Plant Cell Reports
Plant Cell Reports (1994) 14:137-140
@, Springer-Verlag 1994
In vitro selection of endosulfan-tolerant strains of Brassica compestris (cv. Brown Sarson) K.R. Arya 1, R.C. Gupta 1 B.D. Semwal z, and M.U. Beg a Division of Phytotoxicology, Industrial Toxicology Research Centre, M.G. Marg, L u c k n o w - 226 00l, India 2 D e p a r t m e n t o f Botany, D.A.V. College, K a n p u r , India Received 16 April 1993,/Revised version received 12 May 1994 - C o m m u n i c a t e d by J. W i d h o l m
ABSTR ACT Endosulfan tolerant lines of mustard (Brassiea campestris cv. Brown Sasson) have been developed through tissue culture methods. Cotyledonary explants excised from eight day old in vitro grown seedlings were used for inducing callus. Fast growing friable callus I was then transferred to MS medium containing (0.1-2.0 u g l ' ) endosulfan for selection. Five alternating exposures with and without endosulfan containing medium yielded an endosulfan tolerant cell line (ETL). The plants regenerated from ETL were found to tolerate three fold higher concentrations of endosulfan. Callus induced from randomly selected endos~lfan tolerant regenerated plants were also tolerant to 3.0 ugl" endosulfan, thereby, suggesting that tolerance has been acquired at the gene level. Biochemical investigation revealed higher levels of total free sugar, f r e e amino acids, protein and activity of peroxidase ~n the tolerant cell line. Key wools: Brassica campestris . Brown Sarson - Endosulfan tolerant.
Abbreviations: MS: Murashige and Skoog medium (1962); NSM: non-selective medium; SM: selective medium; BAP: 6. Benzylaminopurine; N A A : ~ - n a p h t h a l e n e a e e t i e acid; Z: zeatin. INTRODUC3"ION Introduction of genetic tolerance for pesticides into crop plants may have an advantage of expanded applicability of a particular pesticide, which could be used to eliminate the pest without any adverse effect on crop quality. The tradi. tional plant breeding techniques have introduced numerous improvements in crop yield during the past centuries. However, plant breeders have several constraints on the ability to introduce new genetic information into plants and to create new plant varieties through conventional plant breeding techniques (Ohyama, 1983). Recent developments and advancements in plant tissue culture technology have provided an approach to select pesticide tolerant plants (Chaleff 1986, Wrather et al. 1991) and has proven its utility in selecting mutants by reducing the space, time and cost involved. Thereby, progress has been made with regard to the development of resistance to phytotoxins (Gengenbach and Green, 1975), herbicides (Chaleff and Parsons, 1978), salinity (Nabors et al. 1980) and chilling (Levitt, 1980). In India Brassica campestris cv. (Brown Sarson), the most important oil seed crop, is susceptible to many diseases and pests. The crop requires more frequent treatment with pesticides often leading to higher residue levels and
Correspondence to. K.R.. A r y a
phytotoxicity problems. Tissue culture techniques have been used to enhance the selection of novel somaclonal variations with several Brassiea species, particularly B. oleracea (Zee et al. 1984), B..= alboglabra (Pua et al. 1989), B._= campestris (3ain et al. 1988), but pesticide tolerant somaclonal variants of Brassieas from callus cultures have not been reported so far. The present study demonstrates the successful development of endosulfa~ tolerant mustard plantlets from callus culture, having the capacity to tolerate higher levels of endosulfan, a broad spectrum, contact, residual insecticide. MATERIALS AND METHODS Callus initiation Healthy cotyledonary leaves measuring about 3-4 mm in diameter were excised from eight day old in vitro grown seedlings of B.~. campestris. Leaves were implanted horizontally on Murashige and S,ktoog (1962) medium supplemented with ~.0% sucrose, I00 m~' myoinositol, 0.8% (w/v) agar 0.1 mgl "~ NAA and 2.0 mgl "~ BAP (NSM). Newly formed callus was isolated and subculturod in the same medium at 30 days interval. Isolation and selet~ion * A stock solution of 1 mgl ~ of endosulfan (technical grade from M/s Hoechst India Ltd, Bombay) was prepared in ethyl alcohol, serially diluted and passed through a 0.22 um pre-sterilized nitrocellulose mem. brane filter. Selective medium (SM) was prepared by mixing aliquotes of stock :solution of endosulfan in ethanol with 100 ml of autoclaved NSM medium to a final 0.1, 0.5, 1.0, 1.5 and 2.0 ugl*- endosulfan concentration. Control medium was supplemented with the same volume of ethyl alcohol. Medium SM was then dispensed into sterilized petri plates (Steriware). Small pieces of friable callus were transferred t~ SM medium. The endosulfan concentration of 1.0 ugl'inhibited about 80% growth (Table 1) and was used for selection studies. The newly developed callus on 1.0 ugf endosulfan concentration was isolated and subeultured on selective medium (SM) for 30 days. The callus was then subcultured alternately at 4 weeks intervals on (SM) and (NSM) till no inhibition of callus growth was observed. After 5 alternative subcultures in NSM and SM medium, the endosulfan treated cell line showed a better growth rate than the control and was considered to be an endosulfan tolerant cell line (ETL). The ETL was further subeultured on non-selective medium for 10 passages with periodic checking for a possible reversibility to its original endosulfan sensitivity. The acquired tolerance after 10 generations on NSM was checked by sub qulturing these ETL cell lines on 1.0, 2.0, 3.0 and 4.0 ugl "~ endosulfan containing medium. The cell lines were obs.~rved to tolerate endosulfan concentrations up to 3.0 ugl "1. These lines were termed as ETL 1 cell lines.
