Planta (Bed.) 106,237--245 (1972) 9 by Springer-Verlag 1972
Adventive Plants from Ovules and Nuceili in Citrus J. K o e h b a , P. S p i e g e l - R o y a n d t I a n n a h Safran The Voleani Center, Agricultural Research Organization, Bet Dagan, Israel Received February 14 / March 28, 1972
Summary. 1- to 8-week-old ovules and nucelli from three Citrus eultivars-Shamouti and Valencia (Citrus sinensi8) oranges and Marsh Seedless (C. paradisi) grapefruit~-were cultured in vitro. No embryo differentiation was observed in the explants prior to culture. The Shamouti ovules had degenerated and were apparently unfertilized. Embryoids formed on Murashige and Tucker nutrient medium supplemented with 500 mg/1 mMt extract. Whole plants developed on the same basal medium supplemented with kinetin and indole-3-acetic acid (IAA), coconut milk or gibberellic acid (GAs). A higher kinetin/IAA ratio or the addition of coconut milk favoured stem elongation more than root formation while a lower kinetin/IAA ratio favoured root formation and inhibited stem elongation. The addition of GAs to the basal medimn stimulated rooting and stem elongation. These results can be of aid in mutation research, allowing irradiation at stages prior to embryonic development. Introduction Nucellar e m b r y o g e n y is of w i d e s p r e a d occurrence in Citrus a n d r e l a t e d genera [5]. B o t h nueellar a n d zygotic e m b r y o s of Citrus spp. have been grown in vitro. Cultured nucelli of p o l y e m b r y o n i e varieties gave rise to a d v e n t i v e embryos, a n d in rare cases, to fully d e v e l o p e d seedlings [7]. R a n g a n et al. [4] raised nucellar e m b r y o s in vitro from t h r e e m o n o e m b r y o n i c varieties, w i t h t h e a i m of p r o d u c i n g virus-free plants. A l a t e r r e p o r t [5] deals with t h e i n d u c t i o n of zygotic e m b r y o d e v e l o p m e n t in p o l y e m b r y o n i c Citrus cultivars. W h i l e little success was o b t a i n e d in the beginning, i m p r o v e m e n t in t h e g r o w t h m e d i u m lead to b e t t e r results [1]. Some i n f o r m a t i o n is available on nucellar e m b r y o f o r m a t i o n in vitro in p r a c t i c a l l y seedless Citrus cultivars. B u t t o n a n d B o r n m a n [2] grew p l a n t s from nucellar e x p l a n t s of unfertilized W a s h i n g t o n Navel, while B i t t e r s et al. [1] succeeded in o b t a i n i n g p l a n t s from nucelli of t h e R o b e r t son N a v e l orange. This p a p e r r e p o r t s on nucellar e m b r y o f o r m a t i o n in different t y p e s of e x p l a n t s a n d on t h e effect of g r o w t h m e d i a on r o o t f o r m a t i o n a n d stem elongation in t h e S h a m o u t i a n d Valencia orange eultivars a n d in t h e Marsh Seedless grapefruit. All these cultivars have a t e n d e n c y for seedlessness, this being m o s t p r o n o u n c e d in Shamouti.
