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Studies on the Interaction of Legume Root Callus with Rhizobium
Originalarbeiten . Original Papers
Department of Botany, Sri Venkateswara College, University of Delhi, New Delhi 110021, India, and Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India
Studies on the Interaction of Legume Root Callus with Rhizobium V. RANGA RAO and N. S. SUBBA RAO
With 8 figures Received August 8,1974 . Accepted August 31,1974
Summary Root calli of three legumes - Arachis hypogaea, Cajanus cajan and Cicer arietinum were inoculated with homologous rhizobia. Measurable nitrogenase activity was detected when inoculated calli of Arachis, Cajanus and Cicer were tested. Light microscopic observations of transections of inoculated calli revealed bacterial penetration in the form of infection thread-like structures and host cells filled with bacteria or bacteroid-like cells. In Cicer, characteristic nodular outgrowths were seen whose cells were also filled with bacteria. None of these observations were made in uninoculated calli indicating the features of possible symbiosis in legume root calli upon inoculation with Rhizobium.
Introduction To overcome the limitations imposed by changes in environmental factors influencing the phenomenon of symbiosis under natural conditions, refined techniques have been recently employed to study legume-Rhizobium interactions. One such technique has been the use of root tissue cultures (either cell aggregates or callus masses) to study the interaction between Rhizobium and legumes under controlled environmental conditions. Despite an initial unsuccessful attempt (VELIKY and LA RUE, 1966), symbiosis has been found to occur in root tissue cultures of soybean (HOLSTEN et aI., 1971; PHILLIPS, 1974; CHILD and LA RUE, 1974) and Pea (RANGA RAO et aI., 1974) inoculated with Rhizobium. The present work relates to root callus-Rhizobium interactions with three tropical legumes - Arachis hypogaea, Cajanus cajan and Cicer arietinum.
Materials and Methods Seeds of three legumes - Arachis hypogea, Cajanus cajan and Cicer arzetznum were surface sterilised with cetavlon (1 0/0) and germinated on White's basal medium. Explants
Z. P/lanzenphysiol. Bd. 80. S. 14-20. 1976.
Interaction of legume root callus with Rhizobium
15
of roots (1-2 cm) were transferred to White's basal medium supplemented with 2,4-D and Kinetin. Profuse callus formation was observed within four weeks. Small, actively growing callus pieces were later transferred to White's basal medium and allowed to remain there for 7 days. Pure cultures of Rhizobium were isolated from nodules of individual legumes using the methods of VINCENT (1970). They were subsequently maintained on Yeast extract mannitol (YEM) agar slants and inoculated to YEM liquid medium and incubated for 3 days on a horizontal shaker rotating at 100 RPM. One ml of the resulting broth containing rhizobial cells was transferred to tubes containing White's basal medium supporting the growth of individual calli. Un inoculated callus cultures on White's basal medium were used as controls. All these steps were carried out under laboratory day light conditions. Upon inoculation, the calli were transferred to darkness and incubated for a further period of one week. Subsequently, they were subjected to two or three washings in White's basal liquid medium before transferring them to White's basal solid medium. After a further period of two weeks incubation, the inoculated and uninoculated calli of Arachis, Cajanus and Cicer were analysed for nitrogenase activity by acetylene reduction technique (HARDY et a!., 1968) by transferring approximately 5 mm size calli to vials and injecting with 0.5 ml of acetylene (C 2 H 2 ). The conversion of C 2 H 2-C 2 H 4 was analysed by gas chromatography. Parallel samples of calli of all three legumes were passed through tertiary butyl alcohol series and embedded in paraffin wax. Microtome sections were cut to 8-10 fl. Slides were stained with safraninFast Green combination and made permanent by mounting in Canada Balsam.
