somatic embryogenesis induction in bolus ex. hook f.studentsrepo.um.edu.my/2125/5/ch_4.pdf ·...

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CHAPTER 4

SOMATIC EMBRYOGENESIS INDUCTION IN Gerbera jamesonii

Bolus ex. Hook f.

4.1 EXPERIMENTAL AIMS

Somatic embryos are formed from somatic or vegetative cells that are not

normally involved in reproduction or the development of embryos. No endosperm or seed

coat is formed around a somatic embryo. There are many applications of somatic

embryogenesis such as clonal propagation of genetically uniform plant materials,

elimination of viruses from plants, generation of plants from single cells through

protoplasts and also development of synthetic seeds technology. Somatic embryogenesis

was first observed by Reinert (1958) and Steward et al. (1958) in Daucus carota cell

suspension cultures. Somatic embryogenesis has proven to be useful for

micropropagation. Sharp et al. (1980) reported that there are two types of somatic

embryogenesis, direct embryogenesis and indirect somatic embryogenesis. Direct somatic

embryogenesis occurs when an embryo is formed directly from a cell or tissue without

callus formation. Meanwhile, indirect somatic embryogenesis form when embryos are

derived from cells of callus phase.

There are several factors that influence the induction of somatic embryogenesis

such as the addition of plant growth regulators in the culture medium, type of explants

used, type of medium, physical and environmental factors and many others. Depending

on the plant species, hormones in the culture medium play very important role in the

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induction of somatic embryogenesis. In some plant species, the addition of auxin with or

without the combination of cytokinin induces the somatic embryogenesis. Nevertheless,

in some cases, the development of an embryo from the embryogenic cells is usually

accomplished by the absence of auxin in the culture medium.

The objectives of the study in this chapter were to achieve direct and indirect

somatic embryogenesis from leaves and petiole explants of aseptic seedlings of Gerbera

jamesonii Bolus ex. Hook f. The most suitable and responsive explant in the induction of

somatic embryogenesis was first identified. Various plant hormones were added in the

culture medium to identify the suitable hormones that could induce whether direct or

indirect somatic embryogenesis. Embryogenic and non-embryogenic callus would be

identified through double staining technique (Gupta et al., 1987). Different phases of

somatic embryos, i.e. globular, heart, torpedo and cotyledonary were identified in this

chapter. Somatic embryo obtained from this experiment would further be used in the

production of synthetic seeds and also for regeneration and acclimatization of plantlets in

the green house.

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4.2 MATERIALS AND METHODS

4.2.1 Preparation of Explants

Leaf explants obtained from 8-week-old aseptic seedlings were used in this

experiment. Secondary leaf explants were cut into 10mm x 10 mm size with midrib

discarded were utilized to initiate cultures. In order to obtain aseptic seedlings, Gerbera

seeds were soaked in distilled water for 30 minutes with addition of 1-2 drops of Tween-

20 followed by 40% (v/v) sodium chloride solution and gently agitated for 10 minutes.

The seeds were then rinsed 3 times in distilled water and then soaked in 70% (v/v)

alcohol for 1 minute. Finally, the seeds were rinsed 3 times in sterile distilled water.

Sterilized seeds were cultured on MS (Murashige and Skoog, 1962) basal medium. pH of

the medium was adjusted to 5.8 prior to autoclaving at 121 oC for 21 minutes.

4.2.2 Preparation of Culture Medium and Callus Induction

MS medium (1962) containing 30 g/l sucrose and 8 g/l technical agar was used in

this experiment. pH of the medium was set to 5.8 prior to autoclaving. In this experiment,

various types of plant hormones such as 2,4-D, BAP, and TDZ were used to study

induction of embryogenic callus. Based on previous research, Bespalhok and Hattori

(1998) used MD medium supplemented with the combination of 2, 4-D and Kinetin to

induce somatic embyogenesis in Tagetes erecta L. (African marigold). Gupta and Conger

(1999) reported that somatic embryogenesis of Panicum virgatum L. was established in

MS medium fortified with 2, 4-D and BAP. Somatic embryogenesis was also observed

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when floret explants of Dendrathema grandiflorum were cultured on MS medium

containing high concentrations of IAA and Kinetin (Tanaka et al., 2000). However, in

this experiment, 2, 4-D, BAP and TDZ were used for the aim of induction of

embryogenic callus.

4.2.3 Identification of Embryogenic Callus

For identification of embryogenic callus, a small piece of callus was placed on a

glass slide and 2-3 drops of 2% acetocarmine solution was dropped onto the callus. The

callus were divided into small pieces and heated over a low flame for a few seconds. The

slide was rinsed with distilled water to remove all liquid. Two to three drops of 0.5% of

Evan’s blue solution was dropped to acetocarmine stained cells. After 30 seconds, the

slide was rinsed again with distilled water and all excessive water was removed. 1-2

drops of glycerol was added to the stained cells, in order to prevent the cells from drying.

