Pl~nta (Berl.) 123, 307--310 (1975) 9 by Springer-Verlag 1975
Short Communication
Organogenesis from Callus Culture of Hordeum vulgare T s a i - Y i n g Cheng* a n d H a r o l d H. S m i t h Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA Received 20 February 1974; accepted 28 January 1975 Summary. Rapidly proliferating callus cultures from the apical meristem of Hordeum vulgate L., cultivars "Himalaya" and "Mari", were established on a defined medium supplemented with auxin and cytokinin. A frequency of organ differentiation of ca. 85% was obtained by transfer to the same medium but without any growth regulators. All regenerated plants had the diploid chromosome number of 14 (2n). Organogeaesis in vitro from callus cultures of crop species of t h e Gramineae h a v e beer1 r e p o r t e d for o a t s [1], rice [5], w h e a t [6] a n d s o r g h u m [3]. W i t h t h e e x c e p t i o n of rice, no q u a n t i t a t i v e d a t a were p r e s e n t e d for callus g r o w t h a n d organ formation. These previous r e p o r t s h a v e s h o w a t h a t a r e l a t i v e l y high a u x i n c o n c e a t r a t i o u is r e q u i r e d for b o t h i n i t i a t i o n a n d m a i n t e n a n c e of callus, a n d t h a t s u b s e q u e a t l y deprivir~g t h e callus of all g r o w t h r e g u l a t o r s results in shoot formation. I n this p a p e r , i m p r o v e m e n t s are r e p o r t e d in m e t h o d s for o b t a i n i n g r a p i d proliferation of b a r l e y ca]li a n d for d e p e n d a b l e high f r e q u e n c y d i f f e r e n t i a t i o n of b a r l e y p l a n t s f r o m these calli. Two cultivars of barley, Hordeum vulgate L., "Himalaya" and "Mari" were used in these studies (seed source: Dr. Paul Bottino from stocks maintained by Dr. R. A. Nilan, Washington State University, Pullman, Wash., USA.) The solid medium was modified from Murashige and Skoog [4] and Cheng [2] to contain, per liter (1) inorganic salts: 1.65 g NH~N03, 1.90 g KN0~ 0.44 g CaCle. 2 H20, 0.37 g FIgSO~. 7H~0, 0.17 g KH2P04, 30 mg FeSO4.7H20, 36 mg Na2EDTA, 6.2 mg H~B03, 22.3 mg MnSQ.4H20, 10.5 mg ZnSO~-7H20 , 0.8 rag KI, 0.3 mg NaMoO4-2H20, 0.03 rag CuSQ-5 H~O, and 0.03rag CoC12-6H20; and (2) organic substances: 500rag myo-inositol, 5rag thiamine HC1, 30 g sucrose and 0.8% agar (pH 5.5). The plant regulators added to the medium were the auxins indole-3-acetic acid (IAA), 2,4-dichlorophenoxyaeetie acid (2,4-D), ~.-naphthaleneacetic acid (NAA) and p-ehlorophenoxyaeetic acid (CPA), and the cy~kinin 6-(3-methyl-2-butenyl-amino)purine (2iP). All media were autoclaved for 8 rain at 121 ~ with 1.41 kg/em 2 pressure. The growth of barley in culture was arbitrarily divided into three stages: callus initiation, callus maintenance, and organ induction. The same basal medium was used throughout but different amounts of plant growth substances were used for each of these stages. The callus-initiating medium (CI-medium) contained 10 btM IAA, 15 btM 2,4-D and 1.5 ~M 2iP. The callus-maintaining medium (CM-medium) contained similar amounts of IAA and 2iP plus one of the following: 5-20 b~H 2,4-D, 2 0 4 0 b~M NAA, or 2 0 4 0 y2r CPA. No growth substances were included in the shoot-inducing medium (SI-medium). For experiments on the initiation of callus in culture, apical meristems dissected from l-week-old seedlings were used. The seedlings were grown from dehusked seeds sterilized for 5 min with 20 fold diluted Clorox (5.25 % sodium hypochlorite) plus 0.2 % Alconox (a detergent used for cleaning glass, Scientific Glass Apparatus, Bloomfield, N.J., USA) and germi* Present address: Chemistry Dep~rtment, Oregon Graduate Center, Beaverton, Oregon 97005, USA.