Results
Visual as well as light microscopic observations revealed the presence of bacterial aggregates around the inoculated calli which remained fresh and continued to grow in the presence of Rhizobium. Nitrogenase activity was detectable in inoculated calli and the acetylene reduction assay data for Arachis hypogaea, Cajanus cajan and eicer arietinum are presented in Table 1. The mode of penetration of Rhizobium and the establishment of bacteroid-like structures in the cells of the callus mass of individual legumes seem to differ and hence have been described under separate headings.
Table 1: Nitrogenase activity in callus samples of Arachis hypogaea, Cajanus cajan and Cicer arietinum inoculated with homologous rhizobia.
Inoculated sample number
1. 2. 3. 4. Control sample
Nitrogenase activity
nmoles C 2 Hig. fresh weight of callus 24 hrs
Arachis hypogaea
Cajanus cajan
178 84 131.5 77.5 No activity
275 275 210 225 No activity
Cicer arietinum 56 105 112 150 No activity
Z. Pjlanzenphysiol. Bd. 80. S. 14-20. 1976.
16
V.
RANGA RAO
and N.
S. SUIlBA RAO
Arachis hypogaea
The appearance of infection thread-like structures differed depending on the plane at which the callus was cut. While in some sections the threads appeared to start intercellularly from the periphery of the callus and enter deep inside continuously (Fig. 1), in others several disconnected threads were seen apparently inducing meristematic activity in cells surrounding them (Fig. 2). Such meristematic cells were filled with bacteroid-like cells and were bigger than their neighbours (Fig. 3). Cajanus cajan
The callus was composed of loose aggregates of cells. The infection thread-like structures were haphazard and very rarely organized and continuous. Meristematic activity was frequently observed around infection threads and meristematic cells were not only filled with bacteroids (Fig. 4) but were also larger in size than the surrounding cells. Cicer arietinum
The aggregation of bacteria in the form of a crust on the periphery of the callus of Cicer (Fig. 5) appeared to be more intense than on the periphery of those of Cajanus and Arachis. At some sites on the callus, peripheral nodular outgrowths (Fig. 6) were observed as a result of intense meristematic activity of callus cells which may be attributed to the activity of bacteria. Nearly half of the peripheral cells of the outgrowths contained bacteroid-like cells (Fig. 7) whereas the central portion of such outgrowths had relatively few cells which contained bacteroid-like cells (Fig. 8).
Discussion
The observed intense growth of bacteria around the callus mass often resulting in crust formation reflects the possible effects of exudates from the callus on the growth of rhizobia and indicates the need for investigations into the nature of substances exuded by actively growing calli. It is, however, well known that root exudates of legumes exert stimulatory influence on the growth of rhizobia (RIVORA, 1969). The first sign of tolerance of the callus to the presence of Rhizobium in its vicinity was the continued growth of the callus mass even after inoculation. The occurrence of infection thread-like structures have been earlier shown in Rhizobium infected soybean tissue cultures (HOLSTEN et ai., 1971) and Pea root callus (RANGA RAO et ai., 1974). The present results revealed that in general, the initial infection was intercellular, either penetrating deep into the callus mass in the form of an infection thread as in Arachis or limited to the peripheral region of the callus tissue as in Cajanus and Cicero In either case, the bacteria probably enter the cells through the
z. Pflanzenphysiol. Bd. 80. S. 14-20. 1976.
Interaction of legume root callus with Rhizobium
17
Figs. 1-3: Arachis hypogaea. 1. A section of the callus showing the linear type of infection thread-like structure X 500. - 2. A section of the callus showing disconnected type of infection thread-like structures X 400. - 3. A number of bacteroid-filled cells adjacent io infection thread··like structures X 500. Fig. 4: Cajanus cajan. A group of (·bacteroid-filled» cells X 700.
