SPATHIPHYLLUM Wallissi_victoria frank_0621160

The Anatomical Structure of the Peace Lily

Spathiphyllum wallisii

Victoria Frank

0621160

BOT ° 3410

Dr. Greenwood

17 December 2009

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Introduction:

The Peace Lily (Spathiphyllum wallisii), also known as White Sail or Spath, is a member of the genus

Spathiphyllum, which contains about 40 species of monocotyledonous flowering herbaceous plants in the

family Araceae. They are native to tropical regions of the Americas and southeastern Asia. Although

there is not a lot of information available on this genus, much more is known about the family, Araceae,

which includes about 110 genera and 1,800 species. Araceae are herbaceous plants that are rhizomatous or

tuberous and many of the species have calcium oxalate crystals (Carr 2005). Some species are poisonous

when consumed due to the calcium oxalate, including S. wallisii (Boyce 1993). Species within this family

range from submerged to free-floating aquatics to terrestrial plants. Some species are epiphytic, others,

hemiepiphytic parasites and others are climbers (International Aroid Society 2009). The genus

Spathiphyllum is herbaceous evergreen with large dark green foliage borne on petioles. They have

underground rhizomes, which produce shoots and roots from their nodes. A large inflorescence is a

characteristic of this family. The spathe is a glossy, slightly fragrant, white and/or green modified leaf with

a long cylindrical spadix consisting of a number of small flowers, which may be either bisexual or

unisexual (Chant SR et al. 1978). The purpose of this paper is to determine the anatomical structure of S.

wallisii using multiple laboratory techniques and experiments.

Scientific Classification:

Family: Araceae

Subfamily: Monsteroideae

Tribe: Spathiphylleae

Genus: Spathiphyllum

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Materials & Methods:

Leaf, petiole, flower stem, rhizome and root samples were selected from a Spathiphyllum wallisii, which

was provided by Dr. J. Greenwood, B. Lee and D. McClellan. These samples were observed in depth using

a variety of staining and observational methods in order to achieve a better understanding of the cellular

construct of the plant tissues and organs. A number of different observational tools and microscopes were

utilized; including a brightfield microscope, stereobinocular microscope, epiflorescence microscope and

cross-polarizing microscopy. The stains that were used include Toluidine Blue O (TBO), which stains

pectin pink to reddish purple, and lignin various shades of blue-green as well as staining phenolic

compounds shades of blue (Peterson et al 2008). Other stains that were used include phlorogucinol (which

stains lignin in secondary cell walls), iodine potassium iodide (I2KI) (which stains starch purple to blue-

black), sudan III/IV (which stains cutin in storage cells and other lipids and should appear orange-red), acid

fuchsin (which stains proteins pink-red) and amido black 10B/Acetic acid (which stains protein deposits

black-blue in some fungal mantles).

Leaf

A leaf sample was selected and observed in order to identify the venation patterns. Phyllotaxy of

the plant was also observed. A fresh leaf epidermal peel was prepared by tearing the leaf cross-wise

exposing epidermal cell layers at torn sections. Nail polish impressions were produced of the abaxial and

adaxial epidermal surfaces of the leaf (L. Peterson et al. 2009) in order to study the epidermal cell

structures as well as the stomatal complexes.

Leaf samples were selected for clearing using 10% aqueous potassium hydroxide (KOH) at 55°C

(Peterson et al). The cleared samples were stained with TBO as well and viewed using polarizing

microscopy.

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Multiple leaves were extracted from the plant and several different stains were applied to transverse

and longitudinal sections of leaf tissue, in order to identify different cellular components and vascular tissue

organization. Stains utilized included TBO, Sudan III/IV, Phloroglucinol, and I2KI.

Petiole & Stem

Sections of mature petiole were removed, and transverse and longitudinal fresh hand sections were

cut. Several transverse cross sections were stained using TBO, in order to observe and identify vascular

and ground tissue organization and structure. Fresh petiole hand sections were also stained with I 2KI to

observe starch and Sudan III/IV to observe cutin in lipids and other storage bodies.