4.2.4 Embryogenic Callus Initiation and Establishment of Cell Suspension Culture

The secondary leaves of aseptic seedlings were cultured on MS medium

supplemented with 0.1- 2.0 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 3.0% sucrose

and 0.8% agar and incubated in the dark at 25 ± 1oC for 2 months. After 2 months, white

friable callus was formed and these callus were transferred into somatic embryo induction

suspension culture medium consisting of 0.1-2.0 mg/l 2, 4-D and 0.1 or 1.0 mg/l α-

Naphthalene acetic acid (NAA) with 3.0% sucrose. Ten 125 ml flasks containing 30 ml

of the medium and 2.0 g of callus tissue were used in two replicates. All cultures were

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incubated on a rotary shaker at 110 rpm and maintained in the dark at 25 ± 1oC. The

cultures were subcultured at 10 days interval for 4 to 6 weeks.

4.2.5 Induction of Somatic Embryos

The cells of suspension cultures were then filtered through 425 µm pore size

woven wire test sieve to separate the cell clumps. The filtrate was rinsed with basal liquid

MS medium. The cell clumps were then transferred into 25 petri dishes containing MS

medium supplemented with 0.1-1.0 mg/l BAP and 0.1-1.0 mg/l NAA with the addition of

0-50 mM Proline in each treatment. All cultures were incubated under 16 hours light and

8 hours dark at 25 ± 1oC. Cultures were observed for 4-6 weeks.

4.2.6 Development of Plantlets from Somatic Embryos

Shoots developed from somatic embryos were transferred to MS basal medium

for further development of plantlet and root growth. All cultures were incubated in the

culture room under 16 hours light and 8 hours dark with 1000 lux light intensity.

4.2.7 Microscopic Studies

Microscopy studies on somatic embryogenesis were done using

Microphotography Microscope and Scanning Electron Microscope (SEM). Somatic

embryogenesis stages like globular, heart shape, torpedo and cotyledonary phases were

further identified in this study.

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4.2.8 Data Analysis

Data obtained were analyzed using Duncan’s Multiple Range Test (DMRT). Mean with

different letters in the same column differ significantly at p=0.05.

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4.3 RESULTS

4.3.1 Induction and Identification of Embryogenic Callus

White-cream friable callus was formed after 6 weeks when leaf explants were

cultured on MS medium supplemented with 0.01-2.0 mg/l 2, 4-D (Table 4.1). Green

compact callus was formed when the same explants were cultured on 0.01-2.0 mg/l TDZ

(Table 4.2). Green and yellowish callus were also observed when leaf explants were

cultured on MS medium supplemented with the combination of 0.1 BAP and 0.01-2.0

mg/l 2,4-D (Table 4.3). Young secondary leaves from aseptic seedling were identified

as the best explant for the induction of embryogenic callus and somatic embryos. These

callus were tested using double staining method to determine the embryogenic and non-

embryogenic features.

Observations made under microscope of the white-cream friable callus (Table

4.1) showed early stage embryos after double staining was done, the embryonal heads

stained red (Figure 4.1 a) and suspensors stained blue. Meanwhile, when the green

callus was double stained, the cells remained blue and did not show any organization of

head and suspensor (Figure 4.1 b). Cross section of embryogenic (Figure 4.2 a) and non-

embryogenic callus (Figure 4.2 b) were made and examined under scanning electron

microscope. The embryogenic callus have thicker cell walls compared to cell walls of

non-embryogenic callus.

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The addition of 2, 4-D at concentration of 0.01-2.0 mg/l in the MS medium has

initiated the growth of embryogenic callus. All callus formed was friable and white-

cream coloured. No callus was formed when explants were cultured on MS basal

medium. Embryogenic callus was obtained (100%) when explants were cultured on MS

medium supplemented with 1.8mg/l and 2.0 mg/l 2, 4-D with the addition of 30% sucrose

and 0.8% technical agar (Table 4.1). However, production of embryogenic callus

decreased as 2, 4-D concentration in medium increased over 2.0 mg/l.

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Table 4.1: Induction of callus from leaf explants cultured on MS medium supplemented with 2, 4-D after 8 weeks.

Hormone (mg/l) (2,4-D)

Callus formation (%) after 8 weeks

Observations

0.0 0.0 No formation of callus

0.01 49.7 ± 1.1 White friable callus Embryogenic callus











1.8 100 ± 0.0 White friable callus Embryogenic callus



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Table 4.2: Induction of callus from leaf explants cultured on MS medium supplemented with TDZ after 8 weeks. Hormone (mg/l) (TDZ)


Observations


0.01 13.2 ± 1.0 Green compact callus Non-embryogenic callus











1.8 58.1± 1.5 Green compact callus Non-embryogenic callus



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Table 4.3: Induction of callus from leaves explants cultured on MS medium supplemented with BAP and 2,4-D after 8 weeks.