138 R e g e n e r a t i o n and t e s t i n g - Cultures developed from the ETL cell line were transferred to regeneration l m e d i u m containin~ MS basal salts s u p l e m e n t e d with 2.0 mgl" Z and 0.1 m g l IAA. R e g e n e r a t e d l S h ~ 1 7 6 were transferred to SM medium containing 3.0 ugl" endosulfan and their growth was visibly compared with that of control shoots on the s a m e medium. Well developed shoots about 2-~ em long were transferred to MS medium containing 0.5 mgt "1 N A A for rooting to develop complete plantlets (Arya et al. 1991). The p e r m a n e n c e of the tolerance towards endosulfan was studied by inducing f r e s h callus from t h e juvenile leaf of t h e s e regenerated ETL plants. The induced callus was again .e3xposed several times to SM medium containing 1.0-4.0 .ugl "~ endosulfan. Callus exhibiting tolerance up to 3.0 ugl "1 endosulfan was termed as the ETL 2 cell line. Biochemical investigation - Weighed s a m p l e s of callus were homogenized in a pestle and m o r t a r with 2 ml of chilled 0.06 M phosphate buffer (pH 6.1). The extract was centrifuged at 600xg (IEC-25 refrigerated centrifuge) for 5 min and the s u p e r n a t a n t was u s e d for the estimation of total free sugars, free amino acids, protein and peroxidase activity following standard methods of Montgomery (1957), Lee and Takahashi (1966), Lowry et ah (1951), Srivastava et al. (1972), respectively. RESULTS AND DISCUSSION Exposure to increasing concentrations of endosulfan resulted in a progression of reduced growth of the eall~s culture (Table 1). Concentrations of 0.5 and 1.0 mgl reduced growth to 61 and 21% respectively, in comparison to the controls. At t h e 1.5.2.0 u g t " level of endosulfan t h e growth was negligible and t h e callus was dark brown after 30 days of incubation. The inhibition of growth in t h e p r e s e n c e of endosulfan m a y b e either d u e to interference in t h e basic metabolic functions, p r e s u m a b l y hormonal control of cell growth and cell elongation (Agarwal and Beg, 1982) or pesticide inhibition of the synthesis of related metabolites (Haliga, 1984). However, p.rpeise targets of endosulfan action are unknown. At 1.0 u g l ' - endosulfan, about 80% growth inhibition was observed after 30 days of incubation, while prolonged incubation (40-45 darts) showed almost complete lethality. In contrast, at 0.5 u g l ' - the general a p p e a r a n c e and growth of t h e callus was similar to that of e~ntrol even after 40.45 days of incubation. Therefore, 1 ugl dose was considered to be the lethal concentration and cells growing after 30 days on this level were used for t h e selection of the r e s i s t a n t phenotype. Selection for growth on medium s u p p l e m e n t e d with pesticides at a concentration toxic to
normal cells has yielded variant cell lines in n u m e r o u s plant species (Hughes, 1983). Isolation of tolerant cell lines from sensitive cell populations by the direct exposure of callus cultures to the t e s t compounds h a s earlier b e e n obtained u s i n g picloram, amitrol, N-phenyl c a r b a m a t e and 2,4.1:) (Chaleff, 1986). Isolated caUi exhibited better growth r e s p o n s e after five alternating s u b c u l t u r e s on SM and NSM media than the callus maintained on NSM medium as a control (Fig. 1). This result s u g g e s t e ~ that the cell line has acquired tolerance up to the 1.0 ugl "1 exposure concentration. Earlier e x p e r i m e n t s conducted in this laboratory with Cicer arietinum callus culture initiated from root explants revealed acquisition of tolerance after only three alternating e x p o s u r e s on SM and NSM medium (Saxena and Beg, 1988). However, in the p r e s e n t study five alternating s u b c u l t u r e s on SM and NSM were required. Oonzales and Widholm (1985) have also suggested alternating selection and nonselective step in a multistep selection procedure. They used sublethal concentrations of an inhibitor and s u b s e q u e n t l y subcultured into media containing higher inhibitor concentrations. This t y p e of gradual selection procedure h a s also b e e n u s e d to select glyphosate resistance in a carrot eeU culture ( N a f z i g e r et al. 1984). Maintenance of the selected endosulfan tolerant (ETL) cell line for over 10 subculture p a9s s a g e s (ETL.) in NSM medium [ showed that the cell line retained a ~-fold elevated tolerance to the lowest concentration (1.0 ug" ). This suggested that the cell line h a s acquired tolerance at the cellular level.
