Planta (Bed.) 124, 1--11 (1975) 9 by Springer-Verlag 1975
Haploid Callus and Regeneration of Plants from Anthers of Digitalis purpurea L. G e r d C o r d u a n a n d Christel S p i x Lehrstuhl fiir Pflanzenphysiologie, 1%uhr-Universitgt Bochum, Postfaeh 2148, D-4630 Bochum, Federal Republic of Germany Received 4 February; accepted 28 February 1975 Summary. Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch ( Science 163, 85-87; 1969) supplemented with 5 mg/12,4-dichloro. phenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct genotype to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions, If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/1 kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28 ~ and 8 h dark at 20 ~ but the survivM was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.
Introduction A s u r v e y of t h e l i t e r a t u r e shows t h a t m a n y m e m b e r s of t h e Solanaceae h a v e p r o v e d to be s u i t a b l e o b j e c t s for t h e p r o d u c t i o n of h a p l o i d p l a n t s b y a n t h e r culture (for references see Smith, 1974). H u n d r e d s of p l a n t s a r e easily o b t a i n e d o u t of one a n t h e r of, e.g., Nicotiana tabaeum ( S u n d e r l a n d a n d Wicks, 1969). H a p l o i d tissues a n d p l a n t s m a y arise from a n t h e r s in two different w a y s : first, b y d i r e c t developm e n t of microspores into e m b r y o i d s a n d s u b s e q u e n t l y into p l a n t l e t s , as has been m a i n l y o b s e r v e d in t h e S o l a n a c e a e ; second, b y f o r m a t i o n of a n undifferent i a t e d callus on a n a u x i n - c o n t a i n i n g m e d i u m b y mitoses in t h e pollen, followed b y d i f f e r e n t i a t i o n of p l a n t s u n d e r certain light conditions on auxin-free media, this m o d e of d e v e l o p m e n t h a v i n g been f o u n d in m o s t families o u t s i d e t h e Solanaceae. I n b o t h instances one also observes p l a n t s w i t h a higher degree of ploidy, which is caused b y either n u c l e a r fusion in t h e microspore (Engvild, 1972) or b y e n d o m i t o s e s in t h e cells of p o l l e n - d e r i v e d callus (Nitsch, 1969). Callus m a y , however, also develop from s o m a t i c tissue, such as t h e connective tissue or t h e f i l a m e n t of t h e a n t h e r . I f t h e r e g e n e r a t e d p l a n t s h a v e a diploid c h r o m o s o m e set a n d if t h e s t a r t i n g 1 Planta (Bed.), Vol. 124
2
G. Corduan and C. Spix
m a t e r i a l is n o t very heterozygous, one should n o t find p h e n o t y p i c a l l y detectable segregation products, a n d it is therefore v e r y difficult to decide if regenerates originated from a generative or a vegetative cell. As Digitalis purpurea is a m e m b e r of the Scrophulariaceae we m a y expect t h a t the second of the two ways described above would be the one for p r o d u c t i o n of haploid p l a n t s in this species. To distinguish between callus derived from generative a n d somatic cells it was desirable to find indicators t h a t would parallel cytological determinations: W e were p a r t i c u l a r l y interested in developing methods to regenerate large n u m b e r s of p l a n t s from a n t h e r cultures. F r o m the c u r r e n t literature it is e v i d e n t t h a t the n u m b e r of regenerates from a n t h e r - d e r i v e d callus is c o m p a r a t i v e l y small. Conditions allowing the regeneration of h u n d r e d s of p l a n t s from one a n t h e r would enable us to propagate a d i s t i n c t g e n o t y p e in a sufficient n u m b e r of plants. F r o m our results with a n t h e r culture in Nicotiana tabacum (Corduan, 1973) we k n e w t h a t the composition of the m e d i u m as well as e n v i r o n m e n t a l conditions are i m p o r t a n t for regeneration of large n u m b e r s of plants. I n this report we shall p r e s e n t our results on p r o d u c t i o n of anther-derived callus a n d regeneration of p l a n t s in Digi-
talis purpurea. Material and Methods Plant Material. Flowering shoots of Digitalis purpurea L. were collected at five different locations in the central and western part of the Federal Republic of Germany, varying in climate. Preparation o/Anthers. The flowering shoots were kept for 2-12 days at 4~ The anthers were then isolated, washed for 15 rain in 70 % ethanol, sterilized in 5 % sodium hypochlorite for 4 rain, and washed 5 times with sterilized distilled water. The stage of pollen development was determined before culturing. Culture Procedures. Media which were used for anther culture are summarized in Table 1. Most of the chemicals used in these media were purchased from Merck (Darmstadt, Germany) except 2,4-dielflorophenoxyacetic acid = 2,4-D and 4-chloro-2-methyl-phenoxyacetic acid 4-Mp which were purchased from Fluka (Buchs, Switzerland) and 2(2,4-diehlorophenoxy)propionie acid = 2,4-Dp and 4-(2,4,5-trichlorophenoxy)-butyricacid = 4-Tb from Schuchardt (Munich/Miinchen, Germany). For regeneration of plants out of the anther-derived callus the same basM media were modified with varying amounts of cytokinins like 6-furfurylaminopurin = kinetin and benzylaminopurin from Fluka and trans-6-(4-hydroxy-3-methylbut-2enyl)aminopurin = zeatin from Calbiochem (San Diego, Calif., USA). The media were autoclaved for 20 min at 120~ and 1.2 arm. Glass Petri dishes of 6 cm diameter were filled with 10 ml of medium in a sterile cabinet. Six anthers were cultured in one l~etri dish. For organogenesis, anther-derived callus was transferred to 50-ml Erlenmeyer flasks filled with 35 ml of medium. The callus was transferred to fresh medium every 4 weeks. Environmental Conditions. Variation of environmental conditions involved: 1) photoperiods of 12, 14 and 16 h with temperatures of 25, 28 and 30~ during the light period and 19 and 20~ during the dark period. Light sources used were HLRG from Philips (Eindhoven, Netherlands), and H QL, ttWL and Fluora fluorescent tubes from Osram (Berlin). The light intensities were 1500 and 5000 Ix, measured at the level of the culture flasks; 2) continuous light from Fluora fluorescent tubes, 1500 lx at 25~ and 3) continuous dark at 25~ Cytological Studies. Orcein staining of chromosomes proved to be superior to acetocarmine or Feulgen staining, giving good results with callus as well as with root-tip preparations. Root tips were fixed in 0.029 % 8-hydroxyquinoline (Merck, Darmstadt) 4 h after onset of the light period, and incubated at 4~ for 4 h. They were then hydrolyzed for 5 min in ethanol : conc. HC1 (2:1, v/v), washed, stained for 3 rain with oreein (2.2 g oreein, Merck, were refluxed in 100 ml acetic acid for 30 min; the solution was then filtered and diluted to give a 45 % solution), and squashed (private communication by 1)rofessor J. Grau, Institut ffir Systematisehe Botanik, University of Munich).
