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ISSN 0931-1890, Volume 24, Number 4

ORIGINAL PAPER

Histocytological changes and reserve accumulationduring somatic embryogenesis in Eucalyptus globulus

Gloria Pinto • Sonia Silva • Lucinda Neves •

Clara Araujo • Conceicao Santos

Received: 14 July 2009 / Revised: 23 April 2010 / Accepted: 11 May 2010 / Published online: 25 May 2010

� Springer-Verlag 2010

Abstract We recently described a protocol for Eucalyptus

globulus somatic embryogenesis (SE). For its immediate use

at industrial levels, some stages of the process require better

control. In particular, SE germination rates are variable,

decreasing SE efficacy. As reserves may play a central role

in embryogenic processes, we followed histocytological

changes and reserve fluctuations, during SE. For SE induc-

tion, explants of mature zygotic embryos were grown

on Murashige and Skoog (MS) medium with 3 mg l-1

a-naphthalene acetic acid and later transferred to MS with-

out growth regulators (MSWH). Samples of zygotic embryo

cotyledons (explants), of globular and dicotyledonar

somatic embryos, and of embling leaves were analysed for

reserve accumulation and histocytological profiles. Cotyle-

don cells of zygotic embryos were rich in lipid and protein

bodies, having almost no starch. After 3 weeks of induction,

starch grain density increased in differentiated mesophyll

regions, while in meristematic regions their occurrence was

diffuse. In globular somatic embryos, starch accumulation

increased with time (in amyloplasts), but protein bodies

were absent. Cotyledonary somatic embryos had lower

density of starch grains and absence of lipid and protein

bodies. Embling leaves showed typical histological

organisation. This is the first comprehensive study on his-

tological and cytological changes during Eucalyptus SE

with emphasis in reserve accumulation. With this work we

demonstrate that the presently available SE protocol for

E. globulus leads to reserve fluctuations during the process.

Moreover, the reserves of somatic embryo cotyledons differ

from those of their zygotic embryo counterparts, which

reinforce the importance of reserves in the embryogenic

process and suggests that manipulating external conditions,

SE may be optimised giving suitable emblings production

for industrial purposes.

Keywords Embryo reserves � Eucalyptus �Histological differentiation � Myrtaceae �Somatic embryos � Ultrastructural studies

Introduction

Somatic embryogenesis (SE) has the potential to produce

large number of genetically identical plants with reduced cost.

SE in the Eucalyptus genus was described in E. citriodora

(Muralidharan and Mascarenhas 1987; Muralidharan et al.

1989), E. nitens (Ruaud et al. 1997), E. dunni (Termignoni

et al. 1996; Watt et al. 1999), E. grandis (Watt et al. 1999) and

E. tereticornis (Prakash and Gurumurthi 2005). Eucalyptus

globulus has been considered highly recalcitrant in SE pro-

cess and only Pinto et al. (2002, 2008a, b) reported the

regeneration of emblings (SE-derived plants), using mature

zygotic embryos. However, this protocol still presents tech-

nical limitations (e.g. low induction rates, somatic embryo

germination) that need to be optimised prior its application in

Eucalyptus breeding programmes.

In zygotic embryogenesis, the accumulation of reserve

substances represents a key stage to provide compounds

Communicated by R. Matyssek.

G. Pinto and S. Silva contributed equally to this work.

G. Pinto (&) � S. Silva � C. Santos

Centre for Environmental and Marine Studies (CESAM),

Department of Biology, Laboratory of Biotechnology

and Cytomics, University of Aveiro, 3810-193 Aveiro, Portugal

e-mail: gpinto@ua.pt

L. Neves � C. Araujo

Silvicaima SA Constancia Sul, 2250-058 Constancia, Portugal

123

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DOI 10.1007/s00468-010-0446-5 Author's personal copy

used during germination until autotrophy. Several authors

suggest that SE rates may increase when conditions are

manipulated to simulate those of zygotic embryogenesis.

Moreover, monitoring changes of reserves in somatic

embryos may allow recognising stages of vigour and sub-

sequent germination (Merkle et al. 1995).

Histodifferentiation was studied in somatic embryos of

several dicotyledonous woody species in order to under-

stand the induction process, and to clarify cytological

aspects of competent cells involved in SE, to histochemi-

cally follow somatic embryo development concerning

starch mobilisation, protein and polyphenol contents, or

even to compare the evolution of somatic and zygotic

embryos (e.g. Canhoto et al. 1996; Dodeman et al. 1997;

Canhoto et al. 1999; Karkonen 2000; Puigderrajols et al.