Callus 5Omg
I
NSM
I
1st passage of subculture
0.69gm
SM (MS + Endosulfon )
(0.078g)
I
NSM NSM~
I I
Ilnd passage of subculture
SM
|
NSM
I f
passageof subculture
Hlrd
[ .
SM
NSM
o
Table 1: Determin[ttion endosulfan on after 30 days. Endosulfal~ Con~fugl "t )
of the
z
inhibitory concentration of basis of % growth of callus
I I I
[Vlh passage of subculture
SM
o~
.c_
NSM
Fresh weight (g/petfi dish~ Inoculated Harvested
% growth of callus
0.0
1.05+0.04
m
14.3+0.5
100.0
0.1
1.06+0.04
13.3+0.7
90.0
0.5
1.06_+0.04
9.4+0.5
61.0
1.0"
1.06+0.03
4.0+0.1
1.5
1.06+0.03
2.01+0.4
7.0
2.0
1.06+0.03
1.12+0.4
0.4
1.38g Control
SM
(t.Ttg] (ET L]
/
10 passage of subcuttuPe on NSM
~Shoo,
regeneration ~nduct;on of Coitus and proliferation
[
(ET L21 (ET L 1)
Testing
22.0
*Endosulfan concentration at which the callus was isolated for selection studies. Figure r e p r e s e n t s t h e m e a n values with + SD of five replicates each.
Vth passage of subculture
Fig. I:
Experimental design for the development of pesticides tolerant plants in B.~. campesttis cv. (Brown Sarson). NSM; Nonselective medium (MS); SM; Selective medium (MS + 1.0 u g l ' l endosulfan). Each p a s s a g e of subculture SM to NSM and NSM to SM contain 30 days interval.
139 similar observation was a l s o reported by Zenk (1974) in a soybean cell suspension culture when the tolerant cell line at 0.3 mM 2,4-D concentration, suhcultured for a period of 6 months in liquid (non-selective) medium, was found to grow in 1.0 mM 2,4-I) containing medium while the non-treated cell line was completely inhibited at 0.3 mM 2,4-19. Tissue culture techniques may also be used to enhance the selection of novel somaclonal variants. N a h o r s et al. (1980) exposed tobacco callus to medium containing higher concentration of NaCI. Plants regenerated from the NaC1 tolerant callus were found to be NaCI tolerant and the trait was heritable in SOlUe
cases.
Vigorously growing
ealli of both the control and ETL
Cell line
Nos. of cultures
Nos.whieh regenerated plant. lets
Height of shoots (cm)
and
Fresh callus, denoted ETL_, was developed from ETL regenerated leaf explants an~ this was re-examined for the retention of tolerance by exposure to increasing concentrations of endosulfan. A three fold higher concentration of endosulfan was found to be tolerated by the selected cell line (Table 3). This revealed that the expression of tolerance
Table 3:
Testing of acquired tolerance by line in comparison to control.
Endosulfan C o.r~eantrati on ugl "~ (SM)
C on trol
the
ETL 2 cell
Fresh weight/Flask* (~) ETL 2
endosulfan
No. of shoot/ plant
Nos. of roots/ plant
Control
20
8 (40%)
4-5
4-5
4-6
ETL
2O
4 (20%)
5-7
3-5
5-7
ETL 1
20
0
The plantlets were counted and measured at the 451h day after callus was transferred to regeneration medium. Number of roots per plant were determined at the 10th day after regenerated shoots were transferred to rooting medium. transferred to rooting mediu~n containing MS basal medium supplemented with 0.5 mgl - NAA, some rooting response was observed in both cell lines. Plantlets regenerated from
Table 4:
the control ce~ line were to 1.0-4.0 ugl endosulfan plan§ showed no phytowith endosulfan up to 3.0 showed severe phytotoxieity
cell
line were separately transferred to regeneration ~edium consisting of .~IS medium supplemented with 2.0 mgl" BAP and 0.1 mgl I A A . Only 15-20% of the ETL cultures regenerated 3-5 multiple shoots about 5-7 cm in length within 40-50 days in comparison with 40% regeneration of shoots in the control eeU line (Table 25. When the plantlets were Table 9. Regeneration potential of control tolerant cell lines (ETL & ETL1).
the ETL cell line as well as tested for tolerance by exposing concentrations. ETL regenerated toxicity symptoms when treated ug level, whelreas control plants at the 1.0 ugl "l exposure level.