Five sections of different petioles were extracted and prepared for maceration (L. Peterson et al.

2009). One drop of the macerated tissue was placed on a slide and stained with TBO, in order to observe

individual vascular tissue structures.

A primary vegetative shoot tip was excised and protective leaves removed using a probe and extra

fine forceps. A stereobinocular microscope was used in order to reveal the shoot apices and to identify

tissue structures.

Sections of flower stem were selected and transverse and longitudinal fresh hand sections were cut.

Several transverse cross sections were stained using Toluidine Blue O (TBO), I2KI, sudan III/IV and

phloroglucinol. Transverse and longitudinal hand sections were cut of a petiole sample and stained using

amido black to observe p-protein plugs.

Rhizome & Root

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Fresh transverse hand sections of rhizome stained with I2KI and acid fuchsin were inspected to

discover presence of starch and protein. Sections stained with TBO were viewed using a stereoscopic

microscope, and the arrangement of the vascular bundles were observed.

Root primary growth tissue was sectioned and unstained transverse hand sections were produced

and examined under an epifluorescence microscope in order to investigate the presence of suberin in the

exodermis and endodermis. Other transverse hand sections were stained with Toluidine Blue O (TBO) (L.

Peterson et al. 2009), internal xylem and phloem structures were examined.

Transverse sections of primary growth tissue were stained with Calcoflour white (M2R) and

Berberine hemi-sulfate (FeCl3) (Laboratory Exercises” L. Peterson et al. 2009). The slides were viewed

under and epiflorescence microscope looking for cellulose and some extracellular mucilage which is

stained pearly blue-white by the M2R. As well as suberin, lignin and callose, whose naturally fluorescent

compounds are enhanced by the FeCl3 staining process. Some transverse hand sections of primary growth

tissue were stained with iodine potassium iodide (I2KI) to reveal starch in the root tip.

Five sections of different root tips were extracted and prepared for maceration (L. Peterson et al.

2009). One drop of the macerated tissue was placed on a slide and stained with Toluidine Blue O (TBO), by

adding one drop to the macerate tissue in order to observe individual vascular tissue structures.

Sections of root tissue were removed and fixed according to the procedure as outlined in L.

Peterson et al. 2009. Upon completion of the paraffin mold, the plastic cover protecting the mold was

removed. The paraffin was shaved down and excess was removed around the root tissue. The paraffin

block was then placed in a microtome and sections were made ~10 µm thick. The sections were then

placed on slides using water to assist in placement. The slides were then placed on a heater overnight to

remove access water and melt the paraffin. Once the paraffin and samples were fixed to the slide the wax

was removed and the samples were stained using TBO (Peterson et al. 2008).

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Results & Discussion

I ) Petiole & Leaf

The leaf arrangement of S. wallisii was found to be alternate. The leaf type was simple and ovate

with an undulate margin and pinnate venation.

Nail polish peels revealed stomatal complexes in high concentration on the abaxial surface of the

leaf. While stomatal complexes were found in the adaxial surface of the leaf, they were found at a lower

concentration. The epidermal pavement cells appeared to be in a sedum arrangement with raised stomata.

The majority of stomatal complexes only contained two subsidiary cells, which were parallel to the guard

cells and randomly angled.

The leaf and petiole tissues of S. wallisii were found to be very similar in structure. Transverse

sections of leaf tissue stained with TBO revealed collateral vascular arrangements (Fig.1.) with metaxylem

and primary xylem facing the adaxial side of the leaf, and phloem and fiber cap facing the abaxial side of

the leaf. The same collateral formation was found in the petiole tissue. The upper and lower epidermal

layers of the leaf and petiole tissue were found to consist of one to two layers of lamellar collenchyma.

Parenchyma cells and intercellular spaces appeared to form the majority of the rest of the structures.

