In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

doi:10.4236/ajps.2011.22023

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American Journal of Plant Sciences, 2011, 2, 217-222

doi:10.4236/ajps.2011.22023 Published Online June 2011 (http://www.SciRP.org/journal/ajps)

In Vitro Somatic Embryogenesis in Some Oil

Yielding Tropical Tree Species

Premananda Das

The Science Foundation for Tribal & Rural Resource Development, Bhubaneswar, India.

Email: [email protected]

Received February 20th, 2011; revised April 11th, 2011; accepted April 16th, 2011.

ABSTRACT

Somatic embryogenesis was achieved in two oil yielding tropical tree species i.e. Simarouba glauca & Azadirachta in-

dica using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.5 - 1.5

mg/l benzylaminopurine (BA) and 2.0 - 3.0 mg/l NAA (1-napthaleneacetic acid) or 2,4-D (2,4-dichlorophenoxyacetic

acid) and 3% sucrose. MS medium containing 1.0 mg/l BA and 2.0 mg/l NAA was noted to be the most effective in in-

ducing friable embryogenic callus (FEC) in Simarouba glauca; the number of somatic embryos per culture varied in

MS medium supplemented with 1.0 - 1.5 mg/l BA and 1.0 mg/l NAA. In Azad irachta ind ica, somatic emb ryos d eveloped

on MS medium supplemented with 0.5 mg/l BA and 1.5 - 2.0 mg/l 2,4-D which were in various shapes and sizes after

the first subculture on MS medium supplemented with 0.25 mg/l abscisic acid. The somatic embryos which developed

shoots were isolated and rooted in 1/2 strength MS medium supplem ented with 0.25 mg/l abscisic acid and 2% sucrose.

About 25% of embryos germinated within 20 days of culture in ca se of Simarouba glauca and 62% in Azadirachta in-

dica. The so matic embryo-derived plantlets were transferred to the field after being hard ened in the climate contro lled

hardening chamber.

Keywords: Somatic Embryogenesis, Immature Zygotic Embryos, Growth Regulators, Oil Yielding Tropical Tree

1. Introduction

Simarouba glauca Linn. (Simaroubaceae) a multipurpose

fast growing tree from Amazon rainforest and other

tropical areas in Mexico, Cuba, Haita, Jamaica and Cen-

tral America is grown for seed oil, medicines, firewood

and revegetation of barren wastelands even under mois-

ture and nutrient stress. Azadirachta indica A.Juss.

(Meliaceae), popularly called the neem tree, is distributed

in the tropical and subtropical regions of the world [1]. A

native of India and Myanmar, Azadirachta indica was

introduced to Africa, the Middle East, South America

and Australia. All parts of these plants including fruit,

seed, leaf, root and bark are used for their medicinal pro-

perties. The neem tree repor tedly contains more than 100

bioactive compounds. The most important bioactive com-

pound is azadirachtin, other compounds are gedunin,

nimbin and sodium nimbinate. Some plants of Simarouba

glauca and Azadirachta indica bear fruits in profusion,

hence were considered as elite candidates for cloning.

Regeneration of plants via somatic embryogenesis has

been preferred as a method for multiplication of valuable

germplasm in many woody species. Somatic embryo-

genesis was reported for a number of dicotyledonous and

monocotyledonous angiosperms but fewer woody species

[2-4]. Woody species were recalcitrant to in vitro cultu re

and regeneration and most of those reports focused on

propagation or multiplication through organogenesis us-

ing various explants. There are number of reports on so-

matic embryogenesis of woody species [5-11]. It was

thought that plants must coordinate the growth of root

and shoot meristems to maintain an appropriate balance

of root and shoot organs, respond and adapt to various

environmental conditions to achieve an inter-meristem

coordination for growth and development involving the

interplay of several long-range signals [12,13]. In this study

the requirements of culture media, including environ-

mental conditions for induction of somatic embryogenesis,

maturation and germination of the somatic embryos in

economically important oil yielding tropical tree species

for their adaptability t o harsh conditi ons were investigated.

