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In vitro regeneration and multiplication of safflower (Carthamus tinctorius L.)
plan cience IIS[VIIR S( [I N I I I I ( Iq HI I S I I I RS I R I I \ N [ )
Plant Science 93 (1993) 151-157
In vitro regeneration and multiplication of safflower (Carthamus tinctorius
L.)
T e r e s a K . O r l i k o w s k a a, W i l l i a m E. D y e r *b .
aResearch Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice. Poland hPlant and Soil Science Department, Montana State University. Bozeman. MT59717, USA (Received 6 April 1993; revision received 7 June 1993: accepted 14 June 1993)
Abstract
We have developed a useful system for direct shoot regeneration from primary seedling explants and immature embryos of the American safflower (Carthamus tinctorius L.) cultivar. Centennial. Direct shoot regeneration from primary explants was optimal on Murashige and Skoog [1] (MS) basal salts medium containing 0.1 mg/l lnaphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzylaminopurine (BAP) or 0.1 rag/1 thidiazuron (TDZ). Numbers of regenerated shoots were comparable on media containing BAP or TDZ, although TDZ medium was superior in reducing shoot hyperhydricity and permitting multiple harvests of regenerated shoots from primary explants. Shoot regeneration from immature embryos was observed on MS medium containing NAA and TDZ. For shoot multiplication, numerous axillary shoots were obtained from 1 cm long internodal seedling shoot sections on MS medium containing 1 mg/1 2-isopentenyladenine (2iP). Shoots regenerated from primary Montola and Centennial seedling explants were rooted on 0.5X MS medium containing 1 mg/1 NAA and successfully transferred to the greenhouse. Key words." Carthamus tinctorius; Safflower; Direct shoot regeneration; Immature embryo culture
1. Introduction
Safflower is an important oilseed crop cultivated in arid environments of India, Mexico, and western and central North America. We recently reported the first successful Agrobacterium tumefaciens-mediated transformation of safflower and regeneration of transgenic shoot buds [2]. Shoot buds were regenerated from cotyledon- and leaf* Corresponding author.
derived calli, although the frequency of bud formation was only about 26°/,,. Attempts to root regenerated shoots were unsuccessful [2]. A similar lack of success in rooting Indian and Turkish safflower varieties has been reported [3,4]. In order to overcome the limitations associated with regeneration and rooting callus-derived shoots, experiments were carried out to determine the optimal conditions for inducing direct shoot regeneration from primary safflower explants. Optimizing the regeneration and rooting frequency of directly
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152
regenerated shoots from explants cocultivated with Agrobacterium tumefaciens should permit more efficient recovery of successful transformants. George and Rao [3] reported direct regeneration of shoot buds from cotyledons of 7-day-old Indian safflower seedlings. About 50% of the explants formed 8-10 shoot buds each on MS medium supplemented with 0.5 mg/1 6-benzylaminopurine (BAP). Likewise, Tejovathi and Anwar [4] observed direct regeneration of multiple shoot buds from Indian cultivars using 2-3-day-old seedlings without root and shoot primordia on MS medium plus 0.5 mg/1 BAP and 0.1 mg/1 1-naphthaleneacetic acid (NAA). However, efficiency of regeneration was not given. Preliminary experiments in our laboratory showed that direct shoot regeneration was highest in explants from immature embryos and less than 15-day-old seedlings. Leaf explants from l-monthold greenhouse plants produced only callus (data not shown). Therefore, efforts to optimize direct shoot regeneration were concentrated on young seedlings and immature embryos. The long term goal was to develop an efficient procedure for mass plantlet production which could be used in conjunction witla gene transfer techniques. 2. Materials and methods
Field-grown Centennial and Montola seeds were surface cleaned by rinsing in 0.01% dishwashing detergent for 1 rain, sterilized in 0.1% HgCI2 for 10 min and rinsed in sterile water (3 x 10 min). Surface sterilized seeds were transferred for germination to 0.5X MS basal salts medium solidified with 6 g/l Bacto agar. Explants were taken from cotyledons, leaves, hypocotyls and roots of 3-20-day-old seedlings. Leaves and cotyledons were cut into basal and apical halves, while hypocotyls and roots were sliced transversely into 3 mm sections after removal of apical meristems. Immature embryos were taken from greenhouse grown plants when flower petals began wilting. Embryos were sterilized as above and dissected into cotyledons and embryonic axes after excising root tips. Regeneration media (pH 5.8) consisted of 1X MS salts, B5 vitamins [5], 30 g/l
T.K. Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