308
Tsai-Ying Cheng and H. H. Smith
Table 1. Increase in fresh weight of barley callus culture The callus was cultured on a CiK-medium containing 2,4-D or NAA. Each mean fresh weight and its standard error were calculated from six samples of 25-day-old cultures. The inoculum for each callus weighed about 4 rag. Cultivar
Growth substance and conc. (tzl~)
Final fr. wt. (mg/piece)
Ratio final/initial
Himalaya
2,4-D (5) IqAA (20)
513~: 64 604• 112
128 152
Marl
2,4-D (5) NAA (20)
704• 164 752• 81
186 188
nated on a solid culture medium lacking all plant growth regulators. The meristems were cultured on CI-medium in a growth chamber at 25~ with a 16-h photoperiod (600 fc and an 8-h dark period). Within 3-5 weeks, sizable callus tissue appeared around the edge of the initial explant. All callus was kept growing in the exponential phase by subculturing it every 3 4 weeks on fresh CM-medium. Measurements of growth of barley callus (Table 1) show that the fresh weight at 25 days was greater by at least two orders of magnitude than the inoeulum weight. The synthetic auxins, 2,4-D, NAA, and CPA, at concentrations of 5 to 40 tzM were sufficient to support rapid proliferation of callus. Growth was similar for both varieties, and exceeded by far that reported for most other in-vitro cultures of Gramineae. The auxin I A A was added to provide growth stimulation during the initial culture period. However, IAA alone was not sufficient to support callus growth, and the synthetic auxins, 2,4-D, NAA or CPA were also incorporated into the media in order to maintain cultures at their maximum growth rate. Although the need for the eytokinin (2iP) at this stage of the culture has not been ascertained, it was always included in the CI- and CMmedia. The 2iP was used because it is as a rule more active than kinetin in promoting both cell growth and organ formation [7]. Organ differentiation in culture was initiated by transferring exponentially growing calli from CM-medium to SI-medium which is devoid of all growth substances. Although kinetin has been used for shoot induction in some monocotyledons such as rice, wheat, and oats, it is not essential at least in barley. Shoots began to emerge from the surfaces of calli in ca. 4 weeks. The differentiated ealli were then transferred to SI-medium diluted 1 : 1 in order to give a lower salt concentration that enhanced root production. A typical plant regenerated from such a callus (Fig. 1 a) is shown in Fig. 1 b. The resulting plants were grown to maturity in soil in pots under greenhouse conditions. They did not differ in either morphology of fertility from control plants grown from seed. Ca. 85% of the calli that had been through 3 consecutive transfers exhibited organogenesis; ca. 60% of those subcultured 5 times did so (Table 2). Ability for short production was completely lost after 4-5 months culture. A similar decline and loss of organogenetie capacity with prolonged culture has also been observed in rice. Cytological examination of root tips stained by the Feulgen method showed that the restored plants had maintained the diploid chromosome number of 14
Organogenesis in Barley Callus Culture
309
Fig. 1. (a) Callus culture of Hordeum vulgare "Himalaya" from which plants were regenerated. (b) Barley plants shown about 8 weeks after first appearance of shoots from callus placed on SI-medium after three consecutive cultivations (originating from the apical meristem) on CM-medium containing 10 aM IAA, 20 [~M NAP_ and 1.5 ~M 2iP
Table 2. Frequency of plants differentiated in vitro from callus cultures of Hordeum vulgate "Himalaya" No. times subcultured
No. calli
No. plants induced
Organogenesis (% of oalli)
3 5
13 15
11 6
85 60
(2n). No chromosomal aberations were detected in a n y of the cells examined. This o b s e r v a t i o n is compatib]e with the lack of a n y e v i d e n t d e v i a t i o a from the n o r m a l p h e n o t y p e a m o n g t h e restored plaats. This does n o t imply, of course, t h a t
310
Tsai-Ying Cheng and H. H. Smith
t h e e n t i r e callus p o p u l a t i o n was of a h o m o g e n e o u s diploid g e n o m e composition. I t does seem h i g h l y p r o b a b l e , however, t h a t t h e s u s t a i n e d r a p i d i t y of cell division a n d t h e brief c u l t i v a t i o n in vitro w o u l d f a v o r n o r m a l m i t o t i c division a n d maintain diploidy. The culture m e t h o d now d e v e l o p e d for Hordeum vulgare m e e t s t h e requirem e a t s of fast g r o w t h r a t e a n d r e t e n t i o n of g e n o m e c o m p o s i t i o n a n d t o t i p o t e n c y . I t should be useful in r e c o v e r i n g m u t a n t cells b y selective t e c h n i q u e s a n d subs e q u e n t l y to restore p l a n t s f r o m such m u t a n t cultures. Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Development Administration (ERDA). We thank Ms. J. Wysoeki, Ms. C. Ferris, and Mr. :B. Peltz for technical assistance.
