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Anatomy of Polyploid Cassava and its Interspecific Hybrids

By

Dalva Graciano-Ribeiro², Nagib M.A. Nassar³, 4, Danielle Yasmin Conceição Hashimoto³, Sandro Freitas

Miranda³ and Lauana Costa Nogueira³

²Departamento de Botânica. Universidade de Brasília, Brasília. Caixa Postal 04477 CEP 70719-000 Brasília, Brasil. ³Departamento de Genética e Morfologia. Universidade de Brasília, Brasília. Caixa Postal 04477 CEP 70719-000 Brasília, Brasil. 4E-mail: nagnassa@rudah.com.br.

Cassava anatomical data are rare in literature. Despite of its economic value, less is found about the anatomical

comparative study related to physiological aspects. The stem anatomy of an interspecific hybrid (Manihot esculenta x M.

oligantha) and its tetraploid was studied. Hand cross sections were applied to stem, colored by safranin and alcian blue,

and mounted in synthetic resin. The tetraploid form displayed larger medulla and absence of growth rings. Vascular

tissues in the tetraploid were larger. Both forms had similar vessel elements and articulated laticifers. The nature of

distribution of different tissues in the two types suggests drought resistance may be imparted to the tetraploid form.

Key words: Manihot, comparative stem anatomy, cassava hybrid, vessel elements, drought resistance.

Cassava, Manihot esculenta Crantz, is the staple food for more than 800 million poor people in the tropics and

subtropics (FAO, 2006). The genus Manihot belongs to family Euphorbiaceae, subfamily Crotonoideae, tribe

Manihoteae (Webster, 1994). It has some 99 species distributed among 19 sections (Rogers and Appan, 1973; Nassar,

2002, 2007; Nassar et al., 2008).

Viègas (1940) contributed with anatomical cassava description of vegetative parts in M. utilissima; but nothing related to

cassava anatomical studies and polyploids has been made to date.

In the present work, stem anatomy was studied in a Manihot interspecific hybrid, M. esculenta x M. oligantha, and its

induced tetraploid. The information obtained may be useful for cassava breeding since it may shed light on anatomical

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base of some economic characters such as tolerance to drought.

Material and Methods

A diploid hybrid of cassava, Manihot esculenta Crantz with M. oligantha Pax, obtained by the second author (Nassar,

1979) was polyploidized by applying 2% aqueous colchicine solution to the lateral buds of the hybrid diploid stem at 24h

intervals for one day. Tetraploid stems were identified by morphological and cytogenetic means. They were then

propagated vegetatively. The material is being maintained at the Experimental Station run by the Biology Department,

Universidade de Brasília.

Stem cuttings 10cm in length were taken from the stem between the third and seventh nodes from the top. They were

fixed in 70% FAA (formaldehyde, acetic acid mixture) (Johansen, 1940) for 24h and then stored in 70% ethanol. Free-

hand cross-sections of the stem internodes were made, clarified using 50% sodium hypochlorite solution (Kraus and

Arduin, 1997), stained with 1% safranin-alcian blue (Luque et al., 1996), passed through ethanol series and butyl

acetate and subsequently mounted in a synthetic resin (Paiva et al., 2006). Approximately thirty cross-sections of each

hybrid were analyzed.

Stem samples of both the diploid and tetraploid plants were macerated to observe vascular elements. The following

steps were taken to this end: small pieces of the samples were immersed in Franklin (1945) at 60° C for 72h, until they

softened and had no pigmentation. They were macerated, and dissociated samples were mounted in a synthetic resin

after passing through an ethanol series. The criterion of classification by size is based on Frost (1930). Small vessel

elements thus measured 0.1-0.3mm, medium elements 0.3-1.3mm, and big ones 1.3-2.0mm.

Histochemical tests were followed to study the presence of crystals: for calcium carbonate crystals, glacial acetic acid

was used and for oxalate crystals, 5% sulfuric acid (Strassburger, 1943). This promotes dissociation of the crystals.

Photomicrographs were taken using a Zeiss Axioskop, images being captured in Motion Image Plus 2.0.

Results

In cross-sections, the hybrid stems appeared to be circular or cylindrical. Presence of phellogen and vascular cambium

characterized secondary growth (Fig. 1 - A and B).

In the stem, the bark epidermis was interrupted by periderm and cork (phellem) that varied in thickness. Sporadic

lenticels were observed. The cortical parenchyma showed idioblasts consisting of druses, prismatic crystals and some

laticifers.

The collenchyma tissue was angular, consisting of up to five cell layers, within which were the parenchyma cells

containing prismatic crystals, druses and starch granules, which form a starch sheath. Later, pericyclic fibers formed an

interrupted cylinder with a cell wall of variable thickness (Fig. 1 - B and E). Diploid stems exhibited more granules.

