- Email: [email protected]
Piriformospora indica: a new biological hardening tool for micropropagated plants
FEMS Microbiology Letters 181 (1999) 297^302
Piriformospora indica: a new biological hardening tool for micropropagated plants N.S. Sahay, A. Varma * School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India Received 25 February 1999; received in revised form 5 October 1999; accepted 11 October 1999
Abstract Piriformospora indica is a novel plant growth promoting root endophyte. Regenerated plantlets of tobacco subjected to two different biological hardening techniques showed 88^94% survival when inoculated with P. indica as compared to 62% survival of uninoculated controls under similar conditions. The tendency of the plantlet to overcome the stress in terms of revival capacity was maximal in the case of P. indica as compared to the control. The fungus has the potential to render protection to the micropropagated plantlets and help them escape the `transient transplant shock'. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : Piriformospora indica; Biological hardening; Micropropagated plantlet ; Survival
1. Introduction The acclimatization phase (physical/chemical) raises problems concerning survival and development of plantlets [1]. The mortality rate and the `transient transplant shock', on transfer of plantlets to the ¢eld, are very high. Stunted growth often leads to non-recovery of the plants and they are often attacked by soil microorganisms. Today, about 50% of £ori-horticultural plants are produced by micro-
* Corresponding author. Tel.: +91 (11) 610 7676; Fax: +91 (11) 618 7338; E-mail: [email protected]
propagation techniques. However, at the weaning stage, about 10^40% ( 6 70%) of plantlets either die or do not attain market standard, thereby causing signi¢cant losses at the commercial level [2,3]. The application of arbuscular mycorrhizal fungi (AMF) as a tool for biological hardening has solved a part of the problem [1]. The lack of an authentic AMF axenic culture is an inherent problem for commercial application. A new root endophyte, Piriformospora indica [4^7], has recently been reported which resembles the AMF with respect to morphological features, physiological characteristics and the mode of inter- and intracellular invasion of the mycelium to the cortical region of the root. Because of its ease of culture and plant growth promotional e¡ect [5], we have evaluated the potential of P. indica to improve the survival and establishment of tissue-cultureraised plants.
0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 4 2 - X
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2. Materials and methods 2.1. Tissue culture of tobacco 2.1.1. Seed sterilization and germination Seeds of tobacco (Nicotiana xanthi) were sterilized following the protocol of Gamborg and Phillip [8]. Sterilized seeds were transferred on 1/2 strength MS medium [9] with 1% sucrose, in a petri plate (90 mm in diameter) for germination. Plates were kept in the dark, at 24 þ 2³C. 2.1.2. Callus initiation and shoot induction Cotyledons and hypocotyls were excised from the germinated seedlings and cultured abaxial side up, or upside down (for cotyledons), on MS medium supplemented with 0.5 mg l31 benzyl amino purine (BAP) and 2 mg l31 naphthalene acetic acid (NAA) for callus initiation in a culture room at 24 þ 2³C and 2000 lux £uorescent light for 24 h. Small pieces of callus (about 0.5 g fresh weight) were excised and sub-cultured on fresh medium of the same composition every month to maintain a callus stock. Calluses obtained by the above procedure were cut into pieces, weighing approximately 0.5 g each. One piece was placed on each culture slant containing MS medium forti¢ed with 0.5 mg l31 BAP and 2 mg l31 NAA, whereas 0.8 mg l31 BAP and 0.1 mg l31 NAA were used for shoot/multiple shoot proliferation [10]. 2.2. Biological hardening of tobacco plantlets 2.2.1. Mass culture of P. indica One agar disc (about 1 cm in diameter) seeded with hyphae and spores of P. indica was placed in a petri plate (90 mm, disposable) containing modi¢ed minimal agar medium (pH 4.8). Inoculated petri plates were incubated for 7 days at 30³C in an incubator in the dark. 2.2.2. Pot preparation and inoculum placement Washed pots of size 10U6 cm were wiped with 70% ethanol. Autoclaved soil and acid-washed sterile sand were mixed in a ratio of 3:1 (w/w) for ¢lling the pots. Soil substratum was added ¢rst up to one third of
