Establishment and maintenance of Soybean Mosaic Virus in soybean ...

379

Virology / Virologie

Establishment and maintenance of Soybean Mosaic Virus in soybean callus culture Pengyin Chen, Glenn R. Buss, Richard E. Veilleux, and Sue A. Tolin

Abstract: Susceptibility to inoculation with Soybean mosaic virus (SMV) and virus activity were investigated in soybean callus cultures growing in vitro. Excised hypocotyls of susceptible soybean, Glycine max (L.) Merr. ‘Essex’, were cultured in Msoy medium in the light at 25 °C. Established calluses were inoculated with SMV in vitro by a soak–prick method. In addition, SMV-infected leaves of soybean ‘Lee 68’ were surface sterilized, excised, and placed on callus-inducing medium. Calluses infected with SMV initiated by either method grew in vitro as well as calluses from uninfected tissues. Callus cultures turned brownish yellow after 6–8 weeks, when the media became depleted of nutrients. However, callus with an active SMV infection could be maintained by regular subculture to fresh medium. Longevity of SMV–callus cultures was increased by storage at 10–15 °C, thus reducing the frequency of transfers. The virus was detected in infected calluses by serological tests. Infectivity assays confirmed the presence and viability of SMV in callus cultures. Most callus cultures induced directly from infected leaves retained virus and high infectivity, whereas infective cultures from in vitro inoculated hypocotyls appeared to decrease in number and infectivity with repeated subcultures. However, selected infective cultures retained high infectivity after 10 successive transfers over a period of 20 months. These results demonstrate that SMV can be cultured and maintained active in callus cultures in vitro. Key words: Glycine max, Soybean mosaic virus, inoculum, tissue culture, virus infectivity. Résumé : La sensibilité à l’inoculation du virus de la mosaïque du soja (SMV) et l’activité du virus furent étudiées sur des cultures de cal de soja in vitro. Des hypocotyles excisés de soja sensible, Glycine max (L.) Merr. ‘Essex’, furent cultivés dans le milieu de culture Msoy à la lumière et à 25 °C. Les cals établis furent inoculés in vitro avec le SMV par une méthode de piqûre–trempage. De plus, des feuilles de soja ‘Lee 68’ infectées par le SMV furent stérilisées superficiellement, excisées et placées sur un milieu favorisant la formation de cals. Les cals infectés par le SMV et amorcés par l’une ou l’autre des méthodes crûrent in vitro aussi bien que les cals provenant de tissus non infectés. Les cultures de cal devirent jaune brunâtre après 6 à 8 semaines, quand les éléments nutritifs du milieu furent épuisés. Cependant, un cal avec infection évolutive par le SMV a pu être maintenu par repiquage régulier sur milieu nutritif frais. La longévité des cultures de cal infectées par le SMV fut augmentée par l’entreposage à une température de 10 à 15 °C, réduisant ainsi la fréquence de repiquage. Le virus fut détecté dans les cals infectés par des tests sérologiques. Des essais d’infectiosité confirmèrent la présence et la viabilité du SMV dans les cultures de cal. La plupart des cultures de cal démarrées directement de feuilles infectées conservèrent le virus et une forte infectiosité, alors que le nombre et l’infectiosité des cultures infectieuses obtenues d’hypocotyles inoculés in vitro semblèrent décroître avec les repiquages répétés. Cependant, certaines cultures infectieuses conservèrent leur forte infectiosité après 10 repiquages successifs sur une période de 20 mois. Ces résultats démontrent que le SMV peut être cultivé et maintenu actif dans des cultures de cal in vitro. Mots clés : Glycine max, virus de la mosaïque du soja, inoculum, culture de tissus, infectiosité du virus. 385

Chen

et al.: Soybean Mosaic Virus / callus culture / maintenance of virus

Accepted 18 March 2004. P. Chen1. Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA. G.R. Buss. Department of Crop and Soil Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. R.E. Veilleux. Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. S.A. Tolin. Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. 1

Corresponding author (e-mail: [email protected]).