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J. Kochba, P. Spiegel-Roy and H. Sultan:
Methods and Materials Ovules and nucelli were excised aseptically with the aid ol a dissecting microscope from fruitlets 1- to 6-week old for Shamouti, 6- to 8-week old for Valencia (Citrus sinensis Osb.) oranges, and 8 week old for Marsh (Citrus paradisi Macf.) grapefruit. Fruit length for each sample was recorded (Table 1). Histological preparations of the tissues were made by fixation in formaldehyde-acetic acidethanol (FAA). Following dehydration and paraffin embedding, the sections were cut to 10-12 ~ thickness, stained with safranin and light green, and mounted in Canada balsam [6]. Explants were cultured on a basal nutrient medium (BM) of inorganic salts and organic substances as suggested by Murashige and Tucker [3]. 5% sucrose and 1% agar were added. The pH of the medium was adjusted to 5.7 and the medium autoclaved for 15 rain at 121 ~ C. The following additives to the basal medium were used during different phases of work: Malt extract (ME), coconut milk (CCM), kinetin, indole-3-aeetic acid (IAA), or gibberellic acid (GA3). Explants were cultured in 200• mm test tubes containing 15 ml of the medium, fitted with plastic caps, and kept at 26~1 ~ C and 16 h light daily. Light was supplied from Gro-Lux fluorescent tubes at 1000 lux. Results Ovules a n d nucelli e x t r a c t e d from 1- to 6-week old S h a m o u t i fruitlets were cultured on a basal m e d i u m containing 500 mg/1 m a l t e x t r a c t (Table 1). The highest percentage of cultures showing e m b r y o n i c developm e n t was o b t a i n e d using ovules from 4-week-old fruitlets. Earlier stages p r o d u c e d a l m o s t no embryos, while only a few cultures from l a t e r stages d e v e l o p e d to embryos. Nucelli y i e l d e d only a few cultures from 3- to 5-week-old fruitlets. Some of t h e cultures from ovules d e v e l o p e d callus, from all stages of fruit d e v e l o p m e n t tested. Nucellar e x p l a n t s showed this response only in rare eases. Ovules from 6 a n d 8-week-old fruitlets, a n d nueellar e x p l a n t s from 8-week-old fruitlets of Valencia a n d Marsh gave rise to cultures with e m b r y o n i c development. A higher percentage of successful cultures with these varieties was i n i t i a t e d from nucelli t h a n from ovules. B o t h Valencia a n d Marsh y i e l d e d a m u c h higher p e r c e n t a g e of cultures w i t h e m b r y o initials from b o t h t y p e s of explants, a n d a higher average n u m b e r of e m b r y o i d s per culture t h a n S h a m o u t i ; in c o n t r a s t to Shamouti, none of these cultures f o r m e d callus. Histological e x a m i n a t i o n of S h a m o u t i ovules showed t h a t a t t h e t i m e of excision t h e y were highly d e g e n e r a t e d a n d p r o b a b l y unfertilized, while t h e Valencia a n d Marsh ovules were well d e v e l o p e d a n d c o n t a i n e d large nueelli (Fig. 1). No f o r m a t i o n of nucellar or zygotic e m b r y o s could be d e t e c t e d in S h a m o u t i ovules a t a n y stage of d e v e l o p m e n t . Ovules from fruitlets older t h a n 6 weeks h a d c o m p l e t e l y degenerated. Nucellar e m b r y o s were d e t e c t e d in Valencia a n d Marsh Seedless ovules as early as 10 weeks after fruit set (Fig. 2) b u t in 8-week-old fruitlets which were used for culture, no e m b r y o n i c d e v e l o p m e n t was found.
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239
Table 1. Embryoid formation in vitro in explants of Citrus ovules and nucelli a Time after anthesis (weeks)
1 1 2 2 3 3 4 4 5 6 6
6 6 8 8
8 8
Length of fruitlets (mm)
6.8• 9.4 ~: 0.6 12.1-4-0.8 14.5• 17.4• 24.7 • 1.3
17.3• 1.3 31.7 ~ 2.1
29.4~2.0
Type Cultures c Cultures of developing developing explant b embryonic initials callus No.
%
Ov. Nuc. Ov. Nuc. Ov. Nuc. Ov. Nuc. Ov. 0v. Nuc.
1 0 1 0 0 1 11 2 2 5 0
Ov. Nuc. Or. Nuc.
Ov. Nuc.
Average no. of embryoids per culture
No.
%
Shamouti 2 0 2 0 0 2 22 4 4 10 0
5 0 7 0 1 0 8 1 7 3 0
10 0 14 0 2 0 16 2 14 6 0
1.0 0.0 1.0 0.0 0.0 1.0 1.4 1.0 1.0 1.4 0.0
11 0 17 21
Valencia 44 0 68 84
0 0 0 0
0 0 0 0
1.2 0.0 1.3 3.2
12 21
Marsh 48 84
0 0
0 0
1.3 3.1
a Counted after 12 weeks of culture. b Ov. = ovule, Nuc. = nucellus. c 50 cultures per treatment for Shamouti, and 25 cultures for Valencia and Marsh.
