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High frequency embryogenesis through isolated microspore culture in Brassica napus L. and B. Carinata braun
Plant Science, 39 (1985) 219--226 Elsevier Scientific Publishers Ireland Ltd.
219
HIGH FREQUENCY EMBRYOGENESIS THROUGH ISOLATED MICROSPORE CULTURE IN B R A S S I C A N A P U S L. A N D B. C A R I N A T A B R A U N
PHAN V. CHUONG and W.D. BEVERSDORF Crop Science Department University of Guelph, Guelph, Ontario N1G 2W1 (Canada) (Received November 15th, 1984) (Revision received March 7th, 1985) (Accepted April 3rd, 1985) Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l naphthaleneacetic acid (NAA). Embryogenic responses were observed at culture temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7--54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium. Key words: Brassica napus; microspore culture; embryogenesis; embryo types; temperature effect
Introduction T h e u t i l i t y o f h a p l o i d p l a n t s in b r e e d i n g a n d genetic r e s e a r c h has e n c o u r a g e d d e v e l o p ment of efficient techniques to produce them. In Brassica, a n t h e r c u l t u r e has b e e n t h e primary source of haploid plants [1--8]. E m b r y o f r e q u e n c i e s f r o m c u l t u r e d Brassica a n t h e r s h a v e varied w i t h t h e species a n d t h e t e c h n i q u e u s e d b u t has generally b e e n in the range of 0--8 embryos per cultured a n t h e r [ 4 , 1 0 - - 1 2 , 1 5 - - 1 7 ] . In a single r e p o r t o f isolated m i c r o s p o r e c u l t u r e o f B. napus, L i c h t e r [9] increased t h e yield o f e m b r y o s u p t o 3.4 e m b r y o s p e r a n t h e r in o n e t r e a t ment and indicated that embryos could be obtained by a simple culture method without l o w t e m p e r a t u r e p r e t r e a t m e n t o f b u d s or preculture of anthers.
Abbreviations: BA, benzyladenine; NAA, naphthalene-acetic acid; NN, Nitsch and Nitsch.
T h e r e s e a r c h r e p o r t e d h e r e was p a r t o f an ongoing program to improve the efficiency of t h e h a p l o i d a p p r o a c h t o Brassica v a r i e t y i m p r o v e m e n t . T h e r e p o r t deals w i t h t h e i m p r o v e d yield a n d r e g e n e r a t i o n p o t e n t i a l o f isolated m i c r o s p o r e - d e r i v e d e m b r y o s o f t h e a m p h i d i p l o i d s , B. n a p u s and B. carinata. Materials a n d m e t h o d s Six varieties o f B. napus L. and o n e o f B. carinata B r a u n e v a l u a t e d in this s t u d y w e r e g r o w n in a c o n t r o l l e d e n v i r o n m e n t r o o m u n d e r a 16-h p h o t o p e r i o d ( a p p r o x . 2 0 0 p m o l m -2 sec -1 o f p h o t o s y n t h e t i c a l l y a c t i v e r a d i a t i o n at t h e b e n c h surface) at 2 5 / 2 0 ° C f o r l i g h t / d a r k p e r i o d s , respectively. Young flower buds from the main raceme and lateral b r a n c h e s w e r e surface sterilized w i t h a 5.25% (v/v) s o l u t i o n o f s o d i u m h y p o c h l o r i t e f o r 10 m i n a n d w e r e t h e n rinsed t h r e e t i m e s w i t h sterile d e i o n i z e d w a t e r . O n l y t h e a n t h e r s f r o m b u d s in w h i c h p e t a l s e x t e n d e d a p p r o x i m a t e l y h a l f t h e length of t h e a n t h e r a n d in
0168-9452/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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which anthers were yellow-green in color were used for microspore isolation. Anthers were gently macerated in a washing solution of Bs medium [13] supplemented with 13% (w/v) sucrose (Keller, personal commun.). The resulting suspension was aseptically filtered through a 44-pm nylon screen and collected by centrifugation at room temperature for 3 min at 100 X g followed by three washes with washing medium. The microspores were resuspended in culture medium, consisting of the macro- and micronutrients and vitamins of NN medium [14] as modified by Lichter [3] devoid of Difco potato extract, but containing 13% sucrose, 0.05 mg/1 BA and 1.00 mg/1 NAA, titrated to pH 6.0 and filter sterilized. The study consisted of seven genotypes {Table I) evaluated at each of six culture temperatures (22, 25, 27, 30, 32 and 35°C). In addition, two of the genotypes, R-2128 (B. carinata) and Isuzu (B. napus) were also evaluated in a split temperature regime consisting of an initial 3 days incubation at 32°C followed by 25 days of culture at 25°C. Each temperature-genotype treatment combination was evaluated by plating (asepti-
