A. annua. In addition to somatic embryo-
genesis, the efficiency of organogenesis from leaves #1
through 6 was very high (Figures 3(c) and 5). All ex-
plants from leaf #6 produced adventitious buds in three
weeks. These results demonstrated the high efficiency of
Open Access AJPS

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants
Open Access AJPS
2214
Figure 7. Selected ion chromatographs showing formation of artemisinin in leaves of regenerated plants. (a) A peak showing
artemisinin in crude leaf extraction characterized by a mass-to-charge of 341 [m/z]+; (b) No peaks at 341 [m/z]+ detected from
crude root extraction; (c) An authentic standard of artemisinin characterized by a mass-to-charge of 341 [m/z]+.
regeneration capacity of leaves of self-pollination plants.
Another goal of this investigation was to compare re-
generation capacity of different positional leaves of seed-
lings and to select explant resources for future genetic
transformation to enhance metabolic engineering of ar-
temisinin. We understand that testing all positional
leaves can provide a comprehensive result showing re-
generation capacities of all leaves. In consideration of
reducing time, labor and spaces, we chose 3-week to
5-week old seedlings grown on agar medium in our in-
vestigation. This time frame allowed us testing regenera-
tion of explants in a relatively short period. Our data
showed that although explants from the 1st to the 6th
leaves of seedlings could efficiently produce embryo-
genic calli and adventitious buds on ME4, the average
values of the 6th leaf were higher than those of other
leaves in nearly 3-week period after inoculation (Figure
3(b)). Actually, after continuous culture to 4 weeks, all
explants of the 6th leaf produced adventitious buds. In
addition, on the medium containing BAP and NAA, ex-
plants from the 6th leaf showed the highest percentage of
adventitious bud induction in approximately 3 weeks
after inoculation (Figure 3(c)). These results indicate
that the 6th leaf tested in our experiments has the highest
regeneration capacity. The possible reason was that on
5-week old seedlings, the 6th leaf was less mature than
others, thus gave the highest efficiency. The other possi-
ble reason was that the 6th leaf itself had higher regenera-
tion capacity than others. This is because the spatial posi-
tions of tissues have been reported to dramatically affect
regeneration of plants, the examples of which are Popu-
lus trichocarpa [47], A. mangium [48] and Cornus cana-
densis [49].
This investigation is to develop self-pollination A. an-
nua plants as a platform to understand genetics of ar-
temisinin biosynthesis and to enhance metabolic engi-
neering for high yield. As we know, the final elucidation
of biosynthetic pathways of natural products essentially
needs genetic evidence. For example, genetic evidence
from Arabidopsis thaliana and other model plants has
helped the intensive understanding of anthocyanin and
proanthocyanidin pathways in the plant kingdom [50-54].
To date, biochemical, molecular and synthetic evidence
has demonstrated the enzymatic steps from amorphor-4,
11-diene to artemisinic acid and dihydroartemisinic acid
[22-26,55] and mapping of F1 hybrid of A. annua has
helped identify loci associated with artemisinin forma-
tion [5], however, genetic evidence, such as knockout of
genes and their impact on artemisinin productions, re-
mains largely lacking. One of crucial reasons has been
the challenge of the heterogeneous progeny resulting
from the cross-hybridization preference of A. annua
[2,9,27,28]. This heterogeneity of progeny increases dif-
ficulty to select mutant plants to identify pathway and
regulatory genes involved in artemisinin biosynthesis. In
addition, genetic transformation of A. annua has been a
challenging hurdle in metabolic engineering of artemisi-
nin most likely due to heterogeneity of progeny [43]. We
have developed self-pollination plants [2]. Progenies of
F5 and F6 generations have not shown any segregation in

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination Artemisia annua
Progeny and Artemisinin Formation in Regenerated Plants 2215
plant growth and morphology as well as artemisinin for-
mation, indicating that they are mostly likely inbred ho-
mozygous plants. We believe that the protocol of effi-
cient regeneration developed in present study will help
accelerate the use of self-pollination plants to develop
genetic approaches such as mutagenesis to elucidate
biosynthetic steps and regulatory mechanism of artemisi-
nin formation.
5. Conclusion
The high regeneration variation of different ecotypes of A.
annua plants has been reported to be a severe hurdle for
the success of genetic transformation. The main reason
likely is the segregation of progeny plants resulting from
natural cross-hybridization. Our experiments demonstrate
a high and reproducible regeneration efficiency of self-
pollinated A. annua progeny through both somatic em-
bryogenesis and organogenesis. Positional effects of
leaves from juvenile seedlings on callus induction and
regeneration are observed in our experiments. In the se-
lected first six leaves of seedlings, the sixth leaf shows
the rapidest response to induction of embryogenic callus
and organogenesis as well as regeneration. Regenerated
plants from both somatic embryogenesis and organo-
genesis produced a valuable level of artemisinin. Our
data show that self-pollinated A. annua plants form an
appropriate platform to genetically understand artemisi-
nin biosynthesis and enhance metabolic engineering.
6. Acknowledgements
This research was funded by Multidisciplinary Research
Grant (MRG) Program at North Carolina Biotechnology
Center. We are grateful to Mrs. Valerie Knowlton at
Center for Electron Microscopy for her assistance in
SEM.
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