the height of pot [2]. A layer of live inoculum of P. indica mixed in a small amount of sterile soil was then layered over it. Above this layer, one layer of soil substratum was added to sandwich the inoculum between the two layers of substratum. The inoculum was added at a rate of 1% of the total soil content in the pot (w/v). Pots were placed in a plastic tray containing one tenth strength Hoagland solution for 20 min to allow absorption of nutrients and water. One plant was placed in the center of each pot. Roots of the plant were adjusted in such a manner that they were in constant touch with the inoculum. 2.2.3. Biological hardening technique (BHT) I and II Pots were kept in the mist chamber, maintained at 90% relative humidity, at 25 þ 2³C and 1000 lux of di¡used light. In the case of BHT-I, pots were not covered with a plastic bag but in BHT-II, pots were tightly covered with plastic bags, other conditions being similar in both techniques. The morphological condition of the whole plant, such as color and nature of the leaves, tensile strength and color of the stem, etc., was recorded at regular intervals. The rate of plantlet survival was evaluated after 8 weeks. Seven plants from each treatment were evaluated. The experiment was repeated a total of ¢ve times. 2.2.4. Staining and quanti¢cation of colonized roots Roots were washed thoroughly under running tap water and cut into 1-cm pieces. Segments were stained following the technique described by Dickson et al. and Phillip and Hayman [11,12]. Di¡erentiated hyphal structures, hyphae, vesicles, arbuscule and spores, were taken as an index for the presence or absence of colonization. Percent colonization was determined by the modi¢ed grid intersect technique [11,12].
3. Results and discussion Callusing from hypocotyls and regeneration of shoots was established on the MS medium. For good callusing and shoot bud regeneration, the concentration of hormone was optimized as NAA 2 mg l31 and BAP 0.5 mg l31 . For the production of large numbers of plantlets, multiple shoot regeneration
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Fig. 1. Biological hardening of tissue-culture-raised tobacco plantlets. Well-regenerated plantlets of tobacco (30 days old) were subjected to biological hardening by transferring them into sterile thermocol pots (10U6 cm) containing autoclaved soil and acid-washed sand in a ratio of 3:1 (w/w). P. indica was inoculated by placing the mycelium obtained from broth culture at a rate of 1% (w/v). Photographs were made after 8 weeks of biological hardening treatment. Left, control; right, treated with P. indica.
Table 1 Morphological features of tobacco plantlets after treatment with mycosymbionts Mycosymbiont
BHT-I Control
Glomus mosseae
P. indica
BHT-II Control Glomus mosseae P. indica
Weeks 2
4
4 lower leaves dried out of total 8 leaves per plant, stem green but lost tensile strength 4 lower leaves dried out of total 10 leaves per plant, stem green but lost tensile strength
5 leaves dried out of total 10 leaves per plant, stem turned brown
4 lower leaves dried out of total 10 leaves, stem green but lost tensile strength leaves lost the tensile leaves lost the tensile leaves lost the tensile
turgor pressure and stem strength turgor pressure and stem strength turgor pressure and stem strength
8
4 leaves dried out of total 12 leaves per plant, stems turned brown, a few still green and regained tensile strength 8^10 leaves dried out of total 16 leaves per plant, stem green and regained tensile strength 2^3 leaves turned yellow, stem turned brown 1^2 leaves dried, stem regained tensile strength none of the leaves was dried, plants were healthy, stem regained tensile strength
8 plants died 4 plants died, the rest were healthy 2 plants died, most were healthy
Micropropagated tobacco plantlets were subjected to biological hardening techniques. Substratum was a mixture of sterile soil and acidwashed sand in a ratio of 3:1. Live inoculum (spores, hyphae, colonized root, etc.) were included at 1% to each pot (10U6 cm). Plantlets in the tissue culture room were free of any microbial contamination and during the transfer from culture bottle/tubes to pots adequate precautions were taken to prevent contamination from outside.