sucrose, 8 g/l Bacto agar and several combinations of BAP or thidiazuron (TDZ) with NAA. Shoot multiplication was carried out on MS medium (pH 5.8) supplemented with 0.1, 0.5, or 1.0 mg/l BAP; 0.5, 1.0 or 2.0 mg/1 2-isopentenyladenine (2iP) or kinetin; or 0.01, 0.05 or 0.1 mg/1 TDZ. All media components except AgNO 3 (see below) were added before autoclaving. Cultures were incubated at 22°C under a 16-h/day photoperiod with a fluorescent light intensity of 165 ~E m -2 sec -~. Cultures were transferred to fresh medium every 2 weeks. For rooting, regenerated leafy structures were first allowed to elongate into 2-3-cm long shoots (about 3 weeks) on elongation medium (pH 5.8) consisting of 0.5X MS salts and MS vitamins [1] with 100 mg/1 inositol, 30 g/l sucrose, 8 g/l Bacto agar, 1 mg/l kinetin, 0.05 mg/1 NAA and 2.5 mg/l AgNO3 (filter sterilized). Rooting was carried out on 0.5X MS medium (pH 5.2) containing 1X MS vitamins, 100 mg/l inositol, 30 g/1 sucrose, 2.8 g/l Phytagel® (Sigma), 2.5 mg/l AgNO3, 1 mg/1 riboflavin and one of five auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA at 1 mg/1; or 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,3,5-trichloro-c~-phenoxypropionic acid (2,4,5CI3POP) at 0.1 mg/1. Shoots were incubated for 5 days in the dark and then transferred to the light conditions described above. 3. Results
Leaf primordia appeared after 7-10 days on the proximal cut surfaces of cotyledons and leaves as well as on hypocotyl sections closest to the apical meristem (Fig. 1A). Explants from 3-7-day-old seedlings produced leafy structures mostly from cotyledons, while older seedlings (10-20-day-old) also produced leafy structures from leaves and hypocotyls. Root sections produced only callus under these experimental conditions. Regeneration of leafy structures was observed on most NAA/cytokinin combinations tested (Table 1). Numbers of regenerated leafy structures generally increased with increasing BAP concentration in combination with NAA and regeneration was more efficient overall on media containing BAP rather than TDZ. Addition of 0.1
1116 Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
153
Fig. 1. In vitro regeneration a n d r o o t i n g of shoots from p r i m a r y safflower explants. (A) Multiple shoots regenerating from basal edge of 7-day-old C e n t e n n i a l c o t y l e d o n (magnification, x 20). (B) E l o n g a t e d s h o o t on 0.5X MS m e d i u m c o n t a i n i n g 1 mg/l kinetin, 0.05 rag/1 N A A a n d 2.5 mg/l A g N O 3 (magnification, x 8). (C) R o o t e d plant after 3 weeks of growth in soil (bar, 10 cm).
Table 1 Direct regeneration of leafy structures from leaves and c o t y l e d o n s (cotyl.) of I 1-day-old C e n t e n n i a l seedlings after 20 days on different auxin and c y t o k i n i n concentrations. D a t a are n u m b e r of leafy structures per seedling ± S.E. (n = 4) Cytokinin (mg/l)
BAP 0.1 0.5 1.0 T D Z 0.001 0.01 0.1
1.0 No cytokinin
( N A A mg/l) 0
0.1
0.5
1.0
leaf
cotyl,
leaf
cotyl,
leaf
cotyl,
leaf
cotyl.
0 30 ± 10 14 ± 5
0 If) ± 5 17 + 2 ---
16 25 44 18 20
72 107 103 28 12
14 + 4 60 ± 18 25 + 4
60 ± 19 180 + 52 104 ± 31 ---
20 30 78 0 27
45 35 120 30 30
__
20 + 6
--
0
-0
± 4 ± 10 ± 14 .4- 6 ± 6
-4- 12 + 21 + 25 ± 9 ± 5
12 ± 4
40 ±
21 0
7 5 ± 21
± 8
0
13
--
0
+ 4 ± 8 ± 29 + 8
56 ±
4 ± 3
0
17
± + ± ± +
16 17 44 8 11 6 0 ± 25 211 ± 86 0
T.K. Orlikowska, l,f~E. Dyer~Plant Sci. 93 (1993) 151-157
154
or 0.5 mg/1 NAA to BAP medium induced the highest levels of regeneration, while NAA at 1.0 mg/l slightly decreased direct regeneration and stimulated callus growth. However, leafy structures regenerated on BAP media suffered from hyperhydricity [7]. On TDZ media, regeneration frequency increased slightly with increasing TDZ concentrations and leafy structures grew vigorously. Leafy structures were generally more abundant on cotyledons than on leaves. Overall, the most effective medium for direct shoot regeneration with a minimum of hyperhydricity contained MS basal salts with 0.1 mg/l NAA and 0.1 mg/1 TDZ. Immature embryos regenerated leafy structures on almost all media tested, accompanied by callus growth especially at higher TDZ concentrations (Table 2). Minimal regeneration was observed on media containing TDZ and 10 mg/1 NAA. Regeneration occurred primarily from basal or wounded edges of cotyledons, although some regeneration occurred from hypocotyls on media with 10 mg/l NAA and no TDZ (data not shown). Hyperhydricity was observed in some leafy struc-
tures regenerating from primary explants and in many structures regenerating from callus. Transfer of explants with regenerating leafy structures onto elongation medium after 2-3 weeks limited callusing and hyperhydricity and permitted normal shoot development (Fig. 1B). When regenerated leafy structures were subcultured repeatedly on media with low concentrations of cytokinins (0.25 rag/1 BAP, 0.01 mg/1 TDZ or 1 mg/l kinetin), shoots grew as rosettes and flower buds were formed which eventually flowered. Even though some regenerated shoots were obtained from immature embryos, direct regeneration from seedling explants was judged superior because of easier explant preparation and better vigor of regenerated shoots. Adventitious shoots were produced on 1 cmlong internodal shoot sections from 20-day-old seedlings on MS media supplemented with varying amounts of BAP, TDZ, 2iP or kinetin (Table 3). None of the media tested stimulated shoot proliferation from 1 cm-long shoot apical meristems. Most shoot proliferation was observed on media
Table 2 Regeneration of leafy structures from immature Centennial embryos cultured on I x MS media with various hormone concentrations a f t e r 20 d a y s Growth
regulators
Germination
(mg/l) TDZ
Callus
Direct shoot
Shoot regeneration
growth
regeneration
from callus
NAA
0
0
-
0
0.1
+(albino)
-
-
-
0
1.0
+
-
-
-
0
10.0
-
+
+
-
0.01
0
-
+
+++(h)
+(h)
0.01
0.1
-
+
+++
-
0.01
1.0
-
+
+++
0.01
10.0
-
+
-
+++(h)
0.1 0.1
0
-
+
+++
+(h)
0.1
-
+
++(h)
+
0.1
1.0
-
+
++
+
0.!
