Plant Cell Reports (1983) 2:252-254
Plant Cell Reports
© Springer-Verlag 1983
Shoot Regeneration in Root Cultures of Solanaceae A. Zelcer,'O. Soferman, and S. Izhar Division of Breeding and Plant Genetics, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50-250, Israel Received May 12, 1983 / August 22, 1983 -Communicated by I. K. Vasil
Abstract Root cultures from several species of the Solanaceae were i n i t i a t e d and subcultured on Murashige and Skooq medium without growth regulators. Direct shoot regeneration was observed in four d i f f e r e n t species. The e f f e c t of several concentrations of auxin (IAA) and cytokinins (BAP, zeatin) on the number of shoots generated by two h i g h l y resoonsive species (Nicotiana exigua, N. debneyi) is described. Abbreviations: RAP, benzylaminoDurine; IAA, i n d o l acetic acid; NAA, naDhtaleneacetic acid. Introduction Numerous studies on mode of action and biosynthesis of growth regulators (Davidonis et a l . , 1978; Koda and Okazawa, 1978; Abou-~andour a-n-d~artung, 1980), production of natural compounds (Lui and Staba, 1979; Zito and Staba, 1982), anatomy of organogenesis (Bonnet and Torrey, 1966; Thomas and Street, 1972) and several other asnects, have made extensive use of plant root cultures over the past 50 years. The work on culture of isolated roots was reviewed in 1964 by Butcher and Street. In several instances, shoot reqeneratipn has been reported to occur in established root cultures. The described de novo shoots were generated e i t h e r endogenously, i . e , from the root Dericycle, and apoarently without involvement of callus tissue (Seeliqer, 1956; Torrey, 1958) Dr else apoeared at l a t e r stages, subseouently to the induction of callus outqrowths (Norton and B o i l , 1954; Danckwardt-Lilliestrom, 1957; Thomas and Street, 1972; Coli.in et a l . , 1979). As part of a general e f f o r t to screen f o r novel systems to be used f o r genetic manioulation of plants, we report here our preliminary results on shoot regeneration in r o o t - c u l t u r e s of several species of the Solanaceae. Materials and Methods Seeds were s u r f a c e - s t e r i l i z e d with a 25% solution of commercial bleach for 30' and germinated on s o l i d i fied MS medium (Murashige and Skoog, 1962; obtained from Flow Lab.) supplemented with 3% sucrose and 1% * Contribution from the A q r i c u l t u r a l Research Organization, The Volcani Center, Bet Dagan, I s r a e l , No. 743-E, 1983 series.
agar (Difco, Bacto-Agar), Seedlings were grown in a c u l t u r e room at 26nC, under a 16h nhotoperiod and a mixed fluorescent and incandescent i l l u m i n a t i o n . Plantlets with well-develooed root systems (2-4 weeks old) served as the source f o r root exDlants. For some snecies, root exDlants were obtained from rooted shoot-cultures that had been established in our laboratory 3-6 months p r i o r to the i n i t i a t i o n of the present work. Three-cm segments of root terminals were transferred to 125-mi Erlenmeyer flasks containing 20 ml of l i a u i d culture media (MS basal medium with 3% sucrose. or as stated otherwise). Roots were maintained under the environmental conditions previously mentioned, as agitated cultures (100 rom) in o r b i t a l shakers (New Brunswick) unless stated otherwise, Subcultures were performed by t r a n s f e r r i n g root segments into new flasks at lO-20-day i n t e r v a l s , In those experiments designed to test the e f f e c t of growth regulators, root segments were cultured in "25 compartment-dishes '~ ( S t e r i l i n Lab,) with 2-3 ml of l i q u i d medium per w e l l , Basal culture medium was s t e r i l i z e d by autoclaving, while qrowth regulators were f i l t e r - s t e r i l i z e d and added subsequently to culture vessels as needed, Results and Discussion Three d i f f e r e n t basal media were i n i t i a l l y assayed in root cultures of several species: MS