T a b l e I. E m b r y o s per a n t h e r o b t a i n e d t h r o u g h i s o l a t e d m i c r o s p o r e c u l t u r e in Brassica. D a t a pres e n t e d h a s b e e n c o n v e r t e d to t h e m e a n n u m b e r o f e m b r y o s p e r 17 0 0 0 p l a t e d m i c r o s p o r e s { a n t h e r equivalent calculated from three replications each consisting o f a b o u t 200 000 microspores). Cultivars
I n d u c t i o n t e m p e r a t u r e (°C) 22
25
27
30
32
35
B. n a p u s Bronowski Altex Regent Isuzu CR-1 ATR Tw
--0.1 0.1 -0
1.3 2.2 1.4 1.0 -0
3.8 2.5 2.7 4.3 1.3 4.4
7.4 7.7 7.3 9.6 7.0 8.3
0 4.3 3.5 1.6 9.4 2.7
0 0 -0 0 0
B. carinata R-2128
0.4
1.7
2.1
54.1
39.1
0
cally) approx. 200 000 microspores (the equivalent of 12 anthers) in 2.5 ml of culture medium in a 60 X 15 mm Falcon petri dish sealed with parafilm. Three replications of each temperature-genotype combination were cultured in darkness for 4 weeks after which embryo frequencies and embryo types were determined. After 4 weeks in culture, samples of embryos from the 30°C temperature treatments were transferred to basal solid Bs medium [13] lacking growth regulators or a basal NN medium [14] supplemented with 2% (w/v) sucrose and 0.8% (w/v) agar but devoid of growth regulators. These subcultures were maintained under a 16-h photoperiod at 25°C for 4 weeks after which the frequency of plantlets produced per subcultured embryo was determined. Results Initiation of successful microspore culture was marked by an increase in microspore volume, usually within 24 h. Within 3 days, some microspore-derived cells burst out of the exine (Fig. la) and underwent cell division (Fig. lb). Microspore cultures incubated at 25, 30, 32 and 35°C behaved similarly for the first 2 days of culture after which cultures incubated at 35°C contained a higher frequency of exine-free microspore-derived cells. Cultures maintained at 22°C contained exinefree microspores although dividing microspores were less frequent than at higher culture temperatures. Clusters of cells resulting from microspore division were apparent after 5 days in culture (Fig. lc). Embryo development occurred more rapidly at 30 and 32°C culture temperatures. Globular (Fig. ld) and heart-shaped embryos (Fig. le) were observed within 10--14 days for cultures maintained at 30 and 32°C. Embryo development was not synchronized within cultures. After 3 weeks in culture, in addition to torpedo-shaped and cotyledonous embryos, small globular embryos and cell clusters were c o m m o n l y observed. The rate of embryo
221
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8eo
6 D
6
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6
0
~
ee
t
~
ol
D
0 0
e ©
c~ e ~ a
Q
~
~
o ~ O
AI
v
j
ld--
i le
Fig. 1. Microspore culture of Bo Carinata var. R-2128 at 30°C. Bar = 50 um. (a) Microspore bursts out of exine, (b) first division after 3 days in culture, (c) cell clusters after 5--7 days in culture, (d) globular embryo, (e) heart-shape after 10 days in culture, (f) embryo production of R-2128 at 27°C (upper left), 30°C (lower center) and 32°C (upper right). Fig. 2. Embryo classification: five types of embryos. (A) type A, globular embryo; (B) type B, abnormal embryo; (C) type C, bipolar torpedo-shaped embryo; (D) bipolar cotyledonous embryos; (E) multilobe abnormal embryos. Bar = 1.0 ram.
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development declined after 4--5 weeks in culture regardless of embryo size, probably as a result of medium nutrient depletion.
and shape into five categories (Fig. 2): Type A: globular embryos with smooth surfaces. Many Type A embryos failed to continue to develop when left in medium. Type B: round or oval abnormal embryos with more than two lobes. These developed abnormally and have not been observed to produce plantlets. Type C: bipolar heart-shaped and torpedoshaped embryos with root end opposite shoot end. This type of embryo appears to be a transitory stage leading to Type D embryos. Type D: cotyledonous embryos possess features similar to true embryos, including a radical connected by a hypocotyl-like structure to an incomplete 2-lobed cotyledonus structure. (Classification of Type C or Type D was somewhat arbitrary for embryos intermediate between these two types.) Type D embryos appeared capable of plantlet formation following subculture. Type E: large abnormal embryos fre-
Temperature effects Embryonic responses occurred within the range of 22--32°C, with the highest frequency occuring at 30°C for all gentoypes except CR-1 which produced more embryos at 32°C. The B. carinata genotype R-2128 produced the highest embryo yield in this study (Fig. lf). At 30°C, embryo frequencies (Table I) ranged from 7 to 54 per anther equivalent (approx. 17 000 microspores). No embryos developed at 35°C and only a few embryos developed for some genotypes at 22°C.