2000; Quiroz-Figueroa et al. 2002).

Though much attention has been paid to conditions

leading to SE in several species of Myrtaceae family (e.g.

Muralidharan and Mascarenhas 1995; Canhoto et al. 1999;

Pinto et al. 2002), little attention was drawn to histocyto-

logical and reserve pattern changes during the whole pro-

cess, from induction to plant conversion. Concerning this

issue, few studies are available in the Eucalyptus genus.

The histological evolution of somatic and zygotic embryos

was compared in E. nitens (Bandyopadhyay and Hamill

2000). Also, Arruda et al. (2000) demonstrated that an

increase of calcium concentration gave higher total protein

and sugar content in E. urophilla calluses. No further

studies are available, probably due to the recalcitrant

characteristics of the genus to SE induction.

As stated above, the available protocol for SE in this

species still leads to low rates of germination (0–40%

depending on the family) (Pinto et al. 2008a). In the present

work, we hypothesised that these low rates may result from

imbalances of reserve accumulation in mature somatic

embryos compared to the zygotic embryos. Adequate

external conditions leading to reserve accumulation in

somatic embryos close to those occurring in zygotic

embryos prior germination may eventually increase SE

germination rates (Canhoto et al. 1996).

The objective of this work was to characterise E. glob-

ulus primary SE for reserve accumulation and histocyto-

logical changes. Particular attention was given to the

cotyledonary stage prior germination by comparing mature

somatic and zygotic embryo cotyledons.

Materials and methods

Induction of SE

Half-sib seeds of E. globulus Labill. (Celbi, Leirosa,

Portugal) collected in the Centre of Portugal from open

pollinated families were surface sterilised as described by

Pinto et al. (2008a). SE cultures were initiated from zygotic

embryos in accordance with the protocol established by

Pinto et al. (2002, 2008b). Briefly, decoated seeds were

inoculated on Murashige and Skoog (1962) medium (MS)

with 3 mg l-1 a-naphthalene acetic acid (NAA), 30 g l-1

sucrose, 2.5 g l-1 Gelrite�, and pH 5.8, and incubated in

the dark at 24 ± 1�C for 3 weeks. Explants were then

transferred to MS hormone-free medium (MSWH) and

incubated under the same conditions described above.

Thereby, they were maintained by subculture on this

medium. Medium culture, NAA, sucrose and Gelrite� were

purchased from Duchefa (Haarlem, The Netherlands). All

other chemicals used in these experiments (unless other-

wise specified) were purchased from Sigma (St. Louis,

MO, USA).

Histocytological studies and reserve accumulation

For histological and cytological characterisation, samples

(n C 3) were collected from: (a) cotyledons of mature

zygotic embryos after imbibition (explant at day 0); (b)

cotyledons after 1 week of culture on induction medium;

(c) cotyledons after 3 weeks of culture on induction med-

ium; (d) globular somatic embryos; (e) dicotyledonar

somatic embryos; and (f) embling leaf (SE-derived plant).

Samples were fixed in 2.5% (v/v) glutaraldehyde in

1.25% (w/v) piperazine-N,N0-bis-(2-ethanesulfonic acid)

(PIPES) buffer (pH 7.4) for 3 h and washed in PIPES. Tis-

sues were then fixed in 1% (w/v) osmium tetroxide in PIPES

buffer for 1 h, rinsed in the same buffer and dehydrated

through a graded ethanol series and embedded in a graded

low-viscosity epoxy resin (Embed-812). The blocks were

polymerised at 60�C for 48 h. Ultra-thin sections were cut

with a LKB ultra-microtome using a diamond knife and

collected on uncoated copper grids. The sections were

contrasted with uranyl acetate for 15 min and lead citrate for

10 min and observed with a Elmiskop-101 transmission

electron microscope (Siemens AG, Germany) at 80 kV.

For light microscopy, semi-thin sections (0.5–1.5 lm)

from the material embedded for electron microscopy were

obtained using glass knife and stained with toluidine blue

(0.1%) (w/v) for general staining, Sudan Black B (0.1%)

(w/v) for lipid staining, bromophenol blue as a protein

stain or by periodic acid-Schiff reaction (PAS) for car-

bohydrate staining (Silva et al. 2010). Samples were

analysed in a Nikon eclipse 80i light microscope (Nikon

Corporation, Kanagawa, Japan) and digital photographs

were taken using a Leica DC 200 digital camera (Leica

Microsystems AG, Wetzlar, Germany). PIPES buffer was

acquired from Duchefa (Haarlem, the Netherlands), while

the remaining chemicals were purchased from Agar

Scientific (Essex, UK).

