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


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 andHaselwandter (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 tempera-ture 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% NH 4 OH and 0.5% H 2 O 2 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 ReseaRch PaPeR supported in accordance to the finding. A positive correlation with organic carbon content in soil coincides with the findings of Mohammad et al.,(2003), who reported the same result while investigating under semi-arid environment of Jordan. Organic matter content in the soil increases the waterholding capacity of the soil (Brady and Weil, 1996;Panwar and tarafdar, 2005;Carrenhoet al., 2007) and, therefore, may facilitate a more favourable soil moisture condition for the AMF population. The phosphorus content (5.3 kg/hec) of the soil came out to be high as compared to Jodhpur soil (3.1 Kg/hec) which again suggested a lower population of AMF in Jaipur soil. The rhizosphere soils collected from field and successive pot cultures in Jodhpur have higher AMF spore densities compared to other sites (Panwar and Tarfdar, 2005). This may be because of poor soil fertility (in terms of available phosphorus) which results in higher AMF populations (Norani, 1996;Panwar and Tarafdar, 2005;Carrenhoet al., 2007 ). In general, soils remained alkaline in reaction, became low in organic matter content and available phosphorus status.
The isolated fungus (Fig: B) was confirmed by S.P.Institute of Biotechnology, Jaipur. Mycelium and spore was mounted in lacto phenol for identification. The identification was based on spore and mycelium colour, size, surface ornamentation and wall structure with reference to the descriptions and pictures provided by the International Collection of Vesicular and Arbuscular Mycorrhizal Fungi (http://invam.caf.wvu.edu) and originally published species descriptions ( Fig: C-D ).
The investigated sample is Scutellospora reticulata which is Arbuscular Mycorrhizal Fungi AMF was thus immobilized for the production of synthetic seed (Fig: E). Alginate was used as a matrix for the immobilization of fungus culture. Alginate beads have been widely used for entrapment of BCAs like PGPR and fungi (Trivedi & Pandey, 2008), including AMF (Declerck et al., 1996;Vega et al., 2003). Vega et al., 2003 also worked on growth of micropropagated bananas colonized by root organ culture produced arbuscular mycorrhiza fungi entrapped in calcium alginate beads. Some findings was also reported on immobilization of Pellets (2 mm) of the fungus T. Viride and their activity of β-glucosidase was investigated using cellbiose and salicin as substrates (Matteau and Saddler1982). Royer et al., (1983) also reported on hyphal outgrowth from beads that contained viable mycelial fragments. Immobilized fungi were first investigated for a role in biotransformations such as that of cortexolone to hydrocortisone by Curvularialunata (Sonomoto et al.,1983) and of glucose to itaconic acid by Aspergillus terreusn (Horitsu et al., 1983). Immobilized fungi are increasingly studied for application in processes which have traditionally only used free mycelia. The artificial seeds germinated on PDA media after 4-5 days ( Fig: F).
The in vitro raised plant was grown on Jaipur soil (Fig: G) inoculated with artificial seeds as well as soil without the seeds. The growth in both the cases was recorded alternatively after 15 days. The growth of 6 cm was observed after 30 days (Fig:  H) which was measured upto 15 cm after 90 days (Table-2) (Fig: I) in soil inoculated with artificial seeds while the plantlets raised on normal soil were unable to sustain (Fig: J). It led us to conclude that artificial seeds can be used to raise Leptadenia reticulata (Retz.)Wight and Arn.in arid as well as semi arid regions with the help of artificial seeds. Positive effect of AMF beads on rice plant has ample of evidences (Seli-cia& Bagyaraj, 1994;Xiao et al., 2010). Similar results were reported in cowpea (Vignaunguiculata) varieties where AM inoculated plants out performed than non-inoculated plants (control) in terms of growth, productivity parameters and nutrient uptake (Yaseen, et al., 2011). Finally in order to make a confirmatory check the roots of the in vitro raised plant was stained and observed at Department of Zoology, Jaipur. Cleared and stained roots showed the presence of vesicles, arbuscules and hyphae which confirmed the infection of in vitro raised plant with AMF. The fungi identified by (Fig: K-L).