Sushma Tamta

In vitro multiplication of Quercus leucotrichophora and Q. glauca: Important Himalayan oaks

Multiple shoots of Quercus leucotrichophora L. and Q. glauca Thunb. were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and Murashige and Skoog (MS; 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA3)/ indole-3-butyric acid (IBA). BA (22.19 μM) was effective for induction of multiple shoots and addition of GA3 to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 μM). Nearly 78% and 67% rooting was obtained in Q. leucotrichophora and Q. glauca microshoots (3–4 cm high), respectively on 1/2 strength WP medium supplemented with IBA (14.76 μM). However, this was associated with basal callus formation. Treatment with IBA (25–100 μM) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 μM for 24 h was most effective (90% and 100% rooting in Q. leucotrichophora and Q. glauca, respectively). In vitro rooted plants were hardened and established in garden soil.Growth performance of 6-month-old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light (0, 600, 900, 1200, 1500 and 2000 μmol m−2s−1) and temperature (20, 25, 30, 35 and 40 °C). Light optimum for photosynthesis was around 2000 μmol m−2s−1 in Q. leucotrichophora and around 1500 μmol m−2s−1 in Q. glauca whereas optimum temperature for photosynthesis was 25 °C in Q. leucotrichophora and 30 °C in Q. glauca. The rate of transpiration at different temperatures (20–40 °C), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 μmol m−2s−1. Temperatures beyond 35 °C adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species. In vitro raised and hardened plants of both the species were comparable to ex vitro plants in terms of gas and water vapour exchange characteristics, within the limits of this study.

Prolonged Laccase Production by a Cold and pH Tolerant Strain of Penicillium pinophilum (MCC 1049) Isolated from a Low Temperature Environment.

Production of laccase by a cold and pH tolerant strain of Penicillium pinophilum has been investigated under different cultural conditions for up to 35 days of incubation. The fungus was originally isolated from a low temperature environment under mountain ecosystem of Indian Himalaya. The estimations were conducted at 3 temperatures (15, 25, and 35°C), a range of pH (3.5–11.5), and in presence of supplements including carbon and nitrogen sources, vitamins, and antibiotics. Optimum production of laccase was recorded at 25°C (optimum temperature for fungal growth) and 7.5 pH. The production of enzyme was recorded maximum on day 28 (11.6 ± 0.52 U/L) following a slow decline at day 35 of incubation (10.6 ± 0.80 U/L). Fructose and potassium nitrate (0.2%) among nutritional supplements, chloramphenicol (0.1%) among antibiotics, and folic acid (0.1%) among vitamins were found to be the best enhancers for production of laccase. Relatively lower but consistent production of laccase for a longer period is likely to be an ecologically important phenomenon under low temperature environment. Further, enhancement in production of enzyme using various supplements will be useful for its use in specific biotechnological applications.

In Vitro Propagation of the Important Tasar Oak (Quercus serrata Thunb.) by Casein Hydrolysate Promoted High Frequency Shoot Proliferation

An efficient micropropagation protocol has been developed for tasar oak. Nodal segments from in vitro grown seedling were used for shoot multiplication. Best shoot multiplication response, in terms of number of shoots per explant as well as shoot length, was obtained in woody plant (WP) medium supplemented with 6-benzyladenine and indole-3-acetic acid (8.88 μM BA + 1.43 μM IAA); but the establishment of cultures was difficult due to basal callus formation and necrosis in due course of time. Out of the two used growth adjuvants, casein hydrolysate (CH, 500 mg L−1) promoted shoot multiplication rate significantly in comparison to silver nitrate and also eliminated the basal callus formation problem and necrosis faced during the later stage of shoot proliferation. In vitro rooting on WP medium supplemented with 100 μM indole-3-butyric acid (IBA) when applied for 48 hr gave the best results in comparison to prolonged exposure. Well-acclimatized plantlets were transferred to the field with 80% survival rate. This protocol could be useful not only to propagate and conserve this oak but can also uplift the socioeconomic status of the Himalayan people as its leaves are used to feed the tasar silk worm during rearing period. This method will also be helpful for propagation of high value trees for a reforestation program.

Influence of kinetin on in vitro rooting and survival of banj oak ( Quercus leucotrichophora L.)

A study concerning the influence of cytokinins on shoot regeneration by using different stem segments derived from in vitro raised seedlings and their subsequent rooting was conducted in banj oak (Quercus leucotrichophora L.). Cytokinins play an important role in shoot regeneration and their multiplication. In the present study, cytokinins particularly kinetin (Kn) influenced in vitro rooting and subsequent survival of these in vitro raised plants in addition to shoot multiplication. It was found that the microshoots raised via kinetin treatment rooted very well (94.44%) without any basal callus formation in comparison to microshoots raised via other cytokinin treatments (that is, BA and 2-iP). In addition to this, these in vitro raised plants showed maximum survival rate (90%) during hardening process. On the basis of available literature this is a unique and significant study regarding the comparative effect of different cytokinins on in vitro propagation study of Q. leucotrichophora by using different stem segments, particularly the influence of kinetin in vitro rooting and survival of in vitro raised plants in addition to shoot multiplication. This significant study could be useful for large scale production of successfully hardened plants so that it would be helpful in conservation of this important Himalayan forest tree species.