Callus and Plants from Anthers of Digitalis
3
Table 1. Media used for culture of anthers of Digitalis purpurea The basal media used were those described by Nitseh and Nitseh (1969)=Nitsch; Linsmaier and Skoog ( 1 9 6 5 ) = L S ; Murashige and Skoog (1962)=MS; and White (1943). For other abbreviations see text. Concentrations are in rag/1 Nitsch ,, ,, ,, ,, ,, ,, ,, ,, ,, ,,
-k 1000 casein hydrolysate -t- 5, 10 or 20 2,4-D -l-5, 10 or 20 NAA -t-5, 10 or 20 2,4-Dp q-5, 10 or 20 4-Mp ~-5, 10 or 20 4-Tb -b 5 2,4-D without myo-inositol nc 5 2,4-D without pyridoxine. HCI -t- 5 2,4-D without thiamine. HC1 -t- 5 2,4-D without relic acid + 5 2,4-D without biotin
MS ,, ,,
-1-5, 10 or 20 2,4-D -]-5, 10 or 20 NAA -t-5, 10 or 20 2,4-Dp
LS ,, ,, ,,
q- 5, § 5, -t-5, + 5,
10 10 10 10
or or or or
20 20 20 20
2,4-D NAA 2,4-Dp 4-Tb
White -k 5, 10 or 20 2,4-D ,, + 5, 10 or 20 NAA ,, q- 5, 10 or 20 2,4-Dp
Table 2. Production of anther-derived callus from cultured anthers of Digitalis purpurea Media and auxin (rag/l)
Cultured anthers forming callus (%) Continuous light
Nitsch ,, ,, ,, "S L
-t- 5 -k 10 -k 20 -t- 5 -t- 5 -t- 5 MS -t- 5 MS -t- 10 White -k 5 ,, q- 10
2,4-D 2,4-D 2,4-D NAA 2,4-Dp 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D
8.0 2.5 2.0 1.5 1.4 0.9 7.5 3.6 1.6 16.6
Continuous dark 11.0 6.0 2.0 0 5.5 0 7.1 3.2 1.4 5.5
In each treatment, 200 anthers were used. Conditions of light: fluorescent tubes, 1500Ix, 25~ of dark: 25 ~ All anthers were cultured in the late tetrad stage of mierospore development
Results Induction o/Haploid Callus D u r i n g 1 9 7 0 - - 1 9 7 4 f l o w e r i n g s h o o t s w e r e c o l l e c t e d f r o m p o p u l a t i o n s of D . purpurea a t d i f f e r e n t r e g i o n s of t h e F e d e r a l R e p u b l i c of G e r m a n y . A t o t a l of 7 0 0 0 a n t h e r s w e r e i n c u b a t e d o n 51 d i f f e r e n t m e d i a u n d e r v a r y i n g e n v i r o n m e n t a l c o n d i t i o n s . M o s t of t h e a n t h e r s w e r e c u l t u r e d b e t w e e n t h e l a t e t e t r a d s t a g e a n d t h e e a r l y m o n o n u c l e a t e s t a g e of t h e m i c r o s p o r e . P r o d u c t i o n of callus f r o m t h e a n t h e r s was o b s e r v e d a f t e r a c u l t u r e p e r i o d of 8 w e e k s u s i n g t h e m e d i a a n d cond i t i o n s s u m m a r i z e d in T a b l e 2. A n t h e r - d e r i v e d callus w a s p r o d u c e d o n l y w h e n t h e f l o w e r i n g s h o o t s w e r e k e p t a t 4 ~ for 2 - 1 2 d a y s b e f o r e t h e a n t h e r s w e r e i s o l a t e d . W e f o u n d no s i g n i f i c a n t d i f f e r e n c e s in callus p r o d u c t i o n f r o m a n t h e r s b e t w e e n p o p u l a t i o n s f r o m d i f f e r e n t regions. B e c a u s e of t h e t M c k n e s s of t h e e x i n e w e w e r e n o t a b l e t o o b s e r v e t h e first m i t o s i s in t h e pollen. T h a t d i v i s i o n s h a d o c c u r r e d w a s 1"
G. Corduanand C. Spix percent of the anthers forming callus
15%
9 early tetrads
.............
~:.~!!?!(-/--~
late tetrads early mono nucleate stage mono nucleate stage ........... mitosis ; , , binucleate stage
12%
9% 6% 3%
IA .A
tG!- ~oj
,~o
50 60 7080ram
~Omm
b
length of the anthers length of the flower buds
Fig. 1. Dependence of callus formation of the stage of microspore development. Out of the 5 anthers of one flower bud, 4 were used for callus production, while the 5th was used for cytological examination of microspore development. A total of 100 anthers of each stage was cultured in the dark a~ 25~ on the medium of Nitsch supplemented with 5 mg/12,4-D
evident only when the pollen had divided twice to form 4 cells. After this stage the exine was broken and the next mitoses produced a white callus with cells smaller than those of the tapetum or connective tissue. Cytological determinations showed that when the anther had a dark brown or black color most of the callus was haploid. When the anthers had a light color most of the callus produced seemed to be derived from somatic tissue. Out of 46 calli, derived from dark-colored anthers we found 39 to be exclusively haploid while 4 had a mixed population of haploid and diploid cells and 3 were entirely diploid. On the other hand, out of 45 calli derived from light-colored anthers 38 were found to be diploid, 2 had diploid and haploid cells and 5 were a mixture of diploid and polyploid cells. Based on these observations we used anther color as an indicator for haploid callus and only purely haploid cell lines were used for subsequent experiments. The dependence of callus formation on the pollen stage was another indicator. We were able to detect haploid chromosome sets in the dividing callus only when the anthers were cultured in the late tetrad stage of microspore development. At this stage we found also the highest production of callus. These results are illustrated in Fig. 1. I n subsequent experiments, therefore, we cultured anthers only in the late tetrad stage. The callus which developed from the anthers was carried through 3 subcultures of 4 weeks each under conditions identical to those used for callus production. The callus was subsequently transferred to various conditions and media to induce organogenesis.
Induction o/Organogenesis Root Formation. We were able to observe root formation from the callus under various conditions and on several media. Subsequent culture of such rooted callus on auxin-free media did not result in bud formation, even after repeated subculturing for up to 20 times (4 weeks each). The only type of organogenesis observed was root production.
Callus and Plants from Anthers of Digitalis
5
Table 3. Conditions controlling regeneration of plants from anther-derived callus of Digitalis purpurea Continuous light
16-h photoperiods
-- Kinetin
+ Kinetin
-- Kinetin
+ Kinetin
No. of calli in which regeneration occurred
8
0
0
8
Viable plants (in % of total regenerates)
80-90
0
0
ca. 30
Callus from 8 different anthers was carried through 3 subcultures of 4 weeks each in the dark on the medium of Nitsch and Nitsch (1969) supplemented with 5 mg/1 2,4-D. Then portions of 0.5 g of each callus were transferred to medium of Nitsch and Nitsch with or without kinetin (2.2 mg/1) and were subsequently cultured either in continuous light (fluorescent tubes, 15001x, 25 ~ or under 16 h light at 28 ~ fluorescent tubes, 1500 lx and 8 h dark at 20 ~ Regeneration started after the 5th subculture and the viability of the regenerates was checked routinely during the following 15 subcultures (4 weeks each).
B u d Formation. W h e n u n d i f f e r e n t i a t e d callus was c u l t u r e d on v a r i o u s m e d i a a n d u n d e r v a r i o u s conditions shoot regenerates were f o r m e d on some m e d i a b u t m o s t of t h e m were either n o t able to form roots, or v i a b i l i t y after t r a n s f e r to soil was low. Viable p l a n t s were o b t a i n e d o n l y when t h e callus d e v e l o p e d from a n t h e r s grown on t h e b a s a l m e d i u m of N i t s c h a n d N i t s c h (1969) s u p p l e m e n t e d w i t h 5 m g / l 2,4-D. I f grown in continuous light from fluorescent tubes, t h e callus was able to r e g e n e r a t e b u d s on t h e m e d i u m of N i t s c h a n d N i t s c h with no a d d i t i o n of kinetin. W h e n t h e m e d i u m was s u p p l e m e n t e d w i t h k i n e t i n r e g e n e r a t e s occurred o n l y u n d e r p h o t o p e r i o d i c conditions consisting of light from fluorescent t u b e s a t 28 ~ a n d 8 h d a r k a t 20 ~ These results are s u m m a r i z e d in T a b l e 3.
To d e t e r m i n e how m a n y p l a n t s could be r e g e n e r a t e d from callus d e r i v e d f r o m single a n t h e r s we c u l t u r e d callus from t h r e e different a n t h e r s which h a d d e v e l o p e d in t h e d a r k , a n d callus from t w o a n t h e r s which h a d d e v e l o p e d in t h e light. T h e callus of t h e d a r k - c u l t u r e d a n t h e r s was grown in t h e d a r k for 3 s u b c u l t u r e s of 4 weeks each, a n d t h e callus of t h e light c u l t u r e d a n t h e r s was s u b c u l t u r e d for t h e s a m e t i m e in t h e light, b o t h on 2 , 4 - D - s u p p l e m e n t e d m e d i u m of N i t s c h a n d Nitsch. A f t e r this t i m e t h e calli were t r a n s f e r r e d to auxin-free m e d i u m of N i t s c h a n d N i t s c h a n d c u l t u r e d in continuous light from fluorescent tubes. The results of this e x p e r i m e n t can be seen in T a b l e 4. U n d e r t h e conditions described we f o u n d c o n t i n u e d g r o w t h of u n d i f f e r e n t i a t e d callus as well as r e g e n e r a t i o n of plants, a n d a close correlation b e t w e e n callus p r o l i f e r a t i o n a n d r e g e n e r a t i o n of plants, as shown in Fig. 2. These results d e m o n s t r a t e t h a t t h e t o t i p o t e n c y of t h e callus is n o t d i m i n i s h e d d u r i n g t h e culture period. To be sure of r e p r o d u c i b i l i t y we c u l t u r e d a n t h e r s 8 t i m e s on 2,4-D-containing m e d i a a n d s u b j e c t e d t h e d e v e l o p e d callus to b u d - f o r m i n g conditions. I n all cases we f o u n d m a n y r e g e n e r a t e s beginning to form b e t w e e n t h e 3rd a n d 10th subculture. Most of these r e g e n e r a t e d p l a n t s grew well u n d e r b o t h greenhouse a n d field conditions a f t e r t r a n s f e r to soil.