2. Materials and Methods

2.1. Plant Material

Immature fruits (drupe) of Simarouba glauca and Aza-

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

218

dirachta indica were collected from elite trees growing

in dry deciduous forests 50 - 60 days after fruit set. The

fruits were washed with 2% (w/v) detergent solution

(Teepol) for 10 min, further ringed with 70% ethanol for

1 min, surface sterilized with 0.1% (w/v) aqueous solu-

tion of mercuric chloride for 15 min, followed by three 5

min rinses in sterile distilled water. Embryonic axis along

with cotyledons were aseptically cultured on Murashige

and Skoog [14] medium supplemented with various con-

centrations of BA or Kn (0, 0.25, 0.5, 1.0, 1.5, 2.0 mg/l),

NAA or 2,4-D (0, 0.5, 1.0, 1.5, 2.0, 3.0 mg/l) alone or in

combinations for callus induction. The pH of the media

was adjusted to 5.7 using 0.1N NaOH or 0.1N HCl prior

to addition of 0.8% (w/v) agar (Qualigen, India). Rou-

tinely, 20 ml of molten medium was dispensed into 25 ×

150 mm glass tubes (Borosil, India), capped with non-

absorbent cotton plugs wrapped in one layer of cheese-

cloth. The cultures were sterilized at 121˚C and 104 kPa

for 15 mi n.

2.2. Induction of Somatic Embryogenesis

Callus mass (500 ± 20 mg) was transferred to MS me-

dium supplemented with different concentrations of BA,

kinetin and 2,4-D or NAA (0, 0.25, 0.5, 1.0, 1.5, 2.0 , 2.5

and 3.0 mg/l) singly or in combinations for induction of

somatic embryogenesis. L-proline or L-glutamine was

added to the culture medium to enhance the embryogenic

potential. The cultures were incubated under 16h photo-

period with light intensity of 55 µmol·m–2·s–1 provided

by cool, white fluorescent lamps (Phillips, India) at 25˚C

± 2˚C. Subculturing was made every 4 week intervals.

The media were solidified with 0.8% agar-agar. Mor-

phological changes were recorded through visual obser-

vations at 3-week intervals. The embryogenic response

and number of somatic embryos per culture were re-

corded. Each treatment had 20 replicates and the experi-

ment was repeated three times. Data were also recorded

in respect to embryogenic frequency, number of embryos

and frequency of normal embryos per culture.

2.3. Maturation and Germination of Somatic

Embryos

The somatic embryos were transferred to various culture

media with (0.1 - 0.25 mg/l abscisic acid and 2% sucrose)

or without growth regulators for maturation and germi-

nation. The cultures were routinely sub-cultured at

4-week intervals. In another experiment, the cultures

were kept in the dark for 2 weeks which were then trans-

ferred to the light for germination of somatic embryos. In

all the experiments, each treatment consisted of 10 repli-

cations and the experiments were repeated three times.

The somatic embryos were transferred to 1/2 MS me-

dium supplemented with 2% sucrose and abscisic acid

(0.1 - 0.25 mg/l) for germination.

3. Results and Discussion

3.1. Induction of Somatic Embryogenesis

The effects of the different growth regulators on em-

bryogenic callus induction are indicated in Table 1. Fri-

able calli developed from immature zygotic embryos

within 3 - 4 weeks of culture on MS medium supple-

mented with various concentrations of auxins and cyto-

kinins. The maximum proliferation of callu s was noted in

the medium containing BA and NAA or 2,4-D. Kinetin

was not as effective in callus induction as BA. BA with

2,4-D in the medium was observed to be better for callus

proliferation. The proliferated calli were subsequently

sub-cultured on various media for embryogenesis. Em-

bryogenic callus mass developed on MS medium sup-

plemented with 0.5 - 2.0 mg/l BA and 2.0 - 3.0 mg/l

NAA in S. glauca and 0.5 mg/l BA and 1.0 - 2.0 mg/l

2,4-D in case of A. indica.. The medium devoid of

growth regulators did not help in proliferation of em-

bryogenic calli. The maximum rate of embryogenic cal-

lus proliferation was noted on MS medium supplemented

with 1.0 - 1.5 mg/l BA and 2.0 mg/l NAA in Simarouba

glauca and 0.5 - 1.0 mg/l BA and 1.0 - 2.0 mg/l 2,4-D in

Azadirachta indica (Figures 1(a)-(b) and 2(a), Tabl e 1).

Proliferation of friable embryogenic calli in terms of

fresh weight was better in the medium having BA as

compared to Kn. Similar responses were observed when

2,4-D was replaced with NAA. BA at a concentration of

1.0 mg/l along with 2.0 mg/l NAA improved the rate of

embryogenic callus proliferation and improved the pro-

duction of somatic embryos per culture; though NAA

helped in th e proliferation of embryogenic callu s as good

as the 2,4-D, few somatic embryos developed in Sima-

rouba glauca. However, the medium supplemented with

0.5 mg/l BA and 1.5 mg/l 2,4-D showed a higher rate of

proliferation of embryogenic calli in A. indica (Table 2).

