Analisis Morfologico Granadilla

7/30/2019 Analisis Morfologico Granadilla

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PROPAGATION AND TISSUE CULTURE

Received or publication: 15 June, 2011. Accepted or publication: 2 November, 2011.

1 Department o Agronomy, Faculty o Agronomy, Universidad Nacional de Colombia. Bogota (Colombia).2 Corresponding author. [email protected]

Agronoma Colombiana 29(3), 377-385, 2011

Morphological and anatomical analyses of the seed coats ofsweet granadilla (Passiflora ligularis Juss.) seeds

Anlisis morfolgico y anatmico de las cubiertas desemillas de granadilla (Passiflora ligularis Juss.)

Julin Crdenas-Hernndez1, 2, Diego Miranda L.1, Stanislav Magnitskiy1, and Carlos Carranza1

ABSTRACT RESUMEN

Te study o histology and morphology o seeds o genus Pas-sifora has been o high utility or the classication o species.In seeds o sweet granadilla, the histological characteristicsand methodologies or their study are unknown. Tis studywas aimed to know the tissue and morphological character-istics o the seed coats o seeds o sweet granadilla and to beable to determine its value in the dierentiation o accessions.Five accessions collected in producing zones o the Provinceo Huila, Colombia, were analyzed. In morphological analysis,all accessions presented alsioveate ornamentation and entiremargin. Te seeds presented high change in weight and size orevery accession; there stood out the seeds o the accession PmNor presenting major size (7.42 mm long), weight (35.62 mg),homogeneity in these variables and a typical orange color. Forthe histological analysis, a protocol was adapted to realize sec-tions o seed coats in paran, by means o which one managed toobtain sections (7 ) that in the optical microscope show clearlythree well dierentiated layers, belonging, possibly, to exoteg-men (internal layer), mesotesta (medium layer) and exotesta(external layer). Every layer presented dierences in the orm othe cells, color and thickness, between the sections o the basal

and medium parts o the seeds, but the dierences between theanalyzed accessions were not observed. Te majority o seedshad a thickness o seed coats that varied among 235 and 475 .

El estudio de la histologa y morologa de las semillas delgnero Passifora ha sido de gran utilidad para la clasicacinde especies. Sin embargo, las semillas de granadilla no hansido investigadas. Con este estudio se busc conocer lascaractersticas tisulares y morolgicas de la cubierta de lassemillas de granadilla y as poder determinar su valor en ladierenciacin de accesiones. Se analizaron cinco accesionescolectadas en zonas productoras del departamento del Huila(Colombia). En el anlisis morolgico, todas las accesionespresentaron ornamentacin alsioveada y borde entero. Lassemillas presentaron gran variacin en peso y tamao dentrode cada accesin; se destacaron las semillas de la accesinPmN por presentar mayor tamao (7,42 mm de longitud),peso (35,62 mg), homogeneidad en estas variables y un colornaranja caracterst ico. Para el anlisis histolgico se adapt unprotocolo para realizar cortes de la cubierta de las semillas enparana, mediante el cual se logr obtener cortes (7 ) que almicroscopio ptico muestran claramente tres capas bien die-renciadas, pertenecientes posiblemente a la exotesta (externa),mesotesta (media) y exotegmen (interna). Cada capa presentdierencias en la orma de sus clulas, color y espesor, entre los

cortes basales y los cortes medios de las semillas, sin dierenciasclaras entre las accesiones analizadas. La mayora de las semillasmostraron un grosor de la cubierta entre 235 y 475 .

Key words: exotegmen, mesotesta, exotesta, macrosclereids,tanierous cells.

Palabras clave: exotegmen, mesotesta, exotesta, macroesclereids,clulas taneras

Introduction

Morphology and anatomy seed research contributes to

knowledge o taxonomy, evolution, and ecology o Angio-

spermae species (Cortez and Carmello-Guerreir, 2008).

Passifora is the most important genus o the amily Pas-sioraceae and is represented by approximately 500 species.

