ORIGINAL ARTICLE

Morphological and histochemical investigationson Himantoglossum robertianum (Loisel.) P. Delforge(Orchidaceae) seeds

Mehmet Aybeke

Received: 24 January 2013 / Accepted: 3 June 2013 / Published online: 2 July 2013

� Springer-Verlag Wien 2013

Abstract In the present study, the morphological and

histochemical properties of Himantoglossum robertianum

(Orchidaceae) seeds were investigated. According to the

morphological studies, the testa cells are dead and show

reticulations. According to the histochemical tests, mature

embryos were globular and covered with cuticular sheath

in especially on the embryo parts of the seed. Furthermore,

high amounts of soluble carbohydrates, lipid and protein

bodies were found in mature embryos; but very little of

starch was found. Consequently, the present results are

important for both European-Mediterranean terrestrial

orchids and present taxon, H. robertianum; thus they are

discussed from the point of view of seed germination

physiology and orchid embryology.

Keywords Himantoglossum � Embryo � Orchid � Seed �Histochemistry

Introduction

Orchid seeds are very small and transparent. Embryos in

seeds can hardly be seen by means of a microscope.

Embryos take up little space in the seeds. Thus, orchid

seeds may travel great distances in the air due to their

fusiform aerodynamic features and lightweight properties

(Dockrill 1969). Furthermore, the color, shape of the testa,

and the embryo are systematically significant. The walls of

the testa cells can be smooth or reticulated, and when

reticulation is present, its pattern may be distinctive

(Arditti et al. 1980). Therefore to date, several systematic

studies related to seed surface morphology have been

conducted, especially on epiphytic orchids. In addition,

seed germination, seed viability, mass propagation and

seedling development studies have mostly been conducted

(Vinogradova and Andronova 2002; Huehnea and Bhinijaa

2011; Nontachaiyapooma et al. 2011; Hay et al. 2010; Roy

et al. 2011). This is because in nature, a symbiotic rela-

tionship with a fungus, mainly the Rhizoctonia species, is

essential for at least the later stages of germination. Even

in this mycorrhizal symbiosis case, germination may take

a long time, sometimes months or years (Handley 1982;

Clements 1988). In other words, orchid seed germination

without a fungus is impossible under natural conditions. It

has been hoped that symbiotic germination utilizing orchid

mycorrhizal fungi might be a way to overcome the diffi-

culty of germination and to enhance the growth of the

seedling (Tomita 1995). Although different morphologi-

cal, palynological, embryological, caryological, and ana-

tomical studies have been carried out on several European

orchid taxa (Delforge 2006; Sezik 1984; Aybeke et al.

2010; Aybeke 1997, 2000, 2002, 2004, 2007a, b, 2012a, b,

2013), very few efforts have been directed toward inves-

tigating the histochemistry of the seed. This is because

working with orchid seeds is very difficult due to their

small morphology. For example, Ophrys mammosa seeds

are 557 lm in length and 138 lm in width. Its embryos

are smaller than the seeds: 107 lm in length and 62 lm in

width (Aybeke 2007a). Furthermore, the seed surface

features of Himantoglossum robertianum, a European-

Turkish terrestrial orchid, are unknown. Therefore, inves-

tigating not only the seed surface morphology but also the

histochemical properties of orchid seeds will undoubtedly

enlighten us to its developmental process, germination

biology and physiology (Lee et al. 2006). The present

M. Aybeke (&)

Department of Biology, Faculty of Science, Trakya University,

Balkan Campus, 22030 Edirne, Turkey

e-mail: mehmetaybeke@yahoo.com

123

Plant Syst Evol (2014) 300:91–97

DOI 10.1007/s00606-013-0862-2

study was designed to fill in the gap by investigating

the morphological and histochemical properties of

H. robertianum seeds.

Materials and methods

The seeds of H. robertianum (Loisel.) and P. Delforge

(Orchidaceae) were collected during field work from the

region of Mugla in the towns of Ula and Kızılagac in

Turkey. For the morphological investigations, mature seeds

of natural plants were prepared for the scanning electron

microscopy (SEM), and observed as previously described

by Arditti et al. (1979, 1980). The colors and dimensions

(length and width) were recorded for seeds and embryos

under an Olympus stereomicroscope and a Zeiss Jena light

microscope. For SEM preparations, specimens were

mounted on double-sided cellophane tape on aluminum

stubs, and coated with gold/palladium. The coated grains

were then observed and photographed with a Jeol-JSMT-

330 scanning electron microscope.