Embryogenic callus induction was faster in S. glauca in

the media containing NAA as compared to those having

2,4-D; 2,4-D, however, showed better results in A. indica.

Embryo development in somatic cells was often accom-

panied with cellular stress. Moreover, NAA, the most

frequently used compound for induction of somatic em-

bryogenesis (Figure 2(b)), was known to activate many

stress related genes supporting the hypothesis that somati c

embryogenesis resulted due to extreme stress response of

cultured cells. Proline acted as a potential antioxidant,

which helped in ameliorating the stress [15]. Moon et al

[8] reported that MS medium containing 3% sucrose, 1.0

g/l glutamine along with 2,4-D helped in development of

somatic embryos in Oplopanax elatus. ABA and acti-

ated charcoal appeared to be very important agents for v

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

219

Table 1. Effect of cytokinins and auxins on induction of embryogenic callus from immature zygotic embryos of Simarouba

glauca (SG) and Azadirchta indica (AI).

MS + growth regulator (mg/l) Percent of Explant Response(Mean  SE)*

Kn BA 2,4-D NAA SG AI

0 0.25 0.5 0

18.2  0.8 (NE) 29.4  0.6 (NE)

0 0.25 1.0 0

26.4  0.6 (NE) 32.2  0.8 (E)

0 0.25 2.0 0

42.1  0.8 (NE) 45.6  0.7 (E)

0 0.5 2.0 0

49.8  0.7 (NE) 66.8  0.6 (E)

0 0.5 3.0 0

60.2  0.8 (NE) 51.2  0.8 (E)

0.25 0 1.0 0

38.4  0.8 (NE) 34.4  0.9 (NE)

0.50 0 2.0 0

38.2  0.5 (NE) 50.1  1.0 (NE)

1.00 0 3.0 0

54.2  0.7 (NE) 56.6  0.4 (NE)

0 0.25 0 1.0

32.8  0.9(E) 25.6  0.7 (NE)

0 0.25 0 2.0

45.8  0.6 (E) 36.4  0.8 (NE)

0 0.5 0 3.0

68.8  0.8 (E) 58.4  0.8 (NE)

0 1.0 0 2.0

75.2  0.6 (E) 56.2  0.7 (NE)

0 1.5 0 2.0

76.2  0.8 (E) 66.6  1.0 (NE)

0.25 0 0 20

56.6  0.7 (NE) 56.9  1.1 (NE)

0.50 0 0 3.0

64.4  0.6 (NE) 68.4  0.5 (NE)

*20 Replicates per culture; repeated thrice. NE—Non-embryogenic Ca l li, E—E mbryogenic Calli.

(a) (b)

Figure 1. In vitro somatic embryogenesis in Azadirchta indica. (a)-(b) Development of somatic embryos from immature zygotic

embryos after 4 weeks of culture. (c) Somatic embryos with cotyledons and radicle. (d) Embryo derived plantlets established

in pots.

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

220

Table 2. Development of somatic embryos in embryogenic callus cultures of Simarouba glauca (SG) and Azadirchta indica (AI)

cultured on different induction media after 8 weeks of subculture.

No of Somatic Em bryos p er 200 m g Embr yogenic Cal li (Mean ± SE) *

Culture Medium + 3% ( w/v) Sucrose SG AI

MS + 0.25 mg/l Kn + 1. 0 mg/l NAA 50.4  0.7 0

MS + 0.5 mg/l Kn + 1.0 mg/l NAA 62.6  0.8 8.6  0.9

MS + 0.5 mg/l Kn + 1.5 mg/l NAA 58.2  0.6 22.8  0.7

MS + 0.5 mg/l BA + 1.5 mg/l 2,4-D 14.8  0.4 75.2  0.8

MS + 1.0 mg/l BA + 2.0 mg/l 2,4-D 20.6  0.7 122.2  0.8

MS + 1.5 mg/l Kn + 2.0 mg/l NAA 98.7  0.9 41.6  0.6

MS + 1.5 mg/l BA + 2.0 mg/l 2,4-D 38.7  0.8 80.6  0.9

MS + 1.5 mg/l BA + 2.0 mg/l 2,4-D + 400 mg/l L-proline 42.4  1.1 121.7  1.2

MS + 1.5 mg/l BA + 2.0 mg/l 2,4-D + 600 mg/l L-proline 46.3  0.6 128.4  1.1

MS + 1.0 mg/l Kn + 2.0 mg/l NAA + 600 mg/l L-proline 126.3  0.6 28.4  1.1

(10 replicates per treatment; repeated thrice). *Data collected after four weeks of culture on proliferation medium.