6
G. Corduan and C. Spix Fresh weight of callus in mg -- x
number of
regenerates = 9 X
l0 G`,
X
105
X X
9
X
10 5 9
9
10/,
X X
i0/. ,
x
e 9
103
X
103 .
•
x
X
9
I9
102 .
•
10 2.
x x
9
10
X
10
xx .....
eee
1
i 9
I
15
20
subcultures
Fig. 2. Production of callus and regenerates b y a single a n t h e r of Digitalis purpurea. For further explanation see Table 3 from which the data for this figure were t a k e n
Table 4. Regeneration of plants from callus derived from single anthers of Digitalis purpurea in course of 20 4-week subcultures in continuous light or dark on the medium of Nitsch a n d Nitsch (1969) Subculture
No. of plants produced b y callus from a n t h e r No. 1 (dark)
2 (dark)
3 (dark)
1 (light)
2 (light)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
0 0 0 0 0 3 13 14 14 212 1067 2411 3388 5000 10000 20000 40000 80000 160000 320000
0 0 0 0 0 1 1 1 7 508 1413 2105 2565 3000 6000 12000 24000 48000 96000 192000
0 0 0 0 0 0 0 0 0 0 0 10 9 12 14 15 60 120 210 405
0 0 4 5 12 107 385 586 1440 1390 2000 4000 8000 16000 32000 64000 128000 256000 512000 1000000
0 0 0 2 3 3 7 15 49 120 210 360 1000 2000 4000 8000 16000 32000 64000 128000
The erossline under t h e numbers indicates the limit between counted plants a n d calculated numbers. F r o m this point on only a p a r t of t h e callus was transferred to the next subculture a n d t h e n u m b e r of plants was estimated b y multiplying calli and regenerated plants b y the reducing factor.
Callus and Plants from Anthers of Digitalis
7
Table 5. Chromosome numbers of plants regenerated from anther-derived callus of Digitalis
purpurea Callus was grown on the medium of Nitsch and Nitsch (1969) containing 5 rag/1 2,4-D in either continuous light (fluorescent tubes, 1500 lx, 25~ or in darkness (25~ Chromosome :No.
No. of plants having chromosome number indicated Light
Dark
52 53 56 (= 2n) 92 94 95 100 102 103 106 108 110 112 (~4n)
0 0 0 1 2
1 1 51 0 0
1
0
6 2 2 1 2 4 12
0 0 0 0 0 0 0
Total
33
53
Regeneration o/Diploid and Tetraploid Plants Cytological determinations showed that most of those plants which regenerated from anther-derived callus that had developed in the dark were diploid with 56 chromosomes, whereas most of those plants which regenerated from callus that had developed in the light were tetraploid with 112 chromosomes. These data are compiled in Table 5. All the euploid plants flowered and fruited normally and the seeds germinated as well as seeds of the starting material. We were able to differentiate between diploid and tetraploid plants by the thickness and size of the leaves. We did not find a single haploid plant in this initial series of experiments.
Regeneration o/Haploid Plants I n a repetition of the experiment described above we shortened the time of callus growth on the 2,4-D-containing medium. The anthers were cultured in the dark. When the callus was breaking out of the anther wall it was immediately transferred to auxin-flee medium of Nitsch and Nitsch for proliferation; after a culture period of 2 months it was transferred to continuous light for bud formation. B y this method we were able to find some plants with 28 chromosomes among a large number of diploid plants. The five haploid plants that were observed did not survive the transfer to soil. Other experiments to obtain haploid plants were unsuccessful, including use of the method of Sharp et al. (1972) with isolated pollen on a nurse culture, and of the method described by Debergh and Nitsch (1973) with macerated antherderived plants of Nicotiana or Digitalis as a component in the medium.
8
G. Corduan and C. Spix
Diploidization during Organogenesis To resolve the question at which stage of development the haploid chromosome number changes to the diploid one we started with selected haploid callus. During the first step of bud formation we found chromosome numbers between 28 and 34. In the next stage of development small leaves could be observed and rootlets were visible. We found chromosome numbers between 38 and 42 in the root tips of these plantlets. When the plantlets had developed in the culture flasks and were ready to be transferred to soil we found chromosome numbers between 44 and 48. Some weeks after transfer to soil we found only the diploid set of 56 chromosomes. We have repeated this experiment several times in 1974 and always found that the multiplication of chromosomes starts only during organogenesis and not in the undifferentiated callus.
Modi/ication by Kinetin I t is seen in Table 3 that callus treated with kinetin will form buds only under certain photoperiodic conditions and that only one third of the surviving plantlets could be grown potted in soil. The kinetin-treated regenerates produced narrow, elongated leaves and large double flowers with an altered pigmentation. In some eases a new shoot developed out of these flowers. We checked 20 plants and found that they all had a chromosome number of 56. The plants fruited normally and set seed. The leaf morphology of the seed progeny of these plants was again normal and identical with the control and the starting material. We can assume, therefore, that the observed abnormalities were due to modification. We are now growing these plants under field conditions to determine if the flowers will also show the normal form. Discussion We have found that callus formation out of anthers can be induced on several basal media provided they are supplemented with 2,4-D. Subsequent regeneration of plants occurred only on the basal medium of Nitsch and Nitsch (1969) (which does not contain 2,4-D) and depended critically on the concentration of 2,4-D in the media used for the production of anther-derived callus : callus grown previously on media with 2,4-D concentrations higher than 5 rag/1 was no longer able to regenerate plants upon transfer to the basal medium. Furthermore, regeneration at high frequencies occurred only in continuous light, or under certain photoperiodic conditions when kinetin at 2.2 rag/1 was added to the basal medium. The regenerated plants were diploid when the callus developed in the dark, and tetraploid when the callus was cultured in continuous light. Most of the regenerated plants and their progeny were euploid; they flowered and fruited normally, and grew well under field conditions. In the following we want to discuss two of the main questions arising from the work presented here: 1) which are the factors determining regeneration, and 2) do the diploid regenerates originate from generative cells ?
Factors Determining Regeneration In dicotyledons, outside the Solanaceae, haploid callus was obtained from the hybrid of Brassica oleracea • alboglabra (Kameya and Itinata, 1970), Arabidopsis thaliana (Gresshoff and Doy, 1972), Geranium varieties (Abo El-Nil and Itilde-
Callus and Plants from Anthers of Digitalis
9
brandt, 1973), and Vitis vini/era (Gresshoff and Doy, 1974). Subsequent regeneration of plants was either not achieved (Vitis vini/era) or the number of regenerates was small. Most of the media used by the workers cited above were complex and high in hormones. I n the present study we also observed poor regeneration when media with high auxin and kinetin concentrations were used. Our results indicate t h a t the totipotency of the cells is not lowered when the callus is cultured on the medium of Nitsch and Nitsch (1969). The only hormone in this medium is IAA at a low concentration (0.1 mg/1). We were also able to observe regeneration of plants in the complete absence of IAA but less than in its presence. We found a high sensitivity of the cells to kinetin. At a concentration of 2.2 rag/1 the viability of the regenerates was reduced to one third, and modifications were found in leaves and flowers. No regeneration at all, during up to 20 subcultures, was found when the concentration of kinetin was raised to 5 rag/l, and only poor regeneration was obtained in various combinations of auxins and cytokinins. When the callus was cultured for 3 months on media enriched with kinetin and auxins and then transferred to the hormone-flee medium no viable plants were obtained during 29 subcultures. We assume t h a t the reason for the sensitivity of our callus cells to exogenous hormones is the well-balanced level of endogenous hormones established b y the environmental conditions used. Especially the light sources used seem to be of importance. Fluora fluorescent lamps have a different emission spectrum, with a m a x i m u m at 665 nm, as compared to H QL and H W L lamps with maxima at 550 nm, and their light is thus closer to the absorption of chlorophyll. When using these fluorescent tubes and in the absence of exogenous hormones the callus was dark green whereas addition of hormones or using other light sources reduced the green color.
