Punjab Agricultural University, Ludhiana, , India. Received 20 April 2009; revised 4 November 2009; accepted 20 January 2010

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1 Indian Journal of Biotechnology Vol 9 July 2010, pp Biotization with Piriformospora indica and Pseudomonas fluorescens improves survival rate, nutrient acquisition, field performance and saponin content of micropropagated Chlorophytum sp. S K Gosal 1, A Karlupia 1, S S Gosal 2 *, I M Chhibba 3 and A Varma 4 1 Department of Microbiology, 2 School of Agricultural Biotechnology & 3 Department of Soils Punjab Agricultural University, Ludhiana, , India 4 Amity Institute of Microbial Technology, Amity University, Noida, , India Received 20 April 2009; revised 4 November 2009; accepted 20 January 2010 Micropropagated plantlets usually exhibit high mortality rate upon their transfer to soil as a result of transplantation shock caused by abiotic and biotic stresses and weak root system in the absence of beneficial microflora. Biotization of micropropagated Chlorophytum sp. with the fungus, Piriformospora indica and the bacterium, Pseudomonas fluorescens, improved plantlet survival rate, growth parameters, field performance, P content and the micronutrient acquisition. Biotized plants showed root colonization of P. indica in cortical cells of roots and exhibited the presence of pear shaped spores and hyphae as well as rhizospheric colonization of P. fluorescens. Microbial biotization enhanced plant survival up to 91.2% by dual inoculation over uninoculated control (78.8%), on transfer from laboratory to green house. Biotized field grown plants exhibited increase in root length, number of lateral roots, shoot dry weight, leaf length, number and dry weight of fleshy roots in dual inoculation at P F which were significantly better over single as well as uninoculated control. Plants inoculated with P. indica exhibited maximum chlorophyll content (8.76 mg g -1 ) while maximum P content (0.26%) was observed in dual inoculated plants, which was at par with P. indica alone even at low phosphorus. Higher saponin content was observed with both, P. indica alone as well as dual inoculations. Maximum acquisition of Cu (40 µg g -1 ) was observed in P. indica inoculated plants at P F level, which was at par with dual inoculation. In dual inoculated plants, the highest contents of Fe (4405 µg g -1 ), Zn (135 µg g -1 ) and Mn (160 µg g -1 ) were observed. Thus, microbial biotization improved survival and nutrient uptake by micropropagated Chlorophytum plants. Keywords: Chlorophytum, micropropagation, micronutrient, Piriformospora indica, Pseudomonas fluorescens, saponin Introduction Diverse biotic and abiotic challenges endanger the survival of plants that have sedentary life style; therefore, these plants of terrestrial ecosystem establish mutually beneficial relationship with useful microorganisms like bacteria and fungi. Microorganisms colonize the plants both externally and internally in their natural environment. Interaction of beneficial bacteria and fungi with plants can improve plant performance under stress environment, and consequently enhances yield. Biotization is the metabolic response of in vitro grown plant material to a microbial inoculum(s), leading to development and physiological changes enhancing biotic and abiotic stress resistance of the derived propagules. Studies with different crops like potato, onion, tomato, *Author for correspondence: Tel: Ext. 270; Fax: , ssgosal@rediffmail.com watermelon, cucumber and pepper have shown evidence of interaction between plantlets and microorganisms 1. The inoculation of seeds with beneficial microorganisms has been practiced for more than 50 years, but the inoculation of tissue culture raised plantlets is relatively a new aspect. Plant tissue culture is based on aseptic conditions, hence microorganisms including beneficial endophytes are treated as problem causing contaminants. But now-a-days microbial inoculants, primarily bacterial and mycorrhizal, are being evaluated as propagule priming agents for successful transplanting, also called biopriming. Biotization could be achieved during in vitro rooting or under ex vitro conditions. Chlorophytum sp., which is commonly called as safed musli, is an important medicinal herb belonging to family Liliaceae, having fleshy medicinal roots that contain saponins and alkaloids as curative for physical weakness, diabetes and arthritis 2.