Tis taxon presents a wide natural distribution, with plants

grown in the wild in the Americas, Western India, Galapa-

gos Islands, Australia, Southeast Asia, Malaysia, Polynesia,

and some islands o the Pacic Ocean (Vanderplank, 1990).

Fruits oPassifora sp. possess multiply seeds adhered to

the uniculus on the ovary wall and surrounded by an aril

that covers the seed and constitutes the eatable part o the

ruit (Werker, 1997). Te seeds o the amily Passioraceae

present seed coats with generally lignied cells that not

only aect water absorption but also oer a resistance tothe embryo growth (Cardozo, 1988).

Seed coats develop rom the integuments that surround

the ovule prior to ertilization. Beore ertilization, cells o

the integuments are relatively undierentiated. However,


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378 Agron. Colomb. 29(3) 2011

development aer ertilization can include extensive di-

erentiation o the cell layers into specialized cell types. In

addition, some cell layers in the seed coats may accumulate

large quantities o certain substances, such as mucilage or

pigments that can also contribute to overall seed morphol-

ogy (Mosa et al., 2005).

Te layers o the seed coats in seeds oPassifora sp. aredeveloped rom bitegmical ovary, in which a radial unequal

elongation o cells could be observed (Werker, 1997). Te

aril begins o a meristematic margin continued around the

distal part o the raphe and includes the exostomal region.

Te pigments o the aril are located in the chromoplasts

(Werker, 1997).

MacDougal (1994) realized a taxonomical review o the

section Pseudodysosmia o subgenus Decaloba o genus

Passifora, and included the ornamentation o seeds among

the characters that he used in its descriptions. Tis allowed

considering the species o genus Passifora to be an excel-

lent object o study to characterize the seed morphology

and, thereore, illustrate the taxonomical potential that the

above mentioned structure presents or this genus (Prez-

Cortz et al., 2005).

On this matter, Prez-Cortz et al. (2005) realized an

anatomical description o seed coats o eight species o

Passifora sp., ound eight anatomically dierent patterns

and observed a layer o macrosclereid cells and a layer o

obliterate cells in all the studied species. Nevertheless, P.

ligularis was not among the analyzed species.

Te seeds oP. edulis, in the external tissues, present cells

o palisade parenchyma, prismatic and lignied with thick

reticulate wal ls, which orm conical projections inside the

endosperm. Te endotesta has thick cell walls, prismatic

elongated cells in the depressions o the tegmen and cuboids

in the channels (Werker, 1997).

Te seeds oP. ligularis are black, compressed, relatively

small, and hard. Te seeds and the aril contain about 17.6%

carbohydrates (rujillo, 1983; Werker, 1997). Prez-Cortz

et al. (2002) describe the seeds oP. ligularis with transverseaverage section o elliptical orm, basal o sharp orm or

sharply truncated, having ornamentation oveated or al-

sioveated and entire margin.

Te objective o the present research was to characterize

the anatomy o seed coats o seeds o sweet granadillausing

ve accessions collected in producing zones corresponding

to the South o Province o Huila, Colombia.

Materials and methods

Te collection o ve accessions was realized in the pro-

ducing zone o sweet granadilla(Passifora ligularis Juss.)

in the South o Huila in the municipalities La Argentina,

San Agustn and Palestina, the last one being the largest

producer o sweet granadilla at national level.

Te ruits were taken to the Laboratory o Crop Physiology

o Faculty o Agronomy, Universidad Nacional de Colombia

(Bogota), or their physico-chemical characterization. For

the analysis o seeds, ve accessions ound in two commer-

cial cultures were collected, one in the district La Mensura

(2,012 m a.s.l.) and the other one in the district El Roble

(1,807 m a.s.l.) o the municipality Palestina. Te chosen

accessions were named PrJ1, PrJ2, PrJJ, PmFD, and PmN;

the rst three were rom the district El Roble and two last

ones were rom the district La Mensura.

