Veena Agrawal

IN VITRO CLONAL PROPAGATION OF HOLARRHENA ANTIDYSENTERICA (L.) WALL. THROUGH NODAL EXPLANTS FROM MATURE TREES

SummaryIn vitro clonal propagation of 18–20-yr-old Holarrhena antidysenterica tress has been achieved by employing nodal explants. The tree explants showed marked seasonal variation in their morphogenic response under in vitro conditions. Maximum response was obtained from the beginning of May to the end of July, followed by a gradual decline, finally dropping to zero from October to February. The explants induced multiple shoots only on cytokinin-containing medium. Several cytokinins [N6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2ip), 6-furfuryl aminopurine (Kn), and adenine sulfate (Ads)] were assayed. The best response was achieved with 15 μM BA in which 62.5% of cultures produced 2.75±0.2 shoots per explant with 3.56±0.2 cm average length. Amongsth the three heavy metals assayed, silver nitrate (AgNO3) significantly improved the response. This compound enhanced both the percentage of responding cultures (86.6%) and the average shoot number (4.73±0.2) at a concentration of 20mgl−1. Further improvement in the morphogenic response occurred when explants from in vitro shoots were employed instead of mature trees. In this case, the percentage of morphogenic cultures was increased to 100% at the third subculture with an average of 11.45±0.3 shoots per explant. Regenerated shoots were rooted in half-strength Murashige and Skoog medium with 10 μM indole-3-acetic acid. The plantlets were successfully acclimatized in soil.

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoogs medium supplemented with 20 μM N6-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 μM indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 μM IBA + 0.5% activated charcoal + 1 μM BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoogs (MS) medium augmented with 1 µM N6-benzyladenine (BA) + 1 µM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 µM BA was optimum for eliciting morphogenic response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived calli, respectively. The addition of 0.5 µM α-naphthaleneacetic acid (NAA) to MS + 5 µM BA further elevated the maximum average number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 µM IBA.

Phytotoxic effects of Cu, Zn, Cd and Pb on in vitro regeneration and concomitant protein changes in Holarrhena antidysenterica

The nodal explants of in vitro shoots of Holarrhena antidysenterica L. were cultured on Murashige and Skoogs (MS) medium augmented with 15 μM N6-benzyladenine (BA) alone (control) or supplemented with different concentrations (1, 5, 10 and 20 mg dm−3) of CdCl2, CuSO4, Pb(NO3)2 and ZnSO4. The maximum morphogenic response in terms of average shoot number (4.95 ± 0.17) was seen in control. ZnSO4 proved to be less inhibitory in comparison to CuSO4, Pb(NO3)2 and CdCl2. None of the explants cultured on CdCl2 containing medium induced multiple shoots. Maximum protein content [3.80 ± 0.04 mg g−1(f.m.)] was observed in control and slightly less [3.50 ± 0.02 mg g−1(f.m.)] in tissues exposed to 1 mg dm−3 of CuSO4 and minimum [1.00 ± 0.02 mg g−1(f.m.)] in Zn treated (20 mg dm−3) explants. SDS-PAGE analysis of the treated tissues revealed that two new polypeptides (29 and 20 kDa) in response to Cu and Zn treatment, respectively, have been synthesized.

Influence of some adjuvants on in vitro clonal propagation of male and female jojoba plants

SummaryThe influence of different adjuvants, activated charcoal (AC), casein hydrolyzate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA), has been assessed on the shoot production potential of the nodal explants derived from in vitro-raised male and female jojoba (Simmondsia chinensis) shoots. Nodal explants of each sex were cultured separately on Murashige and Skoog medium supplemented with different levels of AC, CW, CH, PVP, and TIBA either alone or along with optimum levels of N6-benzyladenine (BA; 10 μM for male, 20 μM for female). Some differences in response of the explants of both the sexes have been observed in terms of (1) percentage of explants developing shoots, (2) average shoot number, and (3) average shoot length. AC alone proved beneficial for elevating morphogenic response in male as well as female explants in comparison to basal medium or media containing AC and the optimum level of BA. When used alone, CH proved inhibitory for shoot differentiation in both sexes, especially in male explants. Addition of PVP to MS enhanced shoot proliferation in female explants only, but along with BA it increased the response of male explants. BA in combination with different levels of TIBA promoted shoot multiplication in female explants. Thus, explants of both male and female shoots exhibited differential morphogenic behavior under the influence of various adjuvants. However, BA alone proved to be the best for differentiation of shoots in both male (10 μM) as well as female (20 μM) explants.

In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene

Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 μg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 μg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia.

