Download histological studies on the origin and process of plantlet

Plant Breeding
HISTOLOGICAL STUDIES ON THE ORIGIN AND PROCESS
OF PLANTLET DIFFERENTIATION IN SUGARCANE CALLUS
MASS
Ming-Chin Liu and Wen-Huei Chen
Taiwan Sugar Research Institute, Tainan, Taiwan, Republic of China
ABSTRACT
A histological study has been made of organogenesis in sugarcane callus
grown on t w o kinds of media,:One was that of Murashige-Skoog, containing
3 ppm 2,4-D and designated man-organ-forming medium; the other was the
same, without 2,4-D but with'kinetin, NAA, and casein hydrolysate for shoot
differentiation. Organogenesis did not usually take place under non-organforming conditions unless the callus was in earlier passages. Proembryoids
characterized by smaller, isodiametric cells with densely staining nuclei could
be formed as early as 8 days after subculturing under shoot-forming conditions.
They later developed .a shoot-apex structure which was ontogenetically
comparable with a seed-embryo derived from a zygote but structurally, more
nearly analogous to a vegetative shoot apex such as is found in the cane tip.
Polyembryonies were not uncommon. The cells in the portion of callus, not
far from the area which bore shoot apices, became differentiated into protoxylem elements. Two patterns of differentiation in callus were observed. I n
one shoots appeared first and roots were initiated later. In the other only
roots formed, and there was a complete loss of totipotency. SchenkHildebrandt medium was very effective in promoting root differentiation in
vitro. An embryoid was believed to be of unicellular origin as shown by our
observation as well as the relevant published papers.
INTRODUCTION
The sequential development of whole carrot plantlets from callus cultures
~ ~ than 20 years ago. Reinertz47 26 was
was reported first by W i g g a n ~more
a pioneer in demonstrating the formation of embryoids from higher plant
tissues in vitro. Steward et alz9first generated plants from cell suspension cultures
of carrot. Konar and Nataraja15 initiated a n embryoid from the stem epidermis
of Ranunculus sceleratus L. Heinz and ;Meeg, and Barba and Nickel13 derived
complete sugarcane plantlets from callus6tissue in 1969. Subsequently regeneration of whole plants from cell culture, callus or segmehts from different parts
of the plant body has been a relatively common phenomenon in many higher
plant species.35
Embryo-like structures, embryoids or whole plants, or both, have been in22*
duced from such highly differentiated cells as the pollen grain,'? 139
30 triploid endosperm cells4> l1 and the epidermal cells from leaves3zand
stems.16These achievements constitute an important means of vegetative propagation and are of great importance to both the horticulturist and the plant
breeder.
Reinert et alz7and Johri12 have demonsjrated that the sequence of development of the embryoid in culture is generally comparable to that of the zygotic
embryo. The embryoid is originated from a single cell, among the millions of
cells in callus, which has the exceptional capability of proliferating into a
group of cells before a n embryoid is formed.
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MING-DHIN
LIU
AND WEN-HUEI CHEN
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Mass production of sugarcane plantlets in vitro has been carried out since
the establishment of the Tissue Culture Laboratory a t this Institute in 1970
with a production capacity of 2 to 3 hundred plants per month. For the past
3 years through studies on shoot differentiation from sugarcane callus tissue,
it has been concluded that shoot differentiation is influenced by (a) the donor's
genotype, (b) the previous culture medium, (c) the number of subcultures
(unpublished data of our laboratory). However, the exact origin of a shoot
primordium and the processes of embryogenesis in callus cultures are still
obscure. Considerable endeavor is needed to clarify the precise internal physiological and biochemical changes which must take place before a cell can
exhibit its totipotency, or acquire embryological competence like that of a
zygote. An experiment was therefore started to trace histologically the origin
and the sequential development of a plantlet from the callus mass.
MATERIALS AND METHODS
Initiation of callus from subapical meristems and rolled young leaves was
achieved on a modified Murashige and Skoog (MS) medium as described in
a previous paper.l' At 3rd or 4th passages the callus masses of F 162 or F 170,
growing on a modified MS medium supplemented with 3 ppm 2,4-D, were
separately inoculated on 2 media. The first medium was that mentioned above
and designated non-organ-forming; the second was the same without 2, 4-D
but supplemented by the following substances a t the concentrations indicated,
in milligrams per liter : kinetin, 1; naphthaleneacetic acid (NAA), 1 ; casein
hydrolysate, 400. This was designated organ-forming medium. After inoculation, a piece of callus 2 x 4 x 4 mm in size was taken after 0, 4, 8, 11, 14,
17, 20, 23, 26 days in culture, and fixed in FPA or CRAF fixatives. The fixed
specimens were dehydrated with a series of tertiary butyl alcohol and embedded
in paraffin. Sections were cut at 10 microns, by a rotary microtome, and stained
with safranin-fast green, or safranin, .tannic acid and orange G.