2 290 INDIAN J BIOTECHNOL, JULY 2010 These bioactive compounds are also considered as aphrodisiac, vitalizers and immunity improving agents. The continued collection of these plants has resulted in the fast depletion of its population, may be due to low rate of multiplication through vegetative means and shy flowering behaviours 3. Chlorophytum is propagated through fleshy roots and the conventional propagation rate is rather slow, usually 1 to 10 plants/year. There is a big demand of safed musli with high saponin content in national and international drug market. Micropropagation through tissue culture technology is an attractive alternative for true to type, rapid and mass multiplication of planting material under disease free conditions 4. However, there have been scanty reports on micropropagation of Chlorophytum borivilianum 5,6. Micropropagated plantlets are vulnerable to transplantation shock resulting in higher mortality rate upon their transfer from lab to soil that is largely due to microbial vacuum. Biotization of micropropagated plantlets could be utilized to overcome the microbial vacuum to reduce their mortality rate. Biotization of tobacco 7,8 and cassava 9 plants with active cultures of AM (Arbuscular Mycorrhizal) fungi have improved the transplantation success. Being an obligate symbiont commercial application of AM as biohardening agent has not been substantially successful due to the difficulty in production of its reliable mass inoculum. Piriformospora indica, a root endosymbiotic fungus, mimics AM in many morphological, functional aspects and growth promotion Besides, it is easily cultivable and can be multiplied on synthetic media under axenic conditions 16. Therefore, it can be used as potential biotizing agent for micropropagated plants. Biotization can be successfully achieved under in vitro conditions during rooting where both bacterial and fungal agents can be applied to newly induced roots of tissue culture grown plantlets. Apart from endophytic fungi some soil bacteria also promote plant growth and such beneficial bacteria are referred to as plant growth promoting rhizobacteria (PGPR), which are preferentially associated with the roots 17. PGPR include several bacterial genera like Pseudomonas, Bacillus, Azospirillum, Azotobacter, etc. Among these, Pseudomonas deserves a special mention as P. fluorescens improves the plant growth directly or indirectly by production of plant growth substances, improving the uptake of certain nutrients from the soil and additionally show antagonistic effects against some important plant pathogenic microorganisms. In addition to this Pseudomonas species have been used to enhance tolerance to transplanting stress in potato 18. Considering these properties of P. indica and P. fluorescens attempts have been made for the first time to biotize micropropagated plantlets of Chlorophytum sp. at transplanting stage to study their role in improving survival rate. Further, the role of these biotizing agents in growth promotion as well as nutrient acquisition under field conditions has also been investigated. Materials and Methods Plant and Microbial Materials An experiment was conducted on micropropagated plantlets of Chlorophytum sp. Shoot cultures were established from mature roots, cultured on MS medium 19 supplemented with BAP (0.5 mg L -1 ). Shoot proliferation was achieved on MS medium supplemented with BAP (2.5 mg L -1 ). Shoot clumps were rooted (Fig. 1a) on MS medium supplemented with IBA (2.0 mg L -1 ). Complete plantlets thus obtained were hardened in trays (Fig. 1b) and biotized with P. indica 7 (Fig. 1c) and P. fluorescens. Microbial Cultures The fungus, Piriformospora indica (DSM 11827) used in the present study was grown on Potato Dextrose Broth at 28±2ºC for d. The bacterium, Pseudomonas fluorescens (local strain), used for biotization was procured from the Department of Microbiology, Punjab Agricultural University, Ludhiana, that was grown on King s B medium 20 at 28±2ºC for 24 hours. Biotization Micropropagated plantlets were biotized in polythene bags (one plantlet/bag) each containing 200 g of soil. Biotization with P. indica was done using broth culture containing both chlamydospores (100 spores ml -1 ) + mycelia (1g ml -1 )/polythene bag and P. fluorescens was inoculated with cells/polythene bag. The inoculum was given in the vicinity of the plant roots by soil drenching method at transplanting stage. Initial data on per cent plant survival were recorded after one month of transplantation in greenhouse maintained at 27±2ºC with 13/11 hours light/dark regime with 65-70% RH.

