Roshan Zamir

Molecular cloning and functional analysis of NAC family genes associated with leaf senescence and stresses in Gossypium hirsutum L.

The NAC domain genes encode a large family of plant-specific transcription factors that play diverse roles in plant development and stress regulation. In this study, a total of 60 full-length putative NAC genes were isolated from Gossypium hirsutum L. Based on their phylogeny, all GhNAC genes were clustered into seven distinct subfamilies, which exhibit functional similarity. Similarly, a phylogenetic tree for GhNAC genes and their motif showed close resemblance among the subfamilies. The isolated 60 full-length GhNAC genes were located on 13 different chromosomes of D sub-genome. Majority of the NACs showed specific temporal and spatial expression patterns in tissue-specific (fibers, cotyledon leaf, mature leaf, stem, root and flower) studies based on qRT-PCR analyses. Furthermore, the roles of GhNAC genes were monitored using qRT-PCR during leaf senescence and following treatment with ethylene, abscisic acid, gibberellic acid and drought or salinity. This first comprehensive study of GhNAC family elucidates the essential role of these genes in cotton development and in response to various stresses. This study lays fundamental foundations for future research and development in cotton genome.

Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni).

An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL(-1) agar and 2.0 mgL(-1) 2,4-D. The addition of 7.0 gL(-1) agar and BA (1.0, 2.0 and 4.0 mgL(-1)) significantly (P<0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL(-1) BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL(-1)) and Kin (1.0 mgL(-1)). Lower agar (3.5 gL(-1)), IAA (2.0 mgL(-1)), and NAA (2.0 mgL(-1)) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL(-1)) in combination with Kin (3.0 mgL(-1)) and 3.5 gL(-1) agar increases dulcoside-A content (Dul-A; 71.8 μg/g-DW) in shoots compared to control (50.81 μg/g-DW). Similar PGRs with 7.0 gL(-1) significantly increases the production of steviosides (Stev. 82.48 μg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 μg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL(-1)) and 7.0 gL(-1) agar than in control (07.39 μg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species.

In vitro regeneration of plantlets from unpollinated ovary culture in sweet orange (Citrus sinensis L. Osbeck)

Callogenesis and organogenesis of ovary of sweet orange ( Citrus sinensis ) cv. Blood red was carried on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of N 6 benzyl adenine (BA), 1-napthaleneacetic acid (NAA) and 2,4-D. 1 mg/l BA + 0.5 mg/l NAA on MS medium was the most effective in callus induction and proliferation. Maximum number of shoots (11) was recorded on the medium with 2 mg/l NAA + 3 mg/l BA. The best medium for root induction was MS together with 2.5 mg/l indole-3-acetic acid (IAA) + 2 mg/l indole–3-butyric acid (IBA), where maximum (16) plantlets were rooted. The regenerated plantlets were successfully acclimatized in jiffy pots containing sterilized soil mixture of sand, silt and clay in 1:1:1 ratio to study their response to in vivo conditions. Key words: Citrus, blood red, ovary culture, callus induction, regeneration, plant growth regulators.

Antioxidant activity via DPPH, gram-positive and gram-negative antimicrobial potential in edible mushrooms

Edible mushrooms (EMs) are nutritionally rich source of proteins and essential amino acids. In the present study, the antioxidant activity via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial potential in EMs (Pleurotus ostreatus, Morchella esculenta, P. ostreatus (Black), P. ostreatus (Yellow) and Pleurotus sajor-caju) were investigated. The DPPH radical scavenging activity revealed that the significantly higher activity (66.47%) was observed in Morchella esculenta at a maximum concentration. Similarly, the dose-dependent concentrations (200, 400, 600, 800 and 1000 µg) were also used for other four EMs. Pleurotus ostreatus exhibited 36.13% activity, P. ostreatus (Black (B)) exhibited 30.64%, P. ostreatus (Yellow (Y)) exhibited 40.75% and Pleurotus sajor-caju exhibited 47.39% activity at higher concentrations. Furthermore, the antimicrobial potential were investigated for its toxicity against gram-negative bacterial strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Klebsiella pneumonia, Erwinia carotovora and Agrobacterium tumifaciens), gram-positive bacterial strains (Bacillus subtilis, Bacillus atrophaeus and Staphylococcus aureus) and a fungal strain (Candida albicans) in comparison with standard antibiotics. Antimicrobial screening revealed that the ethanol extract of P. ostreatus was active against all microorganism tested except E. coli. Maximum zone of inhibition (13 mm) was observed against fungus and A. tumifaciens. P. sajor-caju showed best activities (12.5 mm) against B. subtilis, B. atrophaeus and K. pneumonia. P. ostreatus (Y) showed best activities against P. aeroginosa (21.83 mm), B. atrophaeus (20 mm) and C. albicans (21 mm). P. ostreatus (B) exhibited best activities against C. albicans (16 mm) and slightly lower activities against all other microbes except S. typhi. M. esculenta possess maximum activities in terms of inhibition zone against all microorganisms tested except S. typhi.

