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Birsa Agricultural University, Ranchi
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ThesisItem Open Access Protocol Development of In- Vitro Cultivation of Bamboo ( Dendrocolamus As per )(Birsa Agricultural University, Ranchi, 2002) Peddy Srikanth; Z.A. HaitherBamboos are perennial, woody and evergreen monocotyledonous arborescent grasses belonging to the family Poaceae (Graminae) and Sub-family Bambusoideae. There are about 75 genera and 1250 species of bamboos. Dendrocalamus asper is one of the economically important and widely cultivated bamboo species. The tender shoots of this plant can be consumed as food and is a good source of foreign exchange to various countries. Mature culms of this plant are used for pulp and paper manufacture. Bamboos are propagated artificially by different methods, including through seed. But utilization of seeds as propagating material is difficult and unreliable due to long and unpredictable flowering habit, short dormancy period of seed, poor viability, inborn microbial infestation, poor seed set during off-season flowering, seed sterility and large scale. Consumption of seeds by rodents and wild animals. The vegetative methods, on the other hand, are costly, lobour intensive, cumbersome and time. Consuming. These vegetative propagates are bulky, difficult to transport to distant places and their survival rates are also not very high. This limits large scale cultivation of bamboos in general. Under the situation, propagation through tissue culture seem to be a viable method for large scale propagation of the bamboo species. Therefore the present project was undertaken to establish a protocol for in-vitro propagation of Dendrocalamus asper. In the present study nodal segments (3-4cm) with axillary buds from young juvenile mother plant was used as explants. Surface sterilization using 0.1% (w/v) mercuric chloride (Hgcl₂) for 10 minutes followed by 3-4 times subsequent washing with sterile distilled water proved the best as it resulted the highest percentage (92.68%) of bud break after two weeks. The sterilized nodal segments were cultured aseptically on MS medium supplemented with 0-15 mgl-1 BAP and maximum shoot proliferation. (14-15 shoots per propgule) was achieved on medium supplemented with 12mgl¹ BAP. These proliferated axillary shoots were excised and subcultured on MS liquid medium +3 mgl BAP for the first two subcultures to increase the number of shoots. The shoot multiplication was achieved on both MS solid as well as liquid medium supplemented with 1-5 mgl¹! BAP Highest rate of shoot multiplication (fold) i.e., 15.77 was obtained on MS liquid medium supplemented with 3 mg l-¹ BAP in four weeks. MS solid medium supplemented with 3 mgl¹ BAP resulted only 8.55 fold. Incorporation of NAA (0.2-1.0 mgl ¹) to the medium along with BAP did not increase the rate of shoot multiplication and shoot length but it resulted in better quality erect shoots. MS medium in its full strength (1x) was found to be the most effective basal nutrient medium for shoot multiplication. The studies on sucrose concentration in the medium showed that 3% sucrose was essential for rapid multiplication of shoots. The effect of pH reflected that shoot multiplication occured even on acidic medium and highest rate of shoot multiplication (15.88) was obtained at pH 5.8. A regular subculture cycle at an interval of 4 weeks resulted in healthy cultures devoid of brown leaves and high rate of shoot multiplication. For in-vitro root regeneration on MS medium supplemented with 10mgl-¹ IBA yielded 90% rooting, 19.66 roots per propagule in four weeks, while 3 mg NAA supplemented MS medium resulted 91.66% rooting with 10 roots per propagule NAA resulted short roots while IBA resulted long roots. Addition of BAP (0.1-0.5 mgl ¹) to the rooting medium, neither enhanced root regeneration percentage nor improved the number of the in-vitro roots The cultured plantlets were successfully hardened under high humidity on sterilized soil sand FYM(1:1:1) mixtured with 1/2 strength MS nutrient medium irrigations (without organics).ThesisItem Open Access Protocol Development of invitro Clonal Propagation of Orchid ( Vanda Spa)(Birsa Agricultural University, Ranchi, 2002) Ranjeet Kumar Sinha; Z.A HaitherOrchids, one of the most beautiful group of flowering plants belong to the family Orchidaceae (Monocotyledons). The exquisite beauty of Orchid flowers due to brilliance in colour, remarkable range of sizes, manifold shapes, and variation in the form and wide range of distribution has aroused highest admiration throughout the world. The Orchid comprises about 800 genera with around 35,000 species. In India, about 1300 species of Orchids are found in Himalayas and others scattered in eastern and western Ghats. A vast majority of Indian Orchids are confined to mountain where they are distributed from base of hill to the elevation of 4300 m above mean sea level in climates ranging from tropical to temperate. Orchids are terrestrial, epiphytic, lithophytic or saprophytic but no Orchid is aquatic. The cut