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Morphometric studies

Microphotographs were used in morphometric studies and

the relative percentage of occupation of the different tis-

sues (epidermis, undifferentiated parenchyma, palisade

mesophyll, spongy mesophyll and vascular strands) was

measured using ImageTool for Windows (version 3.00,

University of Texas Health Science Center, San Antonio,

TX, USA). In specific microphotograph sections, compar-

ative measurements were performed for lipid and starch

grain occupation rates and number of protein per cell.

At least 15 cells per cutting and three cuttings of each

condition were assessed.

Results

Cotyledonary zygotic embryos

The mesophyll of cotyledons (3-mm long) from mature

zygotic embryo was highly undifferentiated, having 7–9 cell

layers with few and small intercellular spaces (Fig. 1a, b).

One cell layer of palisade parenchyma was present near the

adaxial epidermis (23% of total occupation, Table 1;

Fig. 1a, b). This mesophyll was interspersed with small

vascular strands with a low percentage (3.95%) of total

occupation (Table 1).

On average, 33% of total area (mostly epidermal and

subjacent layer cells) stained intensively for lipids

(Figs. 1a, 2) and had large protein bodies (Fig. 1b). Epi-

dermal cells were rich in lipids, covered with wax and were,

in general, smaller than those of the mesophyll (Fig. 1a).

SE

After 3 weeks on induction medium, explants showed

compact and whitish calluses and no phenolisation

(Fig. 1c). After transfer to medium without growth regu-

lators, browning occurred (first visual signals of oxidation).

By this period, among abundant roots, globular embryos

were observed, later evolving to greenish cotyledonary

embryos in an asynchronous process (Fig. 1d, e). Also,

abnormal somatic embryos were common, usually having

altered number of cotyledons and/or cotyledons of different

sizes. Cotyledonary embryos germinated (Fig. 1f) and

subsequently converted to emblings.

Induction period

During the first week of SE induction, lipid and protein

digestion occurred (Figs. 1g, 2) but an accumulation of

starch was observed (Fig. 1h, arrows). By this time, most

explant tissues showed a typical cotyledonary histological

organisation. However, in some regions, meristematic

activity was present composed with groups of three or

more vacuolated cells with very thin walls surrounded by a

larger cell wall (Fig. 1h). These meristematic regions pre-

sented thin-walled cells, with dense cytoplasm, small

vacuoles, nucleus with prominent nucleolus and abundant

endoplasmatic reticulum (Fig. 1i). After 3 weeks, meri-

stematic regions enlarged (Fig. 1j), and protein (Fig. 2b)

and starch (Fig. 2a) reserves decreased compared to

1-week-old explants.

Globular somatic embryos

Globular embryos emerged as whitish and round protu-

berances from the callus/explant, mostly immediately after

transfer to expression medium (MSWH), and their complete

independence from the underlying cells was histologically

confirmed. Globular embryos had a protoderm that sur-

rounded a mass of parenchymatous-like vacuolated cells,

loosely packed, with meristematic regions (Fig. 3a) with a

procambial zone (Fig. 3b). Newly formed cells had dense

cytoplasm rich in etioplast-amyloplasts (plastids with

prolamellar bodies and abundant starch grains), endoplas-

mic reticulum, mitochondria and vesicles funded with the

plasmalemma (Fig. 3c, d). Ducts composed by channels

delimited by secreting cells were present even in globular

embryos (Fig. 3a). At this stage, starch grains (Fig. 3a), but

no proteins, were present (Fig. 2).

Cotyledonary somatic embryos

After 3 weeks on expression medium, asynchronism was

evident (globular and cotyledonary were dominant).

Transversal sections of the cotyledonary region showed a

well-defined protodermis surrounding parenchyma cells

and a procambial zone in the center (Fig. 3e, e.a). This

procambial region was surrounded at the abaxial side by

large vacuolated cells and at the adaxial margin by smaller

cells (Fig. 3e, e.a). Few stomata were already present. At

this stage, etioplast-amyloplasts (Fig. 3g) were now rare

(Fig. 3f), while etioplasts were abundant.

Leaf from emblings

Leaves from emblings showed a typical dicotyledon leaf

histology (Fig. 3h). Adjacent to the adaxial epidermis, one

layer of palisade parenchyma was evident, with rare and

small intercellular spaces (Fig. 3h). Poorly differentiated

mesophyll was present. Plastids with different thylakoid

organisation levels and with irregular shapes were seen,

although predominantly plano-convex or biconvex form

predominates (Fig. 3i, j). Also, occasionally small starch

grains were observed.