References 1. Carter, 0., Yamada, Y., Takahashi, E.: Tissue culture of oats. Nature (Loud.) 214, 1029-1030 (1967) 2. Cheng, T. Y. : Induction of indoleacetic acid synthetases in tobacco pith explants. Plant Physiol. 50, 723-727 (1972) 3. Masteller, V. J., Holdcn, D. J. : The growth of and organ formation from callus tissue of sorghum. Plant Physiol. 45, 362-364 (1970) 4. )~urashige, T., Skoog, F. : A revised medium for rapid growth and bioassays with tobacco pith cultures. Physiol. Plantarum (Cph.) 15, 473-497 (1962) 5. Nishi, T., u u Takahastfi, E.: Organ redifferentiation and plant restoration in rice callus. Nature (Loud.) 219, 508-509 (1968) 6. Shimada, T., Sasakuma, T., Tsunewaki, K.: In vitro culture of wheat tissues. I. Callus formation and single cell culture. Canad. J. Genet. Cytol. 11, 294-304 (1969) 7. Skoog, F., Hamzi, tt. Q., Szweykowska, A. )/[. : Cytokinins: Structure/activity relationships. Phytochemistry 6, 1169-1192 (1967)
Short Communication
Organogenesis from Callus Culture of Hordeum vulgare T s a i - Y i n g Cheng* a n d H a r o l d H. S m i t h Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA Received 20 February 1974; accepted 28 January 1975 Summary. Rapidly proliferating callus cultures from the apical meristem of Hordeum vulgate L., cultivars "Himalaya" and "Mari", were established on a defined medium supplemented with auxin and cytokinin. A frequency of organ differentiation of ca. 85% was obtained by transfer to the same medium but without any growth regulators. All regenerated plants had the diploid chromosome number of 14 (2n). Organogeaesis in vitro from callus cultures of crop species of t h e Gramineae h a v e beer1 r e p o r t e d for o a t s [1], rice [5], w h e a t [6] a n d s o r g h u m [3]. W i t h t h e e x c e p t i o n of rice, no q u a n t i t a t i v e d a t a were p r e s e n t e d for callus g r o w t h a n d organ formation. These previous r e p o r t s h a v e s h o w a t h a t a r e l a t i v e l y high a u x i n c o n c e a t r a t i o u is r e q u i r e d for b o t h i n i t i a t i o n a n d m a i n t e n a n c e of callus, a n d t h a t s u b s e q u e a t l y deprivir~g t h e callus of all g r o w t h r e g u l a t o r s results in shoot formation. I n this p a p e r , i m p r o v e m e n t s are r e p o r t e d in m e t h o d s for o b t a i n i n g r a p i d proliferation of b a r l e y ca]li a n d for d e p e n d a b l e high f r e q u e n c y d i f f e r e n t i a t i o n of b a r l e y p l a n t s f r o m these calli. Two cultivars of barley, Hordeum vulgate L., "Himalaya" and "Mari" were used in these studies (seed source: Dr. Paul Bottino from stocks maintained by Dr. R. A. Nilan, Washington State University, Pullman, Wash., USA.) The solid medium was modified from Murashige and Skoog [4] and Cheng [2] to contain, per liter (1) inorganic salts: 1.65 g NH~N03, 1.90 g KN0~ 0.44 g CaCle. 2 H20, 0.37 g FIgSO~. 7H~0, 0.17 g KH2P04, 30 mg FeSO4.7H20, 36 mg Na2EDTA, 6.2 mg H~B03, 22.3 mg MnSQ.4H20, 10.5 mg ZnSO~-7H20 , 0.8 rag KI, 0.3 mg NaMoO4-2H20, 0.03 rag CuSQ-5 H~O, and 0.03rag CoC12-6H20; and (2) organic substances: 500rag myo-inositol, 5rag thiamine HC1, 30 g sucrose and 0.8% agar (pH 5.5). The plant regulators added to the medium were the auxins indole-3-acetic acid (IAA), 2,4-dichlorophenoxyaeetie acid (2,4-D), ~.