Fiy seeds o every accession were taken at random; in each

o these, the mass, length, width, thickness, and number

o osets were measured, the uniormity o distribution o

osets was described, the relation lenght-width (L/W) was

calculated, and the presence o appendices in the base and

the apex o the seeds was measured. In order to do this,

scales o precision and a digital gauge were employed.

Te presence o appendices was described in the base and

the apex o the seeds, enumerating them according to their

presence. For this, some descriptors proposed by Prez-

Cortz et al. (1995, 2002) were applied.

Finally, a photographic record o the seeds o every acces-

sion was realized with help o a microscope Nikon SMZ800

with integrated camera DSZMV using the soware ele-

ments, in the Laboratory o Entomology o the Faculty o

Agronomy, Universidad Nacional de Colombia, Bogota.

In order to undertake histological sectioning, rstly, the

protocol or sectioning in microtome in the Laboratory o

Microbiology o Faculty o Agronomy o the Universidad

Nacional de Colombia (Bogota) was adapted, taking into

account the methodology used by Prez-Cortz et al. (2005,2009). Te principal methodological adaptations occurred

in the processes o tissue soening with hydrochloric acid

(18.5%) and xation o tissues with Mayer Albumen.

Using the adapted protocol, sections o 7 o seed coats

were done o the seeds o ve chosen accessions. With help

o the program Scopephoto 3.0 and optical microscope

Micro BM2000 with built-in camera DCMC510, the photo-


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379Crdenas-Hernndez, Miranda L., Magnitskiy, and Carranza: Morphological and anatomical analyses of the seed coats of sweet granadilla (Passiflora ligularis Juss.) seeds

graphic record o sections was realized. Te entire thickness

o seed coat was measured as well as the thickness o each o

three layers identied with help o the program Imagepro

Express 6.3 de Medicybernetic Inc. Both morphological

and histological analyses were descriptive ones.

Results

Morphological description of seeds of sweet granadilla

Among the accessions no valuable dierences in the type

o ornamentation were recorded, since all accessions had

alsioveated type that is to say with asymmetrical osets or

oveas that were deeper and better delimited towards the

central part o the seeds. All seeds presented entire margin

and apical central horn (Fig. 5-9).

When counted the number o appendices present (le and

right) in the apical and basal zones o the seeds, it was ound

that these could possess rom none up to two appendices

or each zone; nevertheless, the presence or exact number

o appendices was not considered an exclusive eature o

every accession.

Te dry seeds (9-11% moisture) presented the ollowing col-

ors: pale yellow, black, orange, and the most common coee

color; it was more common to nd seeds with combination

o these colors than monochrome seeds (Fig. 1-5A). In all

accessions, imbibed seeds (30-34% moisture) presented

black color (Fig. 1-5B). Aer the seed swallowed with the

imbibition, the osets were lost to produce a smooth surace.

Te membranous layer (exotesta) that surrounded the seedscould have been lost partially or completely (Fig. 5-9B).

Internally, the seeds did not present apparent dierences

among the accessions. In seeds o all accessions, the embryo

was straight and located in the central part o the base;

two cotyledons that occupied the highest proportion o

the internal space and a layer o endosperm surrounding

these organs were observed (Fig. 1-5C).

In dry state, the seeds o PrJ1 presented, in al l cases, com-

bined coloration: clear and black coee (Fig. 1A), while in

imbibed state they were o black color (Fig. 1B). In theseseeds, the presence o apical and basal appendices was

common on both sides (right and le) o the surace (Fig.

1A). Te presence o osets was much desuniorm in these

seeds (Fig. 1A). In the Fig. 1C, it is possible to appreciate

the chalaza positioning towards the apical part o the seeds,

which or this case was o coee color. In some seeds, the

presence o basal horn was observed (Fig. 1B); this one was

much smaller than the central basal horn (Fig. 1A).

Some seeds o PrJ2 presented monochrome seed coats o

black color (Fig. 2A), also seeds were observed with partially

or completely yellow colorations. Te distribution o osets

in these seeds was highly uniorm (Fig. 2A). In these seeds,

the presence o apical or basal appendices was not common.