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from the callus is essential. To achieve this goal, the hormone concentration was optimized as NAA 0.1 mg l31 and BAP 0.8 mg l31 . The time taken for regeneration of plantlets was 1^3 weeks for callusing, 1^2 weeks for shoot bud formation and 1^4 weeks for multiple shoot bud development.
multinucleate spores are shown in Fig. 2c. Surface colonization was higher than inter- and intracellular colonization. Percent root colonization is given in Table 2.
3.1. BHT-I and II Well-grown plantlets (about 2 months old) were selected for biological hardening. P. indica served as mycosymbiont. In the case of BHT-I, plants treated with fungus showed the greatest capacity of retaining tensile strength of stem as compared to control plants (Table 1). The survival rate of BHTII plants was always higher ( s 94%) than BHT-I (88%). On average the percent survival of the treated plants over the control was in the range of 117.5% (BHT-I) and 131.6% (BHT-II). The value of the control was considered 100%. The fresh weight of plantlets and percent colonization were also higher (Table 2). The overall growth of the biologically hardened plantlets is shown in Fig. 1. Morphological observations of the inoculated plants showed a better revival and regeneration capacity than untreated controls (Fig. 1). 3.2. Root colonization Fungal colonization commenced at 4^7 days after treatment (Fig. 2a). No appressorium-like structures were observed at the point of contact of hyphae with root epidermis, whereas a broken epidermal layer was observed near the point of contact (Fig. 2b). Mycelium traversing through the root cells is shown in Fig. 2b. Heavily colonized root segments with
C
Fig. 2. Root colonization of tobacco plants by P. indica. Colonized roots of tobacco were thoroughly washed and then stained with 0.05% trypan blue following the procedure of Dickson et al. [10] and Phillip and Hayman [11]. a: Extramatricial hyphae touching the root epidermis to initiate the process of colonization (U212). b: Initiation of root colonization by hyphae and mycelium traversing through the root (U212). Note the ruptured epidermis, which may be due to mycelial pressure. c: Colonized root segment with the formation of asexual spores, a sector of the ¢gure was enlarged to show the number of nuclei inside the spore (U212).
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Table 2 Plant biomass and percent colonization as a result of interaction of the tobacco plantlets with the mycosymbiont P. indica Mycosymbiont
Survival rate
Biomass (g plant31 )
Percent colonization
Control P. indica (BHT-I) P. indica (BHT-II)
62.5 þ 7.58 88 þ 2.7 94.1 þ 6.64
2.18 þ 0.19 3.08 þ 0.26 5.90 þ 0.13
nd 76.00 þ 25.10 86.66 þ 10.32
RM ANOVA ON RANKS test shows M2 = 8.40 with 3 degrees of freedom. P est = 0.0384, P exact = 0.0190. The di¡erence in median value among the treatment groups is greater than would be expected by chance, i.e. there is a statistically signi¢cant di¡erence (P = 0.0190). The root systems of all treated plants were colonized by the fungus. Data represents mean þ S.D. nd, not detected.
Lin [13] reported that in vitro raised cultures of Fragaria sp. inoculated with Glomus sp. had a signi¢cantly increased transplant survival rate and growth of the plants. In a series of publications a 100% survival rate of mycorrhized plantlets of hortensia, strawberry and raspberry was also reported [1,2,14]. Lovato et al. have summarized the work done in this ¢eld [15]. Plants inoculated with P. indica in these studies always did better in terms of plant biomass, percent root colonization and rate of survival. This supports the assumption that the mutualistic symbiosis is useful in the micropropagation technique for good survival and development of a healthy plant. This is only possible with treatment with friendly fungi at an early stage of growth, i.e. fungal treatment immediately after transfer from the closed culture vessel. AMF are undoubtedly useful, but they are strict biotrophs and hence cannot be cultivated on a de¢ned medium. P. indica provides a substitute and this can be multiplied on a synthetic medium and can easily be applied to the micropropagated plantlets. The positive data with treatment of P. indica that we obtained for tobacco were also reported for micropropagated plants of Bacopa manniera and Artemisia annua [16]. The authors hope that P. indica will obviate the present hindrance to mass cultivation of AMF on synthetic medium and this may be used on a commercial scale for the biological hardening of micropropagated plantlets.