A Review on Red Rot: The "Cancer" of Sugarcane

Sugarcane is an important agro industrial crop of the world. India being the largest consumer as well as the second largest producer of sugar, so, it requires sugarcane production on large scale. But diseases are the major concern for the sugarcane, responsible for its low yield. Among all the diseases, fungal disease named red rot of sugarcane is the most threatening disease of sugarcane, rightly called as ‘Cancer’ of sugarcane. It causes severe loss in yield and quality of the sugarcane. As the fungus Colletotrichum falcatum responsible for this disease is highly variable in nature, hence, it causes the frequent breakdown of resistant varieties. Keeping in view the seriousness of this disease, the present review summarizes the distribution, mode and source of infection, description of casual pathogen and disease management

Propagation and conservation of Dactylorhiza hatagirea (D. Don) Soo, an endangered alpine orchid

Dactylorhiza hatagirea (D. Don) Soo; a source of high quality ‘salep’ used as nervine tonic, has been categorized as critically endangered due to over-exploitation from the wild. This report deals with the propagation of this species using both in vitro and conventional methods. For green pod culture, four nutrient media, Knudson C (KC), Murashige and Skoog (MS), Vacin and Went (VW) and Vejsadova (VJ) were tested by adding different growth additives. MS medium supplemented with peptone (P) (1.0 g/L), morphoinoethane sulphonic acid (MES) (1.0 g/L) and activated charcoal (AC) (0.1%) was found to be the most effective medium for the development of protocorm like bodies (PLBs), development of chlorophyll and for the plantlet formation. To improve vegetative multiplication, tubers were treated with α- naphthalene acetic acid (NAA), indole-3- butyric acid (IBA) and indole acetic acid (IAA) before planting. Rooting was observed in only apical segments. Maximum rooting (38.88%) was induced with 50.0 μM IBA. This study is helpful in the propagation of this endangered orchid at lower elevation which could be a successful effort of conservation of this endangered orchid at lower elevation and this could also reduce pressure on its population in the wild. Keywords: In vitro propagation, protocorm like bodies (PLBs), vegetative propagation, conservation

Optimisation and characterisation of the orange pigment produced by a cold adapted strain of Penicillium sp. (GBPI_P155) isolated from mountain ecosystem

ABSTRACT With globalisation and growing health risks of synthetic colourants, search for pigments from natural sources has increased owing to their non-toxic nature. The present study highlights the bioprospection of natural pigment from a cold adapted fungal strain of Penicillium sp. (GBPI_P155), isolated from soil of Indian Himalayan region. The fungus produced insoluble and orange-coloured pigment in liquid medium with maximum production recorded in potato dextrose (PD) broth at 15°C and 3 pH, while maximum biomass was produced at 25°C and pH 3. While examining the effect of different mineral salts, and carbon and nitrogen sources on pigment production, maximum accumulation of pigment was recorded in PD broth supplemented with 2% maltose. Following production, extraction of pigment was performed using chloroform and characterised partially by UV/vis (λmax at 495 nm and a shoulder peak at 530 nm) and Fourier Transform Infrared (FT-IR) spectroscopy. Thin layer chromatography of chloroform extract resulted in separation of pigment in three fractions with Rf values 0.911, 0.852 and 0.808, which were further analysed using Liquid Chromatography Mass Spectrometry (LC/MS). The overall approach resulted in identification of pigment as a mixture of different derivatives of carotenoids. The extracted pigment also possessed antimicrobial activity against Gram-positive and Gram-negative bacteria and actinobacteria.

Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae

The plastid genome regions of two intergenic spacers, psbA–trnH and trnL–trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA–trnH region was higher than trnL–trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA–trnH and trnL–trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study revealed that psbA–trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL–trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.

Assessment of phytochemicals, antioxidant and antimutagenic activity in micropropagated plants of Quercus serrata, a high value tree species of Himalaya

Abstract The major aim of the present study was to: (i) develop an efficient clonal in vitro propagation procedure, and (ii) investigate and compare phytochemicals, antioxidant, and DNA damage protection activity in methanolic extracts of in vitro raised and mother plant leaves of Quercus serrata. The study succeeded in establishing an efficient clonal micropropagation procedure using nodal segments taken from in vivo growing mother plant. The WP medium supplemented with six-benzyleaminopurine (BAP: 8.88 μM) plus indole-3-acetic acid (IAA: 1.43μM) produced the highest number of shoots/explant having maximum average shoot length. In vitro derived shoots exhibited best rooting upon application of 100μM indole-3-butyric acid for 48 h. Among studied phytochemicals, the total phenol content was recorded higher from in vitro raised plants and total flavonoid in the mother plant. Antioxidant activities were recorded higher in case of in vitro raised plants. Also, the methanolic leaf extract of in vitro raised plants exhibited higher antimutagenic activity that of mother plant. These findings indicate Q. serrata as a promising source of natural antioxidants that could be utilized commercially for the prevention of photoaging and oxidative stress-mediated skin diseases.

Development of ISSR- and RAPD-derived SCAR markers for identification of Gladiolus germplasm

ABSTRACT Gladiolus is an economically important ornamental crop, cultivated for its beautiful flowers throughout the world. The correct genotype identification of plant material is very significant for the floriculture industry. The aim of this study was to develop sequence-characterised amplified region (SCAR) markers from RAPD and ISSR fragments for identification and authentication of Gladiolus germplasm. The SCAR markers developed could be easily employed as valuable tools to identify newly developed Gladiolus cultivars. The SCAR markers, viz. ScG12, ScG34, and ScG36, are specific to the DNA from all 62 Gladiolus cultivars, as they did not amplify the DNA of other taxa of the family Iridaceae, including Iris, Amaryllis, and Narcissus. All three SCAR markers distinguished Gladiolus from other taxa of the family Iridaceae, whereas marker ScAm was specific to the ‘Amethyst’ cultivar. Our results confirmed that this particular SCAR marker distinguished the ‘Amethyst’ cultivar from the other 62 Gladiolus cultivars investigated in the present study. This development of SCAR markers based on RAPD and ISSR markers seems to be the maiden attempt for Gladiolus cultivars.