-naphthaleneacetic acid (NAA) and p-ehlorophenoxyaeetic acid (CPA), and the cy~kinin 6-(3-methyl-2-butenyl-amino)purine (2iP). All media were autoclaved for 8 rain at 121 ~ with 1.41 kg/em 2 pressure. The growth of barley in culture was arbitrarily divided into three stages: callus initiation, callus maintenance, and organ induction. The same basal medium was used throughout but different amounts of plant growth substances were used for each of these stages. The callus-initiating medium (CI-medium) contained 10 btM IAA, 15 btM 2,4-D and 1.5 ~M 2iP. The callus-maintaining medium (CM-medium) contained similar amounts of IAA and 2iP plus one of the following: 5-20 b~H 2,4-D, 2 0 4 0 b~M NAA, or 2 0 4 0 y2r CPA. No growth substances were included in the shoot-inducing medium (SI-medium). For experiments on the initiation of callus in culture, apical meristems dissected from l-week-old seedlings were used. The seedlings were grown from dehusked seeds sterilized for 5 min with 20 fold diluted Clorox (5.25 % sodium hypochlorite) plus 0.2 % Alconox (a detergent used for cleaning glass, Scientific Glass Apparatus, Bloomfield, N.J., USA) and germi* Present address: Chemistry Dep~rtment, Oregon Graduate Center, Beaverton, Oregon 97005, USA.
308
Tsai-Ying Cheng and H. H. Smith
Table 1. Increase in fresh weight of barley callus culture The callus was cultured on a CiK-medium containing 2,4-D or NAA. Each mean fresh weight and its standard error were calculated from six samples of 25-day-old cultures. The inoculum for each callus weighed about 4 rag. Cultivar
Growth substance and conc. (tzl~)
Final fr. wt. (mg/piece)
Ratio final/initial
Himalaya
2,4-D (5) IqAA (20)
513~: 64 604• 112
128 152
Marl
2,4-D (5) NAA (20)
704• 164 752• 81
186 188
nated on a solid culture medium lacking all plant growth regulators. The meristems were cultured on CI-medium in a growth chamber at 25~ with a 16-h photoperiod (600 fc and an 8-h dark period). Within 3-5 weeks, sizable callus tissue appeared around the edge of the initial explant. All callus was kept growing in the exponential phase by subculturing it every 3 4 weeks on fresh CM-medium. Measurements of growth of barley callus (Table 1) show that the fresh weight at 25 days was greater by at least two orders of magnitude than the inoeulum weight. The synthetic auxins, 2,4-D, NAA, and CPA, at concentrations of 5 to 40 tzM were sufficient to support rapid proliferation of callus. Growth was similar for both varieties, and exceeded by far that reported for most other in-vitro cultures of Gramineae. The auxin I A A was added to provide growth stimulation during the initial culture period. However, IAA alone was not sufficient to support callus growth, and the synthetic auxins, 2,4-D, NAA or CPA were also incorporated into the media in order to maintain cultures at their maximum growth rate. Although the need for the eytokinin (2iP) at this stage of the culture has not been ascertained, it was always included in the CI- and CMmedia. The 2iP was used because it is as a rule more active than kinetin in promoting both cell growth and organ formation [7]. Organ differentiation in culture was initiated by transferring exponentially growing calli from CM-medium to SI-medium which is devoid of all growth substances. Although kinetin has been used for shoot induction in some monocotyledons such as rice, wheat, and oats, it is not essential at least in barley. Shoots began to emerge from the surfaces of calli in ca. 4 weeks. The differentiated ealli were then transferred to SI-medium diluted 1 : 1 in order to give a lower salt concentration that enhanced root production. A typical plant