Acknowledgements The authors are grateful to UGC and DBT, Government of India for partial funding of the research work. Figure 2C was made by Dr. T. Hurek at MPI, Marburg, Germany.
References [1] Varma, A. and Schuepp, H. (1994) Infectivity and e¡ectiveness of Glomus intraradices on micropropagated plants. Mycorrhiza 5, 29^37. [2] Varma, A. and Schuepp, H. (1995) Mycorrhization of commercially important micropropagated plants. Crit. Rev. Biotechnol. 15, 313^328. [3] Cassells, A.C., Mark, G.L. and Periappuram, C. (1996) Establishment of arbuscular mycorrhizal fungi in autotrophic strawberry cultures in vitro comparison with inoculation of microplants in vivo. Agronomie 16, 625^632. [4] Verma, S., Varma, A., Rexer, K.H., Hassel, A., Kost, G., Sarbhoy, A., Bisen, P.S., Butehorn, B. and Franken, P. (1998) Piriformospora indica gen. nov. sp. nov., a new root colonizing fungus. Mycologia 90, 896^903. [5] Varma, A., Sahay, N.S., Butehorn, B. and Franken, P. (1999) Piriformospora indica a cultivatable plant growth-promoting root endophyte with similarities to arbuscular mycorrhizal fungi. Appl. Environ. Microbiol. 65, 2741^2744. [6] Blechert, O., Kost. G., Hassel, A., Rexer, K.H. and Varma, A. (1999) In: Mycorrhiza Structure, Function, Molecular Biology and Biotechnology (Varma, A., Ed.), pp. 684^688. Springer-Verlag, Berlin. [7] Varma, A., Singh, A., Sudha, Sahay, N.S., Sharma, J., Roy, A., Kumari, M., Rana, D., Thakran, S., Bharti, K, Franken, P., Hurek, T., Blechert, O., Rexer, K.H., Kost, G., Hahn, A., Hock, B., Maier, W., Walter, M., Strack, D. and Kranner, I. (1999) In: Mycota IX (Esser, K. and Lemke, P.A., Eds.). Springer-Verlag, Berlin (in press). [8] Gamborg, O.L. and Phillips, G.C. (1996) Sterile techniques. In: Plant Cell Tissue and Organ Culture (Gamborg, O.L. and Phillips, G.C., Eds.), pp. 35^42. Narosa, New Delhi. [9] Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 431^497. [10] Phillips, G.C., Hubstenberger, J.F. and Elizabeth, E.H. (1996) Plant regeneration by organogenesis from callus and cell suspension cultures. In: Plant Cell, Tissue and Organ Culture (Gamborg, O.L. and Phillips, G.C., Eds.), pp. 67^79. Narosa, New Delhi. [11] Phillip, J.M. and Hayman, D.S. (1970) Improved procedures for clearing roots and staining parasitic and VAM fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158^ 161.
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[12] Dickson, S., Mandeep and Smith, S.M. (1998) Evaluation of vesicular arbuscular mycorrhizal colonization by staining. In: Mycorrhiza Manual (Varma, A., Ed.), pp. 77^84. SpringerVerlag, Berlin. [13] Lin, C.H. (1986) E¡ect of Three Glomus Endomycorrhizal Fungi on the Growth of Micropropagated Bananas and Asparagus Plantlets. M.S. Thesis, Horticulture Institute, National Taiwan University, Taipei. [14] Varma, A. and Schuepp, H. (1996) In£uence of mycorrhization on the growth of micropropagated plants. In: Concepts
in Mycorrhizal Research (Mukerji, K.G., Ed.), pp. 113^132. Kluwer Academic, London. [15] Lovato, P.E., Schuepp, H., Trouvelot, A. and Gianinazzi, S. (1999) Application of arbuscular mycorrhizal fungi in orchard and ornamental plants. In: Mycorrhiza, 2nd edn. (Varma, A. and Hock, B., Eds.), pp. 443^467. Springer-Verlag, Berlin. [16] Sudha (1998) In Vitro Study of Endosymbionts Associated with Tissue Culture Raised Medicinal Plants. Ph.D. Thesis, Hamdard University, New Delhi.