flower trade of Orchid involves 3% internationally. Major suppliers, like, Thailand, Netherlands and Singapore export flowers worth of US $ 80.0, 77.4 and 20.0 millions, in order per year. Due to their alkaloid, flavanoid, glycosides and other phytochemical constituents Orchids have high therapeutic value. The flower juice of Vanda coerulea is used to cure eye diseases. Cymbidium elegans, Cymbidium pubescens, Epicactic latifolia are used as local medicines. for treatment of nervous disorders. Orchids are also used in many countries as food or for making refreshing drinks. Unfortunately the natural population of Orchid is fast declining due to excessive collection and over harvesting by traders and botanical explorers. So there is need to cultivate and conserve the endangered Orchids. The conventional method of propagation is tedious and time taking. The alternative means of propagation is in vitro clonal propagation. Keeping this in mind the present experiment. on developing a viable protocol for in-vitro clonal propagation of Orchid (Vanda Miss Joaquim) was undertaken. The explant, like, shoot apex and shoot node were washed with detergent and teepol and then sterilized with 0.2% mercurio chloride for 10 minutes. The explants were cut in small pieces under laminar flow hood and subsequently inoculated in Murashige and Skoog (1962) medium modified with different plant growth regulators. The inoculated materials were cultured under aseptic condition at 25+2°C with 16 hours photoperiod of 3000 lux. The medium containing 2% sucrose, 2 mgl¹ BA+ 0.2 mgl¹ NAA was best for shoot node culture and developed 10 shoots/node and 4 leaves per shoot. Protocorm like bodies were developed in cytokinins combination. The combination 1 mgl BA + 0.3 mgl kinetin proved better for getting higher number of buds. However, 7.67 buds/node were found with 1mgl kinetin + 0.1 mgl¹ 2,4-D in around 46 days. It is worthy to note here that 2 mg1¹ kinetin in absence of 2,4-D yielded 7.56 buds/node which is statistically at par with the combination treatment 1mgl kinetin + 0.1 mgl 2,4-D. The shoot apex culture gave significant results on MS. medium supplemented with 2 mgl¹ BA + 0.5 mgl'¹ NAA, 1 mgl¹ BA + 0.2mgl kinetin and 1mgl kinetin + 0.1 mgl 2,4-D. Sub-culturing of plantlet on 2 mgl¹ BA and 0.5 mgl¹ NAA gave about 70-100 shoots. Best result on rooting was achieved on MS medium supplemented with 1 mgr¹ IBA+ 0.5 mgl¹ NAA, 1 mgr¹ NAA + 0.1mgl¹ BA. The maximum root length (49.5 mm and 60 mm) was obtained on medium supplemented with 1 mgr¹ NAA+ 0.1mgr¹ BA and 2 mgl¹¹ NAA + 1 mg/¹¹ IBA respectively. The cultured shoots were hardened successfully in pots containing bark, brick pieces and charcoal in 1:1:1 ratio.ThesisItem Open Access Optimization of protocol for in vitro regeneration in Hylocereus polyrhyzus (Weber) Britton and rose (Dragon fruit)(Birsa Agricultural University, Ranchi, 2019) RANI, NEHA; Banerjee, MadhuparnaDragon fruit is also known as pitaya or the strawberry pear, and is a beautiful tropical fruit that is sweet and crunchy in taste. Dragon fruit is actually a type of cactus which includes about 20 different species. Hylocereus polyrhyzus is one of the species in which the fruit is red fleshed. This plant is mainly popular in Southeast Asia and Latin America, however, dragon fruit is now grown and enjoyed all over the world. Dragon fruit was named after its appearance which is somewhat similar to an artichoke. The pointy scales around the oval shaped fruit is reminiscent of a dragon. The fruit comes in four varieties three are pink-skinned, one with white flesh, one with red flesh, and the third with purple flesh. The fourth variety has yellow skin with white flesh. All have tiny black seeds that are edible, just like kiwifruit. It is also rich with potassium, protein, fiber, Sodium and Calcium which is good for health than other fruit. Dragon fruit is usually propagated by seeds or cuttings. The in vitro culture is one of the best techniques for mass propagation and crop improvement to increase productivity leading to full supply of the demand. In the present work, an efficient in vitro method for plantlet regeneration explants of H. polyrhyzus (Variety- Red Singhal) was developed. The best survival percentage during surface sterilization of H. polyrhyzus (Variety- Red Singhal) explants were achieved by treating explant with 0.05% of HgCl2 for 20 minutes. Shoot multiplication was induced on MS media supplemented with 3.0 mg/l of BAP, 100 mg/l AdSO4 and 100 mg/l ascorbic acid. Highest number of shoots per explant (7 shoots) was observed after 45 days of inoculation in the same media.ThesisItem Open Access Growth curves of finger millet endophytes and maximum antimicrobial substance production(Birsa Agricultural University, Ranchi, 2019) ., ANURADHA; Pande, AnitaThe growth curves generated for the fourteen endophytes will serve as a reference point for any future studies on these endophytes. The endophytes should be preserved at harvest and not subjected to frequent subculture to prevent loss of production of important substances. Alternatively, the genes