I
RESULTS AND DISCUSSION
Fig. 1 is a general view of callus growing on a non-organ-forming medium.
Such a piece of callus was composed of relative large parenchymatous cells
which were randomly distributed in the central portion, and numerous smaller
and isodiametric cells with conspicuous nuclei which usually occurred in the
peripheral region. Organogenesis in callus did not usually take place under
non-organ-forming condition except in earlier passages in which the callus was
strongly influenced by the differentiated tissues of the donor. Therefore, all
meristemoids or embryo-like structures discussed in the following paragraphs
were products of the organ-forming medium.
A callus sample taken from a 14-day-old culture demonstrated nodulelike meristemoids, conforming to Torrey's term31, on its outer surface (Fig. 2).
The meristemoid was composed of numerous cells in orderly development.
The cells were smaller, isodiametric in appearance, lacking apparent vacuoles,
and with densely staining nuclei. The meristemoids were virtually proembryoids
which might develop as early as the 8th day after subculturing. Their crosssections showed cells arranged concentrically (Fig. 3). Meristemoids could later
develop into multi-shoot-apex structures as shown in Fig. 4.
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PLANT BREEDING
FIGURE 1. Parenchymatous callus mass on a non-organ-forming medium.
FIGURE 2. Longitudinal section of nodule-like meristemoids. m = meristemoid. '
FIGURE 3. Cross section of meristemoids seen in Fig. 2.
FIGURE 4. Multiple shoot apices, arising from callus 20 days after subculturing. s = shoot
apex, m = meristemoid.
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MING-CHIN LIU AND WEN-HUE1 CHEN
Figs. 5 and 6 show two typical shoot apices developed from a callus mass.
The structure in Fig. 5 is analogous to that of an embryo as seen in sagittal
sections of a mature sugarcane seed, developed from a zygote, as illustrated
by Artschwager et ~ 1 Like
. ~ the apical structure of a seed-embryo, the apical
meristem, which usually protruded into an empty space, was in the center
and flanked by embryonic leaves which were, in turn, enclosed by a coleoptilelike structure (Fig. 6). The developmental stage in Fig. 6 was much later
than that of Fig. 5, as demonstrated by the mature appearance of cells in the
surrounding coleoptile-like structure. Ontogenetically, a callus-embryo is comparable to a n embryo derived from a zygote as discussed by Reinert et a127
Longitudinal section of 2 typical shoot apices with apical meristem a t the center and leaf primordia on both sides: coleoptile-like structure.
FIGURE 7. Surface view of a shoot apex corresponding to the stages of Figs. 5 and 6.
s = shoot apex.
FIGURE 5 and FIGURE 6.
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PLANT BREEDING
and Johri12. Judging from the appearance of Figs. 6 and 11, the coleoptile-like
structure seemed to be formed earlier than the embryonic leaves which is
characteristic of grass embryosGHowever, histologically, it has not been possible
FIGURE 8. Longitudinal section of double apical meristems flanked by embryonic leaves:
d = double apical meristems.
FIGURE 9. Two apical meristems developing from callus under embryonic leaves, or coleoptile-like structures: a = apical meristem.
FIGURE 10. Surface view of a polyembryoid corresponding to the stage of Fig. 11. s = shoot.
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MING-CHIN LIU AND WEN-HUE1 CHEN
to find a shoot-root axis or other structures such as radicle and coleorhiza,
which are integral parts of a seed-embryo. The coleoptile in callus-embryoids
is considered to be functionless because its original role of protecting the delicate
plumule while it penetrates the soil is unnecessary under the circumstance of
sterile culture. As far as the structure is concerned, a callus-shoot-apex is
therefore much simpler than a seed-embryo, and more resembles a vegetative
shoot apex.