3 GOSAL et al: BIOTIZATION WITH PIRIFORMOSPORA INDICA & PSEUDOMONAS FLUORESCENS 291 Fig. 1 Micropropagation and biotization of C. borivilianum with P. indica and P. fluorescens: a. Rooting of shoot cultures of C. borivilianum; b. Hardening of plantlets in trays; c. Piriformospora indica; d. Micropropagated plants in greenhouse; e. Micropropagated plants in field; f. Fleshy/tuberous roots of C. borivilianum; & g. Spores of P. indica in biotized plant roots. Treatments There were four treatments viz. uninoculated control, biotization with P. indica, P. fluorescens individually and dual i.e. P. indica + P. fluorescens. Three phosphorus (P) levels i.e., P 0 (without P application), P 3/4 (37.5 kg ha -1 ) and P F (50 kg ha -1 ) through single super phosphate were used under field conditions in the present study. Field Studies One-month-old micropropagated greenhouse grown (Fig. 1d) plants were transferred in the field (Fig. 1e) using Randomized Block Design (RBD). Field soil was having medium P content (14.2 kg/ha) high K (160 kg/ha) and low organic carbon (0.3%) content. Soil was made porous by several ploughings and disking to facilitate tuberous root development. Effect of P. indica and P. fluorescens on the growth of micropropagated Chlorophytum sp. under field conditions was studied at three different P levels; zero, three-fourth and full recommended dose. Planting was done on raised beds (10-15 cm height) with a planting distance of 30 cm 15 cm. Nitrogen (@ 26 kg ha -1 ) was applied in the form of urea, in three splits in all the treatments and P was kg ha -1. Different growth parameters such as total length of the roots, dry weight, total number of lateral roots, shoot length and shoot dry weight were recorded. Plant Biomass Roots and shoots were washed in tap water and then in 0.1% HCl to remove the adhering soil particles. Dry weights were recorded by drying root and shoot samples in an oven at 70 C for 2 d. Root and shoot length was measured at two intervals of time i.e. after one month of transplantation to the soil under greenhouse. Number of fleshy roots (Fig. 1f), their yield and dry weight, number of leaves and their length were recorded at harvest under field conditions. Colonization Plantlets were microscopically examined for P. indica colonization by staining the roots with trypan blue method 21. Population count of P. fluorescens was noted by using standard serial dilution pour plate method and antibiotic resistance spectra as marker was studied to check the specificity of the same inoculated species. Plant Nutrients Analysis Nutrients The leaves were analyzed for P and micronutrients such as Cu, Fe, Zn and Mn. Properly processed samples were digested with Piper method 22 in diacid mixture consisting of nitric acid and perchloric acid (HNO 3 and HClO 4 in 3:1 ratio). Total P content was estimated colorimetrically using Vanadomolybdate

4 292 INDIAN J BIOTECHNOL, JULY 2010 method 23. Above-mentioned micronutrients were estimated using atomic absorption spectroscopy. Chlorophyll and Saponin Content Chlorophyll content in leaf tissues of plants was estimated spectroscopically 24. Per cent saponin content in roots was studied by following the Okwu and Josiah method 25. Results Plant Survival Survival rate as recorded after one month of transplantation in soil in the greenhouse was improved with microbial biotization. Maximum per cent survival was observed in plantlets with dual inoculation followed by single inoculations with P. indica and P. fluorescens over uninoculated control (Table 1). After one month of growth in the greenhouse, all the treated plants transferred in the field survived. Microbial Colonization Microscopic examination of P. indica infected stained roots revealed the presence of hyphae and large number of pear shaped spores of P. indica (Fig. 1g) in the cortical cells of stained roots. Maximum colonization of P. indica was observed in dual inoculated plants followed by single inoculation with P. indica after 30 d of biotization (Table 1). Colonization increased up to 90 d growth in soil. Maximum establishment of P. fluorescens in root rhizosphere of C. borivilianum after 30 d of transplantation was observed to be in dual inoculated treatment followed by single inoculation with P. fluorescens. Maximum population increased in dual inoculation after 90 d of transplantation (Table 1). Root-Shoot Length and Biomass After 30 d of growth in green house, significant increase in root length and number of lateral roots was observed over uninoculated control (Table 2). However, non-significant increase in root dry weight was recorded. An increase in