0.0
15.2+0.52
15.50+0.62
1.0
4.00+0.08
15.70+0.63
2.0
1.21+0.04
15.69+0.44
3.0
1.03+0.01
15.63+0.35
4.0
1.00+0.01
5.00+0.53
*Fresh weight of each culture was determined after 30 days of inoculation. Data represent the mean values + SD of five replicates derived from 5 plantiets, regenerated from the ETL cell line. was also passed to regenerated plants. When certain constituents were measured on a per unit fresh weight basis in the selected tolerant cell line ETL2, an increase was found in total free sugar (22.5%/ free amino acids (88%), protein (56%) and peroxidase (38%5 in comparison with the control cell line (Table 4). These variations in biochemical constituen+s are the possible indicators of a new genotypic character but it is difficult to know how since the endosulfan toxicity mechanism is unknown. However, the technique
Free sugars, free amino acids, protein and peroxidase levels in control and the ETL 2 cell line callus upon exposure to endosulfan. ETL 2 exposed to endosulfan (ugl "1)
Parameters*
Free sugar
Control
0.0
1.0
2.0
3.0
3.28+0.31 -
4.02+0.01 [+22-5a% ]
3.96+0.10 (-1.49~
3.85+0.02 (-4.33~
3.73+0.02 (.7.2-1%)
[+20.73%]
[+17.37%]
[+13.71%]
Free amino acid
2.61+0.11 -
4.90+0.05 [ + 87-~7g %0]
6.31+0.10 (+2ft.10%0) [+135.05%]
7.14+0.19 (+a5":71%) [+173.60%]
7.80+0.18 (+59-18 %) [+198.96%]
Protein
9.94+0.57
15.50+0.98 [+55.95%]
17.02+1.05 (+9.8~O/o)
17.20+0.27 (+10.'97%5
17.39+0.19 (+12.~9%o)
[+71.24%]
[+73.07%]
[+75.0%]
7.06+0.29
7.18+0.24
7.29+0.33
7.35+0.21
[+38%]
(+01.70% 5
(+0~.53%)
(+0~.I I%)
[+40%]
[+42%]
[+43.55%]
Peroxidase ODIChange/
5.1240.3
rain/rag protein
*Values are expressed as mg/g fresh weight. Values in parenthesis indicate percent increase (+)/decrease (-1 over unexposed ETL2cell line (
) and control cell lines [
], respectively.
140 developed in this study provides a means of identifying eultivars with g e n o t y p i c characters endowing them with increased tolerance towards endosulfan.
Lee YP, Takahashi T (19a6) Anal Bioehem 1 4 : 7 1 - 7 7 Lowry OH, Rosebrough NF, Farr AL, Randall RJ (1951) Biol Chem 193:265-275
J
ACKNOWLEDGEMENT
Maliga P (1984) Ann Rev PI Physiol 35:519-542 The authors are thankful to the Director, ITRC, Lucknow for providing the laboratory facilities and to Mr. Bit Bahadur for technical assistance.
Montgomery R (1957) Arch Biochem Biophys 67:378.386 Murashige T and Skoog F (1962) Physiol Plant 15:473-497
REFERENCES Agarwal S and Beg MU (1982) Ind J Exp Biol 20:319-323
Nabors MW, Gibb SE, Bernstein CS and Meins ME (1980) Z Pflanzenphysiol 9 7 : 1 3 - 1 7
Arya KR, Gupta RC Ecology 9:1028.1031
Nafziger ED, Widholm JM, Steinrucken (1984) P1 Physiol 76:571-574
and
Beg MU (1991)
Environment P,
Chaleff RS (1986) In: Vasil IK (ed) Cell culture and somatic genetics of plants, Academic Press New York, pp 500.511 Chaleff RS and Parsons MF (1978) Proe Nat Acad Sci USA 75:5104-51.07
HC
and
Kilmr JL
Ohyama K (1983) In: Evans DA, Sharp WR, Ammirato PV and Yamda Y (eds) Hand book of plant tissue culture Vol 1, Macmillan Pub Co, New York pp 501.519 Pua E-C, T d n h TH and Chua N-H (1989) Plant Cell Tissue Org Cult 17:143.151
Gengenbach BG and Green CE (1975) Crop Sci 15:645-649 Gonzalcs RA and Widholm JM (1985) In: Dixon RA (ed) Plant tissue culture: Vol 4 a practical approach, Information and Retrieval Ltd Press Hughes KW (1983) In: Sharp WR, Ammirato PV and Yamada Y (eds) Hand book of plant tissue culture, Macmillan, New York, pp 442-460
Saxena RP and Beg MU (1988) Plant Cell Tissue Org Cult 12:9~73.277 Srivastava AK, Azhar S, Krishnamurti CR (1972) 11:318-385 Wrather J$
Phytochem
and Frey-tag AH (1991) Plant Cell Rep 10:44-47
Jain PK, Chowdhury JB, Sharma DR and Friedt W (1988) Plant cell Tissue Org Cult 14:197-206
Zee SY and Johnson BB (1984) In: Evans D, Sharp WR, Ammirato PV and Yamda Y (eds) Hand book of plant cell culture, Macmillan Pub Co, New York, pp 22%246
Levitt J (1980) In: Response of plants to stresses 2rid ed Academic Press, New York.