Z. P/lanzenphysiol. Bd. 80. S. 14-20. 1976.
18
V. RANGA RAO and N. S. SUBBA RAO
pores or gaps in the cell wall. We do not know whether the observed features of bacterial penetration and establishment of bacteria inside the cells are genetically controlled phenomenon or morphological artifacts created by simple mechanical penetration of bacteria. Liberation of bacteria into the cells by bursting of «infection
Figs. 5-8: eicer arietinum. 5. Bacterial crust around the callus mass X 750. - 6. Nodular outgrowths from the callus X 400. - 7. Peripheral cells of nodular outgrowth filled with bacteroid-like cells X 1000. - 8. Some cells of the central portion of outgrowths filled with bacteroid-like cells X 1000.
Z. Pjlanzenphysiol. Bd. BO. S. 11-20. 1976.
Interaction of legume root callus with Rhizobium
19
threads» was reported in tissue cultures of soybean (HOLSTEN et a!., 1971) and Pea (RANGA RAO et a!., 1974). Meristematic activity in the infected calli of the three legumes studied here could be due to cytokinins synthesized by Rhizobium (PHILLIPS and TORREY, 1970). The ability of some of the infected calli to reduce acetylene to ethylene is a point in favour of assuming the establishment of symbiosis in Rhizobium infected callus cultures. However, LA RUE and GAMBORG (1971) found that normal tissue cultures of several plants showed endogenous ethylene production, which ought to be distinguished from acetylene dependent ethylene production which alone reflects true nitrogenase activity (PHILLIPS, 1974). It was also observed by PHILLIPS (1974) that 2,4-D and Kinetin substantially increased the endogenous ethylene production and inhibited acetylene dependent ethylene production. The tissue cultures of plants used in this investigation received growth regulators only to initiate and develop the callus formation. Therefore, the calli were allowed to grow and acclimatize for one week in fresh White's basal medium (minus growth regulators) prior to Rhizobium inoculation. They were then incubated for 2 more weeks on growth regulator free medium with Rhizobium before testing for nitrogenase activity. These precautions may rule out or minimise the influence of 2,4-D and Kinetin on endogeneous ethylene production. Moreover, while estimating nitrogenase activity in both inoculated and uninoculated calli, necessary correction was invariably made for back ground activity. An added advantage of White's basal medium which has been used in the present study is the relatively low nitrate content in comparison to Murashige-Skoog's medium used in earlier investigations (HOLSTEN et a!., 1971; RANGA RAO et a!., 1974). PHILLIPS (1974) obtained considerable nitrogenase activity by incubating the infected calli on a medium completely devoid of mineral nitrogen. CHILD and LA RUE (1974) using a medium with low levels of inorganic nitrogen found activity up to 250 moles ,umoles C 2H4/g dry weight 24 hrs. of soybean cells. It is, therefore, essential to take into account the levels of inorganic nitrogen while designing any medium for determining the nitrogenase activity in Rhizobium inoculated tissue cultures. PHILLIPS (1974) observed that Rhizobium in association with very small aggregates of cells of soybean did not reduce acetylene and suggested the use of fairly large aggregates, as a prerequisite to protect the highly oxygen sensitive nitrogenase from being exposed to air. In the present investigation we have used relatively large callus clumps (5 mm size) so as to protect the enzyme from oxygen. In the light of our observations which indicate the establishment of certain features of symbiosis in callus cultures of three legumes infected with homologous rhizobia, it remains to be seen if such interactions occur in calli from non-leguminous roots inoculated with Rhizobium. Work on these lines and the qualitative and quantitative nature of callus exudates would indeed be rewarding. Acknowledgements We are grateful to Dr. R. W. F.
HARDY,
Associate Research Director, E. I. Du Pont
Z. Pjlanzenphysiol. Bd. 80. S. 14-20. 1976.
20
V. RANGA RAO and N. S. SUBBA RAO
De Nemours Private Limited, Delaware, U.S.A. for encouragement which helped initiate this project.