proliferation of somatic embryos., Singh and Chaturvedi

[9] reported that 76% of cultures showed somatic embryo

development directly from immature zygotic embryos in

neem on MS medium having 0.1 µM TDZ and 4 µM

ABA. Globular embryos developed into heart and tor-

pedo shaped embryos faster in media containing NAA

and BA (Figures 1(c) and 2(c)-(d)). In woody species,

explants from immature tissues either seed or seedlings

generally exhibited greater ability for somatic embryo-

genesis as compared to mature tissues [16]. 2,4-D was

reported to induce embryogenic callus in a number of

plant species including woody perennials [17,18]. In

A.indica, the highest number of somatic embryos per 200

mg of callus was 152.0 after 8 weeks of culture on the

medium containing 0.5 mg/l BA and 1.0 mg/l 2,4-D.

Murthy and Saxena [19] included BA in the culture me-

dium for induction of somatic embryogenesis in callus

cultures obtained from mature seeds of the neem; in their

study, BA was used alone during induction of somatic

embryos. Inclusion of L-proline or L-glutamine (600

mg/l) in the induction medium enhanced proliferation of

embryogenic calli. Auxin-induced somatic embryogene-

sis in presence of proline was well documented [20].

Free proline was suggested to act as an osmoticum, a

nitrogen storage pool and source of NADP+, necessary

for rapidly growing embryos. The mediation of the cel-

lular redox potential that resulted fro m proline accumula-

tion likely had a significant effect on the flux through

redox-sensitive biochemical pathways like the pentose

phosphate pathway [21].

3.2. Germination of Somatic Embryos

Both globular and cotyledonary somatic embryos devel-

oped on 1/2 strength MS medium supplemented with

0.25 mg/l abscisic acid (Figure 2(d)). The medium de-

void of growth regulators did not promote germination.

Many of the embryos were morphologically normal and

showed distinct cotyledons and radicles. But the abnor-

mal embryos varied in shape and structure, having one,

two or even more unequal cotyledons which sometimes

looked like a cup (Figure 2(c)). Most of the somatic em-

bryos were loosely attached to form an aggregate of em-

bryos that could be easily separated; some somatic em-

bryos arose from the base of other embryos to form clus-

ters indicating secondary somatic embryogenesis. High

percentage of cotyledonary embryos were recovered

when 0.25 mg/l abscisic acid was added to the medium.

The efficiency of somatic embryogenesis was strongly

dependent on the auxin and cytokinin balance during the

initial phase. Though in most of the woody species, it

was difficult to stop proliferation of somatic embryos

once initiated to sh ift to maturation phase, it was possible

to change over to the maturation phase in most of the

cultures using abscisic acid at 1.0 mg/l. For subsequent

growth and development of the embryos into plantlets,

auxin was not required [22]. Within 20 days of transfer to

maturation medium, about 25% of the somatic embryos

germinated which developed roots without showing sec-

ondary callusing (Figures 1(c), (d) and 2(e), (f)). The

somatic embryos so developed due to exogenous applica-

ion of ABA closely resembled zygotic embryos in both t

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species221

(a) (b) (c)

(d) (e) (f)

Figure 2. In Vitro somatic embryogenesis in Simarouba glauca. (a) Development of embry ogenic callus from immature zygotic

embryos after 4 weeks of culture. (b) and (c) Primary and secondary somatic embryogenesis after 4 we eks of subculture. (d)

Somatic embryos in different shapes & sizes. (e) Germination of somatic embryos on 1/2 strength MS medium supplemented

with 1 mg/l abscisic acid and 2% sucrose after 2 weeks of culture. (f) Somatic embryo derived plantlet.

structure and behaviour. About 50% of the plantlets sur-

vived unde r greenhouse cond i t ions.

4. Conclusions

The somatic embryogenesis reported here in two oil

yielding tropical tree species i.e. Simarouba glauca &

Azadirachta indica can be employed for mass cloning of

superior and elite candidates lines without resorting to

gametes fusion. This protocol can be utilized for genetic

improvement and development of stress tolerant lines

through in vitro selections for adoption to stressful envi-

ronments.

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