Zenk MH (1974) In: Kasha KJ (ed) Haploid in higher plants Uni of Guelph Press, Guelph, Canada, pp 339-353.
environmental
Plant Cell Reports (1994) 14:137-140
@, Springer-Verlag 1994
In vitro selection of endosulfan-tolerant strains of Brassica compestris (cv. Brown Sarson) K.R. Arya 1, R.C. Gupta 1 B.D. Semwal z, and M.U. Beg a Division of Phytotoxicology, Industrial Toxicology Research Centre, M.G. Marg, L u c k n o w - 226 00l, India 2 D e p a r t m e n t o f Botany, D.A.V. College, K a n p u r , India Received 16 April 1993,/Revised version received 12 May 1994 - C o m m u n i c a t e d by J. W i d h o l m
ABSTR ACT Endosulfan tolerant lines of mustard (Brassiea campestris cv. Brown Sasson) have been developed through tissue culture methods. Cotyledonary explants excised from eight day old in vitro grown seedlings were used for inducing callus. Fast growing friable callus I was then transferred to MS medium containing (0.1-2.0 u g l ' ) endosulfan for selection. Five alternating exposures with and without endosulfan containing medium yielded an endosulfan tolerant cell line (ETL). The plants regenerated from ETL were found to tolerate three fold higher concentrations of endosulfan. Callus induced from randomly selected endos~lfan tolerant regenerated plants were also tolerant to 3.0 ugl" endosulfan, thereby, suggesting that tolerance has been acquired at the gene level. Biochemical investigation revealed higher levels of total free sugar, f r e e amino acids, protein and activity of peroxidase ~n the tolerant cell line. Key wools: Brassica campestris . Brown Sarson - Endosulfan tolerant.
Abbreviations: MS: Murashige and Skoog medium (1962); NSM: non-selective medium; SM: selective medium; BAP: 6. Benzylaminopurine; N A A : ~ - n a p h t h a l e n e a e e t i e acid; Z: zeatin. INTRODUC3"ION Introduction of genetic tolerance for pesticides into crop plants may have an advantage of expanded applicability of a particular pesticide, which could be used to eliminate the pest without any adverse effect on crop quality. The tradi. tional plant breeding techniques have introduced numerous improvements in crop yield during the past centuries. However, plant breeders have several constraints on the ability to introduce new genetic information into plants and to create new plant varieties through conventional plant breeding techniques (Ohyama, 1983). Recent developments and advancements in plant tissue culture technology have provided an approach to select pesticide tolerant plants (Chaleff 1986, Wrather et al. 1991) and has proven its utility in selecting mutants by reducing the space, time and cost involved. Thereby, progress has been made with regard to the development of resistance to phytotoxins (Gengenbach and Green, 1975), herbicides (Chaleff and Parsons, 1978), salinity (Nabors et al. 1980) and chilling (Levitt, 1980). In India Brassica campestris cv. (Brown Sarson), the most important oil seed crop, is susceptible to many diseases and pests. The crop requires more frequent treatment with pesticides often leading to higher residue levels and
Correspondence to. K.R.. A r y a
phytotoxicity problems. Tissue culture techniques have been used to enhance the selection of novel somaclonal variations with several Brassiea species, particularly B. oleracea (Zee et al. 1984), B..= alboglabra (Pua et al. 1989), B._= campestris (3ain et al. 1988), but pesticide tolerant somaclonal variants of Brassieas from callus cultures have not been reported so far. The present study demonstrates the successful development of endosulfa~ tolerant mustard plantlets from callus culture, having the capacity to tolerate higher levels of endosulfan, a broad spectrum, contact, residual insecticide. MATERIALS AND METHODS Callus initiation Healthy cotyledonary leaves measuring about 3-4 mm in diameter were excised from eight day old in vitro grown seedlings of B.~. campestris. Leaves were implanted horizontally on Murashige and S,ktoog (1962) medium supplemented with ~.0% sucrose, I00 m~' myoinositol, 0.8% (w/v) agar 0.1 mgl "~ NAA and 2.0 mgl "~ BAP (NSM). Newly formed callus was isolated and subculturod in the same medium at 30 days interval. Isolation and selet~ion * A stock solution of 1 mgl ~ of endosulfan (technical grade from M/s Hoechst India Ltd, Bombay) was prepared in ethyl alcohol, serially diluted and passed through a 0.22 um pre-sterilized nitrocellulose mem. brane filter. Selective medium (SM) was prepared by mixing aliquotes of stock :solution of endosulfan in ethanol with 100 ml of autoclaved NSM medium to a final 0.1, 0.5, 1.0, 1.5 and 2.0 ugl*- endosulfan concentration. Control medium was supplemented with the same volume of ethyl alcohol. Medium SM was then dispensed into sterilized petri plates (Steriware). Small pieces of friable callus were transferred t~ SM medium. The endosulfan concentration of 1.0 ugl'inhibited about 80% growth (Table 1) and was used for selection studies. The newly developed callus on 1.0 ugf endosulfan concentration was isolated and subeultured on selective medium (SM) for 30 days. The callus was then subcultured alternately at 4 weeks intervals on (SM) and (NSM) till no inhibition of callus growth was observed. After 5 alternative subcultures in NSM and SM medium, the endosulfan treated cell line showed a better growth rate than the control and was considered to be an endosulfan tolerant cell line (ETL). The ETL was further subeultured on non-selective medium for 10 passages with periodic checking for a possible reversibility to its original endosulfan sensitivity. The acquired tolerance after 10 generations on NSM was checked by sub qulturing these ETL cell lines on 1.0, 2.0, 3.0 and 4.0 ugl "~ endosulfan containing medium. The cell lines were obs.~rved to tolerate endosulfan concentrations up to 3.0 ugl "1. These lines were termed as ETL 1 cell lines.