Probably due to less restriction in the development of a shoot-meristem
in callus than in the intact cane tip, the leaf primordia may not be arranged
as regularly as those of a vegetative shoot apex, so that there may be more
leaf primordia, or more apical meristems in a shoot-apex-unit as seen in the
lower parts of Figs. 4, 8 and 9.
Fig. 7 is a surface view of a shoot apex protruded out of a callus mass.
Its age corresponded to those shown in Figs. 5 and 6. Some layers of green
cells can clearly be seen in the embryonic leaves.
Fig. 8 is a longitudinal section of a double apical meristem whose cells
were densely stained and are flanked by two embryonic leaves. Presumably
any cell at the -periphery of a callus could develop into a shoot primordium.
Therefore polyembryonies were not uncommon ii the process ofshoot differentiation. Johri12 reported polyembryony in the callus culture of Ranunculus
sceleratus L. He attributed this phenomenon to cleavage and/or budding of the
embryonic mass a t different stages of callus development. Sometimes several
embryonic leaves could be seen a t the same time in & shoot-apex unit (Fig. 8).
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PLANT BREEDING
This phenomenon could be caused by the overlapping of several apical meristems.
Two apical meristems (deeply stained) were able to develop from the
callus mass under the covering of embryonic leaves, or the coleoptile-like
structure in the outer layer which grew earlier than the apical meristem (Fig.
9). This shows that the development of a callus-shoot-meristem was not as
regular as that of a vegetative shoot apex in a cane tip.
A surface view of polyembryoids is presented in Fig. 10, the interior
structure of which is shown in longitudinal sect@ in Fig. 11. This is a model
of a triple shoot apex in which each apical meristem is flanked by leaf primordia
and covered by the coleoptile-like structure, giving it the shape of an arch.
Cross sections in the portion of a callus not far from the area where a
shoot apex had developed, showed that some of the callus cells had differentiated into typical protoxylem cells (Fig. 12). Some of them demonstrated
secondary wall thickening in a scalariform-reticulate pattern which could be
clearly distinguished a t a higher magnification (Fig. 13). Tracheid elements
and phloem cells have also been reported in a callus mass.lO~
l4
Fig. 14 is a cross section of a shoot apex. The apical meristem with cells
in an orderly arrangement and deeply stained is in the center and surrounded
by leaves. Some vascular bundles have already formed in the outer layers of
leaves.
A general view of a young shoot is shown in Fig. 15, which was photographed in the developmental stage several days later than that of Fig. 10.
I t appeared to be growing vigorously as shown by the broad, green leaves.
Such a growth pattern was especially common when callus was in its earlier
passages. However, in some sugarcane varieties newly-emerged shoots had a
needle-like form which prevailed after the seventh passage.
Another differentiation pattern in callus is the formation of roots (Figs.
16-18). Fig. 16 is a root primordium just initiated from a callus mass. The
center cells are still meristematic in appearance, and arranged in several rows
indicating a typical root tip. The initiation pattern was analogous to that of
a lateral branch root derived from the taproot .of a normal plant, as discussed
by EsauG.Fig. 17 shows a root system developingofrom callus mass grown on
a Schenk-Hildebrandt (SH) medium (28) whidh is effective in stimulating
root initiation and is now in routine use in our laboratory for rooting plantlets
in vitro. Some plantlets, although they had grown for 6-8 weeks after initiation
still lacked a root system. Consequently they would not survive after having
been transferred to vermiculite. Poor rooting caused considerable difficulty in
raising seedlings although S H medium helped to solve this problem and contributed a great deal to our scheme of mass production of sugarcane plantlets
through callus culture. Fig. 18 is a surface view of some roots which were
developing from callus mass. They were very tender in texture and white in
color.
Organogenesis in sugarcane callus appears to proceed in either of 2 ways,
according to our laboratory results. One pattern was that a shoot appeared
first and a root initiated later, usually after plantlets had grown on sterile
culture for 4-6 weeks. Some sugarcane genotypes failed to form roots at all
(Fig. 15). Another pattern was for a callus piece to form only a root. Such
roots usually appeared on the surface of a callus mass (Fig. 18). The callus
FIGURE 12. Cross section of a piece of callus showing that the cells have differentiated into
protoxylem elements.
FIGURE 13. Magnified view of tracheid elements characterized by the secondary wall
thickening: t = tracheid.
FIGURE 14. Cross section of a shoot apex in later developmental stage. Some vascular
bundles appear: 1 = leaf.