Histochemical protocol

Mature seeds were fixed with 2 % paraformaldehyde and

2.5 % glutaraldehyde in a 0.1 M sodium phosphate buffer,

pH 6.8, at 4 �C overnight. After three 15-min buffer rinses,

the material was dehydrated in a graded ethanol series, and

processed for historesin embedding according to Yeung

(1999). Historesin sections of 3 lm were cut using a dis-

posable blade on a Leica RM 2255 Autocut microtome.

These sections were stained with the periodic acid–Schiff

(PAS) procedure and counterstained with toluidine blue O

for general histological examination. A red color indicates

carbohydrates. For lipid fluorescence staining, neutral red-

rhodamine B was applied to sections. Sections were stained

with a 0.1 % mixture of neutral red-rhodamine B in 0.1 M

potassium phosphate, pH 6.5, for 1 min, then rinsed briefly

in a buffer. Furthermore, Evans blue fluorescence (excita-

tion at 620 nm, emission at 680 nm) was used for the

microscopic investigation of embryo proteins. The pres-

ence of a cuticle was detected using nile red as detailed in

Yeung et al. (1996). The sections were stained with

1 mg ml-1 nile red (Sigma Chemical Co., St Louis, MO,

USA) for 10 min, briefly washed in distilled water and

mounted in water containing 1 % n-propyl gallate (Sigma),

an anti-fading compound. The fluorescence pattern was

examined using an epifluorescence microscope equipped

with the filter set Olympus 68 (546/12 nm excitation filter

and 590 nm emission barrier filter). Also microscopic

sections were stained with Lugol’s iodine and tested for

starch. All images were recorded with an attached digital

camera (Micropublisher Camera and QCapture software;

QImaging, Surrey, British Columbia, Canada). The via-

bility of seed-embryo tissues was determined based on

staining with diacetate of fluorescein. For this test, the

sections were incubated at room temperature for 3–5 min.

The result was observed using an inverted optic micro-

scope 409 (Olympus IMT 2) under UV light (with a blue

filter). The viable tissues were indicated by a green

fluorescence.

Transmission electron microscopy

Seeds were fixed with 2 % paraformaldehyde and 2.5 %

glutaraldehyde in a 0.1 M sodium phosphate buffer, pH

6.8, at 4 �C overnight. After three 15-min buffer rinses, the

material was post-fixed with 1 % OsO4 in the same buffer

for 4 h at room temperature and then rinsed in three 15-min

changes of buffer. Following fixation, the material was

dehydrated in a graded acetone series, and embedded in

Spurr’s resin. Ultrathin sections 60–70 nm thick were cut

using a glass knife on an ultramicrotome (Richert-Jung

Ultracut E, Vienna, Austria). These sections were stained

with uranyl acetate and lead citrate. The sections were

examined and photographed using an EM 10, Zeiss trans-

mission electron microscope at 80 kV.

Results and discussion

Seed morphology

The surface was irregularly reticulated on the periclinal

walls. The testa cells are generally rectangular, although

they are pentagonal or hexagonal at the chalazal tips. Seeds

and embryos are brown (Figs. 1–5).

Morphometric data

Seeds, 531 ± 54 lm in length and 135 ± 25 lm in width,

number of cells at the longest axis of testa were 4.581, avg.

length of testa cell (mm) = 4.86 cells mm-1 length of

testa = 5.57, volume (mm 9 10 - 3) = 1.547.