10.0
-
+
+
++(h)
1.0
0
-
1.0
0.1
-
++ ++
+++ +++
_
1.0
1.0
-
++
+++
+(h)
1.0
10.0
-
++
+
+
h, H y p e r h y d r i c i t y ;
- , n o r e s p o n s e ; +, s l i g h t r e s p o n s e ; + + , i n t e r m e d i a t e
+(h)
response: +++, massive response.
T.K. Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
155
Table 3 Shoot multiplication from 20-day-old Centennial seedling explants after 30 days on IX MS medium with one of four cytokinins Cytokinin (mg/1)
Shoot apex (1 cm)
BAP 0.1 0.5 1.0 T D Z 0.01 0.05 0.1 2iP 0.5 1.0 2.0 KIN 0.5 1.0 2.0 No cytokinin
Shoot section (1 cm below apex)
Shoots (No.)
Hyperhydricity
Yellowing/ necrosis
Shoots (No.)
Hyperhydricity
Shoot elongation
Yellowing/ necrosis
0
+
+
0 0 0 0 0 0 0 0 0 0 0 0
+ + + -
+ + + + + + + + -
8-10
+
+
+
8-12 8-12 0-4 3-7 1-2 2-4 7-13 7-15 0-3 0-3 0-2 0-2
+ + + + + -
+ + + + + + + +
+ + + +
Data are numbers of adventitious shoots obtained per explant (n = 3). +, present; - , absent.
containing
BAP or 2iP, but shoots on BAP medi-
um became numbers ing TDZ
yellow and
eventually
of shoots were formed and
useful
tious shoots. Numerous 2iP medium
especially
shoots remained Rooting
healthy
of elongated
Kinetin
numbers
shoots at
but
and elongated
soft
of adventi-
2 mg/l and
IBA
elongated
a few roots
callus
and
on
only on media containing On
medium
containing
42% of Montola on
roots,
respectively,
but
overgrown
with
4). C a l l u s p r o d u c t i o n
Also, roots produced
gradually
turned
or rooting was not observed
dark
brown.
Shoots
on
Montola
media
and
Centennial
white,
chlorotic
and
of roots were formed
NAA
or 2,4,5-CI3POP.
2,4,5-CIsPOP, shoot callus medium, shoots
89%
shoots bases
and
produced and
within
on this medium
mally swollen. On NAA
IAA and the basal shoot ends
healthy
In contrast,
2,4-D produced became
and Centennial
became
remained
formed.
numbers
the
0.5X MS media with several auxin sources (Table in m e d i a c o n t a i n i n g
and
were
leaves
stunted. Substantial
readily.
was attempted
only
shoots on media containing
was not
were obtained
1 and
shoots
containing
Low
on media contain-
did not elongate.
e f f e c t i v e in i n d u c i n g
necrotic.
roots
3 weeks.
