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

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


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

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

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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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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

Callus response on the maintenance medium: Sections

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

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wheat. Embryogenic callus is a callus that has a capacity to 5. Kott, L. S. and Kasha, K. L. (1984). Canadian Journal of Botany
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8. Magnusson, I and Borrmann, C. H. (1985). Physiologia
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9. Ruiz, M. L., Rueda, M, I., Pelaez, F. J., Candela, M., Sendino,
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