Sieve elements, companion cells and laticifers in rows of small groups between other cells compounded the secondary

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phloem. Rays of phloem parenchyma were continuous with those in the secondary xylem (Fig. 1 - C and F). The diploid

sample presented more secondary phloem cell layers (11 layers), and its laticifers occurred in groups (2-7) distributed

among vessel elements and companion cells. The tetraploid sample had about 7-9 layers and its laticifers were

encountered in groups of 3-5, being more abundant than in the diploid sample. Vascular region was easily identified

with many cell layers undergoing differentiation (Fig. 1 - A and B).

Vessel elements, fibers, tracheids, radial parenchyma and vasicentric axial or scanty paratracheal parenchyma were

observed in the secondary xylem. Solitary vessels and vessel groupings were observed, sometimes with solitary ones

displaying a circular shape while clusters were elongated. Cells of radial parenchyma were rectangular in rows. Xylem

fibers had slightly lignified walls, large cell lumen and rectangular shape. In addition to these, the secondary xylem

exhibited tissue that appeared to be secondary growth rings (Fig. 2 - G and K).

Protoxylem and metaxylem elements were evident in the primary xylem, being surrounded by parenchyma cells (Fig. 2 -

I and L). These enveloped the inner primary phloem with sieve tube elements, companion cells and laticifers.

The medullar area appeared to be divided into two regions, as evidenced by cell shape, content and number of layers.

The peripheral area, close to the primary xylem, contained isodiametric cells, whereas the central area displayed 5-6-

sided polygonal cells. Content and the number of cell layers differed according to stem ploidy (Fig. 3 – M and N).

The peripheral medulla of the tetraploid had about six layers of isodiametric to polygonal (4-7 faces) parenchyma cells,

which contained little starch. Meanwhile, up to seven parenchyma layers were noted in the diploid, and they contained

both starch and druses. In the central medulla of the tetraploid, parenchyma cells apparently void of cytoplasm were

polygonal, hexagonal ones prevailing, these being narrower and more elongated with rare cytoplasm content (Fig. 3 -

N). In the diploid hybrid, these cells were broader but smaller, containing druses, starch and rare prismatic crystals (Fig.

3 - M).

The difference between the diploid and tetraploid stems was apparent in the cortex region. The tetraploid hybrid showed

more prismatic crystals and druses in the cortical parenchyma. The starch sheath contained starch and, rarely, druses.

Pericyclic fibers had slender walls. However, the main structural differences were noted in the primary and secondary

phloems, primary and secondary xylems and in the medulla. The abundance of prismatic crystals in the diploid hybrid

starch sheath (Fig. 1 – B and E) deserves special mention.

The primary and secondary phloem cells were 3-4 folds bigger in the tetraploids (Fig. 1 – C and F). The number of

layers and starch content were also higher in the tetraploid plant than in the diploid (Fig. 1 – A and D).

The tetraploid, radial parenchyma cells of the secondary xylem were rectangular, sometimes elongated, the walls being

thinner and containing abundant starch. Vessel elements were observed in solitary groupings exhibiting tyloses (Fig. 2 –

J and K). The primary xylem was reduced, and contained no starch (Fig. 2 - L). The fibers were rectangular with thin

walls and not apparently empty (Fig. 2 - K). The radial parenchyma cells of the secondary xylem are normally elongated

in the diploid, with narrow thick cell walls and abundant starch. Vessel elements were more abundant with tyloses

present (Fig. 2 – G and H). The primary xylem contained starch and had no tyloses (Fig. 2 - I). The fibers were about

50% smaller with densely thickened walls and they contained starch (Fig. 2 - H). While growth rings were present in the

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diploid, they were absent in the tetraploid (Fig. 1 – A and D).

The main differences, in quantitative terms, were found in the tetraploid, that had more developed secondary vascular

tissues than the diploid, as much in relation to the secondary phloem as to the secondary xylem (Fig. 1 – A and D).

Longitudinal cross-sections of the diploid showed articulated branched laticifers in the cortex of the primary and

secondary phloems as well as in the inner primary phloem (Figs. 3 and 4 – O to T). Laticifers were scarce in the cortical

parenchyma and abundant in the secondary phloem. In contrast, the tetraploid hybrid displayed more laticifers in all the

tissues.

Vessel elements were observed for the type of perforation, size and extremity. In the diploid hybrid, the vessel elements

were of the reticulate type and medium in size; perforation plates were generally simple with an oblique end (Fig. 4 - U);

the fibers were mostly libriform with abundant starch content (Fig. 4 - Y); radial and axial parenchyma likewise had

abundant starch content (Fig. 4 - X). In the tetraploid, the vessel elements were of the reticulate type, being of all sizes,

medium and big ones predominating. Starch content was low (Fig. 4 – V and W).

