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medium supplemented with Zeatin (9.12 µM), adenine (148 µM) and Lactoal-
bumin hydrolysate (10 mg/l). On this medium, the calli continued to proliferate
either into shiny mass or shoots. These shoots were subsequently rooted on me-
dium containing IAA (0.57 µM). While three out of four plants were haploids, the
rest were aneuploids with a chromosome number 2n = 18. Later, in (
1992
, Saha and
Bhattacharya reported formation of globular structures in tea which failed to dif-
ferentiate further on MS medium (with 7 % sucrose). This was supplemented with
NAA (0.53 µM), 2, 4-D (0.45 µM), Kinetin (0.46 µM), and Glutamine (400 mg/l).
However, the differentiation of true pollen embryos and regeneration of haploid
plants were described by Raina and Iyer in (
1992
and Shimokado et al. (
1986
).
Pedroso and Pais (
1994
) tested 17 different media combinations based on MS and
N
6
with various concentrations of carbon source, growth regulators and amino
acids, such as Serine and Glutamine for
C. japonica
. The embryogenic calli from
microspores were obtained when isolated microspores were cultured on 2, 4-D
(4.53 µM), and Kinetin (0.46 µM) and subsequently on MS medium supplemented
with BAP (2.22 µM). However, further growth ceased at maturation stage. Seran
et al. (
1999
) reported the highest response in terms of micro calli formation in a
Sri Lankan tea clone TRI-2043 (78-98 %) out of five different clones selected for
study. On ½ MS + 2, 4-D (9.06 µM) + Kinetin (4.65 µM) + IAA (5.71 µM) under
dark condition, 98 % anther response was achieved. Determination of ploidy levels
in the callus cells showed that the frequency of haploid cells was greater (68 %)
in comparison to diploid cells (6 %). However, plantlets could not be regenerated.
Mishra and Chaturvedi (unpublished) has obtained for the first time microspore
embryogenesis in anther cultures of TV clones of tea (TV19 and TV21) via an in-
tervening callus phase. Within three weeks of culture, the anthers were swollen and
after six weeks longitudinal dehiscence occurred along the anther wall (Fig.
8.2a
),
followed by emergence of transparent white callus from within (Fig.
8.2b
). In both
TV19 and TV21, calli were obtained when anthers were subjected to dark incuba-
tion at 25 °C. After successful induction of callus, embryogenesis in these calli and
embryo germination occurred when cultures were transferred to light conditions
(Fig.
8.2c
,
d
).
So far, there have been no reports on in vitro gynogenesis in tea. Hazarika and
Chaturvedi (unpublished data) have obtained for the first time, successful callus
induction from unpollinated ovules of tea when thin sections of ovary cultures
were subjected to various regimes of temperature and light treatments. Within
a week of the culture, the ovules swelled to almost double their original size
(Fig.
8.3a
) and callusing was observed. In a few cultures, white, friable callus tis-
sue emerged from within the cultured ovules (Fig.
8.3b
) whereas in others profuse
callusing from the entire sections occurred. The nature of callus obtained from
within the ovules was friable and it was white in colour. Histological study of the
ovary slice sections at the time of culture showed the presence of a mature egg
cell within the embryo sac of the unpollinated ovules. Following successful callus
induction, formation of dark green nodulated structures occurred on various media
compositions. Histological investigations revealed the formation of tracheids in
the callus tissue.