Can. J. Plant Pathol. 26: 379–385 (2004)

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Introduction Maintenance of pathogens is essential for research on host–parasite interactions. Unlike other microorganisms, viruses cannot be directly cultured on synthetic medium in vitro since they do not replicate in the absence of host cells (Ingram 1973). Maintenance of virus in vivo by frequent transfer to susceptible host plants is commonly used, but is laborious, time- and space-consuming, and subject to contamination, mutation, attenuation, and accidental loss of viral strains. Other than for Tobacco mosaic virus (TMV), little effort has been directed towards developing methods for growing plant viruses in cell and tissue cultures. Infection of isolated cells and tissue cultures of tobacco with TMV has been demonstrated (Hildebrandt 1973; Ingram 1973; Kassanis 1967). Hildebrandt (1977) proposed that virus-infected, undifferentiated tobacco callus originating from TMV-infected plants could be cultured for unlimited periods in vitro. Tobacco callus cultures growing on agar or liquid media have been successfully inoculated with TMV by mechanical methods (Kassanis et al. 1958; Murakishi 1968; Murakishi et al. 1971; Nims et al. 1967). Infection, synthesis, and distribution of Southern bean mosaic virus (SBMV) have been studied in soybean cell suspensions (White et al. 1977; Wu and Murakishi 1978; Wu et al. 1982). In spite of the advances in tissue culture techniques since these early studies, the use of tissue culture to maintain virus cultures in isolation has received little attention. We speculated that soybean tissue culture might be a useful method to maintain SMV in vitro, to aid host–pathogen interaction studies that require pure virus inoculum of specific pathotypes. Culture of SMV in soybean callus has potential value for long-term maintenance and production of aseptic inoculum, assurance of pure viral strains, and avoidance of contamination between different strains or isolates and with other viruses. In this paper, we report the successful establishment of a tissue culture system for SMV preservation in vitro.

Materials and methods Establishment of soybean hypocotyl callus Seeds of soybean ‘Essex’ were germinated on moist filter paper in petri dishes (100 mm × 15 mm) for 3–5 days. Hypocotyl sections, 0.5 cm in length, were excised and surface sterilized by immersion for 1 min in 70% ethanol, followed by 20 min in 20% bleach containing one drop of Tween® 20 per 100 mL (Sigma Chemical, St. Louis, Mo.), and then rinsed three times in sterile distilled water. Disinfected hypocotyl explants were placed in 25 mm × 150 mm culture tubes containing 20 mL of autoclaved (20 min at 1.2 kg/cm2, 120 °C) Msoy agar medium (Reinert and Yeoman 1982). Cultures were incubated at 25 °C under light (75 µmol m–2 s–1) provided by cool-white fluorescent lamps with a 16-h day length. Established calluses were subcultured at 6- to 8-week intervals by excising actively growing sections (approx. 2-mm cube) onto fresh Msoy agar medium.