Origin o/the Regenerates We have shown t h a t under certain conditions haploid callus can be obtained from anthers but the regenerates were generally diploid or had higher degrees of ploidy. The question therefore arises whether they are really derived from microspores via callus or whether, after all, regeneration originates from somatic tissue of the anther. That the callus does originate from generative cells (microspores) is evidenced by the following observations:
a) The Dependence o/ Callus Formation on a De/inite Stage o/ Microspore Development. No such dependence was found when the callus originated from somatic cells, as reported for Chrysanthemum by Watanebe et al. (1972), for varieties of Lotus by Niizeki and Grant (1971), and for cucumber by I t a r n et al. (1969). These authors all observed a high frequency of callus production independent of the stage of microspore development.
b) The Dark Color o/the Anther. The authors mentioned above reported t h a t in Lotus, Chrysanthemum and cucumber production of callus from somatic tissue occurred only when the anthers showed a light color. c) The Production o/ Haploid Callus when the Anthers were Cultured in the Late Tetrad Stage o/ Microspore Development. This is in agreement by work of Gresshoff and Doy (1974) with Vitis vini]era.
10
G. Corduan and C. Spix
d) The Shi/t /tom Haploid to Diploid Chromosome Numbers during Organogenesis. Apart from studies on Nicotiana and monocotyledonous plants there is, to our knowledge, only the report of Raina and Iyer (1973) concerning production of haploid callus in the eggplant and subsequent differentiation to diploid plants only. These authors assumed, however, that the transition from the haploid to the diploid chromosome number occurred while the callus was in the undifferentiated stage. Chromosome doubling by endomitoses in pollen-callus cells has been reported by Nitsch (1969). Chromosome doubling during growth and differentiation, as a common phenomenon in plant tissue cultures, was described by D'Amato (1964) and Bennici et al. (1968). Our results are in agreement with these observations; in Digitalis chromosome duplication occurs not only in the undifferentiated stage of callus growth but also during differentiation. e) The Production o/Some Haploid Plants. The fact that we have found some haploid plants is the best evidence that callus and regenerated plants originated from generative cells. We assume that prolonged exposure to 2,4-D is responsible for the endoreduplication of the chromosomes. I t is well known that 2,4-D causes chromosomal aberrations and duplications in higher plants (see Mohandas and Grant, 1972, for references). This assumption is further strengthened by our finding that some haploid plants are produced when the 2,4-D treatment is shortened. For practical purposes it is not a disadvantage that nearly all of our regenerates are diploid. It is thus not necessary to treat haploid regenerates with colchicine to obtain fertile plants for crossing experiments or seed production by selfing.
[) Segregation o/Anther-derived Plants as Compared to the Starting Material. Digitalis purpurea is pollinated by insects and is a heterozygous plant but one finds nevertheless little if any differences in morphology between individuals of a given population. Therefore we could not expect that anther regenerates would differ morphologically from the starting material to a greater extent. We did find however in the regenerated diploid plants, apart from the modifications caused by kinetin, in some cases smaller leaves and in all cases smaller shoots. The differences were small but examining the sexual progeny we found them to be stable, as compared to the starting material, both under greenhouse and field conditions. The shoot length of 30 plants of the starting material, when measured at the end of the flowering period, was 83.2=k2.1 cm while the comparable measurements of 80 plants of the sexual progeny of regenerates were 75.1• cm (p>0.01). Summarizing all these observations we can conclude that our diploid regenerates are genotypically different from the starting material and originated from pollen-derived callus. From the point of view of genetic and breeding work the production of haploid callus is preferable to the direct development of embryoids out of the mierospores because the produced genotype can be preserved and multiplied, provided that totipotency is preserved. We feel that our results may contribute to the elaboration of methods for obtaining haploid callus and regenerates from various other plants to a considerably greater extent than presently possible. This work was supported in 1970 and 1971 by the Gesellschaft ffir Strahlen- und Umweltforschung, 1V[unich/Mfinchen and 1972--1974 by a grant from the Bundesministerium fiir Forschung und Technologie to Professor M. H. Zenk. We thank Mrs. B. Entringer and Mrs.
Callus and Plants from Anthers of Digitalis
11
E. Lchmann for accurate technical assistance, Professor J. Grau, institut ffir Systematische Botanik, University of Munich, for check of the cytological determinations, Dr. W. Keller, Canada Department of Agriculture, Ottawa, for help in translation and Dr. N. Amrhein from our Institute for critical reading and further improvement of the manuscript.
References Abo El-Nil, M. M., Hildebrandt, A. C. : Origin of androgenetic callus and haploid geranium plants. Canad. J. Bot. 51, 2107-2109 (1973) Bennici, A., Buiatti, M., D'Amato, F. : Nuclear conditions in haploid Pelargouium in vivo and in vitro. Chromosoma (Ber].) 24, 194-201 (1968) Corduan, G.: t ~ e r die Wirkung unterschiedlicher Kulturbedingungen auf die Entstehung haploider Pflanzen aus Antheren yon hricotiaua tabacum-Variet~ten. Z. Pflanzenphysiol. 69, 64-67 (1973) D'Amato, F. : Endepolyploidy as a factor in plant tissue development. Caryologia 17, 41-51 (1964) Debergh, P., Nitsch, C. : Premiers r4sultats sur la culture in vitro de grains de pollen isol6s chez la tomate. C. R. Acad. Sci. (Paris) 276 D, 1281-1284 (1973) Engvild, K. C., Linde-Laursen, I., Lundquist, A.: Anther cultures of Datura innoxia: flower bud stage and embryoid level of ploidy. Hereditas (Lund) 72,331-332 (1972) Gresshoff, P. M., Doy, C. H. : :Haploid Arabidopsis thaliana callus and plants from anther culture. Aust. J. biol. Sci. 25, 259-265 (1972) Gresshoff, P. M., Doy, C. H.: Derivation of a haploid cell line from Vitis vini/era and the importance of the stage of meiotic development of anthers for haploid culture of this and other genera. Z. Pflanzenphysiol. 73, 132-141 (1974) Ham, C., Koh, Y. S., Chung, D., Kim, B. : Studies on the anther culture of vegetable crops. I. Diploidal liquid callus of cucumber. Korean J. hort. Sci. 6, 25-27 (1969) Kamcya, T., Hinata, K. : Induction of haploid plants from pollen grains of Brassica. Jap. J. Breeding 20, 14-19 (1970) Linsmaier, E. M., Skoog, F. : Organic growth factor requirements of tobacco tissue cultures. Physiol. P]antarum 18, 100-127 (1965) Mohandas, T., Grant, W. F. : Cytogcnetic effects of 2,4-D and amitrole in relation to nuclear volume and DNA content in some higher plants. Canad. J. Genet. Cytol. 14, 773-783 (1972) Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15, 479-497 (1962) Niizeki, M., Grant, W. F. : Callus, plantlet formation, and polyploidy from cultured anthers of Lotus and Nicotiana. Canad. J. Bot. 49, 2041-2051 (1971) Nitseh, J. P. : Experimental androgenesis in Nicotiana. Phytomorphology 19, 389-404 (1969) Nitseh, J. P., Nitsch, C. : Haploid plants from pollen grains. Science 163, 85-87 (1969) Raina, S. K., Iyer, R. D. : Differentiation of diploid plants from pollen callus in anther cultures of Solanum melongena L. Z. Pflanzenzfichtg. 70, 275-280 (1973) Sharp, W. R., Raskin, R. S., Sommer, H. E. : The use of nurse culture in the development of haploid clones in tomato. Planta (Berl.) 104, 357-361 (1972) Smith, H. H. : Model systems for somatic cell plant genetic. BioScience 24, 269-276 (1974) Sunderland, N., Wicks, F. M.: Cultivation of haploid plants from tobacco pollen. Nature (Lond.) 224, 1227-1229 (1969) Watanebe, K., Nishii, Y., Tanaka, R. : Anatomical observations on high frequency callus formation from anther culture of Chrysanthemum. Jap. J. Genet. 47, 249-255 (1972) White, P. R. : A handbook of plant tissue culture. Tempe, Ariz., USA: Jaques Cattell Press 1943
Haploid Callus and Regeneration of Plants from Anthers of Digitalis purpurea L. G e r d C o r d u a n a n d Christel S p i x Lehrstuhl fiir Pflanzenphysiologie, 1%uhr-Universitgt Bochum, Postfaeh 2148, D-4630 Bochum, Federal Republic of Germany Received 4 February; accepted 28 February 1975 Summary. Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch ( Science 163, 85-87; 1969) supplemented with 5 mg/12,4-dichloro. phenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct genotype to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions, If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/1 kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28 ~ and 8 h dark at 20 ~ but the survivM was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.