Hormone (mg/l)

BAP 2,4-D


Observation

0.0


0.1 0.0 0.0 Green compact callus Non-embrogenic callus

0.1 0.01 4.5 ± 0.9 Green compact callus Non-embrogenic callus

0.1 0.1 10.3 ± 0.5 Green compactcallus Non-embrogenic callus

0.1 0.5 11.0 ± 1.2 Light green friable callus Non-embrogenic callus






0.1 1.5 33.5 ± 0.9 Yellowish green friable callus Non-embrogenic callus






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Figure 4.1 (a): Embryogenic callus cells stained red with acetocarmine. Figure 4.1 (b): Non-embryogenic callus cells stained blue with Evan’s blue stain.

X 100

X 100

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Figure 4.2 (a): Cross section of embryogenic callus observed under Scanning Electron Microscope. Embryogenic callus shows ticker cell wall and friable structure.

Figure 4.2 (b): Cross section of non-embryogenic callus observed under Scanning

Electron Microscope. Non-embryogenic cell wall shows compact and thin cell wall.

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4.3.2 Induction and Development of Somatic Embryos

Embryogenic callus formed were then transferred into MS cell suspension

medium containing 0.1-2.0 mg/l 2, 4-D and 0.1 or 1.0 mg/l NAA for 1 month. All

cultured callus began to dissociate into single cells and small cell clumps within the

period. Embryogenesis was observed after 1 week in cell suspension culture. Cell clusters

and aggregates from cell suspension culture were observed (Figure 4.3). Cells were

sieved and transferred into embryo induction medium containing 0.1-2.0 mg/l BAP and

0.1 or 1.0 mg/l NAA with the addition of 0 or 50 mM L-Proline.

During this phase, stages of somatic embryos were developed (Figure 4.4).

Embryogenic callus which has recovered from suspension medium started to develop on

agar solidified medium. Three weeks after the transfer of embryogenic callus to the

embryo induction medium, 15.7 ± 1.4 embryos were developed in culture medium

supplemented with 1.0 mg/l BAP and 0.1 mg/l NAA without the addition of L-proline

(Table 4.4). Meanwhile, medium supplemented with the same concentration of growth

regulators, with the addition of 50 mM L-proline produced higher embryo at 29.8 ± 1.2

embryos. Morphological observation of the embryo stages was done and stages from

globular (Figure 4.5 a, 4.6 a), heart (Figure 4.5 b, 4.6 a), torpedo (Figure 4.5 c, 4.6 c) and

cotyledonary (Figure 4.5 d, 4.6 d) were successfully identified through microphotography

microscope and scanning electron microscope (SEM). Addition of L-proline in the

induction medium (Table 4.4) promoted the development of somatic embryos to form

shoots and plantlets.

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All embryos were then transferred to MS basal medium for further development

of shoots (Figure 4.7) and root growth (Table 4.5). Plantlets formed were then transferred

to soil and grown in the green house for further growth and development.

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Table 4.4: Effects of BAP , NAA and L-Proline on formation of somatic embryos of Gerbera jamesonii Bolus ex. Hook f.

Number of Somatic Embryos per Explant

NAA (mg/l) NAA (mg/l) + 50 mM L-Proline

BAP

(mg/l)