Embryo classification After 4 weeks of microspore culture, a broad array of embryo types were apparent. At this time, embryos were classified by size 80
80 25% n~20
60
6o
4O
4o
20
20
0 0
MF1 A
B
C-D
27°C n~3o
rq
E TYPE
A
B
C-D
B
C-D
E TYPE
w < 0 8o
8O
30°C
&P
n=5oo 6O
6O
40
4O
20
2O
A
B
C-D
E
TYPE
32°C n:400
A
TYPE
Fig. 3. E f f e c t s o f four culture t e m p e r a t u r e s on t h e f r e q u e n c i e s o f e m b r y o t y p e s w h i c h d e v e l o p e d in B. carinata cv. R-2128 (n = t o t a l n u m b e r o f e m b r y o s classified).
223
embryos were normal, while only 19% were normal in cultures maintained at 32°C. The percentage of Type E (large abnormal) embryos increased with increasing incubation temperatures. No Type E embryos developed at 25°C while 55% of the embryos produced at 32°C were Type E. A similar relationship between temperature and embryo type was observed in B. napus but low embryo frequencies precluded meaningful comparisons among B. napus genotypes. The split temperature regime (3 days at 32°C followed by maintenance at 25°C) resulted in a dramatic improvement in embryo frequency compared to the cultures kept at 25°C for the entire culture period, similar percentages of Type C and D (normal) embryos were found (Fig. 4).
quently similar in shape to Type B embryos but larger in size, have not been observed to produce plantlets following subculture. Type C and D embryos were classified as normal embryos due to well developed bipolar features, whereas all other embryo types were classified as abnormal. Due to the complete range of embryos between Type C and Type D, these categories are combined for data presentation hereafter. Influence o f temperature on embryo type Culture temperature had a significant effect on the frequency of embryo types which developed from microspores of B. carinata (Fig. 3). The percentage of normal embryos (Types C and D) decreased with increasing temperature. At 25°C, 70% of the 80
80
32°J2~%
2 5 °C n= 25
N=206 6O
60
R-2128
40
40
20
29
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25°C
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"--i
60
60
ISUZU
aO
40
20
20
.n A
B
L
C-D
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A
B
C-O
Fig. 4. C o m p a r i s o n of split ( 3 2 / 2 5 ° C ) a n d c o n s t a n t (25°C) i n c u b a t i o n t e m p e r a t u r e s o n t h e f r e q u e n c i e s of e m b r y o t y p e s w h i c h d e v e l o p e d in B. carinata var. R - 2 1 2 8 a n d B. n a p u s var. Isuzu (n = t o t a l n u m b e r of e m b r y o s classified ).
224
Fig. 5. Plantlet development from embryo, vat. Isuzu. (a) Normal plantlet on NN medium. (b) Abnormal masses of tissue on B s medium. t u r e survival r a t e t h a n T y p e A a n d B e m b r y o s (80% vs. 15%). D e v e l o p m e n t o f p l a n t l e t s varied w i t h genot y p e a n d s u b c u l t u r e m e d i a . On average, n e a r l y 9% o f the T y p e C a n d D e m b r y o s subcultured to the NN medium developed into p l a n t l e t s while o n l y 1% o f t h o s e subcult u r e d to t h e basal B s - p r o d u c e d p l a n t l e t s ( T a b l e II). The frequency of normal embryos that p r o d u c e d p l a n t l e t s (Fig. 5a) varied f r o m 2.8% f o r var. R e g e n t t o 22.2% f o r Isuzu w h e n s u b c u l t u r e d to t h e N N m e d i u m ( T a b l e II).
Plantlet development
T y p e C, D a n d E e m b r y o s s u b c u l t u r e d f r o m t h e 30°C t e m p e r a t u r e t r e a t m e n t s t o basal Bs a n d basal N N m e d i a t u r n e d green in c o l o r a n d initiated active r o o t g r o w t h w i t h i n 5 d a y s . T y p e C a n d D e m b r y o s g r e w larger w i t h visible increases in h y p o c o t y l a n d cotyledonous structures. The majority of T y p e C a n d D e m b r y o s d e v e l o p e d into irregular masses o f tissue w i t h s o m e leaf-like s t r u c t u r e s (Fig. 5b) similar to a n t h e r - d e r i v e d e m b r y o s f r o m liquid m e d i u m a n t h e r cultures. N o r m a l e m b r y o s h a d a m u c h higher subculTable II.
Percent of normal embryos that developed into plantlets on two enbryo-subculture media.
Cultivars
NN medium
%
B s medium Embryos subcultured (no.)
%
Embryos Subcultured (no.)