were abnor-
67°/,, a n d 3 1 % o f produced
roots,
Table 4 Rooting response of shoots regenerated from Montola and Centennial safflower explants after 21 days on 0.5X MS media containing IAA, IBA, or N A A at 1 mg/l; or 2 , 4 - D or 2,4,5-ClsPOP at 0.1 mg/I Auxin
IAA IBA NAA 2,4-D 2,4,5-CI3POP
% Rooting Montola
Centennial
0 n.t. 67 0 89
0 11 31 0 42
n, 10-15; n.t., not tested
Callus growth
Chlorosis/necrosis
++ ++
+ ++ -
156
respectively. Shoots rooted on this medium were successfully transferred to greenhouse soil mix, with better survival of larger (3-4 cm) shoots (Fig. 1C). About 80% of 4-cm-long rooted shoots survived transplanting into soil (data not shown). 4. Discussion
Safflower is well adapted to low humidity and soil water conditions, and can withstand extended drought conditions. Because of these characteristics, safflower explants and regenerated shoots seem to be particularly sensitive to media water content and high relative humidity in culture vessels. An early symptom of excess water content is hyperhydricity [7], which can be followed by chlorosis and necrosis. Hyperhydricity has been observed in tissue culture of many plant species, and is commonly counteracted by optimization of temperature, media components and vessel ventilation [7]. In this case, hyperhydricity of regenerated safflower shoots was successfully prevented by early transfer to medium containing 0.5X MS salts, weak cytokinin (1 mg/l kinetin) and 2.5 mg/1 AgNO3. Subculturing leafy structures on the same medium but without AgNO3 did not completely eliminate hyperhydricity and often caused flower bud formation on rosette-like shoots. Tejovathi and Anwar [6] reported a similar flowering response on media containing BAP and kinetin. Ag + ions inhibit ethylene action [10] and AgNO 3 has been successfully used to enhance regeneration efficiency from sunflower [9] and Brassica [8] cotyledon cultures. AgNO3 when added at 2.5 mg/l to safflower regeneration media did not affect the percentage of organogenetic cotyledons (almost 100%), but slightly reduced the number of regenerating leafy structures per explant and consequently the number of shoots (data not shown). The presence of AgNO3 in safflower elongation medium generally improved shoot health and growth. Hyperhydricity was also decreased during direct shoot regeneration by the substitution of TDZ for BAP in regeneration media. TDZ was superior to BAP in allowing extended direct shoot regeneration from primary explants, even after transfer of primary explants with shoots to elongation medium. The possibility of obtaining a second and even
T.K. Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
third yield of regenerated shoots from transformed primary explants may be particularly helpful in recovering transgenic plants. Although multiplication of directly regenerated safflower shoots has not yet been attempted in our laboratory, multiple shoots were successfully obtained from seedling hypocotyl sections using 1 mg/1 2iP, indicating that this medium may also be useful for multiplication of regenerated shoots. Rooting of regenerated Centennial and Montola shoots was strongly dependent on the auxin source in 0.5X MS basal salts medium containing riboflavin and AgNO 3. Most rooted shoots were obtained on medium containing 2,4,5-C13POP (89% and 42% for Montola and Centennial, respectively); however, rooting was accompanied by a high incidence of callusing and root morphology was abnormal. Even though rooting on NAA medium was slightly less efficient than on 2,4,5-CI3POP (31% vs. 42% for Centennial, respectively), roots were healthy and plants survived transfer to soil. George and Rao [3] reported that rooting of regenerated shoots from Indian safflower varieties required the use of MS medium and elevated sucrose concentrations. However, this treatment induced only 8% rooting in shoots regenerated from explants of Turkish cultivars [4] and was not successful with Centennial shoots derived from callus [2]. In summary, these results demonstrate an efficient protocol for the direct regeneration of shoots from primary safflower explants. This methodology may be now tested in combination with introduction of foreign genes by Agrobacterium tumefaciens-mediated transformation or biolistic transfer to obtain useful numbers of transgenic safflower plants expressing novel traits. 5. Acknowledgments
We thank Carla Stringfellow for technical assistance and Drs. Richard Stout and Charleen Baker for critically reviewing the manuscript. TDZ was kindly provided by NOR-AM Chemical Co. 6. References
T. Murashigeand F. Skoog, A revised mediumfor rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15 (1962) 473-497.
157
72K. Orlikowska, IEE. Dyer~Plant Sci. 93 (1993) 151-157
2
3
4
5
6
M. Ying, W.E. Dyer and J.W. Bergman, Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. Centennial. Plant Cell Rep., 11 (1992) 581-585. L. George and P.S. Rao, In vitro multiplication of safflower (Carthamus tinctorius L.) through tissue culture. Proc. Ind. Natl. Sci. Acad., B48(6) (1982) 791-794. G. Tejovathi and S.Y. Anwar, Plantlet regeneration from cotyledonary cultures of safflower (Carthamus tinctorius L.), in: G.M. Reddy (Ed.), Proceedings Symposium, Plant Cell and Tissue Culture of Economically Important Plants, Hyderabad, India, 1987, pp. 347-353. Gamborg O.L., R.A. Miller and K. Ojima, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50 (1968) 148-151. G. Tejovathi and S.Y. Anwar, In vitro induction of capitula from cotyledons of Carthamus tinctorius (safflower). Plant Sci. Lett., 36 (1984) 165-168.
7
P. Debergh, J. Aitken-Christie, D. Cohen, B. Grout, S. yon Arnold, R. Zimmerman and M. Ziv, Reconsideration of the term 'vitrification' as used in micropropagation. Plant Cell Tiss. Org. Cult., 30 (1992) 135-140. 8 G.-L. Chi, D.G. Barfield, G.-E. Sim and E.-Ch. Pua, Effect of AgNO 3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes. Plant Cell Rep., 9 (1990) 195-198. 9 K.M. Chraibi, A. Latche, J.-P. Roustan and J. Fallot, Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors, silver and cobalt. Plant Cell Rep., 10 (1991) 204-207. 10 N.L. Biddington, The influence of ethylene in plant tissue culture. Plant Growth Regul., 11 (1992) 173-187.
