Smita Rastogi

Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis

The Dof (DNA binding with One Finger) family represents a classic zinc-finger transcription factors involved with multifarious roles exclusively in plants. There exists great diversity in terms of number of Dof genes observed in different crops. In current study, a total of 28 putative Dof genes have been predicted in silico from the recently available whole genome shotgun sequence of Sorghum bicolor (L.) Moench (with assigned accession numbers TPA:BK006983–BK007006 and TPA:BK007079–BK007082). The predicted SbDof genes are distributed on nine out of ten chromosomes of sorghum and most of these genes lack introns based on canonical intron/exon structure. Phylogenetic analysis of 28 SbDof proteins resulted in four subgroups constituting six clusters. The comparative phylogenetic analysis of these Dof proteins along with 30 rice and 36 Arabidopsis Dof proteins revealed six major groups similar to what has been observed earlier for rice and Arabidopsis. Motif analysis revealed the presence of conserved 50–52 amino acids Dof domain uniformly distributed across all the 28 Dof proteins of sorghum. The in silico cis-regulatory elements analysis of these SbDof genes suggested its diverse functions associated with light responsiveness, endosperm specific gene expression, hormone responsiveness, meristem specific expression and stress responsiveness.

In vitro regeneration of Leucaena leucocephala by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 100 mg dm−3 glutamine, 20.9 µM N6-benzylamino-purine (BAP) and 5.37 µM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm−3 myoinositol, 14.76 µM indole-3-butyric acid (IBA) and 0.23 µM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 40.28 µM NAA and 12.24 µM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 15.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 µM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 10.0 µM BAP, 2.5 to 5.0 µM IBA and 0.5 mM spermidine.

Phylogenetic diversity analysis of Trichoderma species based on internal transcribed spacer (ITS) marker

The phylogeny of Trichoderma and the phylogenetic relationships of its species was investigated by maximum parsimony analysis and distance analysis of DNA sequences from multiple genetic loci 18S rDNA sequence analysis suggests that the genus Trichoderma evolved at the same time as Hypomyces and Fusarium and thus about 110 Myr ago 28S rDNA sequence analysis shows that the genus Trichoderma is part of a monophyletic branch within the Hypocreaceae. Most isolates of the genus Trichoderma were found to act as mycoparasites of many economically important aerial and soil-borne plant pathogens. Trichoderma has attained importance as a substitute for chemical pesticides and hence an attempt was intended to corroborate the positive relatedness of molecular and morphological characters. Two fungal strains, Trichoderma koningii Tk-5201/CSAU and Trichoderma virens Tvi4177/CSAU were isolated from a soil sample collected from CSA Farm, Kanpur district of Uttar Pradesh, India. The universal primers (internal transcribed spacer, ITS) were used for the amplification of 18S rRNA gene fragment and strains were thus characterized with the help of ITS marker. It is proposed that the identified strains T. koningii Tk-5201/CSAU and T. virens Tvi-4177/CSAU be assigned as the type strains of a species of genus Trichoderma based on phylogenetic tree analysis together with the 18S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases. The sequence was deposited in GenBank with the accession numbers KC800923 and KC800924, respectively. Thus an integrated approach of morphological and molecular markers can be employed to identify a superior strain of Trichoderma for its commercial exploitation.

Sequencing of 28SrRNA Gene for Identification of Trichoderma longibrachiatum 28CP/7444 Species in Soil Sample

Most of the Trichoderma species are morphologically very similar and were considered for many years as a single species. Since new species were discovered, a consolidated taxonomical scheme was needed and proposed and defined nine morphological species aggregates. DNA methods brought additional valuable criteria to the taxonomy of Trichoderma which are being used today for studies that include identification and phylogenetic classification. Most isolates of the genus Trichoderma that were found to act as mycoparasites of many economically important aerial and soil-borne plant pathogens. Trichoderma has attained importance for substitute of chemical pesticides and hence an attempt was intended to corroborate the positive relatedness of molecular and morphological characters. A fungal strain of Trichoderma longibrachiatum 28CP/7444 was isolated from a soil sample collected from Barabanki district of Uttar Pradesh, India. The universal primers were used for amplification of the 28S rRNA gene fragment and strain characterized by using 28S rRNA gene sequence with the help of ITS marker. It is proposed that the identified strain Trichoderma longibrachiatum 28CP be assigned as the type strain of a species of the genus Trichoderma based on phylogenetic tree analysis together with the 28S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases. The sequence was deposited in GenBank with the accession number JX978541. Thus an integrated approach of morphological and molecular markers can be employed to identify a superior strain of Trichoderma for its commercial exploitation.

Comparative Study of Biological Agents, Trichoderma harzianum (Th-Azad) and Trichoderma viride (01PP) for Controlling Wilt Disease inPigeon Pea

The paper aims to study the morphological, physiological, molecular characterization and bio-formulation of Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315, an effective fungicide and a biological control agent too that protects the plants and seeds from other pathogenic fungi. The physiologic study is done in an attempt to find the effective management of the disease caused by soil borne pathogens. Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315 isolated from the infected soil samples of Pigeon pea fields and grown at preferable temperatures, pH and different solid and liquid culture media. Most preferable temperature for the growth and sporulation of Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315 has been observed up to 30oC (210.5 mg dry weight of mycelium). A detailed morphology of the strain is done in this study including colony growth rate, colony color, colony edge, mycelial form, conidiation, conidiophore branching, conidial wall, conidial color, etc. The molecular characterization of the strain is carried out using an 18S rRNA gene sequence with the help of a universal Internal Transcribed Spacer marker that gives an amplicon of a total of 1173 base pairs and 546 bp of the 18S rRNA gene was sequenced and used for the identification of isolated fungal strains that is later sequenced and allotted with Gene Bank Accession no. JX119211 and KC800922 respectively. To check the presence of endochitinase gene in two of the potential strains of Trichoderma species viz. T. harzianum (Th Azad) and T. viride 01PP an ech42 primer was used. A talc based bio-formulation has been prepared with this strain where the population of the spores was found to decline after 180 days.