Literature CHILD, J. J., and T.A. LA RUE: A simple technique for the establishment of nitrogenase in soybean callus cultures. Plant Physio!. 53, 88-90 (1974). HARDY, R. W. F., R. D. HOLSTEN, E. K. JACKSON, and R. C. BURNS: The acetyleneethylene assays for N2 fixation: Laboratory and field envaluation. Plant Physio!. 43, 1185-1207 (1968). HOLSTEN, R. D., R. C. BURNS, R. W. F. HARDY, and R. R. HEBERT: Establishment of symbiosis between Rbizobium and plant cells in vitro. Nature. 232, 173-176 (1971). LA RUE, T. A. G., and O. L. G_~MBORG: Ethylene production by cell cultures. Variations in production during growing cycle and in different plant species. Plant Physio!. 48, 396-398 (1971). PHILLIPS, D. A., and J. G. TORREY: Cytokinin production by Rhizobium japonicum. Physio!. Plant. 23, 1057-1063 (1970). PHILLIPS, D. A.: Factors affecting the reduction of acetylene by Rhizobium-Soybean cell associations in vitro. Plant Physio!. 53,67-72 (1974). RANGA RAO, V., S. K. SOPORY, and N. S. SUBBA RAO: Establishment of symbiosis in vitro between Rhizobium and Pea root callus. Curro Sci. (1974) (in Press). ROVIRA, A. D.: Plant root exudates. Bot. Rev. 35, 35-57 (1969). VELIKY, I., and T. A. LA RUE: Changes in soybean root cultures induced by Rhizobium japonicum. Naturwissenschaften. H, 96 (1966). VINCENT, J. M.: I.B.P. Hand Book No. 15, Blackwell Scientific Publishers (1970). Dr. N. S. SUBBA RAO, Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 110012, India. Note added to the proof The literature in this publication is covered only upto August 1, 1974, the date on which the paper was sent for publication. At the time the proof is corrected (August, 1976), several laboratories have demonstrated the nitrogenase activity of rhizobia in the absence of the host plant, notably on well defined media.
Z. Pjlanzenphysiol. Bd. 80. S. 14-20. 1976.
Department of Botany, Sri Venkateswara College, University of Delhi, New Delhi 110021, India, and Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India
Studies on the Interaction of Legume Root Callus with Rhizobium V. RANGA RAO and N. S. SUBBA RAO
With 8 figures Received August 8,1974 . Accepted August 31,1974
Summary Root calli of three legumes - Arachis hypogaea, Cajanus cajan and Cicer arietinum were inoculated with homologous rhizobia. Measurable nitrogenase activity was detected when inoculated calli of Arachis, Cajanus and Cicer were tested. Light microscopic observations of transections of inoculated calli revealed bacterial penetration in the form of infection thread-like structures and host cells filled with bacteria or bacteroid-like cells. In Cicer, characteristic nodular outgrowths were seen whose cells were also filled with bacteria. None of these observations were made in uninoculated calli indicating the features of possible symbiosis in legume root calli upon inoculation with Rhizobium.
Introduction To overcome the limitations imposed by changes in environmental factors influencing the phenomenon of symbiosis under natural conditions, refined techniques have been recently employed to study legume-Rhizobium interactions. One such technique has been the use of root tissue cultures (either cell aggregates or callus masses) to study the interaction between Rhizobium and legumes under controlled environmental conditions. Despite an initial unsuccessful attempt (VELIKY and LA RUE, 1966), symbiosis has been found to occur in root tissue cultures of soybean (HOLSTEN et aI., 1971; PHILLIPS, 1974; CHILD and LA RUE, 1974) and Pea (RANGA RAO et aI., 1974) inoculated with Rhizobium. The present work relates to root callus-Rhizobium interactions with three tropical legumes - Arachis hypogaea, Cajanus cajan and Cicer arietinum.