Can. J. Plant Pathol. Vol. 26, 2004

Inoculation of callus culture with SMV Trifoliolate leaves exhibiting mosaic symptoms were collected from greenhouse-grown soybean ‘Lee 68’ plants previously inoculated mechanically at the unifoliolate stage with SMV-VA/G1 (Hunst and Tolin 1982). Collected leaves were washed with tap water, disinfected as above, and ground in autoclaved sodium phosphate buffer, 0.01 mol/L, at pH 7.0, with 10 mL/g of leaf tissue. The homogenate was filtered though four layers of sterile cheesecloth and placed in a petri dish. Inoculation of callus was done aseptically by excising actively growing callus, soaking callus pieces in inoculum for 30 or 60 min, and pricking them 20 times each with a fine sterile needle. Inoculated callus sections were washed in sterile distilled water and placed onto Msoy medium. Established callus cultures were maintained by regular transfers. Uninoculated calluses were maintained as healthy controls. Direct initiation of infected callus cultures Greenhouse-grown plants of susceptible soybean ‘Lee 68’ were mechanically inoculated with SMV at the unifoliolate stage. Trifoliolate leaves showing mosaic symptoms were collected at 2 and 4 weeks after inoculation. The infected leaves were washed with tap water and surface sterilized as above. Leaf sections (approx. 1 cm × 1 cm) containing midvein were excised and placed in culture tubes containing 20 mL of Msoy agar medium. Similar sections from leaves of uninoculated ‘Lee 68’ plants served as controls in culture. The resulting callus cultures were maintained by bimonthly transfer. Detection of virus in callus cultures and infectivity assay The virus was detected serologically, in callus cultures, using the Ouchterlony double-diffusion test and enzymelinked immunosorbent assay (ELISA). The double-diffusion test was performed according to Hunst and Tolin (1982). The enzyme-linked immunosorbent assay was conducted according to Copeland (1998) using polyclonal rabbit antiserum to SMV-VA/G1 (Hunst and Tolin 1982). For infectivity assay, callus samples (approx. 1 g) from each culture were ground with a chilled mortar and pestle in sodium phosphate buffer, 0.01 mol/L, at ph 7.0, with 5 mL/g of tissue. Inoculum from each callus culture was applied with a pestle onto 75%-expanded unifoliolate leaves of 6 to 12 plants per pot (three replications) of both ‘Lee 68’ and ‘Essex’, lightly dusted with Carborundum®. Inoculated plants were maintained in the greenhouse and examined for virus symptoms 2–3 weeks after inoculation. Dilution endpoint infectivity assays were also conducted using inoculum from 20-month-old cultures prepared as above and diluted with buffer into a series of six 10-fold dilutions. Inoculation was made by applying approximately 100 µL of inoculum from each dilution to each unifoliolate leaf of the test plants. Each inoculation included three replications each with 6 to 12 plants per pot. For the ELISA dilution assays, extract from each culture was diluted with buffer into a series of 12 twofold dilutions (1:5 to 1:1024). Infected and healthy leaves from greenhouse plants were used as controls in the serological tests and infectivity assay.

Chen et al.: Soybean Mosaic Virus / callus culture / maintenance of virus

Data analysis All the data collected from ELISA and infectivity assays were subjected to the analysis of variance (ANOVA) procedure of the SAS system (SAS Institute Inc., Cary, N.C.). Data from similar treatments were subjected to separate and combined analyses of variance. Treatment means were compared by an ANOVA protected least significant difference (LSD) test or Duncan’s multiple range test where applicable. In general, differences were considered significant when detected at P ≤ 0.05. A contingency χ2 test was also used, as appropriate, to compare similar or different treatments and to examine the effect of combined treatments. Preservation of callus cultures and verification of virus strain identity The effect of low temperatures on the longevity of callus cultures was assessed. Calluses from 10 different cultures were each divided into four pieces and transferred into four different tubes containing fresh media. These 40 freshly transferred cultures were further redistributed into four groups of 10. Each group contained one each of the 10 parental cultures. Three groups of the subcultures were allowed to grow at 25 °C for 1 week and then incubated at 5, 10, or 15 °C, respectively, with a light intensity of 75 µmol m–2 s–1 and a 16-h day length. The remaining group was maintained at 25 °C with the same light intensity and day length. Longevity of cultures was determined by the number of days before the next transfer was required, when a viable subculture could still be obtained. The growth of cultures at different temperatures was visually assessed during storage. When a brownish discoloration appeared, calluses were subcultured to fresh media and tested for virus infectivity. Virus strain identity was verified by inoculation of SMVstrain-differentiating genotypes of soybean ‘Ogden’, ‘York’, ‘Marshall’, ‘Kwanggyo’, ‘Lee 68’, and ‘PI96983’ (Chen et al. 1994; Cho and Goodman 1979). Inoculum was made from a mixture of one leaflet from each ‘Lee 68’ plant inoculated previously with each of 15 individual callus cultures. The reaction on each differential cultivar was scored 3 weeks after inoculation.

Results Induction and maintenance of soybean callus Msoy agar medium was suitable for inducing calluses from leaves and hypocotyls of soybean. Calluses generally formed within 10 days and started proliferating after 2 weeks in culture. Those induced from healthy explants were dark green, whereas those that were infected by virus were yellow green. Some of the SMV-infected calluses showed brownish sectors, a sign of deterioration, after a 4week incubation at 25 °C. However, no differences were observed in growth rate between infected and uninfected callus cultures, and both types of culture grew actively for 6–8 weeks without transfer. Some of the cultures exhibited rhizogenesis on aging and were viable for 35 weeks without transfer. Most cultures ceased growing after 8 weeks and required subculture to fresh medium.

