Tapan Kumar Mondal

Recent Advances of Tea (Camellia Sinensis) Biotechnology

Tea is one of the most important non-alcoholic beverage drinks worldwide and gaining further popularity as an important ‘health drink’. It is served as morning drink for 2/3rd of world population daily. Although conventional breeding and propagation contributed significantly for last several decades for varietal improvement, due to the limitations of conventional breeding coupled with the demand for increasing productivity with lower cost of production, application of biotechnology becomes an alternative approach. Therefore, apart from a dozen of tea research institutes globally, several other groups are working on tea and related genera that have registered many valuable information with several achievements. The present review deals with progress in-depth of various aspects of biotechnological works such as micropropagation and alternative approaches, cell and organ culture techniques, genetic transformation, DNA markers as well as organelle genome and gene cloned from tea and related genera which will be valuable information for the workers working on various aspects of Camellia biotechnology.

Identification and characterization of salt responsive miRNA-SSR markers in rice (Oryza sativa)

Salinity is an important abiotic stress that affects agricultural production and productivity. It is a complex trait that is regulated by different molecular mechanisms. miRNAs are non-coding RNAs which are highly conserved and regulate gene expression. Simple sequence repeats (SSRs) are robust molecular markers for studying genetic diversity. Although several SSR markers are available now, challenge remains to identify the trait-specific SSRs which can be used for marker assisted breeding. In order to understand the genetic diversity of salt responsive-miRNA genes in rice, SSR markers were mined from 130 members of salt-responsive miRNA genes of rice and validated among the contrasting panels of tolerant as well as susceptible rice genotypes, each with 12 genotypes. Although 12 miR-SSRs were found to be polymorphic, only miR172b-SSR was able to differentiate the tolerant and susceptible genotypes in 2 different groups. It had also been found that miRNA genes were more diverse in susceptible genotypes than the tolerant one (as indicated by polymorphic index content) which might interfere to form the stem-loop structure of premature miRNA and their subsequent synthesis in susceptible genotypes. Thus, we concluded that length variations of the repeats in salt responsive miRNA genes may be responsible for a possible sensitivity to salinity adaptation. This is the first report of characterization of trait specific miRNA derived SSRs in plants.

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

Summary 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.

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 (1–10μM) 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.

Identification and analysis of novel salt responsive candidate gene based SSRs (cgSSRs) from rice (Oryza sativa L.).

BackgroundMajority of the Asian people depend on rice for nutritional energy. Rice cultivation and yield are severely affected by soil salinity stress worldwide. Marker assisted breeding is a rapid and efficient way to develop improved variety for salinity stress tolerance. Genomic microsatellite markers are an elite group of markers, but there is possible uncertainty of linkage with the important genes. In contrast, there are better possibilities of linkage detection with important genes if SSRs are developed from candidate genes. To the best of our knowledge, there is no such report on SSR markers development from candidate gene sequences in rice. So the present study was aimed to identify and analyse SSRs from salt responsive candidate genes of rice.ResultsIn the present study, based on the comprehensive literature survey, we selected 220 different salt responsive genes of rice. Out of them, 106 genes were found to contain 180 microsatellite loci with, tri-nucleotide motifs (56%) being most abundant, followed by di-(41%) and tetra nucleotide (2.8%) motifs. Maximum loci were found in the coding sequences (37.2%), followed by in 5′UTR (26%), intron (21.6%) and 3′UTR (15%). For validation, 19 primer sets were evaluated to detect polymorphism in diversity analysis among the two panels consisting of 17 salt tolerant and 17 susceptible rice genotypes. Except one, all primer sets exhibited polymorphic nature with an average of 21.8 alleles/primer and with a mean PIC value of 0.28. Calculated genetic similarity among genotypes was ranged from 19%-89%. The generated dendrogram showed 3 clusters of which one contained entire 17 susceptible genotypes and another two clusters contained all tolerant genotypes.ConclusionThe present study represents the potential of salt responsive candidate gene based SSR (cgSSR) markers to be utilized as novel and remarkable candidate for diversity analysis among rice genotypes differing in salinity response.

INDUCTION OF SYNCHRONOUS SECONDARY SOMATIC EMBRYOGENESIS IN CAMELLIA SINENSIS (L.) O. KUNTZE

Summary 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.

