Factors affecting germination and conversion frequency of somatic embryos of Tea [Camellia sinensis (L.) O. Kuntze]

The cause of poor and abnormal germination of tea somatic embryos was investigated with respect to (a) the different factors that affect reserve mobilisation viz. chilling, desiccation or GA₃ and (b) those that affect the maturation process and reserve accumulation viz. ABA.

Tea somatic embryos were sensitive to desiccation and their normal development or germination could not be evoked by external agents like chilling and GA₃. Supplementation with external sources of nutrient precursors and readily available forms of carbohydrates like sucrose or maltose together with trans-cinnamic acid improved the germination of the somatic embryos significantly.     

Induction of synchronous secondary somatic embryogenesis in Camellia sinensis (L.) O. Kuntze

Nutritional effect of nitrate salts of potassium and ammonium, together with different concentrations of sulphate salts of aluminium, potassium, magnesium, and ammonium on secondary somatic embryogenesis, wereinvestigated. Nitrate salts of potassium (9.39 mmol/L) and ammonium (10.31 mmol/L) with only 1.5 mmol/L potassium sulphate produced maximum number of synchronous secondary embryos (i.e. 20-25 secondary embryos per primary embryo in 91.6 percnt; responsive explants).Of the different factorial combinations of glutamine, BAP, and IBA tested, maximum number of synchronous secondary embryos developed on a medium supplemented with 8.88 μmol/L BAP, 0.98 μmol/L IBA and 10 mmol/L glutamine.Synchronous and normal development of secondary embryos could thus be obtained when optimal concentrations of PGRs, glutamine, nitrates, and salts of potassium sulphate were combined together.Germination of the embryos (up to 52 percnt;) was acheived only when sulphate salts of potassium were removed from the medium.     

Assessment of liquid culture procedures for in vitro propagation of Rheum emodi

Micropropagation of an endangered Indian medicinal plant, Rheum emodi Wall., was achieved on Murashige and Skoog's medium using different liquid culture procedures. Liquid static (submerged, semi-submerged and with filter paper bridge) and shake (80 and 120 rpm) culture procedures were assessed for their effects on growth and multiplication rates. Best results were obtained using liquid shake cultures, which resulted in 50% reduction in medium requirement, 37.5% reduction in time and 1.5–2.2 fold increase in growth and multiplication rate. Liquid culture-raised plantlets facilitated easy transplantation and 90–95% survived transfer to potting mix in glasshouse.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Starch deposition and amylase accumulation during somatic embryogenesis in bamboo (Dendrocalamus hamiltonii)

In monocots, the zygotic embryo is protected and nourished by an endosperm. In the present study starch deposition and amylase accumulation was noticed during somatic embryogenesis in stem callus of a bamboo, Dendrocalamus hamiltonii. SEM studies revealed that starch grains were clearly visible in the scutellum during the maturation stage of the somatic embryo. As the somatic embryo developed further, the scutellum got reduced with corresponding increase in amylase. The amylase activity was tested periodically at different developmental stages of embryos. The role of scutellum in somatic embryos for starch deposition and amylase accumulation is discussed.     

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron

The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10M) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate.     

5S rDNA gene diversity in tea (Camellia sinensis (L.) O. Kuntze) and its use for variety identification.

Variability in the organization of repeats of 5S rDNA is useful for phylogenetic studies in various crops. We found variable repeats of 5S rDNA gene in the genome of tea (Camellia sinensis (L.) O. Kuntze) during Southern hybridization. Variability in the repeats of 5S rDNA with specific restriction endonucleases (Sau3AI, BamHI, and ApoI) was analyzed in 28 different tea clones representing 3 types of tea. Our results clearly show that the 5S rDNA gene in tea could be used as a molecular marker to distinguish C. sinensis Chinary tea from the other important types of tea, namely Assamica and Cambod. Upon analysis with restriction endonucleases, the 5S rDNA gene in the tea genome was found to be heavily methylated.     

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA₃) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.     

Characterization of gene expression of <Emphasis Type="Italic">QM</Emphasis> from <Emphasis Type="Italic">Caragana jubata</Emphasis>, a plant species that grows under extreme cold

Caragana [Caragana jubata (Pall.) Poir] is a temperate plant that thrives well under extremes of cold in high altitude of Himalaya and hence the plant is expected to be a source of genes that might play an important role in tolerance to low temperature (LT). In order to identify LT inducible gene(s), differential display of mRNA (DD) was performed using the apical buds growing under snow as well as growing in the near vicinity without snow, and a LT inducible QM gene (CjQM) homologue was identified. Realizing the importance of QM gene (which encodes human Wilms’ tumor suppressor QM protein) in aggregation of 40 and 60S ribosomal subunit and that not much has been reported on this gene in plant systems in relation to its relationship with LT, full length cDNA of CjQM was cloned through rapid amplification of cDNA ends. The gene (977 bp), encoded by small gene family, had an open reading frame of 651 bp and was found to be intronless. The gene exhibited up-regulation within 20 min of exposure to LT and abscisic acid (ABA), but no significant change in gene expression was observed in response to drought stress (DS), salicylic acid (SA) and methyl jasmonate (MJ) application. Up-regulation of CjQM was obtained in the tissues growing in situ under snow. Non-responsiveness of CjQM towards DS, SA and MJ, but up-regulation in response to LT and ABA suggested a specific regulation of the gene in Caragana under varied cues.