In no case, basal horn was observed.

All dry seeds o PrJJ presented combined coloration: coeeand black (Fig. 3A). Te osets were distributed uniormly in

the majority o seeds (Fig. 3A). In Fig. 3B, the mucilaginous

layer (exotesta) could be appreciated that surrounds the

whole seeds; it was observed that this layer was removed

in imbibed seeds, entirely or partially, by supercial con-

tact. In these seeds, the presence o apical appendices was

common, while the basal ones were present in a minor

proportion.

It was very common to nd seeds o PmFD with dierent

tonalities o coee color and without black color (Fig. 4A).

owards the basal and apical parts the presence o osetswas not common in these seeds (Fig. 4A). Te presence o

appendices was not typical or this accession being the one

that had a minor number o appendices in seeds. In no case,

basal horn was observed.

PmN was the only accession, which seeds, in dry state,

presented orange color (Fig. 5A); this coloration was pres-

ent in all seeds o this accession, alone or combined with

coee and/or black color. In these seeds, the presence o ap-

pendices was more common or apical parts than or basal

ones (Fig. 5A). Te osets presented high uniormity on the

seed surace (Fig. 5A). Te basal horn observed previouslyin PrJ1 and PrJJ was not observed in this accession.

Te accession with the highest average length o seeds (7.42

mm) was PmN (ab. 1) that presented values between 7.11

and 7.88 mm. Also, the latter accession had the highest

values o width and thickness, presenting values between

4.12 and 4.7 mm, and 1.73 and 2.07 mm, respectively. Te

rest o the accessions had width o seeds between 3.38 and

4.38 mm; both extreme values were registered in the acces-

sion PmFD. Te minimal thickness was reported or the

seeds o accession PrJJ with 1.42 mm and the maximum one

was reported or the seeds o accession PrJ2 with 1.86 mm.

Te accession PrJJ had very variable L/W values, very

rounded (1.01) and also lengthened (1.86) seeds, with major

tendency to be lengthened, since it presented the highest

average value (1.73); in the rest o the accessions no value

was minor than 1.56 (ab. 1). It is interesting that the seeds

rom the district El Roble (PrJ1, PrJ2, and PrJJ) had aver-

age L/W values higher than those in the seeds rom the


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380 Agron. Colomb. 29(3) 2011

FIGURE 2. Microscopic view of seeds of PrJ2 dry (A), imbibed (B) andtheir internal part (C).

FIGURE 3. Microscopic view of seeds of PrJJ dry (A), imbibed (B) andtheir internal par t (C). Exta = exotesta, bh = basal central horn, chal= chalaza.

FIGURE 4. Microscopic view of seeds of PmFD dry (A), imbibed (B) andtheir internal part (C).

FIGURE 5. Seeds of PmN dry (A), imbibed (B) and their internal par t (C).

A B C

Extra

bch

FIGURE 1. Microscopic view of seeds of PrJ1 dry (A), imbibed (B) andtheir internal part (C). cbh = central basal horn, ApA = apical appendix,ApB = basal appendix, bh = basal horn, Chal = chalaza.

A B Cch

ApA

BpAbh

ChalA B C

A B C

A B C

district La Mensura (PmFD and PmN) located at major

altitude (ab. 1).

Meanwhile the mass o every seed in other accessions was

between 12.6 (PmFD) and 33.0 mg (PrJ1), PmN presented

seed mass that varied rom 30.1 up to 39.6 mg, with an

average o 35.62 mg (ab. 1).

Te number o osets was very variable between the seeds o

every accession, presenting a minimum o 21 osets (PrJ1)

and a maximum o 51 osets (PmFD) (ab. 1). Te number

o osets was least variable in the accession PmN (ab. 1).