Introduction A s u r v e y of t h e l i t e r a t u r e shows t h a t m a n y m e m b e r s of t h e Solanaceae h a v e p r o v e d to be s u i t a b l e o b j e c t s for t h e p r o d u c t i o n of h a p l o i d p l a n t s b y a n t h e r culture (for references see Smith, 1974). H u n d r e d s of p l a n t s a r e easily o b t a i n e d o u t of one a n t h e r of, e.g., Nicotiana tabaeum ( S u n d e r l a n d a n d Wicks, 1969). H a p l o i d tissues a n d p l a n t s m a y arise from a n t h e r s in two different w a y s : first, b y d i r e c t developm e n t of microspores into e m b r y o i d s a n d s u b s e q u e n t l y into p l a n t l e t s , as has been m a i n l y o b s e r v e d in t h e S o l a n a c e a e ; second, b y f o r m a t i o n of a n undifferent i a t e d callus on a n a u x i n - c o n t a i n i n g m e d i u m b y mitoses in t h e pollen, followed b y d i f f e r e n t i a t i o n of p l a n t s u n d e r certain light conditions on auxin-free media, this m o d e of d e v e l o p m e n t h a v i n g been f o u n d in m o s t families o u t s i d e t h e Solanaceae. I n b o t h instances one also observes p l a n t s w i t h a higher degree of ploidy, which is caused b y either n u c l e a r fusion in t h e microspore (Engvild, 1972) or b y e n d o m i t o s e s in t h e cells of p o l l e n - d e r i v e d callus (Nitsch, 1969). Callus m a y , however, also develop from s o m a t i c tissue, such as t h e connective tissue or t h e f i l a m e n t of t h e a n t h e r . I f t h e r e g e n e r a t e d p l a n t s h a v e a diploid c h r o m o s o m e set a n d if t h e s t a r t i n g 1 Planta (Bed.), Vol. 124
2
G. Corduan and C. Spix
m a t e r i a l is n o t very heterozygous, one should n o t find p h e n o t y p i c a l l y detectable segregation products, a n d it is therefore v e r y difficult to decide if regenerates originated from a generative or a vegetative cell. As Digitalis purpurea is a m e m b e r of the Scrophulariaceae we m a y expect t h a t the second of the two ways described above would be the one for p r o d u c t i o n of haploid p l a n t s in this species. To distinguish between callus derived from generative a n d somatic cells it was desirable to find indicators t h a t would parallel cytological determinations: W e were p a r t i c u l a r l y interested in developing methods to regenerate large n u m b e r s of p l a n t s from a n t h e r cultures. F r o m the c u r r e n t literature it is e v i d e n t t h a t the n u m b e r of regenerates from a n t h e r - d e r i v e d callus is c o m p a r a t i v e l y small. Conditions allowing the regeneration of h u n d r e d s of p l a n t s from one a n t h e r would enable us to propagate a d i s t i n c t g e n o t y p e in a sufficient n u m b e r of plants. F r o m our results with a n t h e r culture in Nicotiana tabacum (Corduan, 1973) we k n e w t h a t the composition of the m e d i u m as well as e n v i r o n m e n t a l conditions are i m p o r t a n t for regeneration of large n u m b e r s of plants. I n this report we shall p r e s e n t our results on p r o d u c t i o n of anther-derived callus a n d regeneration of p l a n t s in Digi-
talis purpurea. Material and Methods Plant Material. Flowering shoots of Digitalis purpurea L. were collected at five different locations in the central and western part of the Federal Republic of Germany, varying in climate. Preparation o/Anthers. The flowering shoots were kept for 2-12 days at 4~ The anthers were then isolated, washed for 15 rain in 70 % ethanol, sterilized in 5 % sodium hypochlorite for 4 rain, and washed 5 times with sterilized distilled water. The stage of pollen development was determined before culturing. Culture Procedures. Media which were used for anther culture are summarized in Table 1. Most of the chemicals used in these media were purchased from Merck (Darmstadt, Germany) except 2,4-dielflorophenoxyacetic acid = 2,4-D and 4-chloro-2-methyl-phenoxyacetic acid 4-Mp which were purchased from Fluka (Buchs, Switzerland) and 2(2,4-diehlorophenoxy)propionie acid = 2,4-Dp and 4-(2,4,5-trichlorophenoxy)-butyricacid = 4-Tb from Schuchardt (Munich/Miinchen, Germany). For regeneration of plants out of the anther-derived callus the same basM media were modified with varying amounts of cytokinins like 6-furfurylaminopurin = kinetin and benzylaminopurin from Fluka and trans-6-(4-hydroxy-3-methylbut-2enyl)aminopurin = zeatin from Calbiochem (San Diego, Calif., USA). The media were autoclaved for 20 min at 120~ and 1.2 arm. Glass Petri dishes of 6 cm diameter were filled with 10 ml of medium in a sterile cabinet. Six anthers were cultured in one l~etri dish. For organogenesis, anther-derived callus was transferred to 50-ml Erlenmeyer flasks filled with 35 ml of medium. The callus was transferred to fresh medium every 4 weeks. Environmental Conditions. Variation of environmental conditions involved: 1) photoperiods of 12, 14 and 16 h with temperatures of 25, 28 and 30~ during the light period and 19 and 20~ during the dark period. Light sources used were HLRG from Philips (Eindhoven, Netherlands), and H QL, ttWL and Fluora fluorescent tubes from Osram (Berlin). The light intensities were 1500 and 5000 Ix, measured at the level of the culture flasks; 2) continuous light from Fluora fluorescent tubes, 1500 lx at 25~ and 3) continuous dark at 25~ Cytological Studies. Orcein staining of chromosomes proved to be superior to acetocarmine or Feulgen staining, giving good results with callus as well as with root-tip preparations. Root tips were fixed in 0.029 % 8-hydroxyquinoline (Merck, Darmstadt) 4 h after onset of the light period, and incubated at 4~ for 4 h. They were then hydrolyzed for 5 min in ethanol : conc. HC1 (2:1, v/v), washed, stained for 3 rain with oreein (2.2 g oreein, Merck, were refluxed in 100 ml acetic acid for 30 min; the solution was then filtered and diluted to give a 45 % solution), and squashed (private communication by 1)rofessor J. Grau, Institut ffir Systematisehe Botanik, University of Munich).