Efficient In Vitro Regeneration of Sugarcane (Saccharum Officinarum L.) from Bud Explants

ABSTRACT The regeneration potential of the economically important plant Saccharum officinarum (Sugarcane) was investigated. Callus induction and shoot regeneration along with somatic embryogenesis were induced from bud explants incubated on Murashige and Skoog (MS)-medium supplemented with different plant growth regulators (PGRs) and white sugar. The best callus induction (83.33%) was observed on explants incubated on MS-medium plus 1.0 mg·l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.0 mg·l−1 2,4-D (70%) after 6 weeks of culture. Other combinations (BA, IBA, IAA, NAA and GA3) of PGRs were less effective than 2,4-D. It was observed that lower concentrations of 2,4-D induced somatic embryos in bud explants of Saccharum officinarum, whereas higher concentrations induced non-embryogenic calli. Subsequent sub-culturing of calli onto MS-medium supplemented with BA (6-benzyladenine) induced shoot organogenesis. Highest shoot induction (98%) was recorded for 2.0 mg·l−1 after 3 weeks of culture. With this concentration of BA, maximum number of (178) shoots per explant were recorded, and, when the shoots were transferred to elongation medium, the longest shoots (9.4 cm) were recorded. However, 5.6 cm long shoots were also recorded with 3.0 mg·l−1 zeatin. No root induction hormones were used for rooting. The elongated shoots started rooting upon maturation. A maximum rooting (84%), number of roots/shoot (21) and mean root length of 5.0 cm were observed on medium containing 2.0 mg·l−1 BA along with 1.0 mg·l−1 GA3. The regenerated plantlets were successfully acclimated in field conditions.

Antioxidant activity influenced by in vivo and in vitro mutagenesis in sugarcane ( Saccharum officinarum L.)

The antioxidant potential (1,1-diphenyl-2-picrylhydrazyl (DPPHo)-scavenging activity) of in vitro regenerated and induced mutant sugarcane (Saccharum officinarum L.) was investigated. Efficient callus induction and shoot regeneration were induced in bud explants when incubated on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs). Best callogenesis was observed on MS-medium supplemented with 3 mg L -1 2,4 dichlorophenoxyacetic acid (2,4 D) and on ½ MS medium with 2 mg L -1 2,4 D after 30-days of culture. Almost 85% shoot organogenesis was observed on MS-medium supplemented with 2 mg L -1 6-benzyladenine (BA) and 0.5 mg L -1 gibberellic acid (GA3) within 30 days. Optimum percentage rooting (89%), were obtained for 2 mg L -1 of BA alone. Mother plant setts were irradiated with 60Co mutagen source. Assay of antioxidant activity of in vitro and in vivo grown tissues was evaluated as gross parameter of medicinal efficacy. Significantly higher antioxidant activity (60%) in in vitro regenerated sugarcane was observed as compared to induced mutant (57%) and mother plant (53%). Key words: Saccharum officinarum, in vitro regeneration, induced mutation, antioxidant.

Effect of exposure time and incubation period of various sterilants and antioxidants on the in vitro morphogenesis of guava explants

This experiment was carried out to investigate the effects of various surface sterilants and antioxidants during in vitro propagation of guava (Psidium guajava L.). Different sterilants and exposure time significantly affected survival in shoot tip explants. Maximum survival response (43.3%) was observed when shoot tips were exposed to 2% NaOCl followed by 0.05 % HgCl2 and 4% CaOCl with 36.7 and 33.3 % survival respectively. Similarly 5 and 10 minutes exposure times were statistically at par with each other. The interaction between different sterilants and exposure time was non significant. As compared to soot tips explants, the effect of different sterilants on the survival of nodal explants was also significant. The highest survival response of 31.7% was shown by 0.05% HgCl2 followed by 4% CaOCl and 2% NaOCl (29.2 and 26.7%) respectively. Similarly the response of different exposure times (5 and 10 minutes) in case of nodal explants was non significant. Among all anti oxidants and incubation periods applied, dark incubation of cultures for 24 hours was effective which eliminated (34.3%) browning followed by dip of explants in 75:50 mg l -1 citric acid and ascorbic acid (31.8%) while control was inferior to all and gave 11.7% browning elimination. Similarly the effect of antioxidants on type of explants (shoot tip and nodal explants) was also significant. The highest response of 37.8 % was recorded in shoot tip explants while in nodal explants it was 14.5% only. The inter action between antioxidants and explants types was also significant. In over all, the highest browning elimination of 55% was recorded in shoot tip explants when cultures were kept in dark for 24 hours. Whereas the most inferior results were shown by control treatments where only 6.7 and 16.7% browning was eliminated in tips and nodal explants.

Development of sparse-seeded mutant kinnow (Citrus reticulata Blanco) through budwood irradiation

Kinnow is the major fruit of Pakistan and has a high export potential due to its excellent fruit and juice quality. However, high number of seeds (25 ± 5) per fruit is limiting its export on a large scale. Benefiting from the induced mutations for selectivity, especially in the vegetatively propagated fruit crops like citrus, induced mutation for seedlessness in Kinnow with gamma irradiation of dormant bud which was attempted at the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad. Dormant bud irradiation-cum-grafting technique was employed, using the Citrus jambhiri rootstock for propagation of the scion. A sparse seeded (5 ± 3 seeds/fruit) mutant was evolved from an exposure dose of 20 Gy. The mutant was put to the conventional propagation up to mV 5 , for confirmation of the continuity of the mutation. The sparse seeded mutation was found to be a solid and stable one. The quality parameters in fruit and juice of the mutant were comparable with its parent. Key words : Kinnow, seedlessness, dormant bud, gamma irradiation, mutant kinnow.