J. Env. Bio-Sci., 2015: Vol.

29 (2):413-417
(413) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

DEVELOPMENT OF SOMATIC EMBRYO FROM SINGLE CELL IN BARLEY

(HORDEUM VULGARE L.): HISTOLOGICAL EVIDENCE OF
SOMATIC EMBRYOGENESIS
Tiwari V. K.* and Cocking E. C.
Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, University Park, Nottingham,
NG 7 2 RD, England
[Corresponding author E-mail*: vkt786@rediffmail.com]

Received: 29-09-2015 Accepted: 01-11-2015

Embryogenic callus was successfully initiated by culturing immature embryos of barley cv. Dissa on the agarose-solidified CC2
medium containing 2 mg l-1 2, 4-D, 50 ml l-1 coconut water and 0.2 g l-1 casein hydrolysate. The development of somatic embryos
occurred from single cells on this medium from the nodular, compact, yellow callus that was considered as embryogenic callus
because it has the ability to differentiate; whereas, soft, non-embryogenic callus (watery callus) did not differentiate into shoots
and roots. Many somatic embryos underwent internal cell division to from globular-like structures (meristemoids). These
meristemoids in turn produced secondary embryoids or enlarged considerably to produce shoot buds and / or leafy structures.
Such leafy structures and shoots possessed vascular connections to the ground substance of the callus suggesting organogenesis
and somatic embryogenesis occurred side-by-side. Hence, somatic embryogenesis mainly happens in the scutellar epidermis
followed by the organogenesis.

In vitro cells may differentiate to give rise shoots, root primordia
and embryoids on different media fortified with varying
concentration of auxins and cytokinins. The development of
single embryoid has previously been observed in the region of
scutellum node-derived from young barley embryos1. In wheat,
the development of embryoid was observed in the immature
embryo-derived scutellum or epiblast callus2-8. There are
several reports indicating the induction of embryogenic callus
from immature embryos9. In other study, it is found that the
developm ent of nodular compact callus rather than
embryogenic callus from immature embryos in barley10. In
fact, there is little information available on the systematic
development of somatic embryos in the embryonic-scutellum
tissues of barley. Therefore, the present study was conducted
to detect the embryogenic tissues and to follow somatic
embryo formation and possible organogenesis on different
media with varying concentration of auxins and cytokinins, in
the scutellum-derived tissues of barley. Hence, this is first
study of its kind in barley that provides the systematic
development of somatic embryos, leaves, shoots and roots.
Further, this study will also confirm the basis of plant
regeneration in barley and therefore, the possibility of barley
genetic manipulation at the single cell levels.
MATERIAL AND METHODS
Selection of explants: Callus cultures were initiated from
NAAS Rating (2016)-4.20
immature embryos of barley cv. Dissa. Seeds at milk ripe to
soft dough stage were taken from glass house grown plants.
Dehusked immature seeds were sterilized with 10% Domestos
and then washed three times with sterilized distilled water.
Immature embryos (0.5-0.8 mm) were excised under stereo-
microscope and 8 to 10 immature embryos with scutellum
uppermost were placed on 20 ml agarose-solidified CC2
medium (based on CC medium) containing 2 mg l-1, 2, 4-D,
50 ml l-1 coconut water and 0.5 g l-1 casein hydrolysate (PH
5.8) in the 60 mm x 15 mm Petri-dishes11. These Petri dishes
were incubated at 25 ± 1 °C in the dark condition.
Initiation and selection of tissues derived from the
scutellum used for histological examination: Callus of
21 d old (300-800 mg) from initiation medium was sub-cultured
at the regular interval of 21 d on 20 ml agarose-solidified CC3
medium (based on CC medium) contained 0.5 mg 1-1 casein
hydrolysate, 50 ml 1-1 coconut water, 0.2 mg 1-1 2, 4-D. The
differentiation in callus was induced by plating somatic calli
(20-100 mg) derived from 21 d old callus onto 20 ml agarose-
solidified RM1+ medium 12 ; containing 0.5 mg 1-1 kinetin
and 3 g 1-1 glucose; pH 5.8). Cultures were incubated at 28 ±
1°C under continuous light supplied by fluorescent tubes giving
a light intensity of 40-50 ìmol-1 m 2 s-1. Differentiating callus
from all three media was used for fixation and subsequent
histological studies. The developing scutellum tissues, from