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Detection and infectivity of SMV in callus cultures Extracts from SMV-infected callus and leaves were serologically positive although the intensity of precipitin bands and ELISA values were variable (Table 1). The original leaf callus cultures and infected leaves gave rise to stronger bands than cultures from transferred leaf callus and inoculated hypocotyl callus. Callus cultures from infected leaves showed higher ELISA values than those from inoculated hypocotyl calluses. All SMV-infected cultures gave 90% to 100% infection in infectivity assay. Healthy leaves and callus cultures exhibited negative reactions in serological tests and caused no infection in infectivity assay. In a combined ANOVA, mean infections for original leaf callus, transferred leaf callus, and inoculated hypocotyl callus were not significantly different among each other or from the infected leaf sap, although the ELISA values for transferred leaf callus and inoculated hypocotyl callus were significantly lower than those for the original leaf callus and infected leaf sap. Retention of SMV infectivity in callus cultures with successive transfers A large proportion (92%) of the callus cultures from infected leaves was infective after two successive transfers and produced about 90% infected plants on average in infectivity assay (Table 2). A few cultures showed infection rates lower than 60%, and three of 36 subcultures contained no detectable virus after two transfers. A relatively small proportion (42%) of hypocotyls callus cultures remained infectious after two transfers and gave infection levels ranging from 11% to 100% with an average of 41%. All SMVinfected callus cultures were positive for ELISA, although the values were lower than those of infected leaves. No virus was detected or recovered by ELISA or infectivity assay as expected for all uninfected controls. The combined ANOVA showed that the infectivity of leaf callus cultures was significantly higher than that of inoculated hypocotyl callus cultures, although ELISA values for both types of cultures were not significantly different. After four successive transfers, 85% of the leaf callus cultures remained infectious and averaged 86% infection in infectivity tests (Table 2). A few cultures produced less than 50% infected plants, and 4 of 26 cultures lost the virus. Half of the hypocotyl callus cultures were infective, with infection levels ranging from 8% to 100%, and an average of 36%, in infectivity assay. No infection was caused by extracts from virus-free cultures and healthy leaves. The combined ANOVA showed that the overall infectivity of subcultured calluses from SMV-infected leaves was significantly higher than that of inoculated hypocotyl calluses after two to four transfers. Highly infective callus cultures were selected at the fifth transfer, based on results of the infectivity assays, and propagated through five additional transfers. The results of infectivity tests for these selected cultures after 6, 8, and 10 transfers are shown in Table 3. All selected cultures, except for the healthy controls, were highly infective and gave more than 90% infection. The combined infectivity data for leaf calluses showed no significant difference from that for hypocotyl calluses.

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Table 1. Detection and infectivity of Soybean mosaic virus in initial or first transferred soybean callus cultures and leaves, as determined by Ouchterlony double-diffusion tests, ELISA, and infectivity assays. Culture sample

Culture age (days)*

Ouchterlony band intensity

ELISA (A405)†

Infectivity (%)‡

Original callus from 2-week-infected leaf Original callus from 4-week-infected leaf Callus from 2-week-infected leaf after one transfer Callus from 4-week-infected leaf after one transfer Hypocotyl callus from 30-min soak–prick–inoculation Hypocotyl callus from 60-min soak–prick–inoculation Infected leaf from greenhouse-grown plants Hypocotyl callus from healthy control after one transfer Healthy leaf from greenhouse-grown plants

44 44 7 7 10 10 Fresh 13 Fresh

+++ +++ ++ ++ + ++ +++ – –

1.97 1.78 1.83 1.61 1.63 1.65 1.97 0.03 0.02

100 93 100 100 90 100 100 0 0

a b b c c c a d d

a b a a b a a c c

Note: Means followed by the same letter, within a column, are not significantly different (P ≤ 0.05) according to Duncan’s multiple range test. *Number of days from the date of initiation or transfer to the date of testing. Fresh leaves for infected and healthy controls were collected from 3week-old plants in the greenhouse. † Mean absorbance at 405 nm, recorded 2 h after substrate addition. ‡ Mean percentage of infected plants in greenhouse tests.