Callus and Plants from Anthers of Digitalis
3
Table 1. Media used for culture of anthers of Digitalis purpurea The basal media used were those described by Nitseh and Nitseh (1969)=Nitsch; Linsmaier and Skoog ( 1 9 6 5 ) = L S ; Murashige and Skoog (1962)=MS; and White (1943). For other abbreviations see text. Concentrations are in rag/1 Nitsch ,, ,, ,, ,, ,, ,, ,, ,, ,, ,,
-k 1000 casein hydrolysate -t- 5, 10 or 20 2,4-D -l-5, 10 or 20 NAA -t-5, 10 or 20 2,4-Dp q-5, 10 or 20 4-Mp ~-5, 10 or 20 4-Tb -b 5 2,4-D without myo-inositol nc 5 2,4-D without pyridoxine. HCI -t- 5 2,4-D without thiamine. HC1 -t- 5 2,4-D without relic acid + 5 2,4-D without biotin
MS ,, ,,
-1-5, 10 or 20 2,4-D -]-5, 10 or 20 NAA -t-5, 10 or 20 2,4-Dp
LS ,, ,, ,,
q- 5, § 5, -t-5, + 5,
10 10 10 10
or or or or
20 20 20 20
2,4-D NAA 2,4-Dp 4-Tb
White -k 5, 10 or 20 2,4-D ,, + 5, 10 or 20 NAA ,, q- 5, 10 or 20 2,4-Dp
Table 2. Production of anther-derived callus from cultured anthers of Digitalis purpurea Media and auxin (rag/l)
Cultured anthers forming callus (%) Continuous light
Nitsch ,, ,, ,, "S L
-t- 5 -k 10 -k 20 -t- 5 -t- 5 -t- 5 MS -t- 5 MS -t- 10 White -k 5 ,, q- 10
2,4-D 2,4-D 2,4-D NAA 2,4-Dp 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D
8.0 2.5 2.0 1.5 1.4 0.9 7.5 3.6 1.6 16.6
Continuous dark 11.0 6.0 2.0 0 5.5 0 7.1 3.2 1.4 5.5
In each treatment, 200 anthers were used. Conditions of light: fluorescent tubes, 1500Ix, 25~ of dark: 25 ~ All anthers were cultured in the late tetrad stage of mierospore development
Results Induction o/Haploid Callus D u r i n g 1 9 7 0 - - 1 9 7 4 f l o w e r i n g s h o o t s w e r e c o l l e c t e d f r o m p o p u l a t i o n s of D . purpurea a t d i f f e r e n t r e g i o n s of t h e F e d e r a l R e p u b l i c of G e r m a n y . A t o t a l of 7 0 0 0 a n t h e r s w e r e i n c u b a t e d o n 51 d i f f e r e n t m e d i a u n d e r v a r y i n g e n v i r o n m e n t a l c o n d i t i o n s . M o s t of t h e a n t h e r s w e r e c u l t u r e d b e t w e e n t h e l a t e t e t r a d s t a g e a n d t h e e a r l y m o n o n u c l e a t e s t a g e of t h e m i c r o s p o r e . P r o d u c t i o n of callus f r o m t h e a n t h e r s was o b s e r v e d a f t e r a c u l t u r e p e r i o d of 8 w e e k s u s i n g t h e m e d i a a n d cond i t i o n s s u m m a r i z e d in T a b l e 2. A n t h e r - d e r i v e d callus w a s p r o d u c e d o n l y w h e n t h e f l o w e r i n g s h o o t s w e r e k e p t a t 4 ~ for 2 - 1 2 d a y s b e f o r e t h e a n t h e r s w e r e i s o l a t e d . W e f o u n d no s i g n i f i c a n t d i f f e r e n c e s in callus p r o d u c t i o n f r o m a n t h e r s b e t w e e n p o p u l a t i o n s f r o m d i f f e r e n t regions. B e c a u s e of t h e t M c k n e s s of t h e e x i n e w e w e r e n o t a b l e t o o b s e r v e t h e first m i t o s i s in t h e pollen. T h a t d i v i s i o n s h a d o c c u r r e d w a s 1"
G. Corduanand C. Spix percent of the anthers forming callus
15%
9 early tetrads
.............
~:.~!!?!(-/--~
late tetrads early mono nucleate stage mono nucleate stage ........... mitosis ; , , binucleate stage
12%
9% 6% 3%
IA .A
tG!- ~oj
,~o
50 60 7080ram
~Omm
b
length of the anthers length of the flower buds
Fig. 1. Dependence of callus formation of the stage of microspore development. Out of the 5 anthers of one flower bud, 4 were used for callus production, while the 5th was used for cytological examination of microspore development. A total of 100 anthers of each stage was cultured in the dark a~ 25~ on the medium of Nitsch supplemented with 5 mg/12,4-D
evident only when the pollen had divided twice to form 4 cells. After this stage the exine was broken and the next mitoses produced a white callus with cells smaller than those of the tapetum or connective tissue. Cytological determinations showed that when the anther had a dark brown or black color most of the callus was haploid. When the anthers had a light color most of the callus produced seemed to be derived from somatic tissue. Out of 46 calli, derived from dark-colored anthers we found 39 to be exclusively haploid while 4 had a mixed population of haploid and diploid cells and 3 were entirely diploid. On the other hand, out of 45 calli derived from light-colored anthers 38 were found to be diploid, 2 had diploid and haploid cells and 5 were a mixture of diploid and polyploid cells. Based on these observations we used anther color as an indicator for haploid callus and only purely haploid cell lines were used for subsequent experiments. The dependence of callus formation on the pollen stage was another indicator. We were able to detect haploid chromosome sets in the dividing callus only when the anthers were cultured in the late tetrad stage of microspore development. At this stage we found also the highest production of callus. These results are illustrated in Fig. 1. I n subsequent experiments, therefore, we cultured anthers only in the late tetrad stage. The callus which developed from the anthers was carried through 3 subcultures of 4 weeks each under conditions identical to those used for callus production. The callus was subsequently transferred to various conditions and media to induce organogenesis.
Induction o/Organogenesis Root Formation. We were able to observe root formation from the callus under various conditions and on several media. Subsequent culture of such rooted callus on auxin-free media did not result in bud formation, even after repeated subculturing for up to 20 times (4 weeks each). The only type of organogenesis observed was root production.
Callus and Plants from Anthers of Digitalis
5
Table 3. Conditions controlling regeneration of plants from anther-derived callus of Digitalis purpurea Continuous light
16-h photoperiods
-- Kinetin
+ Kinetin
-- Kinetin
+ Kinetin
No. of calli in which regeneration occurred
8
0
0
8
Viable plants (in % of total regenerates)
80-90
0
0
ca. 30
Callus from 8 different anthers was carried through 3 subcultures of 4 weeks each in the dark on the medium of Nitsch and Nitsch (1969) supplemented with 5 mg/1 2,4-D. Then portions of 0.5 g of each callus were transferred to medium of Nitsch and Nitsch with or without kinetin (2.2 mg/1) and were subsequently cultured either in continuous light (fluorescent tubes, 15001x, 25 ~ or under 16 h light at 28 ~ fluorescent tubes, 1500 lx and 8 h dark at 20 ~ Regeneration started after the 5th subculture and the viability of the regenerates was checked routinely during the following 15 subcultures (4 weeks each).
B u d Formation. W h e n u n d i f f e r e n t i a t e d callus was c u l t u r e d on v a r i o u s m e d i a a n d u n d e r v a r i o u s conditions shoot regenerates were f o r m e d on some m e d i a b u t m o s t of t h e m were either n o t able to form roots, or v i a b i l i t y after t r a n s f e r to soil was low. Viable p l a n t s were o b t a i n e d o n l y when t h e callus d e v e l o p e d from a n t h e r s grown on t h e b a s a l m e d i u m of N i t s c h a n d N i t s c h (1969) s u p p l e m e n t e d w i t h 5 m g / l 2,4-D. I f grown in continuous light from fluorescent tubes, t h e callus was able to r e g e n e r a t e b u d s on t h e m e d i u m of N i t s c h a n d N i t s c h with no a d d i t i o n of kinetin. W h e n t h e m e d i u m was s u p p l e m e n t e d w i t h k i n e t i n r e g e n e r a t e s occurred o n l y u n d e r p h o t o p e r i o d i c conditions consisting of light from fluorescent t u b e s a t 28 ~ a n d 8 h d a r k a t 20 ~ These results are s u m m a r i z e d in T a b l e 3.
To d e t e r m i n e how m a n y p l a n t s could be r e g e n e r a t e d from callus d e r i v e d f r o m single a n t h e r s we c u l t u r e d callus from t h r e e different a n t h e r s which h a d d e v e l o p e d in t h e d a r k , a n d callus from t w o a n t h e r s which h a d d e v e l o p e d in t h e light. T h e callus of t h e d a r k - c u l t u r e d a n t h e r s was grown in t h e d a r k for 3 s u b c u l t u r e s of 4 weeks each, a n d t h e callus of t h e light c u l t u r e d a n t h e r s was s u b c u l t u r e d for t h e s a m e t i m e in t h e light, b o t h on 2 , 4 - D - s u p p l e m e n t e d m e d i u m of N i t s c h a n d Nitsch. A f t e r this t i m e t h e calli were t r a n s f e r r e d to auxin-free m e d i u m of N i t s c h a n d N i t s c h a n d c u l t u r e d in continuous light from fluorescent tubes. The results of this e x p e r i m e n t can be seen in T a b l e 4. U n d e r t h e conditions described we f o u n d c o n t i n u e d g r o w t h of u n d i f f e r e n t i a t e d callus as well as r e g e n e r a t i o n of plants, a n d a close correlation b e t w e e n callus p r o l i f e r a t i o n a n d r e g e n e r a t i o n of plants, as shown in Fig. 2. These results d e m o n s t r a t e t h a t t h e t o t i p o t e n c y of t h e callus is n o t d i m i n i s h e d d u r i n g t h e culture period. To be sure of r e p r o d u c i b i l i t y we c u l t u r e d a n t h e r s 8 t i m e s on 2,4-D-containing m e d i a a n d s u b j e c t e d t h e d e v e l o p e d callus to b u d - f o r m i n g conditions. I n all cases we f o u n d m a n y r e g e n e r a t e s beginning to form b e t w e e n t h e 3rd a n d 10th subculture. Most of these r e g e n e r a t e d p l a n t s grew well u n d e r b o t h greenhouse a n d field conditions a f t e r t r a n s f e r to soil.