Plant Science 93 (1993) 151-157
In vitro regeneration and multiplication of safflower (Carthamus tinctorius
L.)
T e r e s a K . O r l i k o w s k a a, W i l l i a m E. D y e r *b .
aResearch Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice. Poland hPlant and Soil Science Department, Montana State University. Bozeman. MT59717, USA (Received 6 April 1993; revision received 7 June 1993: accepted 14 June 1993)
Abstract
We have developed a useful system for direct shoot regeneration from primary seedling explants and immature embryos of the American safflower (Carthamus tinctorius L.) cultivar. Centennial. Direct shoot regeneration from primary explants was optimal on Murashige and Skoog [1] (MS) basal salts medium containing 0.1 mg/l lnaphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzylaminopurine (BAP) or 0.1 rag/1 thidiazuron (TDZ). Numbers of regenerated shoots were comparable on media containing BAP or TDZ, although TDZ medium was superior in reducing shoot hyperhydricity and permitting multiple harvests of regenerated shoots from primary explants. Shoot regeneration from immature embryos was observed on MS medium containing NAA and TDZ. For shoot multiplication, numerous axillary shoots were obtained from 1 cm long internodal seedling shoot sections on MS medium containing 1 mg/1 2-isopentenyladenine (2iP). Shoots regenerated from primary Montola and Centennial seedling explants were rooted on 0.5X MS medium containing 1 mg/1 NAA and successfully transferred to the greenhouse. Key words." Carthamus tinctorius; Safflower; Direct shoot regeneration; Immature embryo culture
1. Introduction
Safflower is an important oilseed crop cultivated in arid environments of India, Mexico, and western and central North America. We recently reported the first successful Agrobacterium tumefaciens-mediated transformation of safflower and regeneration of transgenic shoot buds [2]. Shoot buds were regenerated from cotyledon- and leaf* Corresponding author.
derived calli, although the frequency of bud formation was only about 26°/,,. Attempts to root regenerated shoots were unsuccessful [2]. A similar lack of success in rooting Indian and Turkish safflower varieties has been reported [3,4]. In order to overcome the limitations associated with regeneration and rooting callus-derived shoots, experiments were carried out to determine the optimal conditions for inducing direct shoot regeneration from primary safflower explants. Optimizing the regeneration and rooting frequency of directly
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03678-O
152
regenerated shoots from explants cocultivated with Agrobacterium tumefaciens should permit more efficient recovery of successful transformants. George and Rao [3] reported direct regeneration of shoot buds from cotyledons of 7-day-old Indian safflower seedlings. About 50% of the explants formed 8-10 shoot buds each on MS medium supplemented with 0.5 mg/1 6-benzylaminopurine (BAP). Likewise, Tejovathi and Anwar [4] observed direct regeneration of multiple shoot buds from Indian cultivars using 2-3-day-old seedlings without root and shoot primordia on MS medium plus 0.5 mg/1 BAP and 0.1 mg/1 1-naphthaleneacetic acid (NAA). However, efficiency of regeneration was not given. Preliminary experiments in our laboratory showed that direct shoot regeneration was highest in explants from immature embryos and less than 15-day-old seedlings. Leaf explants from l-monthold greenhouse plants produced only callus (data not shown). Therefore, efforts to optimize direct shoot regeneration were concentrated on young seedlings and immature embryos. The long term goal was to develop an efficient procedure for mass plantlet production which could be used in conjunction witla gene transfer techniques. 2. Materials and methods
Field-grown Centennial and Montola seeds were surface cleaned by rinsing in 0.01% dishwashing detergent for 1 rain, sterilized in 0.1% HgCI2 for 10 min and rinsed in sterile water (3 x 10 min). Surface sterilized seeds were transferred for germination to 0.5X MS basal salts medium solidified with 6 g/l Bacto agar. Explants were taken from cotyledons, leaves, hypocotyls and roots of 3-20-day-old seedlings. Leaves and cotyledons were cut into basal and apical halves, while hypocotyls and roots were sliced transversely into 3 mm sections after removal of apical meristems. Immature embryos were taken from greenhouse grown plants when flower petals began wilting. Embryos were sterilized as above and dissected into cotyledons and embryonic axes after excising root tips. Regeneration media (pH 5.8) consisted of 1X MS salts, B5 vitamins [5], 30 g/l
T.K. Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