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Discussion

Reserves are crucial for in vitro morphogenesis (Branca

et al. 1994) and some studies correlated reserve con-

sumption patterns with the development of organogenesis

and SE (e.g. Mangat et al. 1990; Martin et al. 2000). In

particular, reserves may play a crucial role during germi-

nation (e.g. Canhoto et al. 1996). Here, we report that, prior

to germination, mature E. globulus zygotic embryos are

rich in lipid and protein reserves lacking starch. This

Fig. 1 Different aspects of SE

induction in E. globulus.

a Cross-section of a zygotic

embryo cotyledon stained with

PAS, showing protein bodies

(arrows) (week 0). Bar 50 lm.

b Ultrastructural view of cells

packed with protein and lipid

bodies (week 0). Bar 1 lm.

c Zygotic embryo after 1 week

on induction medium.

d Globular somatic embryos.

e Cotyledonary somatic embryo.

f Germinated cotyledonary

somatic embryo. g Cotyledon

(zygotic embryo) cross-section

stained with toluidine blue

showing a weakly differentiated

palisade mesophyll,

meristematic regions, vascular

strands and some lipid

deposition (1 week on induction

medium). Bar 50 lm. h The

same as (g) stained with PAS

showing starch grains with

diffuse distribution (arrows).

Bar 50 lm. i Ultrastructural

view of a parenchymatous-like

cell near vascular strands with

small vacuole, prominent

nuclei, dictyosomes and

endoplasmatic reticulum

(1 week after induction).

Bar 1 lm. j Cotyledon (zygotic

embryo) cross-section showing

a reduction in number of starch

grains, cotyledon enlargement

and meristematic region

(3 weeks after induction).

Bar 50 lm. ade adaxial

epiderm, cw cell wall, ddictyosomes, lb lipid bodies,

ld lipid deposition, nunucleolus, pb protein bodies,

pp palisade parenchyma,

rer rough endoplasmatic

reticulum, vs vascular strand

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suggests that in this species, embryos require these types of

reserves to start germination.

In the protocol developed for E. globulus SE, germina-

tion rates are variable within families (Pinto et al. 2008a).

We, therefore, hypothesised that by controlling the reserve

profiles during this SE process, in particular the ones at the

mature somatic embryo cotyledon, we may be able to

better control germination step and emblings yield.

Our data show that starch is an abundant reserve in SE,

probably due to the sucrose present in the culture medium.

Starch begins to accumulate early during induction,

apparently replacing the initial lipid and protein reserves of

the zygotic embryo explant. The lower abundance of starch

found in meristematic regions may be justified by energetic

consumptions. This starch consumption in mitotically

active tissues was reported earlier for other species (e.g.

Barciela and Vieitez 1993; Canhoto and Cruz 1996).

Cangahuala-Inocente et al. (2004) also reported, in Feijoa

sellowiana cotyledonary somatic embryos, an enhanced

synthesis of starch that was associated with the reserve

accumulation to be used during somatic embryo germina-

tion/conversion. Although slight differences were found in

the timing profile of starch accumulation/consump-

tion among other species, a general pattern of starch pre-

accumulation followed by consumption seems to occur in

many dicotyledonous SE processes (e.g. Canhoto et al.

1996; Rodriguez and Wetzstein 1998; Cangahuala-

Inocente et al. 2004), supporting the general pattern

observed in E. globulus.

To our knowledge, this is the first characterisation of

reserve accumulation in mature zygotic embryos for

E. globulus, which are mostly lipids and proteins. During

zygotic embryogenesis, lipase and protease activities play

an important role as reported for other mature zygotic spe-

cies as F. sellowiana (Canhoto et al. 1996). In E. nitens,

somatic embryos and zygotic embryos had, in general,

similar lipid reserves, but in lower contents in somatic

embryos (Bandyopadhyay and Hamill 2000). Interestingly,

E. nitens seems to differ from E. globulus concerning

reserve profiles during SE. However, those authors do not

give further information of subsequent steps for germination

success. These data support the hypothesis of Merkle et al.

(1995) who stated that differences in lipid composition

profiles between zygotic embryos and somatic embryos

probably reflect the type of maturation protocol used.