No f u r t h e r d e v e l o p m e n t occurred on t he original n u t r i e n t m e d i u m after 12 weeks of culture, a n d t h e size of t h e e m b r y o i d s r e m a i n e d 1-2 m m for S h a m o u t i a n d 3-6 m m for Marsh Seedless an d Valencia. T h e y were, therefore, t r a n s p l a n t e d s e p a r a t e l y to m e d i a eontailfing B M ddifferent levels of k i n e t i n a n d I A A , or coconut milk, for Valencia an d Marsh, while S a m o u t i e m b r y o id s were t r a n s p l a n t e d to only one m e d i u m due to t h e low n u m b e r of e m b r y o id s available. Th e d e v e l o p m e n t of t h e cultures on fresh n u t r i e n t m e d i a was r ap i d (Table 2). I n m a n y cases more t h a n one p l a n t d ev el o p ed per culture. Their av er ag e n u m b e r was highest on t h e lower k i n et i n -b higher I A A concentrations for Valencia a n d on CCM for Marsh. M a n y cultures of Marsh an d S h a m o u t i a n d some of Valencia f o r m e d new em b r y o i d s f r o m existing ones. I n t h e S h a m o u t i cultures t her e was no stem elongation,
240
J. Kochba, P. Spiegel-Roy and H. Safran:
II
III
1V
Fig. 1. Micrographs of Citrus ovules. Ovules at explanting; no differentiation of embryos visible. I - I I Shamouti orange (4 weeks after anthesis); I I I Valencia orange (6 weeks after anthesis); I V Marsh Seedless grapefruit (8 weeks after anthesis)
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241
Fig. 2. ~r of Citrus ovules. Ovules 10 weeks after anthesis. Nueellar embryos evident. I Valencia orange; I I Marsh Seedless grapefruit
and only few roots were formed. More Marsh than Valencia embryoids formed elongated stems and roots. Several combinations of kinetin -IAA or CCM stimulated plant development more than did ME. The lower level of kinetin combined with the higher level of IAA favoured root formation in both eultivars, but inhibited stem elongation. On most media, more cultures had roots than elongated stems, but the higher level of kinetin ,-~ the lower level of IAA or CCM was better than the other kinetin/IAA ratios for the development of both organs. Cultures in which stems did not elongate showed a "rosette" type of growth with short internodes and many small, thick leaves (Fig. 3). Newly formed embryoids of all three cultivars were planted on media containing GAs, IAA or kinetin (Table 3). Both GAs and IAA greatly improved rooting of Shamouti embryoids, while Valencia embryoids rooted best with the addition of GAs and Marsh with addition of IAA. More stems from all eultivars elongated on GAa than on any other medium. A higher percentage of Marsh and Valencia than of Shamouti eultnres yielded fully developed plants which could be transferred to the greenhouse for further growth.
242
J. Kochba, P. Spiegel-Roy and H. Safran:
Table 2. Developmenta of transplanted embryoids on media containing different levels of kinetin and IAA or coconut milk (CCM) b Addition to basal medium c
Developing plantlets per culture Avg. No.
Cultures forming new embryoids (%)
0.1 mg/1 Kinetin + 1.0 rag/1 IAA
Shamouti 1.08
31
0
3
1.0 mg/1 Kinetin + 1.0 rag/1 IAA 1.0 rag/1 K i n e t i n + 0.1 rag/1 IAA 0.1 mg/1 Kinetin + 0.1 rag/1 IAA 0.1 rag/1 Kinetin + 1.0 rag/1 IAA CCM 5 % ME 500 rag/1
Valencia 1.04 1.16 1.24 1.84 1.24 1.11
8 12 8 12 4 26
0 12 0 8 20 0
19 16 44 52 44 42
32 32 52 42 26 20
20 32 16 4 26 10
48 42 36 71 26 10
1.0 rag/1 Kinetin ~- 1.0 rag/1 IAA 1.0 mg/1 Kinetin + 0.1 rag/1 IAA 0.1 mg/1 Kinetin ~- 0.1 rag/1 IAA 0.1 mg/1 Kinetin-t- 1.0 rag/1 IAA CCM 5% ME 500 mg/1
Marsh leedless 1.40 1.53 1.44 1.63 2.11 1.00
Cultures with stems (%)
roots (%)
a 40 days after transplanting. b 40-45 cultures per treatment. c Murashige's citrus medium.
Discussion Our objective in raising in vitro nucellar seedlings from Shamouti, Valencia a n d Marsh Seedless was to exploit the earliest stages of e m b r y o formation for use in m u t a t i o n research, with the u l t i m a t e aim of producing solid m u t a n t s [9]. The stage of e m b r y o d e v e l o p m e n t in the explants was therefore crucial since nucellar p l a n t p r o d u c t i o n from already existing proembryos i n the explants would be of little use. We were able to show t h a t no embryonic d e v e l o p m e n t had started i n the explants from 1- to 8-week-old fruitlets. I n i t i a t i o n of nucellar embryo f o r m a t i o n
seems to differ with cultivar. While Button and Bornman [2], and Bitters st al. [I] obtained results similar to ours, Singh [7] found globular a d v e n t i v e embryos in 55-day-old ovules of Citrus reticulata cv. N a g p u r i a n d R a n g a n et al. [4] detected no nucellar embryos in seeds from 4-month-old i m m a t u r e fruits of the sour orange (Citrus aurantium) while a zygotic embryo was present. No zygotic e m b r y o could be detected i n our explants or in later stages of seed development which we examined.
