Abstract:
Histological and morphological studies on somatic embryogenesis were carried out for
efficient in vitro plant development of Camellia sinensis (tea). To determine the
suitable stage of cotyledons for direct somatic embryogenesis, the sterilized cotyledons
(6-12 mm diameter) at various developmental stages were inoculated on MS medium
containing 2.0 mg L'1 BAP and 0.2 mg L’1 NAA. Results indicated that immature
cotyledon (10 mm diameter, stage 3) was the most responsive stage for the production
of typical (normal) somatic embryos. Cytological examinations revealed that typical
somatic embryos at different stages exhibit gradual tissue differentiations. Typical
mature somatic and zygotic embryos morphologically and histologically have similar
bipolar structures.
Immature cotyledon segments (stage 3) were placed on different media containing
NAA (0.0 - 3.0 mg L') in combination with BAP (2.0 mg L ) to select suitable
medium for efficient production of typical somatic embryos. The optimum
concentrations of NAA were in the range of 0.2 - 1.0 mg L'1 for direct somatic
embryogenesis (14.6% - 16.7%). Cotyledon-type and seed-like somatic embryos were
developed in 0.2 and 1.0 mg L’1 NAA respectively. Further, the explants of high
yielding cultivars DTI (Estate selection in Sri Lanka) and TRI 2025 (Assam type)
performed well and exhibited embryogenic potential (28.1% and 21.9% respectively).
Several types of explants obtained from in vitro germinated zygotic embryonic axes
cultured to select the suitable explants for the production of typical somatic
embryos. Results indicated that both hypocotyl and small succulent leaf explants were
were
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the best for the production of firm, typical somatic embryos. Histological studies
showed that most somatic embryos were directly originated from the cortex tissue of
the hypocotyl and upper epidermal layer of the succulent leaf explants.
Further, attempts were made to establish in vitro callus culture from field grown leaves.
Results indicated that high frequencies (50% - 58 %) of aseptic cultures were achieved
for 30 min soaking period among varying
concentrations of disinfectant (15% - 75%) and various exposure times (15-60 min).
The rates of initiation (72.2%) and formation (56.9%) of calli from in vitro aseptic
explants were relatively high in MS medium containing BAP (2.0 mg L'1) and NAA
(3.0 mg L'1) among tested combinations of BAP and NAA concentrations.
in 60% and 75% solutions of CloroxTM
Morphological and histological studies were carried out on the development of
embryogenic callus from leaves of in vitro shoots. It was observed that collenchyma
cells were developed from vegetative cells at the cut end and packed within the
swelling tissues. After bursting, parenchyma cells were gradually formed as a result of
cell divisions. These parenchyma cells differentiated as embryogenic cells. The
average number of single embryogenic cells was significantly higher in the liquid than
in solid medium. Further investigations were done to produce somatic embryos from
primary embryogenic calli. Results showed that formation of somatic embryos (11.7%)
was high in MS medium containing BAP (1.0 mg L'1) and NAA (0.1 mg L’1) in
combination with GA3 (0.1 mg L'1).
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Zygotic embryonic axes were coated with 3% and 4% sodium alginate matrices. The
results revealed that 3% sodium alginate with 100 mM calcium chloride dihydrate
provided the most suitable matrix concentrations for encapsulation. Further, naked and
coated axes cultured on MS medium without (MSI) or with (MS2) 3.0 mg L'1 BAP
and 0.5 mg L'1 IBA gave highest rates of survival (100%) and germination (93-100%).
But efficient plant recovery from non-encapsulated (73.3%) and encapsulated (42.2%)
axes was achieved on MS2 medium. A high rate (48%) of in vitro plantlets was
survived at 8th week of transplanting in soil than in a mixture of soil: sand (1:1 v/v).
Feasibility of cold storage of encapsulated embryonic axes was studied to obtain
efficient in vitro conversion of plants and their survival under ex vitro conditions.
Synthetic seeds and natural seeds were stored at 4 °C for 0, 4, 8, 16, and 20 weeks.
Efficient in vitro germination and plant recovery were obtained from synthetic seeds
stored for four weeks than control. Vigorous plantlets were collected from in vitro
cultures and successfully established in soil: coconut coir dust (1:1 v/v).
To perform synchronous germination of plant material into vigorous plants under ex
vitro conditions, sterilized zygotic embryos and zygotic embryonic axes were cultured
on germination medium whereas seeds were sown in sand bed as a control. Consistent
germination was observed on cultured embryonic axes in vitro for uniform growth
where rate of germination was significantly highest (99.0%) at 4th week. Further,
healthy plantlets developed from embryonic axes in vitro had erect shoots with short
internodes as well as taproots with abundance adventitious roots under nursery
conditions for better adaptation in field as compared with seedling raised by
conventional sexual propagation.