138 R e g e n e r a t i o n and t e s t i n g - Cultures developed from the ETL cell line were transferred to regeneration l m e d i u m containin~ MS basal salts s u p l e m e n t e d with 2.0 mgl" Z and 0.1 m g l IAA. R e g e n e r a t e d l S h ~ 1 7 6 were transferred to SM medium containing 3.0 ugl" endosulfan and their growth was visibly compared with that of control shoots on the s a m e medium. Well developed shoots about 2-~ em long were transferred to MS medium containing 0.5 mgt "1 N A A for rooting to develop complete plantlets (Arya et al. 1991). The p e r m a n e n c e of the tolerance towards endosulfan was studied by inducing f r e s h callus from t h e juvenile leaf of t h e s e regenerated ETL plants. The induced callus was again .e3xposed several times to SM medium containing 1.0-4.0 .ugl "~ endosulfan. Callus exhibiting tolerance up to 3.0 ugl "1 endosulfan was termed as the ETL 2 cell line. Biochemical investigation - Weighed s a m p l e s of callus were homogenized in a pestle and m o r t a r with 2 ml of chilled 0.06 M phosphate buffer (pH 6.1). The extract was centrifuged at 600xg (IEC-25 refrigerated centrifuge) for 5 min and the s u p e r n a t a n t was u s e d for the estimation of total free sugars, free amino acids, protein and peroxidase activity following standard methods of Montgomery (1957), Lee and Takahashi (1966), Lowry et ah (1951), Srivastava et al. (1972), respectively. RESULTS AND DISCUSSION Exposure to increasing concentrations of endosulfan resulted in a progression of reduced growth of the eall~s culture (Table 1). Concentrations of 0.5 and 1.0 mgl reduced growth to 61 and 21% respectively, in comparison to the controls. At t h e 1.5.2.0 u g t " level of endosulfan t h e growth was negligible and t h e callus was dark brown after 30 days of incubation. The inhibition of growth in t h e p r e s e n c e of endosulfan m a y b e either d u e to interference in t h e basic metabolic functions, p r e s u m a b l y hormonal control of cell growth and cell elongation (Agarwal and Beg, 1982) or pesticide inhibition of the synthesis of related metabolites (Haliga, 1984). However, p.rpeise targets of endosulfan action are unknown. At 1.0 u g l ' - endosulfan, about 80% growth inhibition was observed after 30 days of incubation, while prolonged incubation (40-45 darts) showed almost complete lethality. In contrast, at 0.5 u g l ' - the general a p p e a r a n c e and growth of t h e callus was similar to that of e~ntrol even after 40.45 days of incubation. Therefore, 1 ugl dose was considered to be the lethal concentration and cells growing after 30 days on this level were used for t h e selection of the r e s i s t a n t phenotype. Selection for growth on medium s u p p l e m e n t e d with pesticides at a concentration toxic to
normal cells has yielded variant cell lines in n u m e r o u s plant species (Hughes, 1983). Isolation of tolerant cell lines from sensitive cell populations by the direct exposure of callus cultures to the t e s t compounds h a s earlier b e e n obtained u s i n g picloram, amitrol, N-phenyl c a r b a m a t e and 2,4.1:) (Chaleff, 1986). Isolated caUi exhibited better growth r e s p o n s e after five alternating s u b c u l t u r e s on SM and NSM media than the callus maintained on NSM medium as a control (Fig. 1). This result s u g g e s t e ~ that the cell line has acquired tolerance up to the 1.0 ugl "1 exposure concentration. Earlier e x p e r i m e n t s conducted in this laboratory with Cicer arietinum callus culture initiated from root explants revealed acquisition of tolerance after only three alternating e x p o s u r e s on SM and NSM medium (Saxena and Beg, 1988). However, in the p r e s e n t study five alternating s u b c u l t u r e s on SM and NSM were required. Oonzales and Widholm (1985) have also suggested alternating selection and nonselective step in a multistep selection procedure. They used sublethal concentrations of an inhibitor and s u b s e q u e n t l y subcultured into media containing higher inhibitor concentrations. This t y p e of gradual selection procedure h a s also b e e n u s e d to select glyphosate resistance in a carrot eeU culture ( N a f z i g e r et al. 1984). Maintenance of the selected endosulfan tolerant (ETL) cell line for over 10 subculture p a9s s a g e s (ETL.) in NSM medium [ showed that the cell line retained a ~-fold elevated tolerance to the lowest concentration (1.0 ug" ). This suggested that the cell line h a s acquired tolerance at the cellular level.