Histological sections

One o the most importantant achievements o this researchwas the adaptation o the protocol which ollowed to obtain

the quality o images observed in the histological cuts. Te

principal modication o the protocol used by Prez-Cortz

et al. (2005)was to keep the blocks o parane, with the

samples, in a solution o 18,5% (hal o the commercial

concentration) o HCl by three days. Aer that, the blocks

were washed with current water by 10 min to eliminate

completely the acid, thus preventing the damage o the

blades. Other important modication, which let a beter

jation o the cuts in the porta, was the application o the

jator Mayer albumen to the porta prior to set the cut on the

porta. Te application o the albumen was made at least 5

min beore seting the cut, aming it culd dry. An importanttip to maintain the tissue continuity was to use solid steel

blades, because with thin disposable blades the results are

not satisactory by the vibration produced at contact with

the hard seed coats.

With the adjustment o the protocol, the images were taken

that are presented in Fig. 6; in this case, three layers o cells

in the seed coats could be clearly dierentiated.


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TABLE 1. Average value and () standard deviation of the length, width, thickness, relation length-width (L/W), weight and number of fosets of seedsof sweet granadilla of five different accessions.

Parameter PrJ1 PrJ2 PrJJ PmFD PmN

Length (mm) 6.98 0.28 6.65 0.21 6.80 0.18 6.89 0.17 7.42 0.14

Width (mm) 4.06 0.19 3.90 0.14 3.96 0.44 4.18 0.16 4.43 0.14

Thickness (mm) 1.73 0.07 1.73 0.06 1.68 0.06 1.63 0.06 1.91 0.10

L/W 1.72 0.08 1.70 0.06 1.73 0.11 1.65 0.05 1.68 0.06

Weight (mg) 28.44 3.18 26.24 1.89 27.02 2.07 23.77 3.69 35.62 2.75

Number of fosets 28.22 3.78 29.12 3.68 31.33 3.63 37.24 5.52 30.72 2.97

FIGURE 6. Microscopic view of cross sections (7 ) of seed coats of central zone (A, C, and E) and basal zone (B, D, and F) of seeds of sweetgranadilla. Exta = exotesta, Mta=Mesotesta and Extn = exotegmen. hC = hialinic cells, Ms = macrosclereids, tC = taniferous cells and oC =obliterate cells.

A B

C D

E F

Exta Mta ExtnMs

tC

Exta Mta Extn

hC tC Ms

Extn Mta

Exta

Extn

Mta

Extn

tC Extn

Mta

Extn oC


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382 Agron. Colomb. 29(3) 2011

Seed coat layers result are originated rom the integument

development, it could be ound the testa and tegmen divide

in three well dierenced layers: exo, meso and endo, each

one, but is more comon to ound a reduced number. In the

sweet granadilla seed coat two most external layers did

part o testa and represented the mesotesta and exotesta

(Fig. 6A, B and D). Te most inner layer corresponds to

the exotegmen.

Fig. 6A, B an C shows that the most external layer, exotesta,

is composed by hialinic cells o mucilaginous type (Fig. 6A,

B and D) that shaped the brilliant and thin layer that could

be observed in seeds o sweet granadilla at simple sight.

Tis layer had been already reported in seeds oPassifora

sp. by Corner (1976). Te thickness o this layer changed

widely, and even, in some sections, it was observed that

this layer was not continued in the medium part o the

seeds (Fig. 6 C and E). owards the basal part this layer

had a very uniorm thickness (Fig. 6B). Te only accession

that presented this layer in all sections in continues ormwas PrJJ (Fig. 6A). Tis layer had maximum thickness o

248 mkm in the medium zone o seeds and 610 mkm in

the base, which indicates the highest development o this

layer towards the part the tissues nearest to the embryo,

as observed in Fig. 6D.

Te intermediate layer, mesotesta, was characterized with

high uctuation in size, since in the zone corresponding to

osets it had high thickness (Fig. 6C and E), whereas in the

rest o the surace, this layer was narrower than the layer o

macrosclereids (Fig. 6A, B and D). Te cells that composed

this medium layer had changes in color, orm and thicknesso cell walls as well as variation in the number o cell layers.