Algae as crucial organisms in advancing nanotechnology: a systematic review

As nanotechnology is expanding to several commercial fields, there is a need of ecofriendly and energy-efficient methods for the synthesis of nanoparticles. Algae have been discovered to reduce metal ions and subsequently for the biosynthesis of nanoparticles. Since algae-mediated biosynthesis of nanoparticles is an ecofriendly, economical, high-yielding, expeditious and energy-efficient method, a large number of studies have been published in the last few years. This review article therefore is focused on recent progress on the utilization of algae of various classes, viz., Cyanophyceae, Chlorophyceae, Phaeophyceae, Rhodophyceae, etc. for the synthesis of nanoparticles, their characterization and the possible mechanisms involved.

Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) markers

To detect genetic variations among different Simmondsia chinensis genotypes, two gene targeted markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) were employed in terms of their informativeness and efficiency in analyzing genetic relationships among different genotypes. A total of 15 SCoT and 17 CBDP primers detected genetic polymorphism among 39 Jojoba genotypes (22 females and 17 males). Comparatively, CBDP markers proved to be more effective than SCoT markers in terms of percentage polymorphism as the former detecting an average of 53.4% and the latter as 49.4%. The Polymorphic information content (PIC) value and marker index (MI) of CBPD were 0.43 and 1.10, respectively which were higher than those of SCoT where the respective values of PIC and MI were 0.38 and 1.09. While comparing male and female genotype populations, the former showed higher variation in respect of polymorphic percentage and PIC, MI and Rp values over female populations. Neis diversity (h) and Shannon index (I) were calculated for each genotype and found that the genotype “MS F” (in both markers) was highly diverse and genotypes “Q104 F” (SCoT) and “82–18 F” (CBDP) were least diverse among the female genotype populations. Among male genotypes, “32 M” (CBDP) and “MS M” (SCoT) revealed highest h and I values while “58-5 M” (both markers) was the least diverse. Jaccards similarity co-efficient of SCoT markers ranged from 0.733 to 0.922 in female genotypes and 0.941 to 0.746 in male genotype population. Likewise, CBDP data analysis also revealed similarity ranging from 0.751 to 0.958 within female genotypes and 0.754 to 0.976 within male genotype populations thereby, indicating genetically diverse Jojoba population. Employing the NTSYS (Numerical taxonomy and multivariate analysis system) Version 2.1 software, both the markers generated dendrograms which revealed that all the Jojoba genotypes were clustered into two major groups, one group consisting of all female genotypes and another group comprising of all male genotypes. During the present investigation, CBDP markers proved more informative in studying genetic diversity among Jojoba. Such genetically diverse genotypes would thus be of great significance for breeding, management and conservation of elite (high yielding) Jojoba germplasm.

Somatic embryogenesis in the woody legume Calliandra tweedii

In vitro somatic embryogenesis was established from 1–1.2 cm long internodal and petiolar segments excised from 20-year-old woody legume, Calliandra tweedii Benth trees. Within 6–7 weeks, globular, heart, torpedo-shaped and dicot embryos differentiated directly. Among the cytokinins and auxins tested, 1 μM 2iP in Murashige and Skoogs (MS) medium induced well-organized normal embryos in 71% of the internodal segments. Petiolar explants responded best on 0.5 μM NAA wherein 30% of the cultures developed normal embryos. Somatic embryos developed roots and shoots when they were retained for 90 days in old desiccated MS medium. Plants ready for transfer to soil were regenerated through somatic embryogenesis in 4–5 months. These grew well after transfer to soil.

Review on different mechanisms of sex determination and sex-linked molecular markers in dioecious crops: a current update

Flowering plants are known to exhibit vast diversity of sexual systems encompassing bisexual, monoecious and dioecious conditions. Dioecy offers opportunities to explore separately the male and female programmes giving an insight to the evolutionary, developmental and molecular processes leading to separate mechanisms for sex expression. Mechanisms controlling sex can either be genetic or epigenetic (physiological and environmental). Plant hormones too influence sex expression. An active Y sex determination system and an X to autosomes ratio systems are common amongst the flowering plants. Advances in our understanding of sex determination has been addressed both by conventional as well as molecular approaches. Using conventional techniques mainly cytogenetics, sex chromosomes in some dioecious plants have been identified and characterized. Surprisingly, the presence of well defined sex chromosomes was found in only few species. Some sex linked genes have also been identified and characterized using molecular approaches but none of these genes have a direct link to sex determination. Molecular markers have been employed to resolve the enigma associated with dioecism to a certain extent. Its application in plant breeding is immensely beneficial. Positively, it would be beneficial for validation of sex prior their sex expression at larger perspectives. The present review therefore emphasizes the mode of sex determination among dioecious plants vis-a-vis summarizes the works related to gender specific markers generated using male and female plants from agriculturally important dioecious crops.