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243
Fig. 3. Development of transplanted Citrus embryoids into plants in culture. Valencia orange, BSI-~-1rag/1 GA3. I Normal stem elongation and root growth; I I l~osette shoot growth and normal root development Nucellar embryo development in the cultivars examined by us clearly preceded zygotic embryo development. As no zygotic embryo was present, we could use whole nucelli as well as ovules for culture. Shamouti ovules, especially, had to be cultured at an early stage of development because of the rapid degeneration of ovules. Great difficulty was encountered in extracting nucelli from the very small ovules. Probably because of the damage caused to the exceedingly small nucellar explants during extraction, ovules yielded more successful cultures. Although nucelli gave better results than ovules for Valencia and Marsh, the use of entire ovules can be more rewarding due to comparative ease of extraction. Rangan et al. [5] and Bitters et al. [1] had no difficulty in raising entire plants of Citrus on the same medium on which nucellar embryo development was initiated. In the cultivars used in our experiments, fm-ther development did not occur on the same nutrient medium, but we were able to overcome this by the addition of growth substances, namely, kinetin, IAA or GA s. I t is well known that kinetin-auxin ratios can affect organ formation in tobacco callus [8]. The stimulating effect
244
J. Kochba, P. Spiegel-Roy and H. Salran:
Table 3. Root and stem formation a in embryoids on medium containing GA3 as compared to additions of kinetin or IAAb Addition to BM c
Cultures with elongated
developed
stems
roots
(%)
(%)
Shamouti
GA3 1 rag/1 IAA 0.1 rag/1
9 6
36 34
Valencia
GA3 1 rag/1 Kinetin 0.1 mg/l IAA 0.1 rag/1
30 21 14
59 25 25
Marsh
GA~ I mg/l Kinetin 0.1 mg/1 IAA 0.1 mg/1
38 5 22
38 34 41
a 42 days after transplantation. b 34-40 cultures per treatment. e Murashige's citrus medium.
of GA a on rooting of C i t r u s embryoids was first reported b y B u t t o n a n d B o r n m a n [2] for W a s h i n g t o n Navel. We f o u n d the same to hold true for S h a m o u t i a n d Valencia. Moreover, a d d i t i o n of GA s s t i m u l a t e d stem elongation in the cultures of all cultivars. This resulted in a higher percentage of p l a n t s from i n - v i t r o cultures successfully established in the greenhouse. The use of our technique, n a m e l y the culture of plants from nueellar a n d ovular explants, allows i n d u c t i o n of m u t a t i o n s i n cells, giving subsequently rise to entire plants. Contribution from the Volcani Center, Agricultural Research Organization, Bet Dagan, Israel, 1972 Series, No. 2057-E.
References 1. Bitters, W.P., Murashige, T., Rangan, T. S., Nauer, E.: Investigations on established virus-free citrus plants through tissue culture. Calif. Citrus Nurserymen's Soc. 9, 27-30 (1970). 2. Button, J., Bornman, C. It. : Development of nucellar plants from unpollinated and unfertilized ovules of the Washington ~avel orange in vitro. J. S. Afr. Bot. 37, 127-134 (1971).