medium, a modified White medium (Butcher, 1980) and STW medium (Orion et a l . , 1980). Although the growth rate of some species seemed higher in other media, the MS basal medium was adonted as the standard medium f o r the sake of comparison in f u r t h e r experiments with a l l species. I t had been observed in early stages of t h i s work that shaking of c u l t u r e flasks c o n s i s t e n t l y generated a better qrowth rate as compared with s t i l l cultures; thus, a l l root cultures were maintained under c o n t i . nuous shaking. I t soon became evident that some species had a tendency to develoo shoot buds i f cultures were maintained long enough (3 weeks or longer) without subculture (Fig. I ) , Table 1 summarizes the shootd i f f e r e n t i a t i o n response of the d i f f e r e n t species included in the experiments. Development of shoots in the four responsive species l i s t e d in Table 1 occurs apparently without any formation of callus as
253 an intermediate step, and is therefore to be t e n t a t i v e l y considered as a new example of " d i r e c t shoot organogenesis." Anatomical studies w i l l be required to determine the o r i g i n of the regenerated shoots. This response has been followed through many passages over several months of subculturing, without any apparent decline in the shoot-forming capabilities. The e f f e c t of growth regulators on shoot organogenesis was monitored in some of the species (Table 1). Several dosage combinations of auxins (IAA or NAA) and cytokinin IBAP) induced a profuse development of callus tissue and subsequent shoot d i f f e r e n t i a t i o n (results not shown). Our observations confirm a previous report on shoot nrganogenesis in Nicotiana glauca root cultures (Solt et a l . , 1960). The e f f e c t of growth regulators on two new highly responsive species (N. debneyi and N. exi~u_a_a)was f u r t h e r characterized and is summarized in Table 2. Several concentrations of cytokinins increased the degree of shoot regeneration in both species, although a better response was observed when several combined levels of auxin (IAA) and a cytokinin (BAP or Zeatin) were utilized. In t h i s case, higher amounts of zeatin than of BAP were required for a s i m i l a r response to be attained. I t should be stressed t h a t , except for the p o s i t i v e responses achieved in MS medium without growth regulators and l i s t e d in Table 1, the development of greenish callus preceded the regeneration of shoots in a l l tested species.
-.~: Shoot regeneration jn rqot-cultwres of ~iglCO~]ana ex~gua on mb Dasa~ mem~um, bnoo~ i n i t i a l s
are g~ate-e-c[continuously in older roots and attained the size shown a f t e r 2 weeks of additional incubation. Table 1:
Shoot d i f f e r e n t i a t i o n in root-cultures of Solanaceae*
SPECIES
SHOOT REGENERATION IN MS MEDIUM WITHOUT GROWTH REGULATORS
OBSERVATIONS
e f f e c t of growth regulators not tested
Lycopersicon esculentum L. esculentum var. cerasiforme L. p i m p i n e l l i f o l i u m Nicotiana tabacum .
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N. debneyi exigua. r / _ : : .............................................................. N. glauca
.
.
.
.
.
.
.
BAP+IAA (several concentrations) induced .
++ ++
.
.
.
.
.
.
.
.
.
.
.
~!!u~_~b_t_~b~_fe~m~i~
.......................
1-3 mg/It BAP induced callus and enhanced ~ Q ~ _ ~ i 2 ~
-
...........
shoot and callus formation enhanced by several concentrations of growth regula~e~_!~_!~!~_TL
++
...................................................................
N. petunoides
.
...........................
BAP+IAA or BPA+NAA induced callus and shoots
Substitution l i n e s of N. tabacum containing the f o l l o w i n g cyto-~sms: I. N. debneyi
..................................................................
~ : ~ f ~ _ : t ~ ! ~ : ~ _ ~ _ f ~
I I . N. glutinosa I I I . N. megalosiphon
..............