shoot length and shoot dry weight was recorded in all the treatments over uninoculated control. Maximum root weight, shoot length and weight were found in dual inoculation, whereas number of lateral roots were maximum with P. fluorescens inoculation. Field Experiment Dual inoculated plants at full dose of recommended P recorded maximum root and shoot biomass, which were significantly higher as compared to single inoculations and uninoculated control plants (Table 3). Table 1 Extent of biotozation after different time intervals and its effect on survival of micropropagated plantlets of C. borivilianum Treatments Colonization of P. indica PGPR count CFU Plant survival (%) ( 10 6 g -1 soil) (%) 30 DAT 90 DAT 30 DAT 90 DAT 30 DAT 90 DAT Control P. indica P. fluorescens P. indica +P. fluorescens CD (5%) NS DAT Days after transplantation Treatments Table 2 Effect of P. indica and P. fluorescens on root and shoot growth of micropropagated C. borivilianum after 30 d of transplantation under greenhouse conditions Root Shoot Root length Root dry wt No. of lateral roots Shoot length Shoot dry wt (cm/plant) (g/plant) (cm/plant) (g/plant) Control P. indica P. fluorescens P. indica +P. fluorescens CD (5%) 1.24 NS

5 GOSAL et al: BIOTIZATION WITH PIRIFORMOSPORA INDICA & PSEUDOMONAS FLUORESCENS 293 Roots Shoots Leaves Table 3 Effect of P. indica and P. fluorescens on growth of micropropagated field grown C. borivilianum at harvest Parameters P-level Control P.indica P.fluorescens P. indica + P. fluorescens Length (cm/plant) No. of lateral roots Multiplication rate Dry wt (g/plant) Leaf length (cm/plant) No. of leaves per plant Mean P P 3/ P F Mean P P 3/ P F Mean P P 3/ P F Mean P P 3/ P F Mean P P 3/ P F Mean P P 3/ P F Mean P O : No P, P ¾ : 75% of recommended dose of P, P F : Full dose of recommended P CD (5%) Root Length Lateral roots Shoot multiplication Shoot dry wt Leaf length No. of leaves Treatments P-levels NS NS Interaction NS NS NS 0.79 Root In dual inoculated plants, maximum root length was observed at P 3/4 -level of P, which is statistically at par with the root length at full P level, followed by single inoculation with P. fluorescens at P F. It has clearly shown the role of P. fluorescens in increasing root length. Microbial inoculation resulted in significant increase in root length over control irrespective of P-levels. It was found that the root length at P 3/4 was statistically at par with P F, irrespective of culture. Maximum numbers of lateral roots at P F were found in dual inoculated plants, which is at par with P 3/4 -level. All the inoculated treatments showed higher number of lateral roots even at low P level, which was greater than the number of lateral roots at P F of control. Dual inoculation followed by single inoculation with P. fluorescens resulted in statistically significant increase in number of lateral roots over control. Application of P 3/4 resulted in total number of lateral roots statistically at par with those of P F. Shoot Dual inoculaton resulted in statistically significant increase in shoot multiplication rate over control. Maximum shoot multiplication rate (SMR) was observed at P 3/4 level, which was significantly higher as compared to SMR at P F -level, indicating that P. indica was more effective at lower P level. Inoculated plants performed better than uninoculated control plants at all the three P levels. In case of shoot dry weight, maximum shoot dry weight was recorded in dual inoculation with P. indica + P. fluorescens, which was significantly better over control at P F -level of P. Leaf Length and Number In dual inoculated plants maximum leaf length was recorded at P F -level followed by single inoculations.

6 294 INDIAN J BIOTECHNOL, JULY 2010 Plants inoculated with P. indica alone showed more leaf length at all P-levels than P F -level of control indicating that P. indica resulted in increase of leaf length. Biotized plants exhibited more number of shoot clumps as compared to control, which resulted in greater number of leaves. Biotized plants also showed a significant increase in number of leaves over the control plants at all P levels (Table 3). Micronutrients Micronutrient acquisition by plants was highly improved by microbial inoculation (Table 4). Dual as well as single inoculation with P. indica resulted in maximum uptake of Cu at P F -level followed by P. fluorescens treated plants, which was significantly higher as compared to control. Similarly, in case of Fe, dual inoculation showed maximum uptake of Fe at P F -level followed by single inoculation with P. indica and P. fluorescens. This was significantly higher as