Te color changed rom clear coee (Fig. 6E) to very dark

tones (Fig. 6B). Tis pigmentation could be related to the

presence o tannins. Te change in the thickness o cell

walls could indicate the lignication o this layer in some

zones (osets) (Fig. 6C), whereas dierent cells seemed to be

tanierous, with thin cell walls and live citosol. Te dark-

est colors and cellular walls o major thickness appeared

towards the basal part (Fig. 6B and D), such as a unique

pattern or every accession was not observed.

In the part o the appendices, the internal layer could be

increased up to 751 mkm, although in the zone o osets,

where its thickness diminished more, it could have up to

152 mkm. Te opposite happened with the medium layer

that thickened towards the osets, where it could reach up

to 378 mkm only in tissues, as observed in the Fig. 6C,

since this layer commonly did not overcome 152 mkm, with

minimal thickness o 16 mkm, such as observed in Fig. 6E.

Te images show a layer o macrosclereids (Ms), in accor-

dance with descriptions o exotegmen, which constitutes

the most internal layer o cells. Tis one was character-

ized with most uniorm thickness o three layers in all

accessions, in the sections o the basal part as well as in

the medium part o the seeds (Fig. 6A, B and E). Tis

layer presented conical very elongated cells arranged in

anticlinal orm, with cell walls thickened towards theinternal part o cytosol, which was arranged towards the

most external layers that ormed large spaces with the

ollowing layer.

Tese cells presented characteristics o high grade o ligni-

cation that leaved little space to the lumen (Fig. 6A). Tis

characteristic was more observable in the medium part o

the seeds than in the basal one, since, in the last one, the

thickness was more uniorm and the cytosol was more

reduced in volume (Fig. 6B and D). Te dierences among

the orm o cells o the internal layer between the gures

2A, 2B and C were owed to the angle at which the seeds

were sectioned. Te multiple circles observed in B and C

corresponded to the same layer, and these were reducing

their size because there were several cross sections o cells

with a conical orm.

In the medium part o the seeds, the exotegmen presented

gradual undulations; these undulations were those that

ormed the osets (Fig. 6C), which is typical or the seed

coats o seeds o amily Passioraceae (Corner, 1976).

Te internal layer presented changes among the centralzone and the basal part o the seeds; in the basal part

even, seemingly, a variation existed in the number o

layers due to a high variation in size and orm o cells o

this layer, increasing towards the appendices o the seed

coats (Fig. 6D).

Te entire thickness o the seed coats was taken as the

thickness o mesotesta plus the thickness o exotegmen,

since the exotesta did not present continuity. Te entire

thickness uctuated highly, being more constant in seed

coats o seeds o PmN compared with other accessions. In

average, the seed coats o seeds o sweet granadilla had athickness among 235 and 475 mkm that could be increased

up to 790 mkm in the region o appendices.

Te layer o obliterate cells was reported in seeds o other

species o Passifora, however, in this study, it was not

observed; nevertheless, a section o basal part o seeds o

accession PmFD showed a similar layer (Fig. 6F).


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Discussion

Te margin (entire) and the precence o central horn, previ-

ously reported by Prez-Cortz et al. (2002) were observed

in all the seeds analized in this study, wich indicates it is a

general characteristic or the species.

Imbibed seeds o sweet granadilla presented a black colorin the entire seed surace, which is in accordance with data

reported by Prez-Cortz et al. (2002), who conducted a

morphological analysis o seeds in 51 species o genus Pas-

sifora and ound that, in all cases, the seeds were mono-

chrome. Nevertheless, in our study, every seed in dry state

presented between one and three colors, being most typical

the orange color or accession PmN that did not appear in

seeds o any other accession.

Te osets in seeds o sweet granadilla independently o

the accession were distributed uniormly on the central

zone o seed surace, but it was common that these were

not present in seed apex, base or margins; when presented

in these zones, the osets were o major length. Tis agrees

with the data reported by Prez-Cortz et al. (2002) in the

description o seeds oP. ligularis. For the species o genus

Passifora, the seminal characteristics could be considered

as taxonomical characters, since these were demonstrated

being a distinctive eature that allows identiying o species

(Prez-Cortz et al., 2002).

