Reduction of Biotic Stress in Leptadenia Reticulata (Retz.) Wight & Arn. Using Synthetic Seeds Prepared Via Immobilizing Arbuscular Mycorrhiza Fungi and To Carry Out its Conservation

The present study reveals the usefulness of Leptadenia reticulata (Retz.) Wight and Arn. for several medicinal purposes and the presence of various phytochemical indicates the potential of this plant as a source of potent drugs. Huge demand and multipurpose uses of this plant in pharmaceutical industries, population bloom, urbanization, over-exploitation and recurring drought and famine make this plant species endangered Hence, there is a need for developing alternative method for quick and efficient method for conservation and utilisation of germplasm of Leptadenia reticulata (Retz.) Wight & Arn. Our work here is on development of a novel method to conserve this endangered medicinal plant by the means of Micropropagation and raising the in vitro grown plant along with artificial seeds prepared by immobilizing AMF. The development of such a protocol will help us to achieve a large number of individual plants even in the areas where they sustain rarely due to poor survival condition. Introduction Leptadenia reticulata (Retz.) Wight and Arn. plant species are found in arid regions of Rajasthan ( Kiran Bala et al., 1989; Pande et al., 1999) which are characterized by poor sandy soils, low organic matter, uncertain and erratic rainfall, high wind velocity and generally experience water deficit during the growth period. Arbuscular mycorrhizal fungi are a major component of rhizosphere microflora in natural ecosystem and play significant role in the re establishment of nutrient cycling in native ecosystem (Peterson et al., 1985). ). The population of AMF varies greatly and their distribution is affected by various biotic and abiotic factors (Mohammad et al., 2003). Koske (1981) also reported that distribution of AM fungi is related to soil and environmental conditions. Hayman (1983) reported better establishment of vegetation in arid areas by using AMF as these fungi may often enhance plant absorption of phosphorus and other elements, improve water uptake and its transport to plants, and enable the plants to withstand high temperatures (Yaseen et al., 2011). Turnau and Haselwandter (2002) also considered AMF as a tool for re-establishment of endangered plant species. In the present investigation, in vitro raised Leptadenia reticulata (Retz.) Wight & Arn. with and without artificial mycorrhiza seeds were grown in the normal soil. The objective of this study is to determine the response of these seeds to plant survivality and growth. Material and methods The work was carried out at Department of Biotechnology, Poddar International College, Jaipur. The soil sampling of both the sites (site where the plant grows i.e. Jodhpur as well as the site where plant does not grow i.e. Jaipur, Fig: A) was done from Durgapura Agricultural Research Centre, Jaipur. The fungus was isolated from the soil in which Leptadenia reticulata (Retz.)Wight & Arn. grows. This was done by spreading the soil on PDA plates and incubating the plates at 26° ± 2°C for growth. The isolated colony was identified by biochemical testing from S.P. Institute of Biotechnology, Jaipur. The fungi from the plate were spread onto a glass slide with the help of inoculation loop and stained with trypan blue and observed under microscope. The liquid culture of the isolated AMF was prepared by procuring the fungi from the culture plate and diluting it in 10 ml of water. Further the culture was kept for incubation at 25° ± 2° C for 48 hrs. Sodium alginate (3%) was mixed with liquid culture of AMF and the solution was singly dipped into a calcium chloride solution for a few seconds. The so-formed beads were kept on PDA plates for germination. The plates were kept at an incubation temperature of 28°± 2°C for 5 – 7 days. The growth was observed. The in vitro raised plant Leptadenia reticulata (Retz.) Wight & Arn was grown in Jaipur soil with the prepared artificial mycorrhizal seeds. Another in vitro raised plant Leptadenia reticulata (Retz.) Wight & Arn was also grown in the same soil but without the inoculation of prepared artificial seeds. The growth of both the plants was checked alternatively after every 15 days. The length of the plant was recorded. Also, the roots of the in vitro raised plant along with the artificial seeds were sampled and stained to confirm the infection of AMF . To determine the colonization, root samples were collected from the in vitro grown plants and were washed under running tap water and staining of roots was done by the method of Phillips and Hayman (1970). The roots were cut into pieces of 1.0 cm length and placed in 10 % KOH solution, which was kept at boiling point for about 10 minutes. The root samples were isolated and rinsed with distilled water until the brown colour disappeared. Post clearing bleaching was done with alkaline hydrogen peroxide (0.5% NH4OH and 0.5% H2O2 v/v in distilled water). Roots were rinsed again with distilled water ,treated with 1% HCL and stained with 0.05% w/v trypan blue in lactic acid – glycerol. Assessment of colonization was done microscopically. A segment of root was counted as infected when hyphae, vesicles or arbuscules were observed. Result and Discussion Arbuscular mycorrhizal fungi are a major component of rhizosphere microflora in natural ecosystem and play significant role in the re establishment of nutrient cycling in native ecosystem (Peterson et al., 1985). Further, Panwar and Vyas (2002) indicated the significance of AMF in re-establishment and conservation of endangered plants in arid areas. During the present study it is evident that the AMF were affected by soil pH, organic carbon and phosphorus content (Table-1). A significant positive correlation with pH and organic carbon was recorded during present investigation. The Jodhpur soil had pH of 8.3 which is slightly higher than Jaipur soil (8.1). This difference may be seen to support the fact that Jodhpur soil is alkaline in nature and thus is favourable for growth of AMF. Blaszkowski (1993) while investigating plant communities in Poland observed a significant positive correlation between AMF and soil pH. The major difference noted out was among the initial organic carbon content of both the soils. The organic matter content found in Jaipur soil (0.15 %) out to be was considerably lower than Jodhpur soil (0.19%) which possesses high AMF population. This conclusion was