In the diploid hybrid, prismatic crystals and druses were abundant in the cortical parenchyma, secondary phloem, radial

parenchyma, xylematic fibers and peripheral medulla; whereas in the tetraploid hybrid their occurrence was rare.

Histochemical tests showed that crystals and druses were made up of calcium oxalate.

Discussion

Relatively few studies have been published on the comparative anatomy of diploids and their tetraploids. In the present

study, the tetraploid type presents a larger number of vessel elements without tyloses in the secondary xylem, as well

as fibers and parenchyma cells with slender walls. The absence or rare occurrence of tyloses in vessel elements

denotes that the vascular tissue is still functional. This fact associated with a larger number of vessel elements probably

indicates more efficient conduction than in the diploid type.

Growth ring presence in the diploid hybrid probably indicates less efficient conduction or less resistance to external

factors such as light, water and temperature, among others.

The increase in cell volume was observed in the tetraploid secondary xylem: diameter of the vessel elements and axial

parenchyma cells, radial parenchyma and vessel elements of the secondary phloem as well as in medulla. These cells

are broader in the tetraploid type than in the diploid. In quantitative terms, it was noted that the tetraploid type has more

developed secondary vascular tissues than the diploid type, in relation to both secondary phloem and xylem.

Greater cell ploidy is often related to increased in organ size (Stebbins, 1971; Sugiyama, 2005). Cavalier-Smith (1985)

discussed the significance of the proportional increase in cell volume with increasing DNA content. He attributed it to

balanced cell growth through maintenance of a constant ratio between the nuclear volume devoted to transcriptions and

the cytoplasm volume devoted to protein synthesis.

Diploid and tetraploid hybrids showed a fiber distribution similar to that found by Dehgan (1982) in the peripheral region,

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the pericycle fibers forming a complete cylinder that varies in cell-wall thickness. The largest quantity of fibers is

observed in the secondary xylem, however.

Cultivars from the same species can have different lignin increments (Akin, 1989; Silva-Lima et al., 2001a, 2001b; Brito

and Rodella, 2002). Our tetraploid type showed varying lignin content and differing cell-wall thickness, while the diploid

sample had a thicker cell wall. Fibers of the diploid type had starch grain content, and some presented septum. The

presence of starch and septum in fibers probably has serves for storage beyond sustenance requirements.

Cultivars from certain species may exhibit differences in density and thickness of parenchyma cells, resulting in different

levels of resistance to microorganisms (Philip et al., 1991). In addition to parenchyma cells, the hybrid we have

analyzed possesses colenchymatic cells in the cortical region, though the number of layers differs from sample to

sample. All these mechanical features lead us to relate to the tetraploid type the more resistance to drought and

diseases.

In tetraploid types, the structures described above clearly correspond to reports about more robust and harder stems.

Moreover, the large number of vessels in the tetraploid type may retain a larger quantity of water than in diploid plants.

Articulated branched laticifers were observed in the material analyzed. According to Esau (1965), latex occurs in 12500

species in 900 genera. However, studies of laticifer arrangements, types and taxonomic significance are limited.

In the hybrids studied, their occurrence was noted in the cortical parenchyma, primary phloem and secondary phloem.

In longitudinal section, we can classify them as articulated and branched. Metcalfe (1967) considered non-articulated

laticifers to be predominant in the family. The articulated type is restricted to a few species such as Manihot glaziovii

and Hevea brasiliensis.

Vanucci (1985) reported laticifers occurring in leaf mesophyl of Manihot caerulescens while Manihot pilosa displayed

laticifers in leaf mesophyl, petiole and phloem. The same author, though, did not classify laticifers. Typical articulated

laticifers were observed in stems of Manihot sp. aff. caerulescens (Rudall, 1994). Mendonça (1983, 1992) reported

articulated and non-branched laticifers in M. caerulescens Pohl and M. glaziovii Muller von Arg.

Prismatic crystals and druses were abundantly observed in the cortical parenchyma, secondary phloem, radial

parenchyma, xylematic fibers and peripheral medulla of the diploid hybrid, while they were rare in the tetraploid hybrid.

Higley (1880) reported the presence of these materials in Euphorbiaceae. Raphides were not detected in the hybrids

analyzed, only prismatic crystals and druses.

The crystals’ function is not fully understood, the functional significance of calcium oxalate crystals varying according to

crystal morphology and distribution within tissues of particular species (Franceschi and Horner, 1980). We can assume

that they contribute to the strength of tissue and act as a storage deposit regulating the concentration of soluble calcium

and/or oxalate. We can likewise posit that the formation of calcium oxalate crystals in plants may also be linked to water

evaporation.