Table 2. Persistence of Soybean mosaic virus in soybean callus cultures after two (4 months in culture) and four (8 months in culture) transfers, as measured by ELISA and infectivity tests. ELISA (A405)‡

No. of cultures*

% Infection†

Culture sample

2 transfers

4 transfers

2 transfers

4 transfers

2 transfers

Infected leaf from greenhouse-grown plants Callus from 2-week-infected leaf Callus from 4-week-infected leaf Hypocotyl callus from 30-min soak–prick–inoculation Hypocotyl callus from 60-min soak–prick–inoculation Callus from healthy leaf Hypocotyl callus from healthy control Healthy leaf from greenhouse-grown plants

1/1 11/12 22/24 7/19 10/22 0/8 0/7 0/2

1/1 10/12 12/14 6/12 7/14 0/4 0/6 0/1

100 94a 86a 39b 43b 0 0 0

100 86a 86a 33b 39b 0 0 0

1.11 0.94 0.98 0.94 0.93 0.03 0.03 0.03

(30–100) (50–100) (11–100) (14–100)

(33–100) (25–100) (10–91) (8–100)

a b b b b c c c

*Number of infective cultures/total number of cultures tested, after two and four transfers. † Mean percentage of infected plants after inoculation with each culture, following two and four transfers. Numbers in parentheses are ranges of percent infection. Means followed by the same letter, within a column, are not significantly different (P = 0.05) according to Duncan’s multiple range test. ‡ Mean absorbance at 405 nm, recorded 1 h after substrate addition. Means followed by the same letter, within a column, are not significantly different (P = 0.05) according to Duncan’s multiple range test.

Table 3. Persistence of Soybean mosaic virus in selected infectious callus cultures after 6, 8, and 10 transfers (respectively 12, 16, and 20 months in culture), as determined by infectivity tests. No. of cultures after no. of transfers*

% Infection after no. of transfers†

Culture sample

6

8

10

6

Callus from 2-week-infected leaf Callus from 4-week-infected leaf Hypocotyl callus from 30-min soak–prick–inoculation Hypocotyl callus from 60-min soak–prick–inoculation Callus from healthy leaf Hypocotyl callus from healthy control

7/7 3/3 1/1 2/2 0/3 0/4

3/3 2/2 1/1 1/1 0/1 0/1

3/3 2/2 1/1 1/1 0/1 0/1

100 96 91 100 0 0

8 a a b a

100 92 100 100 0 0

10 a b a a

100 100 100 100 0 0

a a a a

*Number of infective cultures/total number of cultures tested after 6, 8, and 10 transfers. † Mean percentage of infected plants after inoculation with each culture, following 6, 8 and 10 transfers. Means followed by the same letter, within a column, are not significantly different (P = 0.05) according to Duncan’s multiple range test.

Table 4 shows the results from dilution end-point infectivity assays of callus cultures. Variations in infectivity of individual cultures were observed. However, callus cultures had higher dilution end points although lower ELISA

values, than did fresh leaf sap. The eight cultures averaged 84%, 69%, and 29% infection with dilutions of 10–1, 10–2, and 10–3, respectively, whereas the leaf sap gave rise to 73%, 36%, and 18% infection with similar dilutions. In a

Chen et al.: Soybean Mosaic Virus / callus culture / maintenance of virus

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Table 4. Detection of Soybean mosaic virus in callus cultures after 10 transfers and 20 months of culture at 25 °C, as determined by infectivity and ELISA dilution assays. % Infection with dilution† Sample* Callus from 2-week-infected leaf (A) 2-week-infected leaf (B) 4-week-infected leaf (A) 4-week-infected leaf (B) Hypocotyl callus from 30-min soak–prick–inoculation (A) 30-min soak–prick–inoculation (B) 60-min soak–prick–inoculation (A) 60-min soak–prick–inoculation (B) Infected leaf from greenhouse-grown plants Subculture A Subculture B LSD