Plantlets obtained
Plantlets obtained
45 102 75 72 40 60
10 3 6 2 3 9
22.2 2.9 8.0 2.8 7.5 15.0
30 45 23 37 45 50
1 0 0 0 0 2
3.3 0 0 0 0 4.0
93
10
10.8
84
0
0
487
43
8.8
314
3
1.0
B. napus
Isuzu CR-I Altex Regent Bronowski ATR-Tw B. carinata
R2128 Total
225 The frequency of plantlets from normal e m b r y o s subcultured to the basal B5 medium was t o o low for comparison among genotypes within B. napus. Discussion Brassica microspore culture, first reported b y Lichter [9] met with limited success. In this study, several genotypes of B. napus and one genotype of B. carinata responded with improved e m b r y o yields using an elevated concentration of NAA (1 rag/l) without low temperature pretreatments. Further, microspores of most genotypes tested were embryogenic over a range of temperatures (22--32°C) although maximum e m b r y o yields were obtained at 30°C. Culture temperature influenced b o t h e m b r y o yield and embryo quality. Higher temperatures resulted in a higher proportion of embryos that developed abnormally, while lower culture temperatures were associated with lower e m b r y o yields of which a higher proportion developed normally. Microspore cultures subjected to 32°C for 3 days followed by maintenance at 25°C for 3--4 weeks increased the total number of embryos produced (n) by 800 and 500% for R-2128 and Isuzu, respectively, in comparison to microspores cultured at a constant temperature of 25°C (Fig. 4). These results are similar to the results obtained from high temperature pretreatment of Brassica anther cultures [1,4,10,11]. Microspore cultures in the split temperature regime also resulted in a high proportion of normal embryos, approaching 70 and 80% for R-2128 and Isuzu, respectively (Fig. 4). Plantlet regeneration from subcultured microspore-derived e m b r y o s was dependent on subculture media and genotype. Only the normal e m b r y o s (Type C and D) were observed to develop into plantlets, yet the majority of e m b r y o s grew irregularly into abnormal structures even though these e m b r y o s c o m m o n l y possessed developed apical meristems (Chuong and Beversdorf, unpublished). The development of more
suitable embryo subculture medium with an optimum combination of hormones to stimulate a complete maturation process and continued development of apices will require additional research. Embryos produced from anther culture on M41 medium [5], a modification of Bs medium could regenerate into plantlets upon subculture onto basal Bs medium (Cardy, pers. commun.), yet regeneration of plantlets from isolated microspore-derived embryos in this study was poor on basal Bs medium in comparison to hormone-free NN medium. A shift from the modified NN microspore culture medium to Bs embryo culture medium may have caused a nutrient shock for the developing e m b r y o s in this study. After 4 weeks, a large portion of small embryos, types A and B resulting from nonsynchronous embryo development {Figs. 3 and 4) failed to survive subculture to either Bs or NN basal embryo culture media and usually failed to complete development if left in the microspore culture medium. By transferring t y p e A and B embryos to a rescue medium, these embryos continued development and were later capable of surviving subculture to the regeneration medium (Choung and Beversdorf, in prep.}. As with anther culture, isolated microspore culture shows a high potential for production of haploids in Brassica. Embryo yields obtained from isolated microspore culture were much higher than c o m m o n l y reported from Brassica anther cultures. Moreover, isolated microspore culture eliminates the possibility of non-pollen or non-microspore embryos arising from somatic tissues of the anther. Improved embryo yields could lead to the use of microspore cultures for mutation in vitro, selection and other potential biotechniques. Acknowledgements The suggestions and assistance of W.A. Keller, Agriculture Canada Research Station, Ottawa, are greatfully acknowledged as is
226
technical assistance Deslaurier and B. assistance from the Engineering Research
from L. Polsoni, C. Watson, and financial National Science and Council, Canada.
References 1 L. George and P.S. Rao, Plant Sci. Lett., 26 (1982) 1 l l . 2 W.A. Keller and K.C. Armstrong, Theor. Appl. Genet., 55 (1979) 65. 3 R. Lichter, Z. Pflanzenphysiol., 103 (1981) 229. 4 W.A. Keller and K.C. Armstrong, Z. Pflanzenzuchtg., 80 (1978) 100. 5 W.A. Keller and K.C. Armstrong, Can. J. Bot., 55 (1977) 1383. 6 G. Wenzel, F. Hoffman and E. Thomas, Z. Priam zenzuchtg., 78 (1977) 149. 7 E. Thomas and G. Wenzel, Z. Pflanzenzuchtg., 74 (1975) 77.
8 W.A. Keller, T. Rajhathy and J. Lacapra, Can. J. Genet. Cytol., 17 (1975) 655. 9 R. Litcher, Z. Pflanzenphysiol., 105 (1982) 427. tO K. Klimaszewska and W.A. Keller, Z. Pflanzenphysiol., 109 (1983) 235. ]1 W.A. Keller and K.C. Armstrong, Euphytica, 32 (1983) 151. 12 W.A. Keller and G.R. Stringam, Production and utilization of microspore-derived plants from Brassica n a p u s anther cultures, in: T.A. Thorpe (Ed.), Frontier of Plant Tissue Culture, Proc. I.A.P.T.C., Calgary, 1978, p. 113. 13 O.L. Gamborg, R.A. Miller and L. Ojima, Exp. Cell Res., 50 (1968) 151. 14 C. Nitsch and J.P. Nitsch, Planta (Berl.) 72 (1967) 355. 15 M. Renard and F. Dolba, Ann. Ameliorat. Plantes, 30 (1980) 191. 16 C.S. Loh and D.S. Ingrain, New Phytol., 91 (1982) 507. 17 Z.H. Chen and Z.Z. Chen, Kexue Tongbao, 28 (1983) 1690.