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Piriformospora indica: a new biological hardening tool for micropropagated plants N.S. Sahay, A. Varma * School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India Received 25 February 1999; received in revised form 5 October 1999; accepted 11 October 1999
Abstract Piriformospora indica is a novel plant growth promoting root endophyte. Regenerated plantlets of tobacco subjected to two different biological hardening techniques showed 88^94% survival when inoculated with P. indica as compared to 62% survival of uninoculated controls under similar conditions. The tendency of the plantlet to overcome the stress in terms of revival capacity was maximal in the case of P. indica as compared to the control. The fungus has the potential to render protection to the micropropagated plantlets and help them escape the `transient transplant shock'. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : Piriformospora indica; Biological hardening; Micropropagated plantlet ; Survival
1. Introduction The acclimatization phase (physical/chemical) raises problems concerning survival and development of plantlets [1]. The mortality rate and the `transient transplant shock', on transfer of plantlets to the ¢eld, are very high. Stunted growth often leads to non-recovery of the plants and they are often attacked by soil microorganisms. Today, about 50% of £ori-horticultural plants are produced by micro-
* Corresponding author. Tel.: +91 (11) 610 7676; Fax: +91 (11) 618 7338; E-mail: [email protected]
propagation techniques. However, at the weaning stage, about 10^40% ( 6 70%) of plantlets either die or do not attain market standard, thereby causing signi¢cant losses at the commercial level [2,3]. The application of arbuscular mycorrhizal fungi (AMF) as a tool for biological hardening has solved a part of the problem [1]. The lack of an authentic AMF axenic culture is an inherent problem for commercial application. A new root endophyte, Piriformospora indica [4^7], has recently been reported which resembles the AMF with respect to morphological features, physiological characteristics and the mode of inter- and intracellular invasion of the mycelium to the cortical region of the root. Because of its ease of culture and plant growth promotional e¡ect [5], we have evaluated the potential of P. indica to improve the survival and establishment of tissue-cultureraised plants.
0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 4 2 - X
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2. Materials and methods 2.1. Tissue culture of tobacco 2.1.1. Seed sterilization and germination Seeds of tobacco (Nicotiana xanthi) were sterilized following the protocol of Gamborg and Phillip [8]. Sterilized seeds were transferred on 1/2 strength MS medium [9] with 1% sucrose, in a petri plate (90 mm in diameter) for germination. Plates were kept in the dark, at 24 þ 2³C. 2.1.2. Callus initiation and shoot induction Cotyledons and hypocotyls were excised from the germinated seedlings and cultured abaxial side up, or upside down (for cotyledons), on MS medium supplemented with 0.5 mg l31 benzyl amino purine (BAP) and 2 mg l31 naphthalene acetic acid (NAA) for callus initiation in a culture room at 24 þ 2³C and 2000 lux £uorescent light for 24 h. Small pieces of callus (about 0.5 g fresh weight) were excised and sub-cultured on fresh medium of the same composition every month to maintain a callus stock. Calluses obtained by the above procedure were cut into pieces, weighing approximately 0.5 g each. One piece was placed on each culture slant containing MS medium forti¢ed with 0.5 mg l31 BAP and 2 mg l31 NAA, whereas 0.8 mg l31 BAP and 0.1 mg l31 NAA were used for shoot/multiple shoot proliferation [10]. 2.2. Biological hardening of tobacco plantlets 2.2.1. Mass culture of P. indica One agar disc (about 1 cm in diameter) seeded with hyphae and spores of P. indica was placed in a petri plate (90 mm, disposable) containing modi¢ed minimal agar medium (pH 4.8). Inoculated petri plates were incubated for 7 days at 30³C in an incubator in the dark. 2.2.2. Pot preparation and inoculum placement Washed pots of size 10U6 cm were wiped with 70% ethanol. Autoclaved soil and acid-washed sterile sand were mixed in a ratio of 3:1 (w/w) for ¢lling the pots. Soil substratum was added ¢rst up to one third of