BPA+IAA induced callus growth and shoot formation
Petunia parodii
poor growth of roots in late passages
P. p a r o d i i , cytoplasmic male s t e r i l e
growth regulators not tested
Solanum melongena
+
growth regulators not tested
* Root cultures were maintained under continuous shaking and morphogenetic response was recorded 3 weeks a f t e r subculture ( -, no shoots; +, sporadic shoot formation; ++ consistent shoot formation).
254 Root cultures grown in MS medium without growth regulators e x h i b i t a high degree of chromosomal s t a b i l i t y and have been used in our laboratory as a source of a large number of protoplasts (results not shown). The d i r e c t root-organogenesis observed in some species suggests that root cultures could Table 2:
be used e f f i c i e n t l y as screening systems for the detection and one-step i s o l a t i o n of plant mutants which e x h i b i t variant root-associated t r a i t s of potential economic value (e.g. s a l t tolerance and resistance to other soil environment stresses, or resistance to root pathogens).
Effect of growth regulators (mg/l) on degree of shoot formation in root cultures of two Nicotiana species a
OBSERVED NUMBEROF SHOOTS Nicotiana exigua
0 1 3 5 10
0 1 3 5 I0
Nicotiana debneyi
0
1
3
5
I0
0
1
3
5
I0
3 0 0 0 0
5 25 38 12 0
1 10 19 15 0
0 2 15 5 0
0 2 0 0 0
5 0 0 i 0
10 25 18 20 0
12 20 18 18 0
8 17 15 10 0
2 19 12 7 0
0
1
3
5
I0
0
1
3
5
I0
2 0 0 0 0
5 3 0 0 0
25 15 10 10 0
20 22 25 35 5
10 8 45 40 10
3 0 0 0 0
5 0 0 3 0
4 1 5 8 i
8 9 16 7 1
8 9 18 12 0
aRoot cultures were maintained in 25-compartment dishes, as described in Materials and Methods. The number of shoots was determined under low magnification of a dissecting microscope 4 weeks a f t e r the t r a n s f e r of root i n i t i a l s . Acknowledgements The active p a r t i c i p a t i o n of Osnat Yotko and Rakefet Kochavy in this project is highly appreciated. We thank Dr. D. Lapushner and Dr. R. Frankel for seed samples of Nicotiana spp. and s u b s t i t u t i o n l i n e s . This research was supported by the Binational (US-lsrael) A g r i c u l t u r a l Research and Development Fund (BARD). References Abou-Mandour, AA, Hartung, W (1980) Z. Pflanzenphysiol. 100: 25-33. Bonnet, HT, Torrey, JG (1966) Amer. J. Bot. 53: 496-507. Butcher, DN (1980) In: Ingram, DS, Helgeson, JP (eds.). Tissue Culture Methods f o r Plant Pathologists, Blackwell, Oxford, pp. 13-17. Butcher, DN, Street, HE (1964) Bot. Rev. 30:513-526.
C o l i j n , CM, Kool, AJ, Nijkamp, HJJ (1979) Protoplasma 99: 335-340. Danckwardt-Lilliestrom, C (1957) Physiol. Plant. 10: 794-797. Davidonis, GH, Hamilton, RH, Mumma, RO (1978) Plant Physiol. 62: 80-83. Koda, Y, Okazawa, Y (1978) Physiol. Plant. 44: 412-416. Lui, JHC, Staba, EJ (1979) Phytochemistry 18:1913-1916. Murashige, T, Skoog, F (1962) Physiol. Plant. 15: 473-497. Norton, JP, B o l l , WG (1954) Science 119: 220-221. Orion, D, Wergin, WP, Endo, BY (1980) J. Nematol. 12: 196-203. Seeliger, I (1956) Flora 144: 47-83. Solt, ML, Dawson, RF, Christman, DR (1960) Plant Physiol. 35: 887-894. Thomas, E, Street, HE (1972) Ann. Bot. 36: 239-247. Torrey, JG (1958) Plant Physiol. 33: 258-263. Z i t o , SW, Staba, EJ (1982) Planta Med. 45: 53-54.