compared to uninoculated control. However, in case of Zn uptake, the increase was non significant. In case of Mn, maximum uptake was observed in dual inoculation followed by single inoculation with P. fluorescens and P. indica as compared to control at P F -level. Present study indicated that inoculation with P. indica and P. fluorescens helped in uptake of different micronutrients. Fertilization effect was evident regarding acquisition of all the micronutrients. P Content In dual inoculated plants, P content was observed to be maximum at P F level, which was at par with single inoculated plants with P. indica at P 3/4 -level of P indicating that P. indica alone can help in as much P uptake at P 3/4 -level as by dual inoculated plants at P F -level thus indicating 25 per cent saving of inorganic P fertilizer. It also helped in enhanced uptake of P even at P 0 indicating that P. indica performed better in P deficient soils (Table 5). Saponin and Chlorophyll Biotization with P. indica alone or in combination with P. fluorescens has led to increase in saponin content. Maximum chlorophyll content was observed in plants inoculated with P. indica alone at P F -level (Table 6). Even at P 0 level the chlorophyll content in S.No 1 Cu Table 4 Effect of P. indica and P. fluorescens on micronutrient content (µg/g) of field grown C. borivillianum at different levels of P at harvest Micronutrients (in ppm) P-level Control P. indica P. fluorescens P. indica + P. fluorescens Mean P P 3/ P F Mean Fe 3 Zn P P 3/ P F Mean P P 3/ P F Mean Mn CD (5%) P P 3/ P F Mean Cu Fe Zn Mn Treatments NS 0.50 P level NS 1.33 NS 0.43 Interaction NS 0.87

7 GOSAL et al: BIOTIZATION WITH PIRIFORMOSPORA INDICA & PSEUDOMONAS FLUORESCENS 295 P. indica inoculated plants was statistically higher as compared to uninoculated control and other treatments. Single inoculation with P. fluorescens and dual biotization were at par at P F -level, however, in both treatments chlorophyll content was significantly higher as compared to uninoculated control. Yield Increase in the number of medicinal fleshy roots was found in all inoculated treatments over uninoculated control at all three P-levels. The number of fleshy roots formation was statistically at par at both the P 3/4 as well as at P F level. Maximum weight of fleshy roots was recorded in dual inoculation at P F -level, followed by single inoculated plants with P. fluorescens or P. indica, with same fleshly root weight in both treatments. In all inoculated treatments, fresh weight of fleshy roots was more than roots of uninoculated plants at P F level. Highest root volume was recorded in plants having dual inoculation at P 3/4 -level. At P F -level root volume of P. indica treated plants and dual inoculated plants were found to be at par, whereas, inoculation showed significantly higher root volume than uninoculated control. As the total volume of root increased, the density of root decreased. Although there was decrease in density of roots but this decrease was Table 5 Effect of P. indica and P. fluorescens on P content of field grown C. borivilianum at harvest P-level P content (%) Treatments P O P 3/4 P F Control P. indica P. fluorescens P. indica + P. fluorescens CD (5%) Treatment: 0.35 non-significant with respect to treatments as well as P-levels (Table 7). Discussion In general, micropropagated plants exhibit high mortality rates upon their transfer to soil. Even 5 per cent mortality causes a huge loss during commercial plant production. The glasshouse and field possess relatively lower humidity, higher light intensity and septic environment that are stressful to micropropagated plants as compared to in vitro conditions 26. The benefit of any micropropagation system can be fully realized only by the successful transfer of plantlets from tissue culture vessels to the ambient conditions found ex vitro. Biotization of micropropagated Chlorophytum sp. with P. indica and P. fluorescens increased resistance of plants to such stresses at the time of transplantation, thus protected micropropagated young plantlets from transplantation shock. Plant growth and biomass is greatly influenced by nutrients and environmental conditions. P. indica helped in nutrient uptake by extending its hyphae in the rhizospheric region where even finest roots can not reach. Thus, role of P. indica and P. fluorescens in nutrient uptake and growth of micropropagated Chlorophytum sp. was studied in field conditions. Per cent survival of plantlets was observed maximum in dual inoculation, this must be due to the positive interaction between P. indica and P. fluorescens and their ability to enhance