Epidermal peels of leaf tissue viewed using cross-polarizing microscopy revealed sclarified fibers running

parallel to the midvein of the leaf (Fig 2.1). A large number of raphide crystals and druse crystals were

observed . Crystals are deposits of inorganic materials, specifically calcium oxalate salt, which have

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Fig. 1. Transverse cross section of leaf tissue stained with TBO showing collateral arrangements of vascular bundles with fiber cap and phloem facing abaxial surface and xylem facing the adaxial surface. LE- lower Epdermis, MES – Mesophyll, FC – Fiber Cap, PH – Phloem, PX – Protoxylem, MX – Metaxylem, LC – Lemellar Collenchyma

(Fig. 2.1) Epidermal peel of leaf tissue viewed using cross-polarizing microscopy revealed sclarified fibers, druse crystals and raphide cyrstals (Fig. 2.2) Detail of cleared leaf tissue stained with TBO showing druse crystals (DuCr), xylem tracheary element (XTE) and sclarified fiber.

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crystallized and become hard prismatic structures (Esau 1953). The crystals are unpalatable to herbivores

and provide protection for the plant.

Large air pockets were found in transverse sections of petiole tissue (see Fig 3). This could

possibly be an adaption to allow this terrestrial plant to survive for extended periods of time when exposed

to water in excess such as a heavy downpour. S. wallisii has been known to survive submerged for several

months before degradation occurs (The International Aroid Society 2009). This was also observed in the

laboritory where roots from one specimen remained submerged in water for approximately months. The

large air pockets and intercellular spaces provide protection against drowning for short periods.

Fig. 3. Transvers section of petiole tissue illustrating large air bubble throughout tissue . Fig. 4. Transverse cross sections stained with I2KI showing large concentration of starch grains throughout tissue.

A transverse section of petiole tissue that was stained with I2KI revealed a high concentration of

starch grains (Fig. 4) throughout the tissue. Starch grains are polysaccharides composed of glucose and

used as a main storage compound (R.L. Peterson et al. 2008). Therefore, the petiole of S. wallisii appears to

be a major storage centre for this plant. Starch grains were also found in the leaf, primarily surrounding the

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vascular bundles, and directly below the adaxial epidermis (Fig 5.2). The starch appears to be located in

the same areas as chloroplasts (Fig 5.1) in the leaf. This formation of starch in the same location as the

chlorophyll could function for storage of excess of glucose produced from photosynthesis.

The transverse sections of petiole tissue stained with Sudan III/IV revealed a textured suberized

cuticle covering the epidermis. The cuticle likely function to restrict water loss through transpiration, as

well as offering protection from herbivory by insects. Trichomes were found on petiole tissue stained with

acid fuchsin. The trichomes were mulitcelluar and sparse. Only three were located and were found on no

other organ of the plant. The trichomes did not appear to serve a significant function.

Macerated petiole tissue was observed after being stained with TBO (see Fig. 6.). Secondary cell

wall thickenings appeared to form in an annular and helical structure. No pitted fibers were observed in the

macerate.

Fig. 5.1. Leaf tissue unstained viewed under polarized microscopy. Fig. 5.2. Leaf tissue stained with I2KI showing starch grains covering the abaxial side of the leaf just below the upper epidermal layer and around the vascular bundles within the midvien of the leaf. Starch grains group at the same locations as the chloroplasts.

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Fig.6. Petiole macerate showing parenchyma cells and annular and helical secondary cell wall deposits in xylem tissue.