Materials and Methods Seeds of three legumes - Arachis hypogea, Cajanus cajan and Cicer arzetznum were surface sterilised with cetavlon (1 0/0) and germinated on White's basal medium. Explants
Z. P/lanzenphysiol. Bd. 80. S. 14-20. 1976.
Interaction of legume root callus with Rhizobium
15
of roots (1-2 cm) were transferred to White's basal medium supplemented with 2,4-D and Kinetin. Profuse callus formation was observed within four weeks. Small, actively growing callus pieces were later transferred to White's basal medium and allowed to remain there for 7 days. Pure cultures of Rhizobium were isolated from nodules of individual legumes using the methods of VINCENT (1970). They were subsequently maintained on Yeast extract mannitol (YEM) agar slants and inoculated to YEM liquid medium and incubated for 3 days on a horizontal shaker rotating at 100 RPM. One ml of the resulting broth containing rhizobial cells was transferred to tubes containing White's basal medium supporting the growth of individual calli. Un inoculated callus cultures on White's basal medium were used as controls. All these steps were carried out under laboratory day light conditions. Upon inoculation, the calli were transferred to darkness and incubated for a further period of one week. Subsequently, they were subjected to two or three washings in White's basal liquid medium before transferring them to White's basal solid medium. After a further period of two weeks incubation, the inoculated and uninoculated calli of Arachis, Cajanus and Cicer were analysed for nitrogenase activity by acetylene reduction technique (HARDY et a!., 1968) by transferring approximately 5 mm size calli to vials and injecting with 0.5 ml of acetylene (C 2 H 2 ). The conversion of C 2 H 2-C 2 H 4 was analysed by gas chromatography. Parallel samples of calli of all three legumes were passed through tertiary butyl alcohol series and embedded in paraffin wax. Microtome sections were cut to 8-10 fl. Slides were stained with safraninFast Green combination and made permanent by mounting in Canada Balsam.
Results
Visual as well as light microscopic observations revealed the presence of bacterial aggregates around the inoculated calli which remained fresh and continued to grow in the presence of Rhizobium. Nitrogenase activity was detectable in inoculated calli and the acetylene reduction assay data for Arachis hypogaea, Cajanus cajan and eicer arietinum are presented in Table 1. The mode of penetration of Rhizobium and the establishment of bacteroid-like structures in the cells of the callus mass of individual legumes seem to differ and hence have been described under separate headings.
Table 1: Nitrogenase activity in callus samples of Arachis hypogaea, Cajanus cajan and Cicer arietinum inoculated with homologous rhizobia.
Inoculated sample number
1. 2. 3. 4. Control sample
Nitrogenase activity
nmoles C 2 Hig. fresh weight of callus 24 hrs
Arachis hypogaea
Cajanus cajan
178 84 131.5 77.5 No activity
275 275 210 225 No activity
Cicer arietinum 56 105 112 150 No activity
Z. Pjlanzenphysiol. Bd. 80. S. 14-20. 1976.
16
V.
RANGA RAO
and N.
S. SUIlBA RAO
Arachis hypogaea
The appearance of infection thread-like structures differed depending on the plane at which the callus was cut. While in some sections the threads appeared to start intercellularly from the periphery of the callus and enter deep inside continuously (Fig. 1), in others several disconnected threads were seen apparently inducing meristematic activity in cells surrounding them (Fig. 2). Such meristematic cells were filled with bacteroid-like cells and were bigger than their neighbours (Fig. 3). Cajanus cajan
The callus was composed of loose aggregates of cells. The infection thread-like structures were haphazard and very rarely organized and continuous. Meristematic activity was frequently observed around infection threads and meristematic cells were not only filled with bacteroids (Fig. 4) but were also larger in size than the surrounding cells. Cicer arietinum
The aggregation of bacteria in the form of a crust on the periphery of the callus of Cicer (Fig. 5) appeared to be more intense than on the periphery of those of Cajanus and Arachis. At some sites on the callus, peripheral nodular outgrowths (Fig. 6) were observed as a result of intense meristematic activity of callus cells which may be attributed to the activity of bacteria. Nearly half of the peripheral cells of the outgrowths contained bacteroid-like cells (Fig. 7) whereas the central portion of such outgrowths had relatively few cells which contained bacteroid-like cells (Fig. 8).