Promoter methylation regulates the abundance of osa-miR393a in contrasting rice genotypes under salinity stress

MicroRNAs (miRNAs) are important molecules that regulate gene expression under salinity stress. Despite their evolutionary conservation, these regulatory elements have been shown to behave differently in different plant species under a particular environmental stress. In this study, we investigated the behavior of salt responsive osa-miR393a and its target gene (TIR1, LOC_Os05g05800) in salt-tolerant (FL478) and salt-sensitive (IR29) rice genotypes. It was found that the mature and precursor sequences of osa-miR393a as well as its cleavage site in TIR1 were conserved among salt tolerant and sensitive genotypes. Promoters of different salt-responsive miRNAs were also found to be less variable between salt-tolerant and salt-susceptible genotypes. Analysis of gene expression, promoter methylation, and cis-element abundance showed that osa-miR393a behaves differently in FL478 and IR29. Salt stress altered the expression pattern of osa-miR393a-TIR1 module in a time-dependent manner in the roots and shoots of two genotypes. Promoter methylation of this regulatory module was also altered at different time points under salt stress. Expression analysis in two genotypes indicated the overall down-regulation of osa-miR393a and up-regulation of TIR1 in FL478 and their reciprocal regulation in IR29. The expression results were complemented by the differential promoter methylation and cis-element abundance of this regulatory module. Together, the results of transcript abundance and promoter methylation of osa-miR393a-TIR1 module signified the association between these two processes which is reported for the first time in plants to the best of our knowledge.

Genome-wide development of novel miRNA-based microsatellite markers of rice (Oryza sativa) for genotyping applications

AbstractRice (Oryza sativa L.) plays a crucial role in assuring food security to the world’s population and is recognized as a superior model plant of monocot crops. Molecular markers, especially co-dominant markers such as simple sequence repeats (SSRs), play an important role in marker-assisted breeding of rice. Although a large number of SSRs are available, most of them are either from protein-coding regions or untranslated regions of the rice genome. SSRs from non-coding regions of the rice genome are almost undiscovered, but discovery of new SSR markers remains a challenge for molecular breeders. The development of novel markers from the conserved regions of different genomes will thus be useful for studying the genetic diversity of closely related species or self-pollinated species. In the present study, a genome-wide investigation yielded a total of 129 SSR markers from the conserved microRNA (miRNA) genes of rice, and 20 of them with repeat numbers ≥7, from all the 12 chromosomes, were validated among 24 diverse rice genotypes. The highest number of repeat motifs corresponded to di-nucleotides, while the tetra-nucleotides were repeated the lowest number of times. Our results therefore show that the number of repeats is inversely related to the length of repeat. The genetic diversity was found to be higher among the exotic material than the Indian landraces, as indicated by their polymorphic information content values of 0.31 and 0.29, respectively. The results hence showed that the miRNA-based microsatellite marker system is a very proficient, novel and breeder-friendly source for genetic diversity analysis or genotyping of rice. The present study is the first report on genome-wide identification and characterization of rice miRNA-SSRs.

An efficient protocol for somatic embryogenesis and its use in developing transgenic tea (Camellia sinensis (L.) O. Kuntze) for field transfer

With the increasing popularity of tea (Camellia sinensis (L.) O. Kuntze) as an important beverage drink throughout the world, the demand for high quality and high yielding varieties is also rising. Therefore, genetic improvement of tea through biotechnological means has become a necessity. Improvement via Agrobacterium tumefaciens mediated transformation holds potential. However, there is an urgent requirement for an efficient in vitro system of regeneration for its successful implementation.

Identification of Novel and Conserved miRNAs from Extreme Halophyte, Oryza coarctata, a Wild Relative of Rice

Oryza coarctata, a halophyte and wild relative of rice, is grown normally in saline water. MicroRNAs (miRNAs) are non-coding RNAs that play pivotal roles in every domain of life including stress response. There are very few reports on the discovery of salt-responsive miRNAs from halophytes. In this study, two small RNA libraries, one each from the control and salt-treated (450 mM NaCl for 24 h) leaves of O. coarctata were sequenced, which yielded 338 known and 95 novel miRNAs. Additionally, we used publicly available transcriptomics data of O. coarctata which led to the discovery of additional 48 conserved miRNAs along with their pre-miRNA sequences through in silico analysis. In total, 36 known and 7 novel miRNAs were up-regulated whereas, 12 known and 7 novel miRNAs were down-regulated under salinity stress. Further, 233 and 154 target genes were predicted for 48 known and 14 novel differentially regulated miRNAs respectively. These targets with the help of gene ontology analysis were found to be involved in several important biological processes that could be involved in salinity tolerance. Relative expression trends of majority of the miRNAs as detected by real time-PCR as well as predicted by Illumina sequencing were found to be coherent. Additionally, expression of most of the target genes was negatively correlated with their corresponding miRNAs. Thus, the present study provides an account of miRNA-target networking that is involved in salinity adaption of O. coarctata.