Callus 5Omg
I
NSM
I
1st passage of subculture
0.69gm
SM (MS + Endosulfon )
(0.078g)
I
NSM NSM~
I I
Ilnd passage of subculture
SM
|
NSM
I f
passageof subculture
Hlrd
[ .
SM
NSM
o
Table 1: Determin[ttion endosulfan on after 30 days. Endosulfal~ Con~fugl "t )
of the
z
inhibitory concentration of basis of % growth of callus
I I I
[Vlh passage of subculture
SM
o~
.c_
NSM
Fresh weight (g/petfi dish~ Inoculated Harvested
% growth of callus
0.0
1.05+0.04
m
14.3+0.5
100.0
0.1
1.06+0.04
13.3+0.7
90.0
0.5
1.06_+0.04
9.4+0.5
61.0
1.0"
1.06+0.03
4.0+0.1
1.5
1.06+0.03
2.01+0.4
7.0
2.0
1.06+0.03
1.12+0.4
0.4
1.38g Control
SM
(t.Ttg] (ET L]
/
10 passage of subcuttuPe on NSM
~Shoo,
regeneration ~nduct;on of Coitus and proliferation
[
(ET L21 (ET L 1)
Testing
22.0
*Endosulfan concentration at which the callus was isolated for selection studies. Figure r e p r e s e n t s t h e m e a n values with + SD of five replicates each.
Vth passage of subculture
Fig. I:
Experimental design for the development of pesticides tolerant plants in B.~. campesttis cv. (Brown Sarson). NSM; Nonselective medium (MS); SM; Selective medium (MS + 1.0 u g l ' l endosulfan). Each p a s s a g e of subculture SM to NSM and NSM to SM contain 30 days interval.
139 similar observation was a l s o reported by Zenk (1974) in a soybean cell suspension culture when the tolerant cell line at 0.3 mM 2,4-D concentration, suhcultured for a period of 6 months in liquid (non-selective) medium, was found to grow in 1.0 mM 2,4-I) containing medium while the non-treated cell line was completely inhibited at 0.3 mM 2,4-19. Tissue culture techniques may also be used to enhance the selection of novel somaclonal variants. N a h o r s et al. (1980) exposed tobacco callus to medium containing higher concentration of NaCI. Plants regenerated from the NaC1 tolerant callus were found to be NaCI tolerant and the trait was heritable in SOlUe
cases.
Vigorously growing
ealli of both the control and ETL
Cell line
Nos. of cultures
Nos.whieh regenerated plant. lets
Height of shoots (cm)
and
Fresh callus, denoted ETL_, was developed from ETL regenerated leaf explants an~ this was re-examined for the retention of tolerance by exposure to increasing concentrations of endosulfan. A three fold higher concentration of endosulfan was found to be tolerated by the selected cell line (Table 3). This revealed that the expression of tolerance
Table 3:
Testing of acquired tolerance by line in comparison to control.
Endosulfan C o.r~eantrati on ugl "~ (SM)
C on trol
the
ETL 2 cell
Fresh weight/Flask* (~) ETL 2
endosulfan
No. of shoot/ plant
Nos. of roots/ plant
Control
20
8 (40%)
4-5
4-5
4-6
ETL
2O
4 (20%)
5-7
3-5
5-7
ETL 1
20
0
The plantlets were counted and measured at the 451h day after callus was transferred to regeneration medium. Number of roots per plant were determined at the 10th day after regenerated shoots were transferred to rooting medium. transferred to rooting mediu~n containing MS basal medium supplemented with 0.5 mgl - NAA, some rooting response was observed in both cell lines. Plantlets regenerated from
Table 4:
the control ce~ line were to 1.0-4.0 ugl endosulfan plan§ showed no phytowith endosulfan up to 3.0 showed severe phytotoxieity
cell
line were separately transferred to regeneration ~edium consisting of .~IS medium supplemented with 2.0 mgl" BAP and 0.1 mgl I A A . Only 15-20% of the ETL cultures regenerated 3-5 multiple shoots about 5-7 cm in length within 40-50 days in comparison with 40% regeneration of shoots in the control eeU line (Table 25. When the plantlets were Table 9. Regeneration potential of control tolerant cell lines (ETL & ETL1).
the ETL cell line as well as tested for tolerance by exposing concentrations. ETL regenerated toxicity symptoms when treated ug level, whelreas control plants at the 1.0 ugl "l exposure level.