stress tolerance by protecting them from subsequent transplantation shock. Frommel et al also reported that Pseudomonas strain PsJN enhanced the tolerance to transplanting stress in potato and was found the most effective plant growth promoting bacterium under in vitro conditions 27. Maximum colonization of P. indica and bacterial count was observed in dual inoculated plants which may be due to the synergistic P-level Treatments Table 6 Effect of P. indica and P. fluorescens on saponin and chlorophyll content of field grown C. borivilianum at different levels of P at harvest Saponin content (%) Cholrophyll content (mg of chl g -1 of fresh leaf tissue) P O P 3/4 P F P O P 3/4 P F Control P. indica P. fluorescens P. indica +P. fluorescens CD (5%) ; Treatments: Saponin-NS; Chlorophyll content- 0.55

8 296 INDIAN J BIOTECHNOL, JULY 2010 Table 7 Effect of P. indica and P. fluorescens on yield of field grown micropropagated C. borivilianum at harvest Parameters P-level Control P.indica P.fluorescens P.indica + P.fluorescens Mean P No. of fleshy roots per plant P 3/ P F Mean Fresh wt. of P fleshy roots P 3/ (g/plant) P F Dry wt. of fleshy roots (g/plant) Volume of fleshy roots (cm 3 /plant) Density of fleshy roots (cm 3 /g/plant) effect of P. indica, with increased population of P. fluorescens by altering root exudation in the rhizosphere. This is attributed to the fact that mycorrhizal root tips tend to support slightly higher populations of Pseudomonas than non-mycorrhizal root tips, possibly due to provision of additional colonization sites or altered root exudation in mycorrhizosphere 28. In the present investigation, there was significant increase in root length and laterals of inoculated plants as compared to control due to the presence of beneficial microflora, which may have produced growth-promoting substances. Similar findings have been made using P. fluorescens Aur6 and ectomycorrhizal fungus, Suillus granulatus, as dual inoculants in Pinus halepensis 29. In the present study, dual inoculations showed significantly more number of lateral rootlets as compared to single inoculations. Better root system helped in more nutrient uptake, which resulted in healthy plants with more shoot biomass, as healthy roots result in a healthy plant. Significantly higher root-shoot biomass in different crops like maize, Bacopa, poplar with P. indica inoculation has also been reported 11. Elevated levels of P-content and micronutrients in P. indica inoculated and dual inoculated plants Mean P P 3/ P F Mean P P 3/ P F Mean P P 3/ P F Mean CD (5%) No.of fleshy Fresh wt Dry wt Vol. of fleshy Density of root fleshy roots fleshy roots roots fleshy roots Treatments NS P-levels NS NS NS Interaction NS NS NS NS NS explain their pivotal role in P uptake from soil extending their hyphae in extensive root system of Chlorophytum sp. and by extending their extramatricular hyphae deeper into the soil to overcome the need of P and micronutrients for the plant. Earlier, the role of P. indica in P uptake and providing it to plant for their growth in an energy dependent process 12 was also studied. In the present study, P. indica resulted in more P uptake especially, in the P deficient soils. Maximum enhancement of plant growth and acquisition of mineral nutrients other than P and Zn was observed in mycorrhizal plants in acidic soils 30. The yield of safed musli in terms of production of fleshy roots was observed to be positively affected by single as well as dual inoculations in the present investigation. The endophytic fungus, P. indica, has been found to colonize roots of a range of host plants and has caused increase in biomass and yield 31. Micropropagation has become an indispensable technology for mass production of elite plant material, but also faces a major limitation of inclement field conditions. Microbial biotization of weak roots of micropropagated plants with beneficial microflora as P. indica and P. fluorescens could increase plant survival and growth which clearly explains the