the height of pot [2]. A layer of live inoculum of P. indica mixed in a small amount of sterile soil was then layered over it. Above this layer, one layer of soil substratum was added to sandwich the inoculum between the two layers of substratum. The inoculum was added at a rate of 1% of the total soil content in the pot (w/v). Pots were placed in a plastic tray containing one tenth strength Hoagland solution for 20 min to allow absorption of nutrients and water. One plant was placed in the center of each pot. Roots of the plant were adjusted in such a manner that they were in constant touch with the inoculum. 2.2.3. Biological hardening technique (BHT) I and II Pots were kept in the mist chamber, maintained at 90% relative humidity, at 25 þ 2³C and 1000 lux of di¡used light. In the case of BHT-I, pots were not covered with a plastic bag but in BHT-II, pots were tightly covered with plastic bags, other conditions being similar in both techniques. The morphological condition of the whole plant, such as color and nature of the leaves, tensile strength and color of the stem, etc., was recorded at regular intervals. The rate of plantlet survival was evaluated after 8 weeks. Seven plants from each treatment were evaluated. The experiment was repeated a total of ¢ve times. 2.2.4. Staining and quanti¢cation of colonized roots Roots were washed thoroughly under running tap water and cut into 1-cm pieces. Segments were stained following the technique described by Dickson et al. and Phillip and Hayman [11,12]. Di¡erentiated hyphal structures, hyphae, vesicles, arbuscule and spores, were taken as an index for the presence or absence of colonization. Percent colonization was determined by the modi¢ed grid intersect technique [11,12].
3. Results and discussion Callusing from hypocotyls and regeneration of shoots was established on the MS medium. For good callusing and shoot bud regeneration, the concentration of hormone was optimized as NAA 2 mg l31 and BAP 0.5 mg l31 . For the production of large numbers of plantlets, multiple shoot regeneration
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Fig. 1. Biological hardening of tissue-culture-raised tobacco plantlets. Well-regenerated plantlets of tobacco (30 days old) were subjected to biological hardening by transferring them into sterile thermocol pots (10U6 cm) containing autoclaved soil and acid-washed sand in a ratio of 3:1 (w/w). P. indica was inoculated by placing the mycelium obtained from broth culture at a rate of 1% (w/v). Photographs were made after 8 weeks of biological hardening treatment. Left, control; right, treated with P. indica.
Table 1 Morphological features of tobacco plantlets after treatment with mycosymbionts Mycosymbiont
BHT-I Control
Glomus mosseae
P. indica
BHT-II Control Glomus mosseae P. indica
Weeks 2
4
4 lower leaves dried out of total 8 leaves per plant, stem green but lost tensile strength 4 lower leaves dried out of total 10 leaves per plant, stem green but lost tensile strength
5 leaves dried out of total 10 leaves per plant, stem turned brown
4 lower leaves dried out of total 10 leaves, stem green but lost tensile strength leaves lost the tensile leaves lost the tensile leaves lost the tensile
turgor pressure and stem strength turgor pressure and stem strength turgor pressure and stem strength
8
4 leaves dried out of total 12 leaves per plant, stems turned brown, a few still green and regained tensile strength 8^10 leaves dried out of total 16 leaves per plant, stem green and regained tensile strength 2^3 leaves turned yellow, stem turned brown 1^2 leaves dried, stem regained tensile strength none of the leaves was dried, plants were healthy, stem regained tensile strength
8 plants died 4 plants died, the rest were healthy 2 plants died, most were healthy
Micropropagated tobacco plantlets were subjected to biological hardening techniques. Substratum was a mixture of sterile soil and acidwashed sand in a ratio of 3:1. Live inoculum (spores, hyphae, colonized root, etc.) were included at 1% to each pot (10U6 cm). Plantlets in the tissue culture room were free of any microbial contamination and during the transfer from culture bottle/tubes to pots adequate precautions were taken to prevent contamination from outside.
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from the callus is essential. To achieve this goal, the hormone concentration was optimized as NAA 0.1 mg l31 and BAP 0.8 mg l31 . The time taken for regeneration of plantlets was 1^3 weeks for callusing, 1^2 weeks for shoot bud formation and 1^4 weeks for multiple shoot bud development.
multinucleate spores are shown in Fig. 2c. Surface colonization was higher than inter- and intracellular colonization. Percent root colonization is given in Table 2.