Embryo; length = 0.108/0.014 mm/SD, width = 0.043/

0.0198, length/width = 1.443, vol = 0.1 (mm-3 9 10-3),

seed volume/embryo volume, % = 6.8, percent air space:

(seed volume-embryo) 9 100, seed volume = 89.54.

According to Aybeke (2007a), Eurpean–Mediterranean

terrestrial orchid seeds are usually fusiform, with their

chalazal poles being only slightly reduced in diameter.

The testa cells are generally elongated rectangles or

penta-hexagonal with raised and thickened anticlinal walls

(Arditti et al. 1980). For Himantoglossum seeds, the

values for ratios of ‘‘seed/embryo volume’’ and ‘‘percent

air space’’ are high. The greater seed volume generally

92 M. Aybeke

123

results because of wider rather than longer testa; higher

testa volumes can also come from greater length rather

than width. In this case, the testa cell values were

determined during seed maturation instead of during

radial expansion (Arditti et al. 1980; Arditti and Abdul

Ghani 2000). A greater percentage of air space may

increase seed buoyancy and aid in wind dispersion. A

lighter weight enables orchid seeds to traverse great dis-

tances, logically pointing to buoyancy as an important

factor in the aerial distribution of the Orchidaceae (Arditti

and Abdul Ghani 2000).

Histochemical tests

Soluble carbohydrates were found in embryo cells accord-

ing to PAS staining (Fig. 6). These results were supported

by a recent study (Stewart and Kane 2010) indicating the

wide suitability of simple exogenous carbohydrates in

supporting the germination and protocorm development of

Habenaria macroceratitis. So they suggests that a wide

range of carbohydrates are suitable for the culture of often

difficult-to-germinate terrestrial orchids. In particular,

Knudson (1922) was the first to demonstrate that orchid

Figs. 1–5 Himantoglossum robertianum, seed surface morphology, (1–2) view in light microscopy, (3) view in fluorescence microscopy, in

SEM (4) and its magnified testa cells on which reticulations are seen (5). Scale bars 1 100 lm, 2–3 50 lm, 4 100 lm, 5 10 lm

Morphological and histochemical investigations 93

123

Figs. 6–10 Histochemical tests; 6 PAS stainings for soluble

carbohydrates, 7 evans blue stainings for protein bodies, 8 lipid

fluorescence staining in mixture of neutral red, rhodamine B, 9 in

TEM section, mature embryo cells bearing lipid (L) and protein

bodies (arrows), N: nucleus, 10 cuticle presence seen intensively in

periphery of embryo and paler toward the micropylar end, with nile

red stainings. Scale bars 6–8, 10 50 lm, 9 1 lm

94 M. Aybeke

123

seeds could be germinated in vitro on a defined nutrient

medium supplemented with a simple carbohydrate source.

In addition, histochemical and ultrastructural investigations

showed dense lipid and protein bodies in the mature embryo

cells (Figs. 7, 8, 9). In Lugol preparations very few starch

grains were found. Similarly, previous works indicated lipid

and protein accumulation in contrast to step-by-step

reduction of starch deposits toward the mature globular

embryo stage (Yeung et al. 1996; Yam et al. 2002; Lee et al.

2006). In contrast, Hosomi et al. (2012) mentioned only

lipid reserves in orchid embryos, but not proteins or car-

bohydrates. Although there is no information about the

relation between the deposit material and the properties of

germination metabolism, in the author’s opinion, such

deposit differences in several orchid taxa will potentially

affect their germination physiology/metabolism.