Plant Cell Reports
© Springer-Verlag 1983
Shoot Regeneration in Root Cultures of Solanaceae A. Zelcer,'O. Soferman, and S. Izhar Division of Breeding and Plant Genetics, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50-250, Israel Received May 12, 1983 / August 22, 1983 -Communicated by I. K. Vasil
Abstract Root cultures from several species of the Solanaceae were i n i t i a t e d and subcultured on Murashige and Skooq medium without growth regulators. Direct shoot regeneration was observed in four d i f f e r e n t species. The e f f e c t of several concentrations of auxin (IAA) and cytokinins (BAP, zeatin) on the number of shoots generated by two h i g h l y resoonsive species (Nicotiana exigua, N. debneyi) is described. Abbreviations: RAP, benzylaminoDurine; IAA, i n d o l acetic acid; NAA, naDhtaleneacetic acid. Introduction Numerous studies on mode of action and biosynthesis of growth regulators (Davidonis et a l . , 1978; Koda and Okazawa, 1978; Abou-~andour a-n-d~artung, 1980), production of natural compounds (Lui and Staba, 1979; Zito and Staba, 1982), anatomy of organogenesis (Bonnet and Torrey, 1966; Thomas and Street, 1972) and several other asnects, have made extensive use of plant root cultures over the past 50 years. The work on culture of isolated roots was reviewed in 1964 by Butcher and Street. In several instances, shoot reqeneratipn has been reported to occur in established root cultures. The described de novo shoots were generated e i t h e r endogenously, i . e , from the root Dericycle, and apoarently without involvement of callus tissue (Seeliqer, 1956; Torrey, 1958) Dr else apoeared at l a t e r stages, subseouently to the induction of callus outqrowths (Norton and B o i l , 1954; Danckwardt-Lilliestrom, 1957; Thomas and Street, 1972; Coli.in et a l . , 1979). As part of a general e f f o r t to screen f o r novel systems to be used f o r genetic manioulation of plants, we report here our preliminary results on shoot regeneration in r o o t - c u l t u r e s of several species of the Solanaceae. Materials and Methods Seeds were s u r f a c e - s t e r i l i z e d with a 25% solution of commercial bleach for 30' and germinated on s o l i d i fied MS medium (Murashige and Skoog, 1962; obtained from Flow Lab.) supplemented with 3% sucrose and 1% * Contribution from the A q r i c u l t u r a l Research Organization, The Volcani Center, Bet Dagan, I s r a e l , No. 743-E, 1983 series.
agar (Difco, Bacto-Agar), Seedlings were grown in a c u l t u r e room at 26nC, under a 16h nhotoperiod and a mixed fluorescent and incandescent i l l u m i n a t i o n . Plantlets with well-develooed root systems (2-4 weeks old) served as the source f o r root exDlants. For some snecies, root exDlants were obtained from rooted shoot-cultures that had been established in our laboratory 3-6 months p r i o r to the i n i t i a t i o n of the present work. Three-cm segments of root terminals were transferred to 125-mi Erlenmeyer flasks containing 20 ml of l i a u i d culture media (MS basal medium with 3% sucrose. or as stated otherwise). Roots were maintained under the environmental conditions previously mentioned, as agitated cultures (100 rom) in o r b i t a l shakers (New Brunswick) unless stated otherwise, Subcultures were performed by t r a n s f e r r i n g root segments into new flasks at lO-20-day i n t e r v a l s , In those experiments designed to test the e f f e c t of growth regulators, root segments were cultured in "25 compartment-dishes '~ ( S t e r i l i n Lab,) with 2-3 ml of l i q u i d medium per w e l l , Basal culture medium was s t e r i l i z e d by autoclaving, while qrowth regulators were f i l t e r - s t e r i l i z e d and added subsequently to culture vessels as needed, Results and Discussion Three d i f f e r e n t basal media were i n i t i a l l y assayed in root cultures of several species: MS medium, a modified