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HIGH FREQUENCY EMBRYOGENESIS THROUGH ISOLATED MICROSPORE CULTURE IN B R A S S I C A N A P U S L. A N D B. C A R I N A T A B R A U N
PHAN V. CHUONG and W.D. BEVERSDORF Crop Science Department University of Guelph, Guelph, Ontario N1G 2W1 (Canada) (Received November 15th, 1984) (Revision received March 7th, 1985) (Accepted April 3rd, 1985) Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l naphthaleneacetic acid (NAA). Embryogenic responses were observed at culture temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7--54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium. Key words: Brassica napus; microspore culture; embryogenesis; embryo types; temperature effect
Introduction T h e u t i l i t y o f h a p l o i d p l a n t s in b r e e d i n g a n d genetic r e s e a r c h has e n c o u r a g e d d e v e l o p ment of efficient techniques to produce them. In Brassica, a n t h e r c u l t u r e has b e e n t h e primary source of haploid plants [1--8]. E m b r y o f r e q u e n c i e s f r o m c u l t u r e d Brassica a n t h e r s h a v e varied w i t h t h e species a n d t h e t e c h n i q u e u s e d b u t has generally b e e n in the range of 0--8 embryos per cultured a n t h e r [ 4 , 1 0 - - 1 2 , 1 5 - - 1 7 ] . In a single r e p o r t o f isolated m i c r o s p o r e c u l t u r e o f B. napus, L i c h t e r [9] increased t h e yield o f e m b r y o s u p t o 3.4 e m b r y o s p e r a n t h e r in o n e t r e a t ment and indicated that embryos could be obtained by a simple culture method without l o w t e m p e r a t u r e p r e t r e a t m e n t o f b u d s or preculture of anthers.
Abbreviations: BA, benzyladenine; NAA, naphthalene-acetic acid; NN, Nitsch and Nitsch.
T h e r e s e a r c h r e p o r t e d h e r e was p a r t o f an ongoing program to improve the efficiency of t h e h a p l o i d a p p r o a c h t o Brassica v a r i e t y i m p r o v e m e n t . T h e r e p o r t deals w i t h t h e i m p r o v e d yield a n d r e g e n e r a t i o n p o t e n t i a l o f isolated m i c r o s p o r e - d e r i v e d e m b r y o s o f t h e a m p h i d i p l o i d s , B. n a p u s and B. carinata. Materials a n d m e t h o d s Six varieties o f B. napus L. and o n e o f B. carinata B r a u n e v a l u a t e d in this s t u d y w e r e g r o w n in a c o n t r o l l e d e n v i r o n m e n t r o o m u n d e r a 16-h p h o t o p e r i o d ( a p p r o x . 2 0 0 p m o l m -2 sec -1 o f p h o t o s y n t h e t i c a l l y a c t i v e r a d i a t i o n at t h e b e n c h surface) at 2 5 / 2 0 ° C f o r l i g h t / d a r k p e r i o d s , respectively. Young flower buds from the main raceme and lateral b r a n c h e s w e r e surface sterilized w i t h a 5.25% (v/v) s o l u t i o n o f s o d i u m h y p o c h l o r i t e f o r 10 m i n a n d w e r e t h e n rinsed t h r e e t i m e s w i t h sterile d e i o n i z e d w a t e r . O n l y t h e a n t h e r s f r o m b u d s in w h i c h p e t a l s e x t e n d e d a p p r o x i m a t e l y h a l f t h e length of t h e a n t h e r a n d in
0168-9452/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
220
which anthers were yellow-green in color were used for microspore isolation. Anthers were gently macerated in a washing solution of Bs medium [13] supplemented with 13% (w/v) sucrose (Keller, personal commun.). The resulting suspension was aseptically filtered through a 44-pm nylon screen and collected by centrifugation at room temperature for 3 min at 100 X g followed by three washes with washing medium. The microspores were resuspended in culture medium, consisting of the macro- and micronutrients and vitamins of NN medium [14] as modified by Lichter [3] devoid of Difco potato extract, but containing 13% sucrose, 0.05 mg/1 BA and 1.00 mg/1 NAA, titrated to pH 6.0 and filter sterilized. The study consisted of seven genotypes {Table I) evaluated at each of six culture temperatures (22, 25, 27, 30, 32 and 35°C). In addition, two of the genotypes, R-2128 (B. carinata) and Isuzu (B. napus) were also evaluated in a split temperature regime consisting of an initial 3 days incubation at 32°C followed by 25 days of culture at 25°C. Each temperature-genotype treatment combination was evaluated by plating (asepti-
T a b l e I. E m b r y o s per a n t h e r o b t a i n e d t h r o u g h i s o l a t e d m i c r o s p o r e c u l t u r e in Brassica. D a t a pres e n t e d h a s b e e n c o n v e r t e d to t h e m e a n n u m b e r o f e m b r y o s p e r 17 0 0 0 p l a t e d m i c r o s p o r e s { a n t h e r equivalent calculated from three replications each consisting o f a b o u t 200 000 microspores). Cultivars
I n d u c t i o n t e m p e r a t u r e (°C) 22
25
27
30
32
35