Discussion
The observed intense growth of bacteria around the callus mass often resulting in crust formation reflects the possible effects of exudates from the callus on the growth of rhizobia and indicates the need for investigations into the nature of substances exuded by actively growing calli. It is, however, well known that root exudates of legumes exert stimulatory influence on the growth of rhizobia (RIVORA, 1969). The first sign of tolerance of the callus to the presence of Rhizobium in its vicinity was the continued growth of the callus mass even after inoculation. The occurrence of infection thread-like structures have been earlier shown in Rhizobium infected soybean tissue cultures (HOLSTEN et ai., 1971) and Pea root callus (RANGA RAO et ai., 1974). The present results revealed that in general, the initial infection was intercellular, either penetrating deep into the callus mass in the form of an infection thread as in Arachis or limited to the peripheral region of the callus tissue as in Cajanus and Cicero In either case, the bacteria probably enter the cells through the
z. Pflanzenphysiol. Bd. 80. S. 14-20. 1976.
Interaction of legume root callus with Rhizobium
17
Figs. 1-3: Arachis hypogaea. 1. A section of the callus showing the linear type of infection thread-like structure X 500. - 2. A section of the callus showing disconnected type of infection thread-like structures X 400. - 3. A number of bacteroid-filled cells adjacent io infection thread··like structures X 500. Fig. 4: Cajanus cajan. A group of (·bacteroid-filled» cells X 700.
Z. P/lanzenphysiol. Bd. 80. S. 14-20. 1976.
18
V. RANGA RAO and N. S. SUBBA RAO
pores or gaps in the cell wall. We do not know whether the observed features of bacterial penetration and establishment of bacteria inside the cells are genetically controlled phenomenon or morphological artifacts created by simple mechanical penetration of bacteria. Liberation of bacteria into the cells by bursting of «infection
Figs. 5-8: eicer arietinum. 5. Bacterial crust around the callus mass X 750. - 6. Nodular outgrowths from the callus X 400. - 7. Peripheral cells of nodular outgrowth filled with bacteroid-like cells X 1000. - 8. Some cells of the central portion of outgrowths filled with bacteroid-like cells X 1000.
Z. Pjlanzenphysiol. Bd. BO. S. 11-20. 1976.
Interaction of legume root callus with Rhizobium
19
threads» was reported in tissue cultures of soybean (HOLSTEN et a!., 1971) and Pea (RANGA RAO et a!., 1974). Meristematic activity in the infected calli of the three legumes studied here could be due to cytokinins synthesized by Rhizobium (PHILLIPS and TORREY, 1970). The ability of some of the infected calli to reduce acetylene to ethylene is a point in favour of assuming the establishment of symbiosis in Rhizobium infected callus cultures. However, LA RUE and GAMBORG (1971) found that normal tissue cultures of several plants showed endogenous ethylene production, which ought to be distinguished from acetylene dependent ethylene production which alone reflects true nitrogenase activity (PHILLIPS, 1974). It was also observed by PHILLIPS (1974) that 2,4-D and Kinetin substantially increased the endogenous ethylene production and inhibited acetylene dependent ethylene production. The tissue cultures of plants used in this investigation received growth regulators only to initiate and develop the callus formation. Therefore, the calli were allowed to grow and acclimatize for one week in fresh White's basal medium (minus growth regulators) prior to Rhizobium inoculation. They were then incubated for 2 more weeks on growth regulator free medium with Rhizobium before testing for nitrogenase activity. These precautions may rule out or minimise the influence of 2,4-D and Kinetin on endogeneous ethylene production. Moreover, while