A405 in ELISA dilution‡

10–1

10–2

10–3

10–4

10–5

10–6

1:10

1:80

1:640

43 92 100 63

65 83 87 65

29 33 47 0

16 5 48 0

5 14 14 0

0 10 0 0

0.48 0.48 0.90 0.70

0.20 0.21 0.37 0.31

0.03 0.02 0.07 0.07

100 88 90 95

68 25 91 74

23 38 10 55

18 5 6 19

9 16 0 6

9 7 0 8

0.41 0.51 0.63 0.39

0.13 0.18 0.14 0.15

0.06 0.04 0.04 0.03

75 71

26 47

20 17

0 6

0 0

4 0

1.02 1.72

0.38 0.27

0.12 0.03

32

54

66

28

18

12

0.74

0.13

0.02

*(A) and (B) denote different subcultures from the same original culture. † Mean percentage of infected plants after inoculation with each culture in three replications with a total of 16–27 plants. ‡ Mean absorbance at 405 nm, recorded 1 h after substrate addition. For healthy leaf extracts, A405 = 0.1 at 1:5 (m/v) and A405 = 0.0 at higher dilutions.

contingency χ2 test for the dilution-series data of percent infection from different treatments (culture samples), we detected no significant differences in infectivity between subcultures (A and B) from the same original cultures; no difference among 2-week-infected leaf callus, 4-weekinfected leaf callus, 30-min soak–prick–inoculated hypocotyl callus, and 60-min soak–prick–inoculated hypocotyl callus; no difference between leaf callus cultures combined and hypocotyl callus cultures combined; but higher infectivity in all callus cultures combined than in infected leaves. In the ELISA dilution test, higher ELISA values were obtained for infected leaves than callus cultures. The difference was not statistically significant, but numerically greater at lower dilutions (1:5 to 1:20) than at higher dilutions. Cold storage of callus cultures and confirmation of virus fidelity Cultures maintained at 25 °C proliferated rapidly and remained viable up to 8 weeks, after which subculture was needed. Cultures at 5 °C had little or no growth during storage and turned brown or died after 4 weeks. Virus was recovered from such brownish calluses after 4–6 weeks of storage, but no successful culture transfers were made. Cultures at 10 or 15 °C were reduced in growth rate compared with those at 25 °C. The survival rate of calluses at 10 or 15 °C was similar to those at 25 °C after 8 weeks of storage. The longevity of callus grown at 10 °C was about 15 weeks, whereas the cultures at 15 °C were successfully maintained for 13 weeks. There was no reduction in callus viability and virus activity at 10 or 15 °C. Inoculation of differential soybean cultivars showed that SMV in callus cultures induced typical mosaic symptoms on ‘Lee 68’, but not on PI 96983, ‘Ogden’, ‘York’, ‘Marshall’, or ‘Kwanggyo’, as expected for strain G1 (Chen et al. 1994; Cho and Goodman 1979). Similar reactions were obtained for the inoculation of leaf sap from a carefully

maintained greenhouse G1 culture. The results indicate that SMV in callus culture remains active and stable.