0.1 1.0 0.1 1.0

0.0 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0 a 0.0 ± 0.0a 0.01 0.0 ± 0.0a 0.0 ± 0.0a 3.4 ± 1.0b 0.0 ± 0.0a 0.1 5.2 ± 0.3b 0.0 ± 0.0a 9.7± 1.5b 0.0 ± 0.0a 0.2 5.4 ± 0.2b 0.0 ± 0.0a 8.2 ± 1.3b 0.0 ± 0.0a 0.3 5.8 ± 1.1b 0.0 ± 0.0a 11.6 ± 1.2bc 0.0 ± 0.0a 0.4 7.3 ± 0.5b 0.0 ± 0.0a 14.3 ± 0.4bc 0.0 ± 0.0a 0.5 8.0 ± 1.0b 0.0 ± 0.0a 21.3 ± 0.8cd 0.0 ± 0.0a 0.6 7.7 ± 0.6b 0.0 ± 0.0a 23.2 ± 1.1cd 2.2 ± 0.7b 0.7 10.3 ± 1.3bc 2.1 ± 1.5b 24.6 ± 0.6cd 5.4 ± 1.3b 0.8 10.7 ± 0.8bc 1.8 ± 0.7b 25.1 ± 1.7cd 7.3 ± 1.0b 0.9 12.6 ± 0.5bc 2.9 ± 0.5b 27.3 ± 2.1d 10.2 ± 0.8bc 1.0 15.7 ± 1.4c 3.3 ± 0.1b 29.8 ± 1.2d 11.6 ± 0.9bc 1.1 15.3 ± 1.4c 3.0 ± 0.5b 28.3 ± 0.5d 11.0 ± 1.2bc 1.2 14.9 ± 0.8c 3.8 ± 1.1b 28.0 ± 1.1d 10.8 ± 1.5bc 1.3 15.6 ± 0.5c 4.6 ± 1.0b 26.5 ± 0.8d 11.4 ± 0.5bc 1.4 13.4 ± 1.3bc 4.1 ± 1.6b 26.0 ± 0.3d 10.1 ± 2.3bc 1.5 14.7 ± 2.2c 5.7 ± 0.8b 27.4 ± 1.2d 10.5 ± 1.6bc 1.6 13.0 ± 1.6c 5.0 ± 0.6b 23.7 ± 1.5cd 8.9 ± 1.0b 1.7 12.8 ± 0.5bc 4.8 ± 2.4b 23.1 ± 0.3cd 9.4 ± 0.5b 1.8 11.5 ± 0.7bc 5.5 ± 0.8b 20.6 ± 0.6cd 8.7 ± 0.7b 1.9 10.9 ± 1.7bc 6.0 ± 1.3b 21.9 ± 1.4cd 8.0 ± 0.3b 2.0 10.4 ± 0.5bc 6.2 ± 0.4b 18.5 ± 1.7c 7.2 ± 1.1b

Mean with different letters differ significantly at p= 0.05

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Table 4.5: Composition of culture medium and growth condition for Gerbera jamesonii somatic embryo induction

Growth Regulators (mg/l) Culture stage Culture medium BAP NAA 2,4-D

L- Proline (mM)

Culture duration (week)

Seedling Callus induction Cell suspension Embryo induction Conditioning phase Root growth

MS MS MS MS MS MS

- - - 0.1-2.0 - -

- - 0.1 0.1-1.0 - -

- 0.1-2.0 1.0 - - -

- - - 0-50 - -

8 6 1 3 3 2

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Figure 4.3: Somatic embryogenesis of Gerbera jamesonii from suspension culture

Figure 4.4: Globular and heart shaped stages of somatic embryos formed on embryo induction medium.

2.0 cm

1.0 cm

G

H

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Figure 4.5 (a): Globular- shaped phase somatic embryo observed under

microphotography microscope

Figure 4.5 (b): Heart-shaped phase somatic embryo observed under microphotography microscope

0.05 cm

0.05 cm

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Figure 4.5 (c): Torpedo-shaped somatic embryo observed under microphotography microscope

Figure 4.5 (d): Cotyledonary phase somatic embryo of Gerbera jamesonii

0.05 cm

0.4 cm

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Figure 4.6 (a): Globular-shaped phase somatic embryo observed under Scanning Electron Microscope

Figure 4.6 (b): Heart-shaped phase somatic embryo observed under Scanning Electron Microscope

H

G

G

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Figure 4.6 (c): Torpedo-shaped phase somatic embryo observed under Scanning Electron Microscope

Figure 4.6 (d): Cotyledonary phase somatic embryo observed under Scanning Electron Microscope

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Figure 4.7: Micro shoots developed from somatic embryo of Gerbera jamesonii

0.25 cm

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4.4 SUMMARY OF RESULTS

1. Young secondary leaf explants were the most responsive explant to form embryogenic callus with 100 ± 0.0% callus formation when 1.8-2.0 mg/l 2,4-D was supplemented to culture media. Non- embryogenic callus was induced when TDZ and combination of 2,4-D and BAP were supplemented in the culture media.

2. Double staining method was used to distinguish the embryogenic and non-embryogenic callus. White cream friable callus showed early stage embryos after double staining was done. The embryonal heads stained red and suspensors stained blue. Cells of green callus remained blue and did not show any organization of head and suspensor when stained.

3. Cross section of embryogenic and non-embryogenic callus showed that embryogenic callus had thicker cell walls compared to non-embryogenic callus cells.

4. The highest number of somatic embryos (29.8 ± 1.2) was obtained when embryogenic cell clusters were transferred into embryo induction medium containing 1.0 mg/l BAP and 0.1 mg/l NAA with the addition of 50 mM L-Proline. While 15.7 ± 1.4 somatic embryos were induced when the embryogenic clusters were transferred to 1.0 mg/l BAP and 0.1 mg/l NAA without the addition of L-Proline.

5. Different stages of somatic embryos (globular, heart, torpedo and cotyledonary- shaped) were observed during the culture period through microphotography microscope and scanning electron microscope.

6. The addition of L-Proline in the embryo induction medium enhanced the induction of somatic embryos.

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