estimating nitrogenase activity in both inoculated and uninoculated calli, necessary correction was invariably made for back ground activity. An added advantage of White's basal medium which has been used in the present study is the relatively low nitrate content in comparison to Murashige-Skoog's medium used in earlier investigations (HOLSTEN et a!., 1971; RANGA RAO et a!., 1974). PHILLIPS (1974) obtained considerable nitrogenase activity by incubating the infected calli on a medium completely devoid of mineral nitrogen. CHILD and LA RUE (1974) using a medium with low levels of inorganic nitrogen found activity up to 250 moles ,umoles C 2H4/g dry weight 24 hrs. of soybean cells. It is, therefore, essential to take into account the levels of inorganic nitrogen while designing any medium for determining the nitrogenase activity in Rhizobium inoculated tissue cultures. PHILLIPS (1974) observed that Rhizobium in association with very small aggregates of cells of soybean did not reduce acetylene and suggested the use of fairly large aggregates, as a prerequisite to protect the highly oxygen sensitive nitrogenase from being exposed to air. In the present investigation we have used relatively large callus clumps (5 mm size) so as to protect the enzyme from oxygen. In the light of our observations which indicate the establishment of certain features of symbiosis in callus cultures of three legumes infected with homologous rhizobia, it remains to be seen if such interactions occur in calli from non-leguminous roots inoculated with Rhizobium. Work on these lines and the qualitative and quantitative nature of callus exudates would indeed be rewarding. Acknowledgements We are grateful to Dr. R. W. F.
HARDY,
Associate Research Director, E. I. Du Pont
Z. Pjlanzenphysiol. Bd. 80. S. 14-20. 1976.
20
V. RANGA RAO and N. S. SUBBA RAO
De Nemours Private Limited, Delaware, U.S.A. for encouragement which helped initiate this project.
Literature CHILD, J. J., and T.A. LA RUE: A simple technique for the establishment of nitrogenase in soybean callus cultures. Plant Physio!. 53, 88-90 (1974). HARDY, R. W. F., R. D. HOLSTEN, E. K. JACKSON, and R. C. BURNS: The acetyleneethylene assays for N2 fixation: Laboratory and field envaluation. Plant Physio!. 43, 1185-1207 (1968). HOLSTEN, R. D., R. C. BURNS, R. W. F. HARDY, and R. R. HEBERT: Establishment of symbiosis between Rbizobium and plant cells in vitro. Nature. 232, 173-176 (1971). LA RUE, T. A. G., and O. L. G_~MBORG: Ethylene production by cell cultures. Variations in production during growing cycle and in different plant species. Plant Physio!. 48, 396-398 (1971). PHILLIPS, D. A., and J. G. TORREY: Cytokinin production by Rhizobium japonicum. Physio!. Plant. 23, 1057-1063 (1970). PHILLIPS, D. A.: Factors affecting the reduction of acetylene by Rhizobium-Soybean cell associations in vitro. Plant Physio!. 53,67-72 (1974). RANGA RAO, V., S. K. SOPORY, and N. S. SUBBA RAO: Establishment of symbiosis in vitro between Rhizobium and Pea root callus. Curro Sci. (1974) (in Press). ROVIRA, A. D.: Plant root exudates. Bot. Rev. 35, 35-57 (1969). VELIKY, I., and T. A. LA RUE: Changes in soybean root cultures induced by Rhizobium japonicum. Naturwissenschaften. H, 96 (1966). VINCENT, J. M.: I.B.P. Hand Book No. 15, Blackwell Scientific Publishers (1970). Dr. N. S. SUBBA RAO, Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 110012, India. Note added to the proof The literature in this publication is covered only upto August 1, 1974, the date on which the paper was sent for publication. At the time the proof is corrected (August, 1976), several laboratories have demonstrated the nitrogenase activity of rhizobia in the absence of the host plant, notably on well defined media.
Z. Pjlanzenphysiol. Bd. 80. S. 14-20. 1976.