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 DEVELOPMENT OF SOMATIC EMBRYO FROM SINGLE CELL IN BARLEY
the immature embryos, were fixed following 3,7,14,21 (d on
CC2 initiation medium), 28 (7 d after transfer to RM1+
regeneration medium i.e. 28 d after initiation) and 35 d of
culture; 14 d after transfer to CC3 maintenance medium i.e.
35 d after initiation. Callus was transfer to RM1+ medium and
or CC3 medium were both after 21 d on CC2 initiation medium
using 4-5 samples for each period. 10-20 μ m thick section
were cut and double stained with safranin and fast green and
observed under the microscope.
RESULTS AND DISCUSSION
Initiation of callus from immature embryos: Embryogenic
callus was successfully initiated from immature embryos of
cv. Dissa on CC2 medium. A longitudinal section of the
scutellum 3 d old cultured immature embryos showed the
epidermal and sub-epidermal cells that entered in divisions
(Fig.-1A) which lead to the enlargement of scutellum surface
and shows abandon appearances due to the active cell
division. Microscopic observations showed that cells were
very closely arranged without intercellular spaces. Rapid callus
formation had been occurred at 7 d, with the formation of
distinctive meristematic areas on the scutellum of the original
embryo (Fig.-1B). The longitudinal sections of 14 d old callus
showed that root and shoot axes were considerably
differentiated with well-developed first and second leaf. The
mitotic cell divisions occurred in the scutellum near to the
procambium tissues and epidermis. This showed the
regenerative capability of the cells derived from scutellum in
barley10, 12. The major morphological changes occurred during
the initial period of immature embryo’s culture i.e. a rapid
elongation of coleoptile and seminal roots ane the proliferation
of the parenchyma cell of root tissues.
In developing embryos, leaves started to elongate after 7 d of
culture. The rapid growing eoidermis of scutellum showed
continued growth and became spiral. A large number of
randomly scattered vascular tissues were developed in the
mesocotyl region. The coleoptile continues to grow after 7 d
and swelling occurs in leaves during culture. The extensive
enlargement and disassociation of scutellar parenchyma cells
were observed in the 14 d old callus (Fig.-1C). Mitotic cell
divisions in these cells lead to the formation of somatic embryo
in the 14 d old callus (Fig.-1 D). Epidermal and sub-epidermal
single, densely stained, vacuolated cells with thick walls were
observed. Hence, the development of somatic embryos
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(414)
occurred from the certain epidermal cells8, 13. Embryos were
formed from a group of proembryonic cells in maize 14.
Subsequently, compact nodular, embryogenic callus was
evident with developing somatic embryos. Such somatic
embryos were loosely attached to the surface of callus and
could be separated easily. Development of atypical embryoids
had been reported in wheat, barley, oat, rye and triticale15.
The formation of a typical embryoid from a multiple cell origin
of biaxial and sub-epidermal cells has been reported 15.
Elongated and vacuolated cells developed from the surface of
epidermis of scutellum; originating from soft and non-
embryogenic callus.
The various types of embryogenic structures were observed
in which a root meristem is mainly observed on CC2 medium
after 14 d old culture (Fig.-1C). These structures had vascular
connections to the ground meristem. Thus, after 14 d of
culture, yellow, soft, unorganized masses of tissues were
emerged from the scutellum. Whereas, densely stained small
cells arose in the proliferating epidermal tissues and showed
an appearance of nodular compact yellow callus in cereals
and grasses. However, there are different observations and
opinion on the identification of embryogenic callus, and the
somatic embryos and their origin from single cell. In the
present study, we observed the development of somatic
embryos from scutellum tissue and of leaves and shoots from
developing somatic embryos were clearly distinguishable. The μ
distinction between the scutellum of somatic embryo, leaf
and shoot formation is strenuous, in wheat16, 17. In the developed
atypical embryoid had exceptional germination that resulted
in the formation of leafy scutellum and multiple shoots.
Germinated embryoid leads in the development of multiple
shoots due to the loss of apical dominance in wheat18. Thus,
globular meristemoid were produced on the periphery of callus.
Callus response on maintenance and regeneration
medium: Differentiation in callus occurred after 14 d, due to
the meristematic activity in some of the parenchyma cells,
when 14 d old callus from maintenance medium cultured for 7
d on RM1+ medium. Discrete nodular structures (meristemoid)
developed on the periphery of the callus (Figs.- 1 E & F). We
found that differentiating and subcultured embryogenic callus
had potential to form globular meristemoid and leafy structure
on the 28 d old callus (21 d on CC2 medium and 7 d on CC3
medium). A longitudinal section of such a meristemoid and
 (415) TIWARI AND COCKING

