Arpan R. Modi

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.

Transcriptional profiling of genes involved in steviol glycoside biosynthesis in Stevia rebaudiana bertoni during plant hardening

Background: Stevioside is a diterpene glycoside found in Stevia rebaudiana Bertoni (Asteraceae) and is 200–300 times sweeter than sucrose. It is synthesized through a plastid localized 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway. Fifteen genes are involved in the formation of steviol glycosides (stevioside and rebaudioside A). In the present investigation, micropropagated plants were allowed to harden for one month during which transcriptional profiling of candidate genes was carried out. Sampling from all the plants was carried out during hardening at different time intervals (day 10, 20, and 30) along with control plants (day 0). Stevioside content was also measured. Results: Of 15 genes, 9 were up‐regulated two‐fold or greater. Nine genes were expressed at higher levels after 30 days than in the untreated controls. Moreover, these transcriptional differences were correlated with a significant enhancement in stevioside content from 0‐ (11.48%) to 30‐ (13.57%) day‐old plants. Conclusions: MEP pathway genes in stevia are expressed at higher levels during hardening, a change to vegetative growth from reproductive growth. Although there were higher transcript levels of candidate genes at the initial phase of hardening, the highest stevioside content was found after 30 days of hardening, suggesting translational/posttranslational regulatory mechanisms. Developmental Dynamics 243:1067–1073, 2014.

Analysis of Differentially Expressed Genes Involved in Stevioside Biosynthesis in Cultures of Stevia Rebaudiana Bertoni Treated with Steviol as an Immediate Precursor

Stevia rebaudiana B. is a medicinal herb that accumulates low-caloric sweetening agents generally known as steviol glycosides among which stevioside and rebaudioside-A are predominant. These sweeteners are synthesized through the commonly known 2-C-methyl-d-erythritol-4-phosphate pathway common to steviol glycosides, gibberellic acid, pigments, and other isoprene-based secondary metabolites. The present investigation was designed to elucidate the role of steviol, an immediate precursor of sweetening agents, in the cultures of plants conducting stevioside biosynthesis in the leaf tissue. It was proposed that either the precursor or an elicitor was responsible for stimulating stevioside production in leaves. Transcript profiling of genes involved in stevioside biosynthesis was explored both in the roots and leaves of the plant and it was found that the transcript of the key enzyme kaurenoic acid hydroxylase, producing steviol, was the same in both tissue. However, when these plants were treated with the precursor steviol, most of the genes in the pathway were downregulated. Interestingly, it was found that very minute concentrations of the precursor were required to enhance the transcription level of genes of the pathway.

Studies of Peroxidase and Esterase Enzyme Activity and Their Isoforms at Different Leaf Stages of Stevia rebaudiana

Leaf of Stevia rebaudiana Bertoni contains steviol glycosides which are extremely sweet compounds and are consumed world wide as low caloric sweetener. The compound responsible for the sweetness is a stevioside whose concentration is found different during various leaf stages. Throughout the development of plant the activity as well as isoforms of several enzymes is also found variable. Here, we studied the activity as well as pattern of isoforms of peroxidase and esterase in young leaf, the leaf just before flowering stage and the leaf after flowering stage of Stevia rebaudiana. The results showed significant variation in activity as well as isozyme banding pattern during different stages of growth.

Evaluation of Clonal Fidelity of Micropropagated Date Palm by Random Amplified Polymorphic DNA (RAPD)

Date palm is a fruit-bearing tree commonly found in arid and semiarid regions. It is a dioecious plant, producing fruit on female plants and a limited number of basal offshoots for propagation. To produce large numbers of uniform plantlets, tissue culture techniques are required. It is highly advisable to detect genetic variation that may occur through micropropagation techniques as it may lead to phenotypic alterations. Random amplified polymorphic DNA (RAPD) is a simple and PCR-based molecular marker technique which can be employed to check the somaclonal variation. Screening of markers requires repeated confirmation of the pattern obtained in individual samples.

Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

Micropropagation and Biomass Production of True-to-Type Stevia rebaudiana Bertoni.

Here we describe an efficient micropropagation protocol for Stevia rebaudiana Bertoni. We present experiments carried out to optimize the suitable media for in vitro shoot multiplication and root induction and to study the effect of culture vessel on shoot multiplication. Among all different media tested for in vitro shoot multiplication, hormone-free liquid medium is most suitable. The highest number of nodes per shoot (5.4) and length of shoot (4.76 cm) at 4 weeks after subculturing are observed when single node explants are placed on modified MS medium supplemented with 1 % sucrose and 0.7 % agar. The highest response of multiplication rate (9.56) is observed on half strength of macroelement of MS with full strength of microelement of MS and 170 mg/l KH2PO4, and 185 mg/l MgSO4 in plastic growth container. Further, RAPD marker analysis of in vitro-raised plants maintained their clonal fidelity and true-to-type without showing any somaclonal variation.