sucrose, 8 g/l Bacto agar and several combinations of BAP or thidiazuron (TDZ) with NAA. Shoot multiplication was carried out on MS medium (pH 5.8) supplemented with 0.1, 0.5, or 1.0 mg/l BAP; 0.5, 1.0 or 2.0 mg/1 2-isopentenyladenine (2iP) or kinetin; or 0.01, 0.05 or 0.1 mg/1 TDZ. All media components except AgNO 3 (see below) were added before autoclaving. Cultures were incubated at 22°C under a 16-h/day photoperiod with a fluorescent light intensity of 165 ~E m -2 sec -~. Cultures were transferred to fresh medium every 2 weeks. For rooting, regenerated leafy structures were first allowed to elongate into 2-3-cm long shoots (about 3 weeks) on elongation medium (pH 5.8) consisting of 0.5X MS salts and MS vitamins [1] with 100 mg/1 inositol, 30 g/l sucrose, 8 g/l Bacto agar, 1 mg/l kinetin, 0.05 mg/1 NAA and 2.5 mg/l AgNO3 (filter sterilized). Rooting was carried out on 0.5X MS medium (pH 5.2) containing 1X MS vitamins, 100 mg/l inositol, 30 g/1 sucrose, 2.8 g/l Phytagel® (Sigma), 2.5 mg/l AgNO3, 1 mg/1 riboflavin and one of five auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA at 1 mg/1; or 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,3,5-trichloro-c~-phenoxypropionic acid (2,4,5CI3POP) at 0.1 mg/1. Shoots were incubated for 5 days in the dark and then transferred to the light conditions described above. 3. Results
Leaf primordia appeared after 7-10 days on the proximal cut surfaces of cotyledons and leaves as well as on hypocotyl sections closest to the apical meristem (Fig. 1A). Explants from 3-7-day-old seedlings produced leafy structures mostly from cotyledons, while older seedlings (10-20-day-old) also produced leafy structures from leaves and hypocotyls. Root sections produced only callus under these experimental conditions. Regeneration of leafy structures was observed on most NAA/cytokinin combinations tested (Table 1). Numbers of regenerated leafy structures generally increased with increasing BAP concentration in combination with NAA and regeneration was more efficient overall on media containing BAP rather than TDZ. Addition of 0.1
1116 Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
153
Fig. 1. In vitro regeneration a n d r o o t i n g of shoots from p r i m a r y safflower explants. (A) Multiple shoots regenerating from basal edge of 7-day-old C e n t e n n i a l c o t y l e d o n (magnification, x 20). (B) E l o n g a t e d s h o o t on 0.5X MS m e d i u m c o n t a i n i n g 1 mg/l kinetin, 0.05 rag/1 N A A a n d 2.5 mg/l A g N O 3 (magnification, x 8). (C) R o o t e d plant after 3 weeks of growth in soil (bar, 10 cm).
Table 1 Direct regeneration of leafy structures from leaves and c o t y l e d o n s (cotyl.) of I 1-day-old C e n t e n n i a l seedlings after 20 days on different auxin and c y t o k i n i n concentrations. D a t a are n u m b e r of leafy structures per seedling ± S.E. (n = 4) Cytokinin (mg/l)
BAP 0.1 0.5 1.0 T D Z 0.001 0.01 0.1
1.0 No cytokinin
( N A A mg/l) 0
0.1
0.5
1.0
leaf
cotyl,
leaf
cotyl,
leaf
cotyl,
leaf
cotyl.
0 30 ± 10 14 ± 5
0 If) ± 5 17 + 2 ---
16 25 44 18 20
72 107 103 28 12
14 + 4 60 ± 18 25 + 4
60 ± 19 180 + 52 104 ± 31 ---
20 30 78 0 27
45 35 120 30 30
__
20 + 6
--
0
-0
± 4 ± 10 ± 14 .4- 6 ± 6
-4- 12 + 21 + 25 ± 9 ± 5
12 ± 4
40 ±
21 0
7 5 ± 21
± 8
0
13
--
0
+ 4 ± 8 ± 29 + 8
56 ±
4 ± 3
0
17
± + ± ± +
16 17 44 8 11 6 0 ± 25 211 ± 86 0
T.K. Orlikowska, l,f~E. Dyer~Plant Sci. 93 (1993) 151-157
154
or 0.5 mg/1 NAA to BAP medium induced the highest levels of regeneration, while NAA at 1.0 mg/l slightly decreased direct regeneration and stimulated callus growth. However, leafy structures regenerated on BAP media suffered from hyperhydricity [7]. On TDZ media, regeneration frequency increased slightly with increasing TDZ concentrations and leafy structures grew vigorously. Leafy structures were generally more abundant on cotyledons than on leaves. Overall, the most effective medium for direct shoot regeneration with a minimum of hyperhydricity contained MS basal salts with 0.1 mg/l NAA and 0.1 mg/1 TDZ. Immature embryos regenerated leafy structures on almost all media tested, accompanied by callus growth especially at higher TDZ concentrations (Table 2). Minimal regeneration was observed on media containing TDZ and 10 mg/1 NAA. Regeneration occurred primarily from basal or wounded edges of cotyledons, although some regeneration occurred from hypocotyls on media with 10 mg/l NAA and no TDZ (data not shown). Hyperhydricity was observed in some leafy struc-
tures regenerating from primary explants and in many structures regenerating from callus. Transfer of explants with regenerating leafy structures onto elongation medium after 2-3 weeks limited callusing and hyperhydricity and permitted normal shoot development (Fig. 1B). When regenerated leafy structures were subcultured repeatedly on media with low concentrations of cytokinins (0.25 rag/1 BAP, 0.01 mg/1 TDZ or 1 mg/l kinetin), shoots grew as rosettes and flower buds were formed which eventually flowered. Even though some regenerated shoots were obtained from immature embryos, direct regeneration from seedling explants was judged superior because of easier explant preparation and better vigor of regenerated shoots. Adventitious shoots were produced on 1 cmlong internodal shoot sections from 20-day-old seedlings on MS media supplemented with varying amounts of BAP, TDZ, 2iP or kinetin (Table 3). None of the media tested stimulated shoot proliferation from 1 cm-long shoot apical meristems. Most shoot proliferation was observed on media
Table 2 Regeneration of leafy structures from immature Centennial embryos cultured on I x MS media with various hormone concentrations a f t e r 20 d a y s Growth
regulators
Germination
(mg/l) TDZ
Callus
Direct shoot
Shoot regeneration
growth
regeneration
from callus
NAA
0
0
-
0
0.1
+(albino)
-
-
-
0
1.0
+
-
-
-
0
10.0
-
+
+
-
0.01
0
-
+
+++(h)
+(h)
0.01
0.1
-
+
+++
-
0.01
1.0
-
+
+++
0.01
10.0
-
+
-
+++(h)
0.1 0.1
0
-
+
+++
+(h)
0.1
-
+
++(h)
+
0.1
1.0
-
+
++
+
0.!