Figure-1. Histology of callus derived from immature embryos of barley (cv. Dissa) on CC2 medium. (A) Transverse section of
scutellum of immature embryos after 3 d of culture. Arrow showing embryogenic cells (x20). (B) Embryogenic cells
developing in cultured immature embryos after 7 d of culture (x20). (C) Development of shoot and root meristem after
14 d of immature embryos culture on CC2 medium (x40). (D). Development of somatic embryos from single cell after
14d of culture (x40). (E and F). Development of meristemoids and vascular tissues after 14 d of immature embryos
culture on CC3 medium; 7 d on RM1+ medium (x40). Abbreviations: cl = coleoptile; ep =epidermis, fl = first leaf; M=
meristemoids; rm = root-meristem; s = scutellum, se = somatic embryo; sl = secondary leave; va = vascular tissue.

Figure-2. Histology of differentiating callus. (A and B). Development of leafy structure and meristemoids with weak vascular
connections in ground meristem after 21 d of immature embryos culture on CC2 medium and 7 d on CC3 medium
(x40). (C and D). Development of shoot meristem, root meristem, leaf structure after 28 d of culture (21 d on CC2
medium and 7 don RM1+ medium). (E and F). Longitudinal section of differentiating callus of mature embryos. Arrow
showing the development of meristemoids, leafy structures after 28 d of culture (14 d on CC3 medium and 14 d on
RM1+ medium) (x40).

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 DEVELOPMENT OF SOMATIC EMBRYO FROM SINGLE CELL IN BARLEY (416)

Figure-3 (A & B). Longitudinal sections of differentiating callus. (A and B). Developed meristemoid containing cytoplasmic
embryogenic cells.

directed by the differentiation of a large number of vascular

tissues leading to such structures (Fig.- 2 E and F). On the
contrary, shoot-root axises were occasionally developed in
the old callus that eventually reduces the chances of plant
regeneration.

Callus response on the maintenance medium: Sections

Figure-3 (C). Cells showing disintegration after 35 d of
immature embryo culture (x40). Abbreviation: gm + ground
meristem, m = meristemoid; v = vascular tissue.
associated callus showed that vascular tissues were randomly
scattered (Fig.- 2 A and B). These meristemoid also produced
shoots bud simultaneously whilst some merely enlarged to
produce large numbers of shoot bud primordia. Sectioning of
the more mature somatic embryos showed a well-defined
shoot and root axis after 28 d (21 don CC2 initiation medium
and 7 d on RM1+ medium) of culture (Fig.-2 C and D). The
regeneration medium had enhanced multiple shoot and leafy
structure formation on the surface of callus. This was usually
of subcultured callus held on 21 d on CC2 medium and 14 d
on RM1+ medium (35 d old callus) showed that meristematic
tissues could also be highly developed. The 14 d passage on
regeneration medium restored the embryogenic potential for
cv. Dissa of barley. These meristematic cells near the callus
surface were thin-walled, highly. cytoplasmic and subsequently
lead to the formation of meristemoid after 35 d of immature
embryo culture (21 d on CC2 initiation medium and 14 d on
CC3 maintenance medium). Many meristematic cells of callus
showed thickened walls with associated vascular bundles in
the meristematic cells became vascular and thereafter
appeared in the lack of cytoplasm with some evidence of
disintegration, initiated callus left for 35 d on the CC3
maintenance medium (Fig.-3 C). The ground meristem showed
the development of large vascular cells. Globular embryoid
formations in the differentiating and subcultured calluses were
morphologically similar. This indicates that embryogenic callus
was not induced from differentiating cells, but from existing
shoot-forming tissues. Similar result has been reported in