An advantage o usage o these characteristics in taxonomy

o genus Passifora is a relative acility to study these, takingin account that, in other species, an electronic microscopy

or the study o seed surace is needed, whereas, in case o

seeds oPassifora sp., one has managed to describe the

seeds and create articial keys only with the use o an

optical microscope (Prez-Cortz et al., 2002).

A great part o the advantages that oer these seed char-

acteristics owe to the dierences that exist between the

species, nevertheless its diagnostically value between ac-

cessions o the same species it is not so strict since, the seed

coats o seeds is very similar. In spite o these limitations,

or studied accessions, were clearly diered the seeds oaccession PmN that presented a typical orange color, size

and heavier weight than those o other accessions.

Characteristics o seed coats, such as the ornamentation

or orm o cells o every layer do not undergo high change

with changing climatic conditions and, thereore, these

present a high potential or identication o the species

(Barthlott, 1984). Variables, such as weight and size, could

change highly with climatic conditions o seed development

(Copeland and McDonald, 2004). In this case, the seeds

were coming rom two arms with dierent agricultural

practices and climate. In spite o this, the seeds o accession

PmN presented major weight and size than the seeds o the

rest o the accessions including PmFD that was collected

in the same arm. Te relation L/W was the highest or the

accessions taken rom the district El Roble (PrJ1, PrJ2, andPrJJ) that or those o the district La Mensura (PmFD and

PmN), being very similar the values o two last accessions

in spite o the high dierences in size and weight that was

observed between them.

Te tissues were colored with ast green and saranin; nev-

ertheless those nal sections did not color sufciently to

observe the colorations typical o every layer. Tis allowed

determining that pigmentation was darker in the basal

tissues them in the tissues o medium zone o the seeds,

but clear dierences on this matter did not appear among

the accessions. Prez-Cortz et al. (1995, 2005, 2009), in

spite o having studied in depth the anatomy o seeds o

Passifora sp., could not realize the immersion in colorings

due to the loss o the tissue. Tereore, results o our study

suggest an important advance and contribution not only

to seeds o amily Passioraceae, but also to other species

with hard seed coats.

Studies on the anatomy o seed coats in species o genus

Passiflora point out that these are constituted by one or

two external layers o transparent cells, a medium layer

o macrosclereids, and an internal layer o obliterate cells(Raju, 1955; Martn and Barkley, 1973; Corner, 1976;

Prez-Cortz et al., 1995, 2005, 2009). Te seed coats in

species o genus Passiflora are ormed by two teguments;

the external tegument originates the most external layer

o the seed coats and the multiplicative layer; rom the

internal tegument originates the layer o macrosclereids

and the layer o obliterate cells (Raju, 1955). Te layer o

macrosclereids and the layer o obliterate cells have been

identied in the anatomical prole o seed coats o the spe-

cies in the literature; or this reason, it could be suggested

that these layers are common or all species o this genus

and are characteristically o the same one. Nevertheless,the observed images o the seed coat o sweet granadilla

could be an exception to this characteristic. As it could be

appreciated in the images o histological sections, in seeds

o sweet granadilla there appeared a layer o macroscler-

eids, a layer o tanierous cells, and a layer o transparent

cells, but the layer o obliterate cells reported or the seed

coats o seeds o all species oPassifora sp. earlier studied

was not common (Prez-Cortz et al., 2002, 2009); only a


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384 Agron. Colomb. 29(3) 2011

section o the part basal o the seed o PmFD presented a

discontinuous layer that could correspond to this layer.

Raju (1955), in the anatomical description o the seed

coats oAdenia venenata (Passioraceae), did not report

the presence o a layer o macrosclereids. It is necessary

to highlight that it should be improved the methodology

to obtain a better conservation o the so tissues o seeds,

which could aect the visualization o the most internallayers like oblitate cells.