White medium (Butcher, 1980) and STW medium (Orion et a l . , 1980). Although the growth rate of some species seemed higher in other media, the MS basal medium was adonted as the standard medium f o r the sake of comparison in f u r t h e r experiments with a l l species. I t had been observed in early stages of t h i s work that shaking of c u l t u r e flasks c o n s i s t e n t l y generated a better qrowth rate as compared with s t i l l cultures; thus, a l l root cultures were maintained under c o n t i . nuous shaking. I t soon became evident that some species had a tendency to develoo shoot buds i f cultures were maintained long enough (3 weeks or longer) without subculture (Fig. I ) , Table 1 summarizes the shootd i f f e r e n t i a t i o n response of the d i f f e r e n t species included in the experiments. Development of shoots in the four responsive species l i s t e d in Table 1 occurs apparently without any formation of callus as
253 an intermediate step, and is therefore to be t e n t a t i v e l y considered as a new example of " d i r e c t shoot organogenesis." Anatomical studies w i l l be required to determine the o r i g i n of the regenerated shoots. This response has been followed through many passages over several months of subculturing, without any apparent decline in the shoot-forming capabilities. The e f f e c t of growth regulators on shoot organogenesis was monitored in some of the species (Table 1). Several dosage combinations of auxins (IAA or NAA) and cytokinin IBAP) induced a profuse development of callus tissue and subsequent shoot d i f f e r e n t i a t i o n (results not shown). Our observations confirm a previous report on shoot nrganogenesis in Nicotiana glauca root cultures (Solt et a l . , 1960). The e f f e c t of growth regulators on two new highly responsive species (N. debneyi and N. exi~u_a_a)was f u r t h e r characterized and is summarized in Table 2. Several concentrations of cytokinins increased the degree of shoot regeneration in both species, although a better response was observed when several combined levels of auxin (IAA) and a cytokinin (BAP or Zeatin) were utilized. In t h i s case, higher amounts of zeatin than of BAP were required for a s i m i l a r response to be attained. I t should be stressed t h a t , except for the p o s i t i v e responses achieved in MS medium without growth regulators and l i s t e d in Table 1, the development of greenish callus preceded the regeneration of shoots in a l l tested species.
-.~: Shoot regeneration jn rqot-cultwres of ~iglCO~]ana ex~gua on mb Dasa~ mem~um, bnoo~ i n i t i a l s
are g~ate-e-c[continuously in older roots and attained the size shown a f t e r 2 weeks of additional incubation. Table 1:
Shoot d i f f e r e n t i a t i o n in root-cultures of Solanaceae*
SPECIES
SHOOT REGENERATION IN MS MEDIUM WITHOUT GROWTH REGULATORS
OBSERVATIONS
e f f e c t of growth regulators not tested
Lycopersicon esculentum L. esculentum var. cerasiforme L. p i m p i n e l l i f o l i u m Nicotiana tabacum .
.
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N. debneyi exigua. r / _ : : .............................................................. N. glauca
.
.
.
.
.
.
.
BAP+IAA (several concentrations) induced .
++ ++
.
.
.
.
.
.
.
.
.
.
.
~!!u~_~b_t_~b~_fe~m~i~
.......................
1-3 mg/It BAP induced callus and enhanced ~ Q ~ _ ~ i 2 ~
-
...........
shoot and callus formation enhanced by several concentrations of growth regula~e~_!~_!~!~_TL
++
...................................................................
N. petunoides
.
...........................
BAP+IAA or BPA+NAA induced callus and shoots
Substitution l i n e s of N. tabacum containing the f o l l o w i n g cyto-~sms: I. N. debneyi
..................................................................
~ : ~ f ~ _ : t ~ ! ~ : ~ _ ~ _ f ~
I I . N. glutinosa I I I . N. megalosiphon
..............