involved in the production of economically important substances can be cloned and expressed with a constitutive promoter.ThesisItem Open Access Allele mining for drought tolerance associated DREB genes in finger millet (Eleusine coracana (L.) Gaertn.) genotypes(Birsa Agricultural University, Ranchi, 2019) SINGH, AAKANSHA; Pande, AnitaThe information generated in the study will help discover novel genes involved in drought stress tolerance. It will also help in identification of endophytes which help the host plant combat various stresses. Though finger millet is an inherently drought tolerant crop, in depth studies are required to understand how it combats this important abiotic stress. The present study is a step in this direction.ThesisItem Open Access Characterization of Rhizobium isolates of Pigeon pea collected from the acidic soils of the state of Jharkhand.(Birsa Agricultural University, Ranchi, 2019) KUMARI, RANJEETA; Dubey, HimanshuAgriculture sector is the backbone of Indian economy however; agricultural productivity is very low in some countries. The low productivity is due to decreasing soil fertility many factors responsible for decreasing soil fertility such as soil acidity, soil erosion, and continuous cropping. Soil acidity o.ccurs when there is a buildup of acid forming element. Acidic soil in Jharkhand occupies approximately 90 percent area of the land. Most leguminous plant requires neutral or slightly acidic soil for growth, especially when depending on symbiotic nitrogen fixitation. Legumes, improves soil fertility through biological nitrogen fixation. It is reported to contribute approximately 40 kg N ha-1 soil acidity is one of the serious problem which affect growth of Rhizobia in the soil of Jharkhand Rhizobium is a gram negative bacteria, soil living bacteria. Rhizobium provides organic nitrogenous compound to the plant and plant provide organic compound made by photosynthesis, this relation is known as symbiosis. The present study focuses on the proteomic analysis of Rhizobium isolates of pigeon pea. This objective was achieved by performing the 2 DE using fourteen (14) different isolate of Rhizobium collected from different regimes of state of Jharkhand. We analysed unique spots with the help of Two Dimensional Gel Electrophoresis. ‘Unique' protein spots identified the genes implicated in the acid-soil tolerance. Two Dimensional Polyacrylamide Gel Electrophoresis of proteins is a robust and reproducible technique. It is the most widely used separation tool in proteomics, by utilizing MALDI-TOF-TOF approach subsequently genes which might be implicated to play crucial roles in imparting acid soil-tolerance to Rhizobium isolates of pigeon pea were identified from the isolates collected acid soils of the state of Jharkhand various spots of Rhizobium isolates were analyzed Protein play a major role to cope with abiotic stress. In this study we perform 2-DE electrophoresis to establish the reproducibility of the protein and MALDI TOF/TOF analysis to identify gene identity of the protein spot. Synthetic peptide was prepared against the protein spot which is expressed in the crop in response to the acid-soil tolerance. Subsequently, antibody is produced by against this peptide as a follow-up. This antibody is employed to perform the Western Blot Analysis in order to detect the proteins induced specifically in response to the acid soil tolerance regimes.ThesisItem Open Access Molecular analysis of antagonistic effect of Trichoderma to Fusarium in Grand Nain Banana plant(Birsa Agricultural University, Ranchi, 2019) Kumar Mahato, BharatProduction of banana is highly affected due to Fusarium Wilt also called Panama disease, caused by Fusarium oxysporum all around the world, leading to higher economic loss. The disease now is widespread and destructive in almost all the banana-growing states in India, Trichoderma harzianum was found to be most effective in inhibiting the growth of Fusarium oxysporum . plants treated with Trchoderma harzianum with Fusarium oxysporum at different interval 3 and 5 days ( Treated, Recovery + Fusarium). The result revealed that Trichoderma harzianum is effective in antagonizing Fusarium spp. by mycoparasitic activity and also by competing for rhizosphere colonization and nutrient. Plants treated with Trichoderma have some altered metabolic activities. Thus, any changes was analyzed by visual observation and marker analysis. The OPA series of primer was found to amplify the DNA sample of Trichoderma harzianum, Fusarium oxysporum and Banana. In standardization process the best temperature (annealing and extension) was selected with the better primers which amplified all the DNA samples. Distinct bands were observed. OPA 02 at 72⁰C extension 55⁰C annealing temperature showed some bands but the distinct bands were not observed. However, further refinement of the protocol is needed.ThesisItem Open Access Characterization of Rhizobium isolates of Chickpea from acidic soil of Jharkhand(Birsa Agricultural University, Ranchi, 2019) GOPE, SAPNA; Dubey, HimanshuOut of several gases present in the atmosphere