10.0
-
+
+
++(h)
1.0
0
-
1.0
0.1
-
++ ++
+++ +++
_
1.0
1.0
-
++
+++
+(h)
1.0
10.0
-
++
+
+
h, H y p e r h y d r i c i t y ;
- , n o r e s p o n s e ; +, s l i g h t r e s p o n s e ; + + , i n t e r m e d i a t e
+(h)
response: +++, massive response.
T.K. Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
155
Table 3 Shoot multiplication from 20-day-old Centennial seedling explants after 30 days on IX MS medium with one of four cytokinins Cytokinin (mg/1)
Shoot apex (1 cm)
BAP 0.1 0.5 1.0 T D Z 0.01 0.05 0.1 2iP 0.5 1.0 2.0 KIN 0.5 1.0 2.0 No cytokinin
Shoot section (1 cm below apex)
Shoots (No.)
Hyperhydricity
Yellowing/ necrosis
Shoots (No.)
Hyperhydricity
Shoot elongation
Yellowing/ necrosis
0
+
+
0 0 0 0 0 0 0 0 0 0 0 0
+ + + -
+ + + + + + + + -
8-10
+
+
+
8-12 8-12 0-4 3-7 1-2 2-4 7-13 7-15 0-3 0-3 0-2 0-2
+ + + + + -
+ + + + + + + +
+ + + +
Data are numbers of adventitious shoots obtained per explant (n = 3). +, present; - , absent.
containing
BAP or 2iP, but shoots on BAP medi-
um became numbers ing TDZ
yellow and
eventually
of shoots were formed and
useful
tious shoots. Numerous 2iP medium
especially
shoots remained Rooting
healthy
of elongated
Kinetin
numbers
shoots at
but
and elongated
soft
of adventi-
2 mg/l and
IBA
elongated
a few roots
callus
and
on
only on media containing On
medium
containing
42% of Montola on
roots,
respectively,
but
overgrown
with
4). C a l l u s p r o d u c t i o n
Also, roots produced
gradually
turned
or rooting was not observed
dark
brown.
Shoots
on
Montola
media
and
Centennial
white,
chlorotic
and
of roots were formed
NAA
or 2,4,5-CI3POP.
2,4,5-CIsPOP, shoot callus medium, shoots
89%
shoots bases
and
produced and
within
on this medium
mally swollen. On NAA
IAA and the basal shoot ends
healthy
In contrast,
2,4-D produced became
and Centennial
became
remained
formed.
numbers
the
0.5X MS media with several auxin sources (Table in m e d i a c o n t a i n i n g
and
were
leaves
stunted. Substantial
readily.
was attempted
only
shoots on media containing
was not
were obtained
1 and
shoots
containing
Low
on media contain-
did not elongate.
e f f e c t i v e in i n d u c i n g
necrotic.
roots
3 weeks.