BPA+IAA induced callus growth and shoot formation
Petunia parodii
poor growth of roots in late passages
P. p a r o d i i , cytoplasmic male s t e r i l e
growth regulators not tested
Solanum melongena
+
growth regulators not tested
* Root cultures were maintained under continuous shaking and morphogenetic response was recorded 3 weeks a f t e r subculture ( -, no shoots; +, sporadic shoot formation; ++ consistent shoot formation).
254 Root cultures grown in MS medium without growth regulators e x h i b i t a high degree of chromosomal s t a b i l i t y and have been used in our laboratory as a source of a large number of protoplasts (results not shown). The d i r e c t root-organogenesis observed in some species suggests that root cultures could Table 2:
be used e f f i c i e n t l y as screening systems for the detection and one-step i s o l a t i o n of plant mutants which e x h i b i t variant root-associated t r a i t s of potential economic value (e.g. s a l t tolerance and resistance to other soil environment stresses, or resistance to root pathogens).
Effect of growth regulators (mg/l) on degree of shoot formation in root cultures of two Nicotiana species a
OBSERVED NUMBEROF SHOOTS Nicotiana exigua
0 1 3 5 10
0 1 3 5 I0
Nicotiana debneyi
0
1
3
5
I0
0
1
3
5
I0
3 0 0 0 0
5 25 38 12 0
1 10 19 15 0
0 2 15 5 0
0 2 0 0 0
5 0 0 i 0
10 25 18 20 0
12 20 18 18 0
8 17 15 10 0
2 19 12 7 0
0
1
3
5
I0
0
1
3
5
I0
2 0 0 0 0
5 3 0 0 0
25 15 10 10 0
20 22 25 35 5
10 8 45 40 10
3 0 0 0 0
5 0 0 3 0
4 1 5 8 i
8 9 16 7 1
8 9 18 12 0
aRoot cultures were maintained in 25-compartment dishes, as described in Materials and Methods. The number of shoots was determined under low magnification of a dissecting microscope 4 weeks a f t e r the t r a n s f e r of root i n i t i a l s . Acknowledgements The active p a r t i c i p a t i o n of Osnat Yotko and Rakefet Kochavy in this project is highly appreciated. We thank Dr. D. Lapushner and Dr. R. Frankel for seed samples of Nicotiana spp. and s u b s t i t u t i o n l i n e s . This research was supported by the Binational (US-lsrael) A g r i c u l t u r a l Research and Development Fund (BARD). References Abou-Mandour, AA, Hartung, W (1980) Z. Pflanzenphysiol. 100: 25-33. Bonnet, HT, Torrey, JG (1966) Amer. J. Bot. 53: 496-507. Butcher, DN (1980) In: Ingram, DS, Helgeson, JP (eds.). Tissue Culture Methods f o r Plant Pathologists, Blackwell, Oxford, pp. 13-17. Butcher, DN, Street, HE (1964) Bot. Rev. 30:513-526.
C o l i j n , CM, Kool, AJ, Nijkamp, HJJ (1979) Protoplasma 99: 335-340. Danckwardt-Lilliestrom, C (1957) Physiol. Plant. 10: 794-797. Davidonis, GH, Hamilton, RH, Mumma, RO (1978) Plant Physiol. 62: 80-83. Koda, Y, Okazawa, Y (1978) Physiol. Plant. 44: 412-416. Lui, JHC, Staba, EJ (1979) Phytochemistry 18:1913-1916. Murashige, T, Skoog, F (1962) Physiol. Plant. 15: 473-497. Norton, JP, B o l l , WG (1954) Science 119: 220-221. Orion, D, Wergin, WP, Endo, BY (1980) J. Nematol. 12: 196-203. Seeliger, I (1956) Flora 144: 47-83. Solt, ML, Dawson, RF, Christman, DR (1960) Plant Physiol. 35: 887-894. Thomas, E, Street, HE (1972) Ann. Bot. 36: 239-247. Torrey, JG (1958) Plant Physiol. 33: 258-263. Z i t o , SW, Staba, EJ (1982) Planta Med. 45: 53-54.