3.1. BHT-I and II Well-grown plantlets (about 2 months old) were selected for biological hardening. P. indica served as mycosymbiont. In the case of BHT-I, plants treated with fungus showed the greatest capacity of retaining tensile strength of stem as compared to control plants (Table 1). The survival rate of BHTII plants was always higher ( s 94%) than BHT-I (88%). On average the percent survival of the treated plants over the control was in the range of 117.5% (BHT-I) and 131.6% (BHT-II). The value of the control was considered 100%. The fresh weight of plantlets and percent colonization were also higher (Table 2). The overall growth of the biologically hardened plantlets is shown in Fig. 1. Morphological observations of the inoculated plants showed a better revival and regeneration capacity than untreated controls (Fig. 1). 3.2. Root colonization Fungal colonization commenced at 4^7 days after treatment (Fig. 2a). No appressorium-like structures were observed at the point of contact of hyphae with root epidermis, whereas a broken epidermal layer was observed near the point of contact (Fig. 2b). Mycelium traversing through the root cells is shown in Fig. 2b. Heavily colonized root segments with
C
Fig. 2. Root colonization of tobacco plants by P. indica. Colonized roots of tobacco were thoroughly washed and then stained with 0.05% trypan blue following the procedure of Dickson et al. [10] and Phillip and Hayman [11]. a: Extramatricial hyphae touching the root epidermis to initiate the process of colonization (U212). b: Initiation of root colonization by hyphae and mycelium traversing through the root (U212). Note the ruptured epidermis, which may be due to mycelial pressure. c: Colonized root segment with the formation of asexual spores, a sector of the ¢gure was enlarged to show the number of nuclei inside the spore (U212).
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Table 2 Plant biomass and percent colonization as a result of interaction of the tobacco plantlets with the mycosymbiont P. indica Mycosymbiont
Survival rate
Biomass (g plant31 )
Percent colonization
Control P. indica (BHT-I) P. indica (BHT-II)
62.5 þ 7.58 88 þ 2.7 94.1 þ 6.64
2.18 þ 0.19 3.08 þ 0.26 5.90 þ 0.13
nd 76.00 þ 25.10 86.66 þ 10.32
RM ANOVA ON RANKS test shows M2 = 8.40 with 3 degrees of freedom. P est = 0.0384, P exact = 0.0190. The di¡erence in median value among the treatment groups is greater than would be expected by chance, i.e. there is a statistically signi¢cant di¡erence (P = 0.0190). The root systems of all treated plants were colonized by the fungus. Data represents mean þ S.D. nd, not detected.
Lin [13] reported that in vitro raised cultures of Fragaria sp. inoculated with Glomus sp. had a signi¢cantly increased transplant survival rate and growth of the plants. In a series of publications a 100% survival rate of mycorrhized plantlets of hortensia, strawberry and raspberry was also reported [1,2,14]. Lovato et al. have summarized the work done in this ¢eld [15]. Plants inoculated with P. indica in these studies always did better in terms of plant biomass, percent root colonization and rate of survival. This supports the assumption that the mutualistic symbiosis is useful in the micropropagation technique for good survival and development of a healthy plant. This is only possible with treatment with friendly fungi at an early stage of growth, i.e. fungal treatment immediately after transfer from the closed culture vessel. AMF are undoubtedly useful, but they are strict biotrophs and hence cannot be cultivated on a de¢ned medium. P. indica provides a substitute and this can be multiplied on a synthetic medium and can easily be applied to the micropropagated plantlets. The positive data with treatment of P. indica that we obtained for tobacco were also reported for micropropagated plants of Bacopa manniera and Artemisia annua [16]. The authors hope that P. indica will obviate the present hindrance to mass cultivation of AMF on synthetic medium and this may be used on a commercial scale for the biological hardening of micropropagated plantlets.
Acknowledgements The authors are grateful to UGC and DBT, Government of India for partial funding of the research work. Figure 2C was made by Dr. T. Hurek at MPI, Marburg, Germany.
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