were abnor-
67°/,, a n d 3 1 % o f produced
roots,
Table 4 Rooting response of shoots regenerated from Montola and Centennial safflower explants after 21 days on 0.5X MS media containing IAA, IBA, or N A A at 1 mg/l; or 2 , 4 - D or 2,4,5-ClsPOP at 0.1 mg/I Auxin
IAA IBA NAA 2,4-D 2,4,5-CI3POP
% Rooting Montola
Centennial
0 n.t. 67 0 89
0 11 31 0 42
n, 10-15; n.t., not tested
Callus growth
Chlorosis/necrosis
++ ++
+ ++ -
156
respectively. Shoots rooted on this medium were successfully transferred to greenhouse soil mix, with better survival of larger (3-4 cm) shoots (Fig. 1C). About 80% of 4-cm-long rooted shoots survived transplanting into soil (data not shown). 4. Discussion
Safflower is well adapted to low humidity and soil water conditions, and can withstand extended drought conditions. Because of these characteristics, safflower explants and regenerated shoots seem to be particularly sensitive to media water content and high relative humidity in culture vessels. An early symptom of excess water content is hyperhydricity [7], which can be followed by chlorosis and necrosis. Hyperhydricity has been observed in tissue culture of many plant species, and is commonly counteracted by optimization of temperature, media components and vessel ventilation [7]. In this case, hyperhydricity of regenerated safflower shoots was successfully prevented by early transfer to medium containing 0.5X MS salts, weak cytokinin (1 mg/l kinetin) and 2.5 mg/1 AgNO3. Subculturing leafy structures on the same medium but without AgNO3 did not completely eliminate hyperhydricity and often caused flower bud formation on rosette-like shoots. Tejovathi and Anwar [6] reported a similar flowering response on media containing BAP and kinetin. Ag + ions inhibit ethylene action [10] and AgNO 3 has been successfully used to enhance regeneration efficiency from sunflower [9] and Brassica [8] cotyledon cultures. AgNO3 when added at 2.5 mg/l to safflower regeneration media did not affect the percentage of organogenetic cotyledons (almost 100%), but slightly reduced the number of regenerating leafy structures per explant and consequently the number of shoots (data not shown). The presence of AgNO3 in safflower elongation medium generally improved shoot health and growth. Hyperhydricity was also decreased during direct shoot regeneration by the substitution of TDZ for BAP in regeneration media. TDZ was superior to BAP in allowing extended direct shoot regeneration from primary explants, even after transfer of primary explants with shoots to elongation medium. The possibility of obtaining a second and even
T.K. Orlikowska, W.E. Dyer~Plant Sci. 93 (1993) 151-157
third yield of regenerated shoots from transformed primary explants may be particularly helpful in recovering transgenic plants. Although multiplication of directly regenerated safflower shoots has not yet been attempted in our laboratory, multiple shoots were successfully obtained from seedling hypocotyl sections using 1 mg/1 2iP, indicating that this medium may also be useful for multiplication of regenerated shoots. Rooting of regenerated Centennial and Montola shoots was strongly dependent on the auxin source in 0.5X MS basal salts medium containing riboflavin and AgNO 3. Most rooted shoots were obtained on medium containing 2,4,5-C13POP (89% and 42% for Montola and Centennial, respectively); however, rooting was accompanied by a high incidence of callusing and root morphology was abnormal. Even though rooting on NAA medium was slightly less efficient than on 2,4,5-CI3POP (31% vs. 42% for Centennial, respectively), roots were healthy and plants survived transfer to soil. George and Rao [3] reported that rooting of regenerated shoots from Indian safflower varieties required the use of MS medium and elevated sucrose concentrations. However, this treatment induced only 8% rooting in shoots regenerated from explants of Turkish cultivars [4] and was not successful with Centennial shoots derived from callus [2]. In summary, these results demonstrate an efficient protocol for the direct regeneration of shoots from primary safflower explants. This methodology may be now tested in combination with introduction of foreign genes by Agrobacterium tumefaciens-mediated transformation or biolistic transfer to obtain useful numbers of transgenic safflower plants expressing novel traits. 5. Acknowledgments
We thank Carla Stringfellow for technical assistance and Drs. Richard Stout and Charleen Baker for critically reviewing the manuscript. TDZ was kindly provided by NOR-AM Chemical Co. 6. References
T. Murashigeand F. Skoog, A revised mediumfor rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15 (1962) 473-497.
157
72K. Orlikowska, IEE. Dyer~Plant Sci. 93 (1993) 151-157
2
3
4
5
6
M. Ying, W.E. Dyer and J.W. Bergman, Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. Centennial. Plant Cell Rep., 11 (1992) 581-585. L. George and P.S. Rao, In vitro multiplication of safflower (Carthamus tinctorius L.) through tissue culture. Proc. Ind. Natl. Sci. Acad., B48(6) (1982) 791-794. G. Tejovathi and S.Y. Anwar, Plantlet regeneration from cotyledonary cultures of safflower (Carthamus tinctorius L.), in: G.M. Reddy (Ed.), Proceedings Symposium, Plant Cell and Tissue Culture of Economically Important Plants, Hyderabad, India, 1987, pp. 347-353. Gamborg O.L., R.A. Miller and K. Ojima, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50 (1968) 148-151. G. Tejovathi and S.Y. Anwar, In vitro induction of capitula from cotyledons of Carthamus tinctorius (safflower). Plant Sci. Lett., 36 (1984) 165-168.
7
P. Debergh, J. Aitken-Christie, D. Cohen, B. Grout, S. yon Arnold, R. Zimmerman and M. Ziv, Reconsideration of the term 'vitrification' as used in micropropagation. Plant Cell Tiss. Org. Cult., 30 (1992) 135-140. 8 G.-L. Chi, D.G. Barfield, G.-E. Sim and E.-Ch. Pua, Effect of AgNO 3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes. Plant Cell Rep., 9 (1990) 195-198. 9 K.M. Chraibi, A. Latche, J.-P. Roustan and J. Fallot, Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors, silver and cobalt. Plant Cell Rep., 10 (1991) 204-207. 10 N.L. Biddington, The influence of ethylene in plant tissue culture. Plant Growth Regul., 11 (1992) 173-187.