Stem

Transverse hand sections stained with TBO from a flower stem were sampled and collateral

vascular arrangements were observed. However, in stem tissue the phloem and fiber cap were facing

outwards, towards the epidermal tissue while the metaxylem and primary xylem were facing inwards,

towards the centre of the stem. The vascular bundles, again, appeared to be randomly placed throughout

the ground parenchyma with no central pith suggesting S. wallisii to be a monocotyledonous plant. It was

observed that the farther the vascular bundles were located from the epidermis of the stem the more the size

of the bundle seemed to stretch. Vascular bundles observed farthest from the centre of the stem appeared to

be condensed, with a large fibrous cap surrounding the phloem and there seemed to be little or no fiber

surrounding the xylem. Farther into the cortex, closer to the centre, the vascular bundles appeared

elongated with smaller fiber caps covering the phloem and continuing around to encircle the xylem. These

fibers, which surround the xylem, could be extraxylary fibers. Extraxylary fibers develop from the same

meristematic tissues as the xylem cells (Esau 1953) and function to protect the xylem. The large fiber caps

observed around the outer perimeter of the stem probably protect the vascular bundles; however, the

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vascular bundles located deeper within the tissue do not require as much protection. Therefore, fibers do

not need to be concentrated on one side of the vascular bundles. It appears they would be more useful

functioning as mechanical support in this location. Another observation on the differences occurring in the

vascular bundles based on location is in the phloem and xylem tissues. The vascular bundles furthest from

the centre of the cortex are mainly made up of fiber caps with very little phloem and xylem tissue. Closer

to the centre of the cortex the xylem and phloem appear larger and consist of 2x to 3x more cells than

vascular bundles closer to the epidermis and thus be able to transport a higher volume of materials to and

from the flower. This is possibly another adaptive protective strategy that S. wallisii has evolved.

Faint traces of lignified cells appeared in a ring formation connecting some of the vascular bundles

closest to the outer edge of the stem. This formation was not observed throughout the whole stem and

appeared only partially present (see Fig.7). It is possible that these cells could be a sclarified fibrous band.

This fiber band could function in protection by providing a hard shell close to the epidermis to prevent

penetration from herbivory, or could also function to provide structural support.

Transverse and longitudinal hand sections that were cut from a stem sample and stained using

amido black did not reveal any p-protein plugs. Transverse sections sampled with I2KI stain did not reveal

many starch grains in this area of the plant. There appeared to be a very low concentration of starch located

around the vascular bundles suggesting the stems function more in transportation of materials then in

storage.

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Fig.7. Transverse section of stem tissue stained with TBO showing partially formed lignified fibrous band, which possibly functions to protect the inner contents of stem structure. Vascular bundle closer to epidermis appear to be more compressed with larger fiber caps while bundles located deeper within cortex appear less compressed with more xylem and phloem tissue.

Rhizome

With the exception of flower stems, S. wallisii does not produce other aerial stems. S. wallisii

instead uses a condensed horizontal stem system called a rhizome which sends out shoots from the top of

the nodes and roots from the bottom of the nodes (J.E.Preece et al 1995). Samples of the rhizome tissue

that were stained with acid fuchsin revealed considerable amounts of protein deposits within totipotant

ground tissue composed of parenchyma cells, covering the entire sample. Samples stained with I2KI also

revealed large quantities of starch throughout the entire organ. This suggests that the rhizome is a main

storage site for proteins and starch (see fig). With such a high concentration of energy storage as well as

being the site of both meristematic apices, perhaps producing subterranean rhizomes was a defensive

strategy of S. wallisii to protect the valuable and delicate storage organs from predation, as well as from

extreme climatic conditions.

Vascular bundles in unorganized and random directional formation were observed (see fig) from

transverse sections of rhizome tissue stained with TBO. Lignified xylem tracheary elements were

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observed; however, identifying phloem tissue was very difficult. The purpose for the random organization

of vascular bundles could be due to the compressed nature of the rhizome and the directional changes that

occur in order for the vascular system in the submerged organs to continue transport to the vascular systems

in the areal organs and vise versa.

Cross polarizing microscopy revealed large quantities of raphide and druse crystals throughout the

entire rhizome. The concentration of druse crystals was to be higher than raphide crystals, although the

concentration of both crystals within the rhizome were significantly higher than found in any other plant

parts. This, once again, suggests that S. wallisii has evolved defensive measures to protect the

most valuable storage and growth centre.