One of the notable features is the deposit of cuticular

material in the periphery of the embryo and inner surface of

the testa. Especially toward the micropylar end, cuticular

fluorescents are fewer (Figs. 10, 11, 12). Similar results

were obtained in some previous studies relating to other

epiphytic orchids (Yeung et al. 1996; Lee et al. 2006). In

orchids, an endosperm fails to develop during seed devel-

opment. Furthermore, the seed coat is thin and does not

offer protection to the embryo, especially in terms of

moisture retention. The formation of a prominent cuticle on

the surface of the embryo may ensure moisture retention by

the embryo cells, which lends importance to the cuticula

layers for enhancing the embryo’s durability and cell via-

bility. The cuticular material may also offer physical pro-

tection to the embryo both at the stage of early desiccation

and during germination (Yeung et al. 1996). In addition, the

presence of cuticular material which encloses the embryo

allows it to take in nutrients by the mycorrhizal hyphae

through the micropylar openings of the seed (Rasmussen

1995; Vinogradova and Andronova 2002). Because the

Figs. 11–13 11 In light

microscopy cuticular sheath on

embryo (arrows indicate the

cuticular wrinkles), in

tangential-longitudinal section

of testa, 12 in TEM section

cuticular layers (arrows) of

testa, 13 transversal section of

seed, dead testa cells (arrows),

in contrast to viable embryo.

Scale bars 11, 13 50 lm, 121 lm

Morphological and histochemical investigations 95

123

suspensor, which undergoes programmed cell death during

seed maturation, is absent, it does not contribute to the

development of the next plant generation following seed

germination (Kawashima and Goldberg 2009).

Since the suspensor cells are tightly pressed against the

inner surface of the seed coat during the phase of embryo

development, it indicates that apoplastic transport is pos-

sible between them. It is important to note that a secondary

wall is not present in these seed coat cells, and the cell wall

remains primary in nature. Together, the lack of cuticular

substance in the suspensor cell wall indicates that apo-

plastic transport will not be impeded (Yeung et al. 1996).

Therefore, to guarantee the transport between them, the

cuticular material is either scarcely deposited or absent

toward the micropylar end of the testa, as shown in the

present study and Lee et al’s work (2006). Based on the

seed viability test, the testa cells were dead (Fig. 13)

because during maturation they became dehydrated and

compressed into a thin layer. The mature embryo was

protected by a single layer of seed coat cells; storage

substances were not present within the seed coat cells (Lee

et al. 2006), as described in the present study.

So far, limited studies have dealt with the seed histo-

chemistry of only a few orchid taxa such as the Paphio-

pedilum (Lee et al. 2006), the Cattleya (Harrison 1977) and

the Cymbidium species (Yeung et al. 1996). These taxa

originate in Northern India, the Philippines, New Guinea,

South America, and Southeast Asia, extending as far south

as Australia. According to our literature review, very few

efforts have been made with terrestrial European-Medi-

terranean orchids such as the Dactylorhiza majalis and the

Platanthera hyperborea (Rasmussen 1990; Richardson et al.

1992). Only in Europe and temperate Asia have 40–60

terrestrial orchid genera been determined so far, and with

new taxonomic regulations and records, this number is on

the increase. So studies such as these will enable us to

better comprehend their biology. Also, the present study is

important with regard to: (a) Himantoglossum seed surface

morphology, (b) seed histochemistry in members of the

terrestrial European-Mediterranean-(including Turkish)

orchids, and finally (c) orchid seed germination physiology.

According to Veyret (1974), the orchid embryo is still little

known since only about 60 kinds have been the object of

embryological investigations, while the family numbers

more than 20,000 species. Furthermore, asymbiotic seed

germination is often difficult for fully mature orchid seeds,

especially those of the terrestrial orchids (Lee et al. 2006).

Knowledge of embryo development and/or histochemistry

will help facilitate success in asymbiotic seed germination

and especially the conservation of endangered orchid spe-

cies (Lee et al. 2006). Consequently, the present results

will contribute to a future comprehensive view on embry-

ological features in the different groups.

Conclusion

In the present study, H. robertianum seed surface mor-

phology as well as seed histochemistry was investigated.

Consequently, seeds are fusiform in shape, transparent and

on the periclinal walls transveral reticulations are present.

According to the histochemical results, lipid, protein

droplets and soluble carbohydrates are present in embryos.

Based on the literature review, the present results are

important both for H. robertianum and Euroasian terrestrial

orchid seed histochemistry, where very limited studies

have been conducted. Consequently, the results were dis-

cussed from the point of orchid conservation, asymbiotic

seed germination and orchid embryology.

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