Production and supply of quality planting material is significant to coconut cultivation but is one of the major constraints in coconut productivity. Rapid multiplication of coconut through in vitro techniques, therefore, is of paramount importance. Although somatic embryogenesis in coconut is a promising technique that will allow for the mass production of high quality palms, coconut is highly recalcitrant to in vitro culture. In order to overcome the bottlenecks in coconut somatic embryogenesis and to develop a repeatable protocol, it is imperative to understand, identify, and characterize molecular events involved in coconut somatic embryogenesis pathway. Transcriptome analysis (RNA-Seq) of coconut embryogenic calli, derived from plumular explants of West Coast Tall cultivar, was undertaken on an Illumina HiSeq 2000 platform. After de novo transcriptome assembly and functional annotation, we have obtained 40,367 transcripts which showed significant BLASTx matches with similarity greater than 40 % and E value of ≤10(-5). Fourteen genes known to be involved in somatic embryogenesis were identified. Quantitative real-time PCR (qRT-PCR) analyses of these 14 genes were carried in six developmental stages. The result showed that CLV was upregulated in the initial stage of callogenesis. Transcripts GLP, GST, PKL, WUS, and WRKY were expressed more in somatic embryo stage. The expression of SERK, MAPK, AP2, SAUR, ECP, AGP, LEA, and ANT were higher in the embryogenic callus stage compared to initial culture and somatic embryo stages. This study provides the first insights into the gene expression patterns during somatic embryogenesis in coconut.

An efficient in vitro propagation and in vitro flowering protocols were developed for the medicinal plant Cichorium intybus (Asteraceae) using leaf disc explants. Media supplemented with the growth regulator naphthalene acetic acid (NAA) (1.5 mg/l) + 6-benzyle adenine (0.25 mg/l) was used for the initial induction of the callus and further subcultured to the same media for the proliferation of the callus. Pale yellow and green calli were noticed, which depends on incorporation of the growth hormones and their varying concentrations. Murashige and Skoog medium in addition with 2 mg/l kinetin+ 0.5 mg/l indole-3-acetic acid (IBA) + 500 mg/l casein hydrolysate resulted in maximum regeneration. Media supplemented via IBA (0.5 mg/l) and NAA (0.5 mg/l) (98%) was found to be optimum for rhizogenesis for in vitro regenerated plants. For acclimatization 5-6 weeks mature in vitro regenerated plants were transferred into the greenhouse for acclimatization. The histological study revealed the presence actively dividing meristematic cells in callus. The occurrence of the peripheral meristematic zone associated with callus was noticed in after 20 days, which formed the shoot meristems after 45 days of incubation. To our knowledge, this is the first report on high-frequency plant regeneration which was carried out indirectly from the leaf explants which was grown in controlled environment with varying concentration of the growth regulators and histology of callus of different stages from leaf explants of C. intybus.

An attempt was made to establish highly competent embryogenic cell suspension culture in coconut, a species recalcitrant to in vitro culture. Embryogenic calli were initiated from shoot meristem explants of coconut. Y3 medium supplemented with 2.4-D (4.5 μM) and glutamine (34.2 μM) was found to be the best medium to initiate cell suspension. Growth evaluation was done by packed cell volume (PCV) and it was found that maximum growth volume of 9.9% was reached at 200 days of culture initiation. About 52% of viable cells were detected through fluorescent microscopy. Cell aggregation was noticed in Y3 medium supplemented with glutamine (34.2 μM) , malt extract (100mg/l), biotin (40.9 μM) and kinetin (9.3 μM), but further progress could not be achieved. It was also observed that embryogenic calli were not of a friable type, but were associated with densely aggregated cells. Because of its hard nature, we were unsuccessful to obtain high quality cell suspension.

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A protocol was standardized to maximize yields of embryogenic calli from shoot meristem culture of coconut. Three different shoot meristem excision methods were tested viz., excision of shoot meristem aseptically from in vitro germinated embryo after 10-12 days, excision of shoot meristem from in vitro germinated embryo subjected to GA3 treatment for five days and excision of shoot meristem from fresh embryo. The primary calli induction after 30 days of culture incubation for the three treatments was 21%, 27% and 79% respectively. Further, the primary calli formed from the shoot meristem excised from fresh embryo gave rise to 56% of embryogenic calli. The calli obtained from the shoot meristem, which were excised from in vitro germinated embryo, formed less percentage of embryogenic calli because of the presence of cotyledonary tissues which inhibited the multiplication of meristematic tissues. In the case of shoot meristem extracted from GA3-treated embryos, the percentage of non-embryogenic calli was more compared to the shoot meristem excised from fresh embryo. It was observed that the addition of GA3 in the initial stages of culture inhibited the formation of embryogenic calli and favored direct shoot development. Currently, the shoot meristem excised from fresh embryo is being employed for scaling up the planting material production from released varieties of coconut.