Fig 8.1 Transverse section of rhizome stained with I2KI showing high density of starch grains throughout the tissue. Fig 8.2 Transverse section of rhizome stained with I2KI showing high density of protein staining pink-red. Dark pink-red areas of stain demonstrate the random, unorganized directional pattern of vascular bundles due to compressed nature of rhizome.

Adventitious roots were observed originating from a ring of lignified tissue enclosing the vascular

bundles in the rhizome. Adventitious roots are usually observed in monocots as roots that only arise from

stem tissue (Esau 1953).

Roots

The general structures of S. wallisii roots were long and thin with a large number of root hairs.

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Longitudinal sections of root tissue revealed S. wallisii to possess a typical closed root system. In a closed

root system there are three separate initials that generate different tissue structures. These structures are

dermacalyptrogen which gives rise to the root cap and protoderm, the periblem, which generates the ground

tissue and cortex and the plerome which forms the vascular cylinder (Coughlin & Kokolic 2009). Figure 9.

shows a longitudinal section of a root tip stained with TBO.

Transverse sections of root tissue stained with TBO showed alternating arrangements of xylem,

Fig. 9. Longitudinal section of the root tip stained with TBO showing closed formation. Circle indicates area of initial developments of the root cap, vascular cylinder and the cortex. AM- Aprical Meristem, Cal- Caliptrogen (dermacalyptrogen), Per – Periblem, Plr – Plerome, PD- Protoderm which gives rise to the epidermis, ED – Edodermis, GM - Ground meristem which gives rise to the cortex, PC – Procambium which gives rise to the vascular cylinder, RC - Root cap. Fig. 10. C – Cortex, PH- Phloem, IX – Immature, xylem, MX – Metaxylem, PX – Protoxylem, EN- endodermis, PC – Pericycle, IC – Intercelluar, spaces, EX – Exodermis, E – Epidermis, RH - Root Hairs

metaxylem and phloem in the vascular cylinder (see fig 10). Large unlignified lacunae were also observed

which could potentially be metaxylem elements, which have not yet matured. The endodermis and

pericycle were also apparent when transverse root tissue stained with acid fuchsin was observed. Portions

of the endodermis, exodermis and epidermis stained bright pink, suggested sites of protein deposit. The

lack of a polyarch arrangement of vascular tissue support the fact that this plant is a monocot.

Transverse fresh hand sections of root tissue stained with I2KI revealed starch grains. The

concentration of starch in the roots was found to be higher than that of the stem tissue but less than that of

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the rhizome samples. The starch grains were found in a gradation pattern in the cortex with little to no

starch found within the central cylinder, pericycle and endodermis and increasing in concentration closest

to the epidermis.

Samples stained with FeCl3 and examined with UV light revealed casparian bands in the exodermis

as well as slight traces in the endodermis. Casparian bands are deposits of lignin and suberin that inhibit

the passage of ions into of the vascular cylinder through the cell walls of the endodermis (Peterson et al

2008).

Summary

A study was carried out on the Peace Lily (Spathiphyllum wallisii) showing that it is a

monocotyledonous plant because of the random arrangement of vascular bundles found in the leaves,

petioles and stem tissue. The lack of a polyarch arrangement in the roots shows confirmation of a

monocotyledon. The large air pockets that were found in the leaves and petiole tissue are an adaption

to allow this terrestrial plant to survive for short periods of time when exposed to water in excess. A

defensive adaptation that S. wallisii has evolved are the large number of druse and raphide crystals

found throughout the whole plant, causing it to be unpalatable for many herbivores. Perhaps the most

beneficial adaptation is found in the subterranean rhizome. The densely packed crystals found within

the rhizomes protect the valuable and delicate storage and growth structure from predation, as well as from

extreme climatic conditions.

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