Discussion It has been difficult to obtain high levels of virus infection in cultured cells and tissues (Kassanis 1957, 1967). Previous methods of inoculating tissue cultures with TMV by pouring inoculum over callus pieces (Kassanis et al. 1958), by gentle agitation of virus–cell mixtures on a shaker (Kassanis et al. 1958; Wu et al. 1960), by microinjection (Nims et al. 1967), or by mixing with a glass rod (Murakishi et al. 1971) did not result in consistent infection of a high percentage of cells. With the soak–prick method used with SMV in this study, the mechanical dissociation of callus cells with a scalpel and pricking with a needle results in wounds that permit entry of virus particles and initiation of the infection process. Soaking the needle-pricked callus pieces in the SMV inoculum for 30 or 60 min presumably allowed virus particles to penetrate the wounded callus tissue, thus increasing the probability of virus entry into cells within the callus mass. In the tobacco callus – TMV system, tissue infected by artificial injury usually became virus-free after subculture, whereas those infected by pricking callus with a needle dipped in the inoculum remained infected permanently (Kassanis et al. 1958). However, vortexing of the cell suspension with virus and incubation with shaking resulted in rapid synthesis of TMV and SBMV in a large proportion of cells (White et al. 1977; Wu and Murakishi 1978; Wu et al. 1982). Soybean callus cells inoculated and cultured in this manner maintained viable SBMV for 120 days when the R3 culture medium was changed periodically (Wu and Murakishi 1978). Although the soak–prick inoculated calluses had lower ELISA values and weaker Ouchterlony reaction than infected leaves, the infected callus cultures retained an amount of virus sufficient for infectivity (Tables 1, 2, and

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4). A similar difference in virus concentration between infected plants and callus cultures has been reported for TMV (Ingram 1973; Kassanis 1957), which may be due to difference in inoculum dose, type and number of injuries, age of the cells, physiological condition, replication and cell-tocell movement of the virus, and the proportion of infected cells in the tissue. Although the original callus cultures established by the soak–prick method were highly infective initially, approximately 50% of the cultures lost the virus after two to four transfers (Table 2); this is probably because the cells in callus clumps were not uniformly infected so that the transfers could have been made from uninfected sections of the original callus. In addition, the infectivity was highly variable in subcultures, ranging from 8% to 100%. Similar results were obtained in TMV – tobacco tissue culture systems (Hildebrandt 1973; Kassanis 1957, 1967). Tobacco callus cultures inoculated with TMV were found to consist of a network of infected cells among healthy ones. More than 40% of the cells from TMV-infected calluses were estimated to contain virus. Some cells remained healthy as determined by single-cell clones. The amount of TMV in respective clones varied greatly after four successive monthly transfers. A certain number of cultures lost TMV, whereas others remained infected through seven monthly subcultures (Hildebrandt 1973). Differentiated tissues regenerated from infected calluses are often found to be virus-free (HanusFajerska 2001; Xu et al. 2000). The direct initiation of SMV-infected callus cultures from infected leaves appears to be efficient because the method takes only one step to establish the infected cultures, and presumably all of the cells are infected. The serological tests indicate that callus cultures initiated from infected leaves may have a higher titer of virus than those from the in vitro inoculated hypocotyls (Tables 1 and 4). A high percentage of leaf callus cultures remained infective and gave higher infectivity than hypocotyl cultures in the first few transfers (Table 2). A few subcultures showed relatively low infectivity, probably because of the decreased amount of virus after repeated transfers (Table 2). The decrease in virus content over subcultures has also been observed in the TMV system (Hildebrandt 1973, 1977; Kassanis 1967). However, highly infective cultures can be selected from subcultured cell lines and maintained with high infectivity (Table 3). In vitro storage of callus cultures by growth limitation at reduced temperatures offers an important alternative to the conventional transfer of SMV to greenhouse plants or other storage methods. Reduced temperatures (10–15 °C) apparently slowed the growth of callus, but had no detrimental effect on virus viability and infectivity, and reduced the need for frequent transfer of the callus cultures. Potentially, longevity of callus cultures can be increased by manipulating other factors such as medium constituents, growth regulators or inhibitors, light intensity, or photoperiod. No pathotypic variation was inferred by assay of SMV in callus cultures on a set of differential cultivars after more than a year of preservation in vitro. With this tissue culture system, we have successfully maintained the type strain G1 of SMV in soybean callus for 5 years.


Our experiments demonstrate two approaches for establishing soybean callus cultures and SMV in vitro. The Msoy agar medium is suitable for inducing callus from infected or healthy explants from soybean leaves and hypocotyls. Calluses infected by SMV were obtained by direct culture of virus-infected leaf tissue to agar medium. Also, calluses induced from uninfected hypocotyl tissue were successfully inoculated with SMV in vitro. The soak–prick inoculation method is simple and efficient. Callus cultures, once established, can be used to maintain SMV by regular transfers.

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