According to Bailey (1944), the reduction of vessel element size is a derivative character, as is the presence of

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reticulate vessel elements and simple perforation. The latter two characteristics were observed in both the diploid and

tetraploid types. Frost (1930) and Webster (1994), consider scalariform perforation to be a primitive form in dicotyledon

wood. These cassava hybrids, then, seem to have acquired a developed evolutionary form.

Summary of conclusions:

1. In general, the tetraploid hybrid had bigger cells and larger structures.

2. This fact associated with a larger number of vessel elements probably indicates more efficient conduction in the

tetraploid type.

3. Laticifers were of articulated and branched type, and their occurrence was in cortical parenchyma, primary and

secondary phloems.

4. Prismatic crystals were abundant in the diploid hybrid; they can be involved in the strength of the tissue and as a

storage deposit. They also may be linked to water evaporation.

5. The presence of reticulate vessel elements and simple perforation in both hybrids are related to a more recent

evolutionary form in plants.

Acknowledgements

This work was carried out with financial assistance from the Brazilian National Council for Scientific Research (CNPq)

and the Brazilian Coordination for Qualifying Graduate Personnel (CAPES). The Manihot living collection at

Universidade de Brasília was established in the 1970’s with financial support from the International Development

Research Centre (IDRC), Canada, to whom we are grateful.

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APPENDIX 1

Voucher specimens of both hybrids have been deposited at the Universidade de Brasília Herbarium (numbers 75973

(diploid) and 75974 (tetraploid)).

Figures Legends

Figure 1 – Stem cross-sections of diploid hybrid (A,B,C) and tetraploid hybrid (D,E,F). A – Stem overview, primary

phloem and secondary phloem in detail ( }), and secondary xylem (2) and vessels with tyloses (®); B - Starch sheath in

detail (®) with crystals and thickened cell wall of pericyclic fibers (*) and primary phloem; C - Secondary phloem in

detail with radial parenchyma (pr), sieve elements (se) and laticifers (®); D - Stem overview, showing greater diameter

and vessels without tyloses; E - Starch sheath in detail (®) without crystals and thinned cell wall pericyclic fibers (*) and

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primary phloem. These areas are more developed. F – Secondary phloem in detail with radial parenchyma (pr), sieve

elements (etc) and laticifers (®). These cells are bigger. Bar = 0.1mm.

Figure 2 –Stem cross-sections of diploid hybrid (G,H,I) and tetraploid hybrid (J,K,L). G – Secondary xylem in detail,

showing solitary (v) and vessel grouping (vg) with tyloses (*). H - Secondary xylem in detail, thickened cell wall of fibers

(*) and radial parenchyma cells with starch (®). I – Primary xylem and primary phloem in detail (®); J - Secondary xylem

in detail, showing more solitary vessel and vessel groupings (vg) without tyloses. K - Secondary xylem in detail, thinner

cell wall fibers (*) and parenchyma cells with starch (®). These cells are bigger. L - Primary xylem and primary phloem

in detail (®). Bar = 0.1mm.

Figure 3 – Stem cross-sections of diploid hybrid (M) and tetraploid hybrid (N). M – Medulla with isodiametric (ip)

parenchymatic cells. N – Medulla with polygonal (pp) parenchymatic cells. They are longer and narrower. Bar = 0.1mm.

Figure 3 – Stem longitudinal sections of diploid hybrid (O,R) and tetraploid hybrid (P,Q). O – Secondary phloem in

detail with radial parenchyma (pr), sieve elements (se), companion cells (white®) and laticifers (black ®). P - Secondary

phloem in detail, laticifers (black ®), radial parenchyma (rp), sieve elements (se). These cells are bigger, and there is no

starch inside. Q – Laticifers in detail (®) in secondary phloem tissue. R – Articulated branched laticifers in detail (®) in

secondary phloem, sieve elements (se), companion cells (black®) and radial parenchyma (rp).

Figure 4 – S - Articulated laticifers in detail (®) in primary phloem tissue. T - Articulated branched laticifers in detail (®)

in primary phloem tissue. Bar = 0.1mm.

Figure 4 – Macerated tissue of diploid hybrid stem (U,X,Y) and tetraploid hybrid (V,W). U - Vessel elements of reticulate

type with medium size and simple perforation plates with straight end. V - Small vessel elements of reticulate type;

simple perforation plates with oblique end with appendices at both extremities. W - Bigger vessel elements of reticulate

type; reticulate perforation plates with oblique end. X – Radial parenchyma cells (black ®) and axial parenchyma cells

with starch content ( white®). Y – Libriform fibers with starch content in detail. Bar = 0.1mm.

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Figure 1

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Figure 2

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Figure 3

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Figure 4