Te seeds o sweet granadilla presented an average thick-

ness o 318 mkm, with values ranging between 218 and

474 mkm, with possibility o extending up to 790 mkm in

the part o the appendices. P. alata and P. lauriolia thicker

seed coats presented average thickness o 512 and 407 mkm,

respectively. P. edulis, P. maliormis, P. quadrangularis, and

P. nitida presented low values 123, 188, 267, and 260 mkm,

respectively. Tis suggests that seed coats o sweet granadilla

have average thickness in relation to other Passifora sp.

Between accessions o sweet granadilla the anatomical

prole did not change a lot between accessions, although

the seeds presented dierences in weight and orm between

accessions. Between some related species, the anatomical

prole o seed coats is uniorm, whereas it varies between

others, thereore, this characteristics could be o high

utility in the taxonomical interpretation (Netolitzky,

1926; Werker, 1997; Prez-Cortz et al., 2009). In genus

Passiflora, morpho-anatomical characterization o seed

coats in the studied species o subgenus Passiflora, series

Lobatae (P. gritensis, P. pallens, P. spectabilis, P. subpel-

tata), Dysosmia (P. oetida), Incarnatae (P. incarnata) and

Imbricatae (P. sidiiolia) (Prez-Cortz et al., 2009) shows

that the anatomical prole o seed coats is specic and

ornamentation o seed coats allows orming groups. Ad-

ditionally to this, other authors emphasized the utility o

seeds in the diagnostics o species o this genus (Deginani,

2001; Prez-Cortz et al., 1995, 2002, 2005, 2009), which is

useul or granadilla too, but not or dierenciation between

accessions o the same specie.

Conclusions

Ornamentation o seed coats and margin o seeds did not

present dierences between the analyzed accessions o

sweet granadilla; in all cases, these presented alsioveated

seed coats with entire margin.

Te seeds o all accessions had rom one up to three col-

ors, and only in one accession, PmN, an orange color was

observed.

Te weight and size were highly variable between the seeds

o every accession, except those belonging to PmN that

presented major uniormity in these characters and major

values o the same ones.

Te soening o hard tissues with HCl and the use o jator

in the porta let the improve o the images achived in the

cuts o granadilla seed coats.

Te seed coats o seeds o sweet granadil la had three layers

well dierentiated in orm and color o their cells, size and

undulations.

Te seed coats presented dierences in thickness and cel-

lular thickness between the medium part and the basal part

o the seeds, having denser tissues towards the basal part.

Te layer o obliterate cells, commonly ound in sections

realized in seeds o other species o Passioraceae, was

not common or the sections o seeds o sweet granadilla.

Acknowledgments

Te authors express their gratitude to the National Uni-

versity o Colombia, to Colciencias and to the Corporation

Cepass-Huila or the nancial and logistic support. Tanks

are extended to the Laboratory o Microbiology o the

Faculty o Agronomy, UNAL, principally to Mr. Wadith

de Len or his support in the adaptation o the protocols

o microtomy and to Proessor Xavier Marqunez or his

help in the analysis o sections.

Literature cited

Barthlott, W. 1984. Microstructural eatures o seed suraces. pp.95-105. In: Heywood, V. and D. Moore (eds.). Current conceptsin plant taxonomy. Academic Press, London.

Cardozo, H. 1988. Eecto de la escaricacin y las dosis de cidogiberlico (GA3) en la germinacin de semilla de curuba (Pas-sifora mollisima). Acta Biol. Colomb. 1(4), 127-132.

Corner, E.J.H. 1976. Te seeds o dycotyledons. Vol. I. CambridgeUniversity Press, Cambridge, Uk.

Copeland, L.O. and M.B. McDonald. 2004. Principles o seed sci-ence and technology. 4th ed. Kluwer Academic Publishers,Norwell, MA.

Cortz, P.A. and S.A. Carmello-Guerreiro. 2008. Ontogeny andstructure o the pericarp and the seed coat oMiconia albicans(Sw.) riana (Melastomataceae) rom Cerrado, Brazil. Rev.Bras. Bot. 31(1), 71-79.

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