0.0
15.2+0.52
15.50+0.62
1.0
4.00+0.08
15.70+0.63
2.0
1.21+0.04
15.69+0.44
3.0
1.03+0.01
15.63+0.35
4.0
1.00+0.01
5.00+0.53
*Fresh weight of each culture was determined after 30 days of inoculation. Data represent the mean values + SD of five replicates derived from 5 plantiets, regenerated from the ETL cell line. was also passed to regenerated plants. When certain constituents were measured on a per unit fresh weight basis in the selected tolerant cell line ETL2, an increase was found in total free sugar (22.5%/ free amino acids (88%), protein (56%) and peroxidase (38%5 in comparison with the control cell line (Table 4). These variations in biochemical constituen+s are the possible indicators of a new genotypic character but it is difficult to know how since the endosulfan toxicity mechanism is unknown. However, the technique
Free sugars, free amino acids, protein and peroxidase levels in control and the ETL 2 cell line callus upon exposure to endosulfan. ETL 2 exposed to endosulfan (ugl "1)
Parameters*
Free sugar
Control
0.0
1.0
2.0
3.0
3.28+0.31 -
4.02+0.01 [+22-5a% ]
3.96+0.10 (-1.49~
3.85+0.02 (-4.33~
3.73+0.02 (.7.2-1%)
[+20.73%]
[+17.37%]
[+13.71%]
Free amino acid
2.61+0.11 -
4.90+0.05 [ + 87-~7g %0]
6.31+0.10 (+2ft.10%0) [+135.05%]
7.14+0.19 (+a5":71%) [+173.60%]
7.80+0.18 (+59-18 %) [+198.96%]
Protein
9.94+0.57
15.50+0.98 [+55.95%]
17.02+1.05 (+9.8~O/o)
17.20+0.27 (+10.'97%5
17.39+0.19 (+12.~9%o)
[+71.24%]
[+73.07%]
[+75.0%]
7.06+0.29
7.18+0.24
7.29+0.33
7.35+0.21
[+38%]
(+01.70% 5
(+0~.53%)
(+0~.I I%)
[+40%]
[+42%]
[+43.55%]
Peroxidase ODIChange/
5.1240.3
rain/rag protein
*Values are expressed as mg/g fresh weight. Values in parenthesis indicate percent increase (+)/decrease (-1 over unexposed ETL2cell line (
) and control cell lines [
], respectively.
140 developed in this study provides a means of identifying eultivars with g e n o t y p i c characters endowing them with increased tolerance towards endosulfan.
Lee YP, Takahashi T (19a6) Anal Bioehem 1 4 : 7 1 - 7 7 Lowry OH, Rosebrough NF, Farr AL, Randall RJ (1951) Biol Chem 193:265-275
J
ACKNOWLEDGEMENT
Maliga P (1984) Ann Rev PI Physiol 35:519-542 The authors are thankful to the Director, ITRC, Lucknow for providing the laboratory facilities and to Mr. Bit Bahadur for technical assistance.
Montgomery R (1957) Arch Biochem Biophys 67:378.386 Murashige T and Skoog F (1962) Physiol Plant 15:473-497
REFERENCES Agarwal S and Beg MU (1982) Ind J Exp Biol 20:319-323
Nabors MW, Gibb SE, Bernstein CS and Meins ME (1980) Z Pflanzenphysiol 9 7 : 1 3 - 1 7
Arya KR, Gupta RC Ecology 9:1028.1031
Nafziger ED, Widholm JM, Steinrucken (1984) P1 Physiol 76:571-574
and
Beg MU (1991)
Environment P,
Chaleff RS (1986) In: Vasil IK (ed) Cell culture and somatic genetics of plants, Academic Press New York, pp 500.511 Chaleff RS and Parsons MF (1978) Proe Nat Acad Sci USA 75:5104-51.07
HC
and
Kilmr JL
Ohyama K (1983) In: Evans DA, Sharp WR, Ammirato PV and Yamda Y (eds) Hand book of plant tissue culture Vol 1, Macmillan Pub Co, New York pp 501.519 Pua E-C, T d n h TH and Chua N-H (1989) Plant Cell Tissue Org Cult 17:143.151
Gengenbach BG and Green CE (1975) Crop Sci 15:645-649 Gonzalcs RA and Widholm JM (1985) In: Dixon RA (ed) Plant tissue culture: Vol 4 a practical approach, Information and Retrieval Ltd Press Hughes KW (1983) In: Sharp WR, Ammirato PV and Yamada Y (eds) Hand book of plant tissue culture, Macmillan, New York, pp 442-460
Saxena RP and Beg MU (1988) Plant Cell Tissue Org Cult 12:9~73.277 Srivastava AK, Azhar S, Krishnamurti CR (1972) 11:318-385 Wrather J$
Phytochem
and Frey-tag AH (1991) Plant Cell Rep 10:44-47
Jain PK, Chowdhury JB, Sharma DR and Friedt W (1988) Plant cell Tissue Org Cult 14:197-206
Zee SY and Johnson BB (1984) In: Evans D, Sharp WR, Ammirato PV and Yamda Y (eds) Hand book of plant cell culture, Macmillan Pub Co, New York, pp 22%246
Levitt J (1980) In: Response of plants to stresses 2rid ed Academic Press, New York.
Zenk MH (1974) In: Kasha KJ (ed) Haploid in higher plants Uni of Guelph Press, Guelph, Canada, pp 339-353.
environmental