B. n a p u s Bronowski Altex Regent Isuzu CR-1 ATR Tw
--0.1 0.1 -0
1.3 2.2 1.4 1.0 -0
3.8 2.5 2.7 4.3 1.3 4.4
7.4 7.7 7.3 9.6 7.0 8.3
0 4.3 3.5 1.6 9.4 2.7
0 0 -0 0 0
B. carinata R-2128
0.4
1.7
2.1
54.1
39.1
0
cally) approx. 200 000 microspores (the equivalent of 12 anthers) in 2.5 ml of culture medium in a 60 X 15 mm Falcon petri dish sealed with parafilm. Three replications of each temperature-genotype combination were cultured in darkness for 4 weeks after which embryo frequencies and embryo types were determined. After 4 weeks in culture, samples of embryos from the 30°C temperature treatments were transferred to basal solid Bs medium [13] lacking growth regulators or a basal NN medium [14] supplemented with 2% (w/v) sucrose and 0.8% (w/v) agar but devoid of growth regulators. These subcultures were maintained under a 16-h photoperiod at 25°C for 4 weeks after which the frequency of plantlets produced per subcultured embryo was determined. Results Initiation of successful microspore culture was marked by an increase in microspore volume, usually within 24 h. Within 3 days, some microspore-derived cells burst out of the exine (Fig. la) and underwent cell division (Fig. lb). Microspore cultures incubated at 25, 30, 32 and 35°C behaved similarly for the first 2 days of culture after which cultures incubated at 35°C contained a higher frequency of exine-free microspore-derived cells. Cultures maintained at 22°C contained exinefree microspores although dividing microspores were less frequent than at higher culture temperatures. Clusters of cells resulting from microspore division were apparent after 5 days in culture (Fig. lc). Embryo development occurred more rapidly at 30 and 32°C culture temperatures. Globular (Fig. ld) and heart-shaped embryos (Fig. le) were observed within 10--14 days for cultures maintained at 30 and 32°C. Embryo development was not synchronized within cultures. After 3 weeks in culture, in addition to torpedo-shaped and cotyledonous embryos, small globular embryos and cell clusters were c o m m o n l y observed. The rate of embryo
221
0 0
8eo
6 D
6
p
6
0
~
ee
t
~
ol
D
0 0
e ©
c~ e ~ a
Q
~
~
o ~ O
AI
v
j
ld--
i le
Fig. 1. Microspore culture of Bo Carinata var. R-2128 at 30°C. Bar = 50 um. (a) Microspore bursts out of exine, (b) first division after 3 days in culture, (c) cell clusters after 5--7 days in culture, (d) globular embryo, (e) heart-shape after 10 days in culture, (f) embryo production of R-2128 at 27°C (upper left), 30°C (lower center) and 32°C (upper right). Fig. 2. Embryo classification: five types of embryos. (A) type A, globular embryo; (B) type B, abnormal embryo; (C) type C, bipolar torpedo-shaped embryo; (D) bipolar cotyledonous embryos; (E) multilobe abnormal embryos. Bar = 1.0 ram.
222
development declined after 4--5 weeks in culture regardless of embryo size, probably as a result of medium nutrient depletion.
and shape into five categories (Fig. 2): Type A: globular embryos with smooth surfaces. Many Type A embryos failed to continue to develop when left in medium. Type B: round or oval abnormal embryos with more than two lobes. These developed abnormally and have not been observed to produce plantlets. Type C: bipolar heart-shaped and torpedoshaped embryos with root end opposite shoot end. This type of embryo appears to be a transitory stage leading to Type D embryos. Type D: cotyledonous embryos possess features similar to true embryos, including a radical connected by a hypocotyl-like structure to an incomplete 2-lobed cotyledonus structure. (Classification of Type C or Type D was somewhat arbitrary for embryos intermediate between these two types.) Type D embryos appeared capable of plantlet formation following subculture. Type E: large abnormal embryos fre-
Temperature effects Embryonic responses occurred within the range of 22--32°C, with the highest frequency occuring at 30°C for all gentoypes except CR-1 which produced more embryos at 32°C. The B. carinata genotype R-2128 produced the highest embryo yield in this study (Fig. lf). At 30°C, embryo frequencies (Table I) ranged from 7 to 54 per anther equivalent (approx. 17 000 microspores). No embryos developed at 35°C and only a few embryos developed for some genotypes at 22°C.
Embryo classification After 4 weeks of microspore culture, a broad array of embryo types were apparent. At this time, embryos were classified by size 80
80 25% n~20
60
6o
4O
4o
20
20
0 0
MF1 A
B
C-D
27°C n~3o
rq
E TYPE
A
B
C-D
B
C-D
E TYPE
w < 0 8o
8O
30°C
&P
n=5oo 6O
6O
40
4O
20
2O
A
B
C-D
E
TYPE
32°C n:400
A
TYPE
Fig. 3. E f f e c t s o f four culture t e m p e r a t u r e s on t h e f r e q u e n c i e s o f e m b r y o t y p e s w h i c h d e v e l o p e d in B. carinata cv. R-2128 (n = t o t a l n u m b e r o f e m b r y o s classified).