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3. Murashige, T., Tucker, D. P. H.: Growth factor requirement of Citrus tissue culture. Proc. 1st Int. Citrus Syrup. vol. 3, p. 1155-1161, H. D. Chapman, ed. Univ. Calif. Riverside: Publ. Dept. 1969. 4. Rangan, T. S., Murashige, T., Bitters, W.B.: I n vitro initiation of nucellar embryos in monoembryonie Citrus. Hort. Sci. 3, 226-227 (1968). 5. Rangan, T. S., Murashige, T., Bitters, W. B. : I n vitro studies of zygotic and nucellar embryogenesis in Citrus. Proe. 1st Int. Citrus Syrup. vol. 1, p. 226-229, H. D. Chapman, ed. Univ. Calif. Riverside: PuN. Dept. 1969. 6. Sass, J. E.: Botanical mierotechnique. Ames: Iowa State Univ. Press i958. 7. Singh, V.P.: Raising nucellar seedlings of some Rutaceae in vitro. In: Plant tissue and organ culture, Int. Syrup. Delhi, India, p. 275 277, P. Maheshwari and N. Ranga Swamy, eds. Univ. Delhi: Int. Soc. Plant Morphologists 1963. 8. Skoog, F., Miller, C. 0. : Regulation of growth and organ formation in plant tissues. Symp. Soe. exp. Biol. 11, 118-131 (1957). 9. Swaminathan, M.S.: The discovery of induced mutations. In: Manual on mutation breeding, p. 131-134. Int. Atomic Energy Agency, Teeh. iRep. Ser. No 119, Vienna 1970. P. Spiegel-Roy Inst. of I-Iortieulture J. Kochba Dept. of Fruit Breeding H. Safran Dept. of Citrieulture The Voleani Center Agricultural Research Organization P.O.B. 6 Bet Dagan, Israel
Adventive Plants from Ovules and Nuceili in Citrus J. K o e h b a , P. S p i e g e l - R o y a n d t I a n n a h Safran The Voleani Center, Agricultural Research Organization, Bet Dagan, Israel Received February 14 / March 28, 1972
Summary. 1- to 8-week-old ovules and nucelli from three Citrus eultivars-Shamouti and Valencia (Citrus sinensi8) oranges and Marsh Seedless (C. paradisi) grapefruit~-were cultured in vitro. No embryo differentiation was observed in the explants prior to culture. The Shamouti ovules had degenerated and were apparently unfertilized. Embryoids formed on Murashige and Tucker nutrient medium supplemented with 500 mg/1 mMt extract. Whole plants developed on the same basal medium supplemented with kinetin and indole-3-acetic acid (IAA), coconut milk or gibberellic acid (GAs). A higher kinetin/IAA ratio or the addition of coconut milk favoured stem elongation more than root formation while a lower kinetin/IAA ratio favoured root formation and inhibited stem elongation. The addition of GAs to the basal medimn stimulated rooting and stem elongation. These results can be of aid in mutation research, allowing irradiation at stages prior to embryonic development. Introduction Nucellar e m b r y o g e n y is of w i d e s p r e a d occurrence in Citrus a n d r e l a t e d genera [5]. B o t h nueellar a n d zygotic e m b r y o s of Citrus spp. have been grown in vitro. Cultured nucelli of p o l y e m b r y o n i e varieties gave rise to a d v e n t i v e embryos, a n d in rare cases, to fully d e v e l o p e d seedlings [7]. R a n g a n et al. [4] raised nucellar e m b r y o s in vitro from t h r e e m o n o e m b r y o n i c varieties, w i t h t h e a i m of p r o d u c i n g virus-free plants. A l a t e r r e p o r t [5] deals with t h e i n d u c t i o n of zygotic e m b r y o d e v e l o p m e n t in p o l y e m b r y o n i c Citrus cultivars. W h i l e little success was o b t a i n e d in the beginning, i m p r o v e m e n t in t h e g r o w t h m e d i u m lead to b e t t e r results [1]. Some i n f o r m a t i o n is available on nucellar e m b r y o f o r m a t i o n in vitro in p r a c t i c a l l y seedless Citrus cultivars. B u t t o n a n d B o r n m a n [2] grew p l a n t s from nucellar e x p l a n t s of unfertilized W a s h i n g t o n Navel, while B i t t e r s et al. [1] succeeded in o b t a i n i n g p l a n t s from nucelli of t h e R o b e r t son N a v e l orange. This p a p e r r e p o r t s on nucellar e m b r y o f o r m a t i o n in different t y p e s of e x p l a n t s a n d on t h e effect of g r o w t h m e d i a on r o o t f o r m a t i o n a n d stem elongation in t h e S h a m o u t i a n d Valencia orange eultivars a n d in t h e Marsh Seedless grapefruit. All these cultivars have a t e n d e n c y for seedlessness, this being m o s t p r o n o u n c e d in Shamouti.