6
G. Corduan and C. Spix Fresh weight of callus in mg -- x
number of
regenerates = 9 X
l0 G`,
X
105
X X
9
X
10 5 9
9
10/,
X X
i0/. ,
x
e 9
103
X
103 .
•
x
X
9
I9
102 .
•
10 2.
x x
9
10
X
10
xx .....
eee
1
i 9
I
15
20
subcultures
Fig. 2. Production of callus and regenerates b y a single a n t h e r of Digitalis purpurea. For further explanation see Table 3 from which the data for this figure were t a k e n
Table 4. Regeneration of plants from callus derived from single anthers of Digitalis purpurea in course of 20 4-week subcultures in continuous light or dark on the medium of Nitsch a n d Nitsch (1969) Subculture
No. of plants produced b y callus from a n t h e r No. 1 (dark)
2 (dark)
3 (dark)
1 (light)
2 (light)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
0 0 0 0 0 3 13 14 14 212 1067 2411 3388 5000 10000 20000 40000 80000 160000 320000
0 0 0 0 0 1 1 1 7 508 1413 2105 2565 3000 6000 12000 24000 48000 96000 192000
0 0 0 0 0 0 0 0 0 0 0 10 9 12 14 15 60 120 210 405
0 0 4 5 12 107 385 586 1440 1390 2000 4000 8000 16000 32000 64000 128000 256000 512000 1000000
0 0 0 2 3 3 7 15 49 120 210 360 1000 2000 4000 8000 16000 32000 64000 128000
The erossline under t h e numbers indicates the limit between counted plants a n d calculated numbers. F r o m this point on only a p a r t of t h e callus was transferred to the next subculture a n d t h e n u m b e r of plants was estimated b y multiplying calli and regenerated plants b y the reducing factor.
Callus and Plants from Anthers of Digitalis
7
Table 5. Chromosome numbers of plants regenerated from anther-derived callus of Digitalis
purpurea Callus was grown on the medium of Nitsch and Nitsch (1969) containing 5 rag/1 2,4-D in either continuous light (fluorescent tubes, 1500 lx, 25~ or in darkness (25~ Chromosome :No.
No. of plants having chromosome number indicated Light
Dark
52 53 56 (= 2n) 92 94 95 100 102 103 106 108 110 112 (~4n)
0 0 0 1 2
1 1 51 0 0
1
0
6 2 2 1 2 4 12
0 0 0 0 0 0 0
Total
33
53
Regeneration o/Diploid and Tetraploid Plants Cytological determinations showed that most of those plants which regenerated from anther-derived callus that had developed in the dark were diploid with 56 chromosomes, whereas most of those plants which regenerated from callus that had developed in the light were tetraploid with 112 chromosomes. These data are compiled in Table 5. All the euploid plants flowered and fruited normally and the seeds germinated as well as seeds of the starting material. We were able to differentiate between diploid and tetraploid plants by the thickness and size of the leaves. We did not find a single haploid plant in this initial series of experiments.
Regeneration o/Haploid Plants I n a repetition of the experiment described above we shortened the time of callus growth on the 2,4-D-containing medium. The anthers were cultured in the dark. When the callus was breaking out of the anther wall it was immediately transferred to auxin-flee medium of Nitsch and Nitsch for proliferation; after a culture period of 2 months it was transferred to continuous light for bud formation. B y this method we were able to find some plants with 28 chromosomes among a large number of diploid plants. The five haploid plants that were observed did not survive the transfer to soil. Other experiments to obtain haploid plants were unsuccessful, including use of the method of Sharp et al. (1972) with isolated pollen on a nurse culture, and of the method described by Debergh and Nitsch (1973) with macerated antherderived plants of Nicotiana or Digitalis as a component in the medium.
8
G. Corduan and C. Spix
Diploidization during Organogenesis To resolve the question at which stage of development the haploid chromosome number changes to the diploid one we started with selected haploid callus. During the first step of bud formation we found chromosome numbers between 28 and 34. In the next stage of development small leaves could be observed and rootlets were visible. We found chromosome numbers between 38 and 42 in the root tips of these plantlets. When the plantlets had developed in the culture flasks and were ready to be transferred to soil we found chromosome numbers between 44 and 48. Some weeks after transfer to soil we found only the diploid set of 56 chromosomes. We have repeated this experiment several times in 1974 and always found that the multiplication of chromosomes starts only during organogenesis and not in the undifferentiated callus.
Modi/ication by Kinetin I t is seen in Table 3 that callus treated with kinetin will form buds only under certain photoperiodic conditions and that only one third of the surviving plantlets could be grown potted in soil. The kinetin-treated regenerates produced narrow, elongated leaves and large double flowers with an altered pigmentation. In some eases a new shoot developed out of these flowers. We checked 20 plants and found that they all had a chromosome number of 56. The plants fruited normally and set seed. The leaf morphology of the seed progeny of these plants was again normal and identical with the control and the starting material. We can assume, therefore, that the observed abnormalities were due to modification. We are now growing these plants under field conditions to determine if the flowers will also show the normal form. Discussion We have found that callus formation out of anthers can be induced on several basal media provided they are supplemented with 2,4-D. Subsequent regeneration of plants occurred only on the basal medium of Nitsch and Nitsch (1969) (which does not contain 2,4-D) and depended critically on the concentration of 2,4-D in the media used for the production of anther-derived callus : callus grown previously on media with 2,4-D concentrations higher than 5 rag/1 was no longer able to regenerate plants upon transfer to the basal medium. Furthermore, regeneration at high frequencies occurred only in continuous light, or under certain photoperiodic conditions when kinetin at 2.2 rag/1 was added to the basal medium. The regenerated plants were diploid when the callus developed in the dark, and tetraploid when the callus was cultured in continuous light. Most of the regenerated plants and their progeny were euploid; they flowered and fruited normally, and grew well under field conditions. In the following we want to discuss two of the main questions arising from the work presented here: 1) which are the factors determining regeneration, and 2) do the diploid regenerates originate from generative cells ?
Factors Determining Regeneration In dicotyledons, outside the Solanaceae, haploid callus was obtained from the hybrid of Brassica oleracea • alboglabra (Kameya and Itinata, 1970), Arabidopsis thaliana (Gresshoff and Doy, 1972), Geranium varieties (Abo El-Nil and Itilde-
Callus and Plants from Anthers of Digitalis
9
brandt, 1973), and Vitis vini/era (Gresshoff and Doy, 1974). Subsequent regeneration of plants was either not achieved (Vitis vini/era) or the number of regenerates was small. Most of the media used by the workers cited above were complex and high in hormones. I n the present study we also observed poor regeneration when media with high auxin and kinetin concentrations were used. Our results indicate t h a t the totipotency of the cells is not lowered when the callus is cultured on the medium of Nitsch and Nitsch (1969). The only hormone in this medium is IAA at a low concentration (0.1 mg/1). We were also able to observe regeneration of plants in the complete absence of IAA but less than in its presence. We found a high sensitivity of the cells to kinetin. At a concentration of 2.2 rag/1 the viability of the regenerates was reduced to one third, and modifications were found in leaves and flowers. No regeneration at all, during up to 20 subcultures, was found when the concentration of kinetin was raised to 5 rag/l, and only poor regeneration was obtained in various combinations of auxins and cytokinins. When the callus was cultured for 3 months on media enriched with kinetin and auxins and then transferred to the hormone-flee medium no viable plants were obtained during 29 subcultures. We assume t h a t the reason for the sensitivity of our callus cells to exogenous hormones is the well-balanced level of endogenous hormones established b y the environmental conditions used. Especially the light sources used seem to be of importance. Fluora fluorescent lamps have a different emission spectrum, with a m a x i m u m at 665 nm, as compared to H QL and H W L lamps with maxima at 550 nm, and their light is thus closer to the absorption of chlorophyll. When using these fluorescent tubes and in the absence of exogenous hormones the callus was dark green whereas addition of hormones or using other light sources reduced the green color.