223
embryos were normal, while only 19% were normal in cultures maintained at 32°C. The percentage of Type E (large abnormal) embryos increased with increasing incubation temperatures. No Type E embryos developed at 25°C while 55% of the embryos produced at 32°C were Type E. A similar relationship between temperature and embryo type was observed in B. napus but low embryo frequencies precluded meaningful comparisons among B. napus genotypes. The split temperature regime (3 days at 32°C followed by maintenance at 25°C) resulted in a dramatic improvement in embryo frequency compared to the cultures kept at 25°C for the entire culture period, similar percentages of Type C and D (normal) embryos were found (Fig. 4).
quently similar in shape to Type B embryos but larger in size, have not been observed to produce plantlets following subculture. Type C and D embryos were classified as normal embryos due to well developed bipolar features, whereas all other embryo types were classified as abnormal. Due to the complete range of embryos between Type C and Type D, these categories are combined for data presentation hereafter. Influence o f temperature on embryo type Culture temperature had a significant effect on the frequency of embryo types which developed from microspores of B. carinata (Fig. 3). The percentage of normal embryos (Types C and D) decreased with increasing temperature. At 25°C, 70% of the 80
80
32°J2~%
2 5 °C n= 25
N=206 6O
60
R-2128
40
40
20
29
b 2
0 cc a_
A
B
C-D
E
A
TYPE
B
C-D
E
TYPE
E
TYPE
Q >a:
~ ~r
100
1 '9 @
32o/25°C
25°C
~ 8o
D=12
n= 60
8o
"--i
60
60
ISUZU
aO
40
20
20
.n A
B
L
C-D
E
TYPE
A
B
C-O
Fig. 4. C o m p a r i s o n of split ( 3 2 / 2 5 ° C ) a n d c o n s t a n t (25°C) i n c u b a t i o n t e m p e r a t u r e s o n t h e f r e q u e n c i e s of e m b r y o t y p e s w h i c h d e v e l o p e d in B. carinata var. R - 2 1 2 8 a n d B. n a p u s var. Isuzu (n = t o t a l n u m b e r of e m b r y o s classified ).
224
Fig. 5. Plantlet development from embryo, vat. Isuzu. (a) Normal plantlet on NN medium. (b) Abnormal masses of tissue on B s medium. t u r e survival r a t e t h a n T y p e A a n d B e m b r y o s (80% vs. 15%). D e v e l o p m e n t o f p l a n t l e t s varied w i t h genot y p e a n d s u b c u l t u r e m e d i a . On average, n e a r l y 9% o f the T y p e C a n d D e m b r y o s subcultured to the NN medium developed into p l a n t l e t s while o n l y 1% o f t h o s e subcult u r e d to t h e basal B s - p r o d u c e d p l a n t l e t s ( T a b l e II). The frequency of normal embryos that p r o d u c e d p l a n t l e t s (Fig. 5a) varied f r o m 2.8% f o r var. R e g e n t t o 22.2% f o r Isuzu w h e n s u b c u l t u r e d to t h e N N m e d i u m ( T a b l e II).
Plantlet development
T y p e C, D a n d E e m b r y o s s u b c u l t u r e d f r o m t h e 30°C t e m p e r a t u r e t r e a t m e n t s t o basal Bs a n d basal N N m e d i a t u r n e d green in c o l o r a n d initiated active r o o t g r o w t h w i t h i n 5 d a y s . T y p e C a n d D e m b r y o s g r e w larger w i t h visible increases in h y p o c o t y l a n d cotyledonous structures. The majority of T y p e C a n d D e m b r y o s d e v e l o p e d into irregular masses o f tissue w i t h s o m e leaf-like s t r u c t u r e s (Fig. 5b) similar to a n t h e r - d e r i v e d e m b r y o s f r o m liquid m e d i u m a n t h e r cultures. N o r m a l e m b r y o s h a d a m u c h higher subculTable II.
Percent of normal embryos that developed into plantlets on two enbryo-subculture media.
Cultivars
NN medium
%
B s medium Embryos subcultured (no.)
%
Embryos Subcultured (no.)