nitrogen share the major portion (about 71%) and is found in the di-nitrogen (an inert) form. It is the component of many bio-molecules required for the growth and development of all organisms. Most of the eukaryotes are incapable of utilizing nitrogen directly from the environment; only a certain group of prokaryotes are genetically feasible to fix the atmospheric nitrogen into the biologically useful form like ammonia which is further utilized by eukaryotes. Rhizobium a gram negative bacteria associates symbiotically with legume crop and are genetically feasible in reducing (fixing) atmospheric nitrogen for leguminous crop. Legumes in turn provide shelter and energy to them. The specificity of Rhizobia to inoculate legume falls either in broad range host specificity to narrow range host specificity. Several abiotic stresses adversely influence the activity of Rhizobium. Soil pH is one of the stresses which hamper the symbiotic association between the two. As per the reports soil pH in the range of 6.5-7.0 are considered best in the case of leguminous crop for the optimal activity of the bacteria. Soil pH below or above this range minimizes the Biological Nitrogen Fixation (BNF) through Rhizobia. Two-dimensional electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique sort’s protein according to two independent properties in two discrete steps: the first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights. Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Thousands of different proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein is obtained. The aim of my work is to perform 2-D Electrophoretic profiling of Rhizobium isolates of Chickpea collected from various soil regimes. Chickpea plant has been taken as a model system for the collection of nodules. Chickpea , as a legume, improves soil fertility through (BNF) biological nitrogen fixation. Chickpea is a crop that provides cash income from its grain. It requires no N fertilizers owing to its ability to fix atmospheric N, and in rotation can improve the N nutrition and yield of subsequent cereals, one of the most important factors that affect the efficiency of symbiosis between Rhizobia and plants is the Ph of the soil in which they interact. The host plant to any symbiotic Rhizobium appears to be the limiting factor for growth in extreme pH, as most legumes require a neutral or slightly acidic soil for growth especially when they depend on symbiotic nitrogen fixation. The present work is associated with the comparison of the soluble protein fraction of the Rhizobium from the nodules of the legume chickpea grown in the normal environmental condition to the soluble protein fraction of the Rhizobium from the nodules of the legume chickpea grown in the acidic soil by employing Two- Dimensional (2-D) Gel Electrophoresis. Gel analysis shows differences in the expression of protein between the two types of Rhizobia.ThesisItem Open Access Characterization of Rhizobium isolates of Soybean collected from the acidic soils of the state of Jharkhand(Birsa Agricultural University, Ranchi, 2019) KUMAR, MIRTYUNJAY; Dubey, HimanshuOut of several gases present in the atmosphere nitrogen share the major portion (about 71%) and is found in the di-nitrogen (an inert) form. It is the component of many bio-molecules required for the growth and development of all organisms. Most of the eukaryotes are incapable of utilizing nitrogen directly from the environment; only a certain group of prokaryotes are genetically feasible to fix the atmospheric nitrogen into the biologically useful form like ammonia which is further utilized by eukaryotes. Rhizobium, a gram negative bacteria associates symbiotically with legume crop and are genetically feasible in reducing (fixing) atmospheric nitrogen for leguminous crop. Legumes in turn provide shelter and energy to them. The specificity of Rhizobia to inoculate legume falls either in broad range host specificity to narrow range host specificity. Several abiotic stresses adversely influence the activity of Rhizobium. Soil pH is one of the stresses which hamper the symbiotic association between the two. As per the reports soil pH in the range of 6.5-7.0 are considered best in the case of leguminous crop for the optimal activity of the bacteria. Soil pH below or above this range minimizes the Biological Nitrogen Fixation (BNF) through Rhizobia. My work is to characterize the Rhizobium isolates of soybean collected from acidic soils of the state of Jharkhand. Through studying the proteome of Rhizobium in acidic soil condition, the response of the isolates towards acidity of soil is being analyzed. At the molecular level, we find that the two dimensional gel analysis reveals a host of proteins which are found to be up-regulated or down regulated in response to different pH conditions. We hypothesize that the protein changes observed on two-dimensional electrophoresis in response to different pH of acidic soil reflected the molecular adaptation mechanism taking place in progress in soybean to combat and recover in response to abiotic stress such as acidic soil.