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 (417) TIWARI AND COCKING

wheat. Embryogenic callus is a callus that has a capacity to 5. Kott, L. S. and Kasha, K. L. (1984). Canadian Journal of Botany
regenerate plants by both organogenesis and embryogenesis19. 62: 1245.
It was found that some out-growths had vascular connection 6. Lührs R and Lörz H (1987) Theoretical Applied Genetics.75:16.

to the ground meristem of the callus. Therefore, plant 7.
regeneration occurred through organogenesis. Similar
observations were reported in sugarcane; proving that both
8.
embryogenesis and organogenesis occurred in scutellar
tissues of barley20. Therefore, embryogenic callus may be
9.
defined as callus that has the totipotency for forming
embryoids and regenerate plants by both embryogenesis and
organogenesis. In conclusion, the regenerative tissues from 10.
immature embryos are easily produced embryogenic callus.
In these calli, regeneration occurs through somatic embryos 11.
and organogenesis.
12.
In this study, we have observed the well-developed leaf, shoot 13.
and root meristem structures when calli were transferred onto 14.
the regeneration medium. Therefore, we consider compact,
nodular, organized structures are embryogenic calli with the
developed somatic embryos, first and second leaf, root and 15.
Maddock S E (1985) In: Cereal Tissue and Cell Culture,
(Bright SWJ, Jones MJK, eds) Dordrecht: Martinus Nijhoff/
Dr W Junk Publishers, 131.
Magnusson, I and Borrmann, C. H. (1985). Physiologia
Plantarum 63:137.
Ruiz, M. L., Rueda, M, I., Pelaez, F. J., Candela, M., Sendino,
A. M and Vazquez, A. M. (1992). Plant Cell Tiss and Org
Culture 28: 97
Oka, S., Saito, N and Kawaguchi, H. (1995). Annals of Botany
76:487.
Potrykus, I., Harms, C. T and Lörz, H. (1979). Theoretical
Applied Genetics 54: 209.
Racchi, M. L and Terragnna, C. (1993). Plant Science 93:195.
Vasil, I. K. (1987). Jour. Pla.Physio. 128: 193.
Vasil, I. K. (1985) In: Tissue Culture in Forestry and Agriculture,
Henke RR, Hughes KW, Constanin MJ, Hollander A(eds.)
New York: Plenum Press, 31.
Ozias-Akins, P and Vasil, I. K. (1983). Protopla. 117: 40.

shoot m eristem on the regeneration medium. Such 16. Ahloowalia, B. S. (1982). Sci. 22: 405.
17. Wernicke, W and Milkovits L. (1986). Protopla.13: 131.
embryogenic callus could be used for the initiation of
18. Ozias-Akins, P and Vasil, I. K. (1982). Protopla. 110:95.
embryogenic cell suspension and the isolation of totipotent
19. Tomes, D. T. (1985). In: Cereal Tissue and Cell Culture, Bright
protoplasts for the plant regeneration from single cell that
SW J, Jones MGK, (eds.) Dordrecht: Martinus Nijhoff/Dr. W
propitiate the high frequency of plant regeneration for the
Junk Publishers.
genetic manipulation studies. 20. Chen, W. H., Davey, M. R., Power, J. B and Cocking, E. C. (1988).
Jou. Experi. Bot. 39: 251.
REFERENCES

1. Norstog, K. (1970). Dev. Bio., 23: 655.

2. He, D. G., Yang, Y. M., Bertram, J and Scott, K. J. (1990). Plant
Sci. 68: 103.
3. Ho, W and Vasil, I. K. (1983). Protoplasma 118: 169.
4. Johansen, D. A, (1940). In: Plant Micro-technique. New York:
Mc Hill Company.

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