FIGURE 15. General view of a young shoot a t a stage several days later than that of Fig. 10.
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PLANT BREEDING
FIGURE16 and FIGURE 17.
Lopgitudinal sections of root
primordia.
FIGURE 16. Root primordium
just initiated from acallus 14m
days after subculturing: r=root
primordium.
FIGURE 17. Root system,
originating from callus cells,
effectively promoted b y
Schenk-Hildebrandt medium.
r = root system.
FIGURE 18. Surface view of
a developing root system derived from a callus 21 days
after subculturing.
MING-CHIN LIU AND WEN-HUEI CHEN
127
appeared to have lost its capability to initiate shoots and was generally regarded as useless because it would never be differentiated into shoots.
Concerning the origin of embryoid in callus, although it is difficult to
determine whether a single cell or a n aggregate could develop into a complete
embryoid, some indirect evidence in the literature led us to believe that a
meristemoid is originated from a particular single cell in a callus mass. The
available data which support this hypothesis are as follows: Muir et all8 reported
that isolated single cells could successfully be grown on a nurse culture, separated by a filter paper, and these gave rise to a callus mass. Later, Visil and
Hildebrandt33134 demonstrated that completely isolated single cells of tobacco
could grow in microchambers to form small clumps of cells which could then
be differentiated to form adult tobacco plants. Konar and Nataraja15 stated
that epidermal cells of Ranunculus sceleratus L. were able to form an embryoid.
Reinert et a127 also found that the formation of an embryoid from a single
carrot cell could be followed directly by serial microscopical observations.
These, and other data, would support the hypothesis that an embryoid in
callus arises originally from a single cell. Still to be determined is what kind
of cells can exhibit this totipotency, and how much physiological and biochemical change occurs in the cell before initiation of an embryoid. I t is suggested that techniques of histochemistry and electron microscopy might be
employed to identify the proembryonic cells in callus masses.
ACKNOWLEDGEMENT
The authors wish to thank Dr Soh-Chao Shih, Director, Taiwan Sugar
Research Institute, for his help in preparing this manuscript.
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All photographs were taken with either 35 mm photo head or luminars of Zeiss Ultraphot
11. Each scale division equals 100 y.
MING-CHIN LIU AND WEN-HUEI CHEN
129
ESTUDIOS HISTOLOGICOS SOBRE EL ORIGEN Y
DESARROLLO DE LA DIFERENCIACION DE PLANTULAS
EN CALLOS DE C A ~ ADE AZUCAR
Ming-Chin Liu y Wen-Huei Chen ,
RESUMEN
Se efectud un estudio de organogenesis en callos de caiia de azdcar
desarrollados en dos tipos de medios. Uno de ellos fu6 el de MurashigeSkoog sumplementado con 2,4-D 3 ppm, y designado corno un medio no
formador de drganos; el otro medio fu6 el mismo anterior per0 sin 2,4-D y
con agredado de kinetina, Bcido naftalen acetic0 y caseina hidrolisada para
diferenciacidn de brotes. La organog6nesis no tuvo lugar usualmente en la
condicidn de no formacidn de organos, except0 el callo en 10s primeros
pasajes. En la condicidn de formacidn de brotes y a 10s 8 dias desde el subcultivo se formaron tejidos proembriodes caracterizados por c6lulas m6s
pequeiias, isodiam6tricas y con nucleos muy tefiidos. Posteriormente ellos
desarrollarian en estructuras de apice, la cual es comparable desde el punto
de vista ontogen6tico al embrion semilla, per0 m6s parecido estructuralmente
al apice vegetativo en el punto de crecimiento de la caiia de azdcar. El
fenomeno de poliembrina fu6 bastante comirn. Las celulas en la porcidn del
callo cercana al nacimiento de 10s apices diferenciaron en elementos del
protoxilema. Se observaron 2 patrones en la diferenciacidn de 10s callos. Uno
de ellos fu6 brotes primeros, y raices iniciadas m5s tarde. El otro fu6 la formacidn sdlamente de raices en el callo, con la p6rdida absoluta de la capacidad
de formar brotes. El medio de Schenk-Hildebrandt fu6 muy efectivo en
provocar diferenciacidn radicular in vitro. Un embroide fu6 considerado
corno originado de una sola celula corno evidenciaron nuestras observaciones
corno asi la literatura disponible.