9 GOSAL et al: BIOTIZATION WITH PIRIFORMOSPORA INDICA & PSEUDOMONAS FLUORESCENS 297 potential of these organisms to act as growth promoting agents. In the present study, biotization of micropropagated C. borivilianum with P. indica and P. fluorescens improved plant survival ex vitro and additionally increased nutrient acquisition, improved growth, fleshy medicinal roots i.e. yield and as well as the alkaloids (secondary metabolites), saponin content of field grown plants. References 1 Nowak J, Review benefits of in vitro "biotization" of plant tissue cultures with microbial inoculants, In vitro Cell Dev Biol Plant, 34 (1998) Kirtikar K R & Basu B D, Liliaceae, Chlorophytum, in Indian Medical Plants (L M Basu, Allahabad, India) 1975, Singh A, Singh S, Patra D D, Singh M, Arya S J K & Khanuja S P S, Cultivation and processing technologies of safed musli (Chlorophytum borivillianum), J Med Arom Plant Sci, 26 (2004) Chahal G S & Gosal S S, Principles and procedures of plant breeding: Biotechnological and conventional approaches (Narosa Publishing House, New Delhi) Purohit S D, Dave A & Kukda G, Micropropagation of safed musli (Chlorophytum borivilianum) a rare Indian medicinal herb, Plant Cell Tissue Organ Cult, 39 (1994) Sharma U & Mohan J S, In vitro clonal propagation of Chlorophytum borivilianum Sant.et Fernand. a rare medicinal herb, from immature floral buds along with inflorescence axis, Indian J Exp Biol, 44 (2006) Varma A & Schuepp H, Mycorrhization of the commercially important plants, their applications in micropropagated plantlets, Crit Rev Biotechnol, 15 (1995) Lovato P E, Schuepp H, Trouvelot A & Gianinazzi A, Application of arbuscular mycorrhizal fungi (AMF) in orchard and ornamental plants, in Mycorrhiza- structures, function, molecular biology and biotechnology, 2 nd edn (Springer, Berlin) 1999, Azcon Anguilar C, Cantos M, Troncoso A, & Barea J, Beneficial effect of arbuscular mycorrhizas on acclimatization of micropropagated cassava plantlets, Sci Hortic, 72 (1997) Verma S, Varma A, Karl-Heinz R, Hasse I A, Kost G et al, Piriformospora indica gen. nov. a new root-colonizing fungus, Mycologia USA, 90 (1998) Varma A, Sahay N S, Butehom B & Franken P, Piriformospora indica a cultivable plant growth promoting root endophyte with similarities to arbuscular mycorrhizal fungi, Appl Environ Microbiol, 65 (1999) Varma A, Singh A, Sahay S, Sharma M S, Roy J et al, Piriformospora indica: An axenically culturable mycorrhizalike endosymbiotic fungus, in Mycota IX, edited by Hock Bled (Springer, Berlin) 2001, Rai M K, Acharya D, Singh A & Varma A, A positive growth response of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial, Mycorrhiza, 11 (2001) Pham G H, Singh A, Malla R, Kumari R, Prasad R et al, Interaction of P. indica with other microorganisms and plants, in Plant surface microbiology, edited by A Varma, L Abbott, D Werner & R Hampp (Springer-Verlag, Germany) 2004, Shahollari B, Varma A & Oelmüller R, A leucine rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana, Plant J, 50 (2007) Pham G H, Kumari R, Singh A, Sachdev M, Prasad R et al, Axenic cultures of Piriformospora indica, in Plant surface microbiology, edited by A Varma, L Abbott, D Werner & R Hampp (Springer-Verlag, Germany) 2004, Kloepper J W, Leong J, Teitze M & Schroth M N, Free living bacterial inocula for enhancing crop productivity, Trends Biotecnol, 7 (1989) Bashan Y, Inoculants of plant growth promoting bacteria for use in agriculture, Biotechnol Adv, 16 (1998) Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol Plant, 15 (1962) King O E, Ward M K & Raney D E, Two simple media for the demonstration of pyocyanin and fluorescin, J Lab Clin Med, 44 (1954) Phillips J M & Hayman D S, Improved procedures for clearing and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection, Trans Br Mycol Soc, 55 (1970) Piper C S, Soil and plant analysis (Ham Publishers, Bombay) Jackson M L, Soil chemical analysis (Prentice Hall, New Delhi) Anderson J M & Boardman M K, Studies on greening of dark brown bean plants II. Development of photochemical activity, Aust J Biol Sci, 17 (1964) Okwu D E & Josiah C, Evaluation of the chemical composition of two Nigerian medicinal plants, Afr J Biotechnol, 5 (2006) Hazarika B N, Acclimatization of tissue-cultured plants, Curr Sci, 85 (2003) Frommel M I, Nowak J & Lazarovits G, Treatment of potato tubers with a growth promoting Pseudomonas spp. Plant growth responses and bacterium distributing in the rhizosphere, Plant Soil, 150 (1993) Linderman R G & Paulitz T C, Mycorrhizal-rhizobacterial interactions, in Biological control of soil-borne plant pathogens, edited by D Homby (CAB International, Wallington) 1990, Rincon A, Diez B R, Fraile G S, Garcia L, Pascual M F et al, Colonization of Pinus halepensis roots by Pseudomonas fluorescens and interaction with the ectomycorrhizal fungus Suillus granulatus, FEMS Microbiol Ecol, 51 (2005) Clark R B, Arbuscular mycorrhizal adaptation, spore germination, root colonization and host plant growth and mineral acquisition at low ph, Plant Soil, 192 (1997) Serfling A, Wirsel S G R, Lind V & Deising H B, Performance of the biocontrol fungus Piriformospora indica on wheat under greenhouse and field conditions, Phytopathology, 97 (2007)