Origin o/the Regenerates We have shown t h a t under certain conditions haploid callus can be obtained from anthers but the regenerates were generally diploid or had higher degrees of ploidy. The question therefore arises whether they are really derived from microspores via callus or whether, after all, regeneration originates from somatic tissue of the anther. That the callus does originate from generative cells (microspores) is evidenced by the following observations:
a) The Dependence o/ Callus Formation on a De/inite Stage o/ Microspore Development. No such dependence was found when the callus originated from somatic cells, as reported for Chrysanthemum by Watanebe et al. (1972), for varieties of Lotus by Niizeki and Grant (1971), and for cucumber by I t a r n et al. (1969). These authors all observed a high frequency of callus production independent of the stage of microspore development.
b) The Dark Color o/the Anther. The authors mentioned above reported t h a t in Lotus, Chrysanthemum and cucumber production of callus from somatic tissue occurred only when the anthers showed a light color. c) The Production o/ Haploid Callus when the Anthers were Cultured in the Late Tetrad Stage o/ Microspore Development. This is in agreement by work of Gresshoff and Doy (1974) with Vitis vini]era.
10
G. Corduan and C. Spix
d) The Shi/t /tom Haploid to Diploid Chromosome Numbers during Organogenesis. Apart from studies on Nicotiana and monocotyledonous plants there is, to our knowledge, only the report of Raina and Iyer (1973) concerning production of haploid callus in the eggplant and subsequent differentiation to diploid plants only. These authors assumed, however, that the transition from the haploid to the diploid chromosome number occurred while the callus was in the undifferentiated stage. Chromosome doubling by endomitoses in pollen-callus cells has been reported by Nitsch (1969). Chromosome doubling during growth and differentiation, as a common phenomenon in plant tissue cultures, was described by D'Amato (1964) and Bennici et al. (1968). Our results are in agreement with these observations; in Digitalis chromosome duplication occurs not only in the undifferentiated stage of callus growth but also during differentiation. e) The Production o/Some Haploid Plants. The fact that we have found some haploid plants is the best evidence that callus and regenerated plants originated from generative cells. We assume that prolonged exposure to 2,4-D is responsible for the endoreduplication of the chromosomes. I t is well known that 2,4-D causes chromosomal aberrations and duplications in higher plants (see Mohandas and Grant, 1972, for references). This assumption is further strengthened by our finding that some haploid plants are produced when the 2,4-D treatment is shortened. For practical purposes it is not a disadvantage that nearly all of our regenerates are diploid. It is thus not necessary to treat haploid regenerates with colchicine to obtain fertile plants for crossing experiments or seed production by selfing.
[) Segregation o/Anther-derived Plants as Compared to the Starting Material. Digitalis purpurea is pollinated by insects and is a heterozygous plant but one finds nevertheless little if any differences in morphology between individuals of a given population. Therefore we could not expect that anther regenerates would differ morphologically from the starting material to a greater extent. We did find however in the regenerated diploid plants, apart from the modifications caused by kinetin, in some cases smaller leaves and in all cases smaller shoots. The differences were small but examining the sexual progeny we found them to be stable, as compared to the starting material, both under greenhouse and field conditions. The shoot length of 30 plants of the starting material, when measured at the end of the flowering period, was 83.2=k2.1 cm while the comparable measurements of 80 plants of the sexual progeny of regenerates were 75.1• cm (p>0.01). Summarizing all these observations we can conclude that our diploid regenerates are genotypically different from the starting material and originated from pollen-derived callus. From the point of view of genetic and breeding work the production of haploid callus is preferable to the direct development of embryoids out of the mierospores because the produced genotype can be preserved and multiplied, provided that totipotency is preserved. We feel that our results may contribute to the elaboration of methods for obtaining haploid callus and regenerates from various other plants to a considerably greater extent than presently possible. This work was supported in 1970 and 1971 by the Gesellschaft ffir Strahlen- und Umweltforschung, 1V[unich/Mfinchen and 1972--1974 by a grant from the Bundesministerium fiir Forschung und Technologie to Professor M. H. Zenk. We thank Mrs. B. Entringer and Mrs.
Callus and Plants from Anthers of Digitalis
11
E. Lchmann for accurate technical assistance, Professor J. Grau, institut ffir Systematische Botanik, University of Munich, for check of the cytological determinations, Dr. W. Keller, Canada Department of Agriculture, Ottawa, for help in translation and Dr. N. Amrhein from our Institute for critical reading and further improvement of the manuscript.
References Abo El-Nil, M. M., Hildebrandt, A. C. : Origin of androgenetic callus and haploid geranium plants. Canad. J. Bot. 51, 2107-2109 (1973) Bennici, A., Buiatti, M., D'Amato, F. : Nuclear conditions in haploid Pelargouium in vivo and in vitro. Chromosoma (Ber].) 24, 194-201 (1968) Corduan, G.: t ~ e r die Wirkung unterschiedlicher Kulturbedingungen auf die Entstehung haploider Pflanzen aus Antheren yon hricotiaua tabacum-Variet~ten. Z. Pflanzenphysiol. 69, 64-67 (1973) D'Amato, F. : Endepolyploidy as a factor in plant tissue development. Caryologia 17, 41-51 (1964) Debergh, P., Nitsch, C. : Premiers r4sultats sur la culture in vitro de grains de pollen isol6s chez la tomate. C. R. Acad. Sci. (Paris) 276 D, 1281-1284 (1973) Engvild, K. C., Linde-Laursen, I., Lundquist, A.: Anther cultures of Datura innoxia: flower bud stage and embryoid level of ploidy. Hereditas (Lund) 72,331-332 (1972) Gresshoff, P. M., Doy, C. H. : :Haploid Arabidopsis thaliana callus and plants from anther culture. Aust. J. biol. Sci. 25, 259-265 (1972) Gresshoff, P. M., Doy, C. H.: Derivation of a haploid cell line from Vitis vini/era and the importance of the stage of meiotic development of anthers for haploid culture of this and other genera. Z. Pflanzenphysiol. 73, 132-141 (1974) Ham, C., Koh, Y. S., Chung, D., Kim, B. : Studies on the anther culture of vegetable crops. I. Diploidal liquid callus of cucumber. Korean J. hort. Sci. 6, 25-27 (1969) Kamcya, T., Hinata, K. : Induction of haploid plants from pollen grains of Brassica. Jap. J. Breeding 20, 14-19 (1970) Linsmaier, E. M., Skoog, F. : Organic growth factor requirements of tobacco tissue cultures. Physiol. P]antarum 18, 100-127 (1965) Mohandas, T., Grant, W. F. : Cytogcnetic effects of 2,4-D and amitrole in relation to nuclear volume and DNA content in some higher plants. Canad. J. Genet. Cytol. 14, 773-783 (1972) Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15, 479-497 (1962) Niizeki, M., Grant, W. F. : Callus, plantlet formation, and polyploidy from cultured anthers of Lotus and Nicotiana. Canad. J. Bot. 49, 2041-2051 (1971) Nitseh, J. P. : Experimental androgenesis in Nicotiana. Phytomorphology 19, 389-404 (1969) Nitseh, J. P., Nitsch, C. : Haploid plants from pollen grains. Science 163, 85-87 (1969) Raina, S. K., Iyer, R. D. : Differentiation of diploid plants from pollen callus in anther cultures of Solanum melongena L. Z. Pflanzenzfichtg. 70, 275-280 (1973) Sharp, W. R., Raskin, R. S., Sommer, H. E. : The use of nurse culture in the development of haploid clones in tomato. Planta (Berl.) 104, 357-361 (1972) Smith, H. H. : Model systems for somatic cell plant genetic. BioScience 24, 269-276 (1974) Sunderland, N., Wicks, F. M.: Cultivation of haploid plants from tobacco pollen. Nature (Lond.) 224, 1227-1229 (1969) Watanebe, K., Nishii, Y., Tanaka, R. : Anatomical observations on high frequency callus formation from anther culture of Chrysanthemum. Jap. J. Genet. 47, 249-255 (1972) White, P. R. : A handbook of plant tissue culture. Tempe, Ariz., USA: Jaques Cattell Press 1943