Plantlets obtained
Plantlets obtained
45 102 75 72 40 60
10 3 6 2 3 9
22.2 2.9 8.0 2.8 7.5 15.0
30 45 23 37 45 50
1 0 0 0 0 2
3.3 0 0 0 0 4.0
93
10
10.8
84
0
0
487
43
8.8
314
3
1.0
B. napus
Isuzu CR-I Altex Regent Bronowski ATR-Tw B. carinata
R2128 Total
225 The frequency of plantlets from normal e m b r y o s subcultured to the basal B5 medium was t o o low for comparison among genotypes within B. napus. Discussion Brassica microspore culture, first reported b y Lichter [9] met with limited success. In this study, several genotypes of B. napus and one genotype of B. carinata responded with improved e m b r y o yields using an elevated concentration of NAA (1 rag/l) without low temperature pretreatments. Further, microspores of most genotypes tested were embryogenic over a range of temperatures (22--32°C) although maximum e m b r y o yields were obtained at 30°C. Culture temperature influenced b o t h e m b r y o yield and embryo quality. Higher temperatures resulted in a higher proportion of embryos that developed abnormally, while lower culture temperatures were associated with lower e m b r y o yields of which a higher proportion developed normally. Microspore cultures subjected to 32°C for 3 days followed by maintenance at 25°C for 3--4 weeks increased the total number of embryos produced (n) by 800 and 500% for R-2128 and Isuzu, respectively, in comparison to microspores cultured at a constant temperature of 25°C (Fig. 4). These results are similar to the results obtained from high temperature pretreatment of Brassica anther cultures [1,4,10,11]. Microspore cultures in the split temperature regime also resulted in a high proportion of normal embryos, approaching 70 and 80% for R-2128 and Isuzu, respectively (Fig. 4). Plantlet regeneration from subcultured microspore-derived e m b r y o s was dependent on subculture media and genotype. Only the normal e m b r y o s (Type C and D) were observed to develop into plantlets, yet the majority of e m b r y o s grew irregularly into abnormal structures even though these e m b r y o s c o m m o n l y possessed developed apical meristems (Chuong and Beversdorf, unpublished). The development of more
suitable embryo subculture medium with an optimum combination of hormones to stimulate a complete maturation process and continued development of apices will require additional research. Embryos produced from anther culture on M41 medium [5], a modification of Bs medium could regenerate into plantlets upon subculture onto basal Bs medium (Cardy, pers. commun.), yet regeneration of plantlets from isolated microspore-derived embryos in this study was poor on basal Bs medium in comparison to hormone-free NN medium. A shift from the modified NN microspore culture medium to Bs embryo culture medium may have caused a nutrient shock for the developing e m b r y o s in this study. After 4 weeks, a large portion of small embryos, types A and B resulting from nonsynchronous embryo development {Figs. 3 and 4) failed to survive subculture to either Bs or NN basal embryo culture media and usually failed to complete development if left in the microspore culture medium. By transferring t y p e A and B embryos to a rescue medium, these embryos continued development and were later capable of surviving subculture to the regeneration medium (Choung and Beversdorf, in prep.}. As with anther culture, isolated microspore culture shows a high potential for production of haploids in Brassica. Embryo yields obtained from isolated microspore culture were much higher than c o m m o n l y reported from Brassica anther cultures. Moreover, isolated microspore culture eliminates the possibility of non-pollen or non-microspore embryos arising from somatic tissues of the anther. Improved embryo yields could lead to the use of microspore cultures for mutation in vitro, selection and other potential biotechniques. Acknowledgements The suggestions and assistance of W.A. Keller, Agriculture Canada Research Station, Ottawa, are greatfully acknowledged as is
226
technical assistance Deslaurier and B. assistance from the Engineering Research
from L. Polsoni, C. Watson, and financial National Science and Council, Canada.
References 1 L. George and P.S. Rao, Plant Sci. Lett., 26 (1982) 1 l l . 2 W.A. Keller and K.C. Armstrong, Theor. Appl. Genet., 55 (1979) 65. 3 R. Lichter, Z. Pflanzenphysiol., 103 (1981) 229. 4 W.A. Keller and K.C. Armstrong, Z. Pflanzenzuchtg., 80 (1978) 100. 5 W.A. Keller and K.C. Armstrong, Can. J. Bot., 55 (1977) 1383. 6 G. Wenzel, F. Hoffman and E. Thomas, Z. Priam zenzuchtg., 78 (1977) 149. 7 E. Thomas and G. Wenzel, Z. Pflanzenzuchtg., 74 (1975) 77.
8 W.A. Keller, T. Rajhathy and J. Lacapra, Can. J. Genet. Cytol., 17 (1975) 655. 9 R. Litcher, Z. Pflanzenphysiol., 105 (1982) 427. tO K. Klimaszewska and W.A. Keller, Z. Pflanzenphysiol., 109 (1983) 235. ]1 W.A. Keller and K.C. Armstrong, Euphytica, 32 (1983) 151. 12 W.A. Keller and G.R. Stringam, Production and utilization of microspore-derived plants from Brassica n a p u s anther cultures, in: T.A. Thorpe (Ed.), Frontier of Plant Tissue Culture, Proc. I.A.P.T.C., Calgary, 1978, p. 113. 13 O.L. Gamborg, R.A. Miller and L. Ojima, Exp. Cell Res., 50 (1968) 151. 14 C. Nitsch and J.P. Nitsch, Planta (Berl.) 72 (1967) 355. 15 M. Renard and F. Dolba, Ann. Ameliorat. Plantes, 30 (1980) 191. 16 C.S. Loh and D.S. Ingrain, New Phytol., 91 (1982) 507. 17 Z.H. Chen and Z.Z. Chen, Kexue Tongbao, 28 (1983) 1690.