regenerated from such a callus (Fig. 1 a) is shown in Fig. 1 b. The resulting plants were grown to maturity in soil in pots under greenhouse conditions. They did not differ in either morphology of fertility from control plants grown from seed. Ca. 85% of the calli that had been through 3 consecutive transfers exhibited organogenesis; ca. 60% of those subcultured 5 times did so (Table 2). Ability for short production was completely lost after 4-5 months culture. A similar decline and loss of organogenetie capacity with prolonged culture has also been observed in rice. Cytological examination of root tips stained by the Feulgen method showed that the restored plants had maintained the diploid chromosome number of 14
Organogenesis in Barley Callus Culture
309
Fig. 1. (a) Callus culture of Hordeum vulgare "Himalaya" from which plants were regenerated. (b) Barley plants shown about 8 weeks after first appearance of shoots from callus placed on SI-medium after three consecutive cultivations (originating from the apical meristem) on CM-medium containing 10 aM IAA, 20 [~M NAP_ and 1.5 ~M 2iP
Table 2. Frequency of plants differentiated in vitro from callus cultures of Hordeum vulgate "Himalaya" No. times subcultured
No. calli
No. plants induced
Organogenesis (% of oalli)
3 5
13 15
11 6
85 60
(2n). No chromosomal aberations were detected in a n y of the cells examined. This o b s e r v a t i o n is compatib]e with the lack of a n y e v i d e n t d e v i a t i o a from the n o r m a l p h e n o t y p e a m o n g t h e restored plaats. This does n o t imply, of course, t h a t
310
Tsai-Ying Cheng and H. H. Smith
t h e e n t i r e callus p o p u l a t i o n was of a h o m o g e n e o u s diploid g e n o m e composition. I t does seem h i g h l y p r o b a b l e , however, t h a t t h e s u s t a i n e d r a p i d i t y of cell division a n d t h e brief c u l t i v a t i o n in vitro w o u l d f a v o r n o r m a l m i t o t i c division a n d maintain diploidy. The culture m e t h o d now d e v e l o p e d for Hordeum vulgare m e e t s t h e requirem e a t s of fast g r o w t h r a t e a n d r e t e n t i o n of g e n o m e c o m p o s i t i o n a n d t o t i p o t e n c y . I t should be useful in r e c o v e r i n g m u t a n t cells b y selective t e c h n i q u e s a n d subs e q u e n t l y to restore p l a n t s f r o m such m u t a n t cultures. Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Development Administration (ERDA). We thank Ms. J. Wysoeki, Ms. C. Ferris, and Mr. :B. Peltz for technical assistance.
References 1. Carter, 0., Yamada, Y., Takahashi, E.: Tissue culture of oats. Nature (Loud.) 214, 1029-1030 (1967) 2. Cheng, T. Y. : Induction of indoleacetic acid synthetases in tobacco pith explants. Plant Physiol. 50, 723-727 (1972) 3. Masteller, V. J., Holdcn, D. J. : The growth of and organ formation from callus tissue of sorghum. Plant Physiol. 45, 362-364 (1970) 4. )~urashige, T., Skoog, F. : A revised medium for rapid growth and bioassays with tobacco pith cultures. Physiol. Plantarum (Cph.) 15, 473-497 (1962) 5. Nishi, T., u u Takahastfi, E.: Organ redifferentiation and plant restoration in rice callus. Nature (Loud.) 219, 508-509 (1968) 6. Shimada, T., Sasakuma, T., Tsunewaki, K.: In vitro culture of wheat tissues. I. Callus formation and single cell culture. Canad. J. Genet. Cytol. 11, 294-304 (1969) 7. Skoog, F., Hamzi, tt. Q., Szweykowska, A. )/[. : Cytokinins: Structure/activity relationships. Phytochemistry 6, 1169-1192 (1967)