The discrepancy in reserve accumulation between

zygotic cotyledons and somatic ones observed in E. glob-

ulus may be, at least in part, responsible for the low

germination rates observed in our protocol. So, by

manipulating external conditions that eventually mimic

zygotic embryo reserve profiles and increase germination

rates, we may adjust this protocol to Eucalyptus breeding

programs. This strategy was successful in other species as,

e.g., in Theobroma cacao L. where somatic embryos lacked

starch and protein reserves in comparison with their

Table 1 Relative percentages (percentages average ± standard error) of mesophyll tissues occupation during induction period (0, 1 and 3 weeks

old)

Vascular strands Palisade parenchyma Undifferentiated parenchyma Spongy parenchyma Lower epidermis Upper epidermis

Week 0 3.95 ± 1.2 23.13 ± 4.3 53.87 ± 2.1 0.00 6.90 ± 0.8 6.70 ± 1.2

Week 1 6.34 ± 2.5 15.04 ± 1.6 54.31 ± 6.0 0.00 5.96 ± 1.8 6.55 ± 0.5

Week 3 16.57 ± 8.8 13.67 ± 3.0 26.78 ± 16.3 26.45 ± 18.4 11.79 ± 9.0 7.34 ± 0.7

0,00

0,50

1,00

1,50

2,00

2,50

3,00

Day 0 1 week 3 weeks* Glob. Cot. Leaf

Day 0 1 week 3 weeks* Glob. Cot. Leaf

aver

age

star

ch g

rain

s/ce

ll

0

2

4

6

8

10

12

aver

age

pro

tein

bo

die

s/ce

ll

0

5

10

15

20

25

30

35

40

Day 0 1 week 3 weeks* Glob. Cot. Leaf

Lip

id a

rea

(%)

A

B

C

Fig. 2 Changes in reserve accumulation during primary somatic

embryogenesis in E. globulus. a Starch, b protein, and c lipids. The

symbol asterisk reports to mitotically responsive regions. Bars are

average ± standard error

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zygotic counterparts. Introducing a growth period into the

culture protocol improved embryo development. In this

species, adding, e.g., abscisic acid to the maturation med-

ium was effective in increasing reserve synthesis and

resulted in higher germination, conversion, and acclimati-

sation rates (Alemano et al. 1997).

In conclusion, this is the first report in Myrtaceae cov-

ering reserve accumulations and histocytological analysis

Fig. 3 Several aspects of somatic embryo development, from

globular to embling stage. a Globular somatic embryo cross-section

showing several starch grains (arrows) and a protoderm surrounding

parenchymatous-like cells. Bar 50 lm. b The same as (a) showing

tracheary element. Bar 1 lm. c Globular somatic embryos cross-

section showing etioplast with crystalline prolamellar body and starch

grains. Rich cytoplasm with numerous mitochondrias and ribosomes.

Bar 1 lm. d Ultrastructural view of a globular somatic embryo cell

with rich cytoplasm with abundant extrusion vesicles to cell wall

synthesis. Bar 1 lm. e Cross-section of a cotyledonary somatic

embryo showing a well-defined protoderm layer. Bar 50 lm. e.a The

same as before highlighting a vascular strand and one palisade cell

layer. Bar 50 lm. f Ultrastructural aspect showing developed

etioplasts in cotyledonary somatic embryo. The thylakoidal structure

in the stroma. Bar 1 lm. g The same as before showing a

ultrastructural detail of etioplasts rich in starch grains. Bar 1 lm.

h Embling leaf cross-section stained with PAS highlighting the

absence of starch grains and typical leaf organisation. Bar 50 lm.

i Ultrastructural embling leaf view showing a cell rich in cytoplas-

matic contents, with a well-defined nucleus and nucleolus and plastids

not completely differentiated. Bar 1 lm. j Ultrastructural embling

leaf showing biconvex chloroplasts in parenchyma cells. Bar 1 lm.

abe abaxial epiderm, ade adaxial epiderm, c chloroplast, du ducts, eetioplast, er endoplasmatic reticulum, ev extrusion vesicle, mmitochondria, n nucleus, nu nucleolus, p protoderm, pp palisade

parenchyma, prb prolamellar bodies, s starch grains, t thylakoid, vvacuole, vs vascular strand

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from induction to embling stages by primary SE following

the only SE available protocol for this species (Pinto et al.

2002, 2008a, b). Sucrose, abscisic acid and polyethylene

glycol are reported to regulate the maturation of somatic

embryos and also to improve storage reserve accumulation

in various systems (e.g. Kumari et al. 2000). We are pres-

ently manipulating these external conditions to increase

lipids and/or proteins in mature somatic embryos to improve

germination rates to acceptable industrial demands.

Acknowledgments Authors thank Celbi for providing the material

and for supporting the project. FCT/MCT project POCI/AGR/60672/

2004 also supported the project. Thanks are also due to Jose Dias and

Armando Costa for technical assistance. Fellowships: Sonia Silva

(SFRH/BD/32257/2006).

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