238
J. Kochba, P. Spiegel-Roy and H. Sultan:
Methods and Materials Ovules and nucelli were excised aseptically with the aid ol a dissecting microscope from fruitlets 1- to 6-week old for Shamouti, 6- to 8-week old for Valencia (Citrus sinensis Osb.) oranges, and 8 week old for Marsh (Citrus paradisi Macf.) grapefruit. Fruit length for each sample was recorded (Table 1). Histological preparations of the tissues were made by fixation in formaldehyde-acetic acidethanol (FAA). Following dehydration and paraffin embedding, the sections were cut to 10-12 ~ thickness, stained with safranin and light green, and mounted in Canada balsam [6]. Explants were cultured on a basal nutrient medium (BM) of inorganic salts and organic substances as suggested by Murashige and Tucker [3]. 5% sucrose and 1% agar were added. The pH of the medium was adjusted to 5.7 and the medium autoclaved for 15 rain at 121 ~ C. The following additives to the basal medium were used during different phases of work: Malt extract (ME), coconut milk (CCM), kinetin, indole-3-aeetic acid (IAA), or gibberellic acid (GA3). Explants were cultured in 200• mm test tubes containing 15 ml of the medium, fitted with plastic caps, and kept at 26~1 ~ C and 16 h light daily. Light was supplied from Gro-Lux fluorescent tubes at 1000 lux. Results Ovules a n d nucelli e x t r a c t e d from 1- to 6-week old S h a m o u t i fruitlets were cultured on a basal m e d i u m containing 500 mg/1 m a l t e x t r a c t (Table 1). The highest percentage of cultures showing e m b r y o n i c developm e n t was o b t a i n e d using ovules from 4-week-old fruitlets. Earlier stages p r o d u c e d a l m o s t no embryos, while only a few cultures from l a t e r stages d e v e l o p e d to embryos. Nucelli y i e l d e d only a few cultures from 3- to 5-week-old fruitlets. Some of t h e cultures from ovules d e v e l o p e d callus, from all stages of fruit d e v e l o p m e n t tested. Nucellar e x p l a n t s showed this response only in rare eases. Ovules from 6 a n d 8-week-old fruitlets, a n d nueellar e x p l a n t s from 8-week-old fruitlets of Valencia a n d Marsh gave rise to cultures with e m b r y o n i c development. A higher percentage of successful cultures with these varieties was i n i t i a t e d from nucelli t h a n from ovules. B o t h Valencia a n d Marsh y i e l d e d a m u c h higher p e r c e n t a g e of cultures w i t h e m b r y o initials from b o t h t y p e s of explants, a n d a higher average n u m b e r of e m b r y o i d s per culture t h a n S h a m o u t i ; in c o n t r a s t to Shamouti, none of these cultures f o r m e d callus. Histological e x a m i n a t i o n of S h a m o u t i ovules showed t h a t a t t h e t i m e of excision t h e y were highly d e g e n e r a t e d a n d p r o b a b l y unfertilized, while t h e Valencia a n d Marsh ovules were well d e v e l o p e d a n d c o n t a i n e d large nueelli (Fig. 1). No f o r m a t i o n of nucellar or zygotic e m b r y o s could be d e t e c t e d in S h a m o u t i ovules a t a n y stage of d e v e l o p m e n t . Ovules from fruitlets older t h a n 6 weeks h a d c o m p l e t e l y degenerated. Nucellar e m b r y o s were d e t e c t e d in Valencia a n d Marsh Seedless ovules as early as 10 weeks after fruit set (Fig. 2) b u t in 8-week-old fruitlets which were used for culture, no e m b r y o n i c d e v e l o p m e n t was found.
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Table 1. Embryoid formation in vitro in explants of Citrus ovules and nucelli a Time after anthesis (weeks)
1 1 2 2 3 3 4 4 5 6 6
6 6 8 8
8 8
Length of fruitlets (mm)
6.8• 9.4 ~: 0.6 12.1-4-0.8 14.5• 17.4• 24.7 • 1.3
17.3• 1.3 31.7 ~ 2.1
29.4~2.0
Type Cultures c Cultures of developing developing explant b embryonic initials callus No.
%
Ov. Nuc. Ov. Nuc. Ov. Nuc. Ov. Nuc. Ov. 0v. Nuc.
1 0 1 0 0 1 11 2 2 5 0
Ov. Nuc. Or. Nuc.
Ov. Nuc.
Average no. of embryoids per culture
No.
%
Shamouti 2 0 2 0 0 2 22 4 4 10 0
5 0 7 0 1 0 8 1 7 3 0
10 0 14 0 2 0 16 2 14 6 0
1.0 0.0 1.0 0.0 0.0 1.0 1.4 1.0 1.0 1.4 0.0
11 0 17 21
Valencia 44 0 68 84
0 0 0 0
0 0 0 0
1.2 0.0 1.3 3.2
12 21
Marsh 48 84
0 0
0 0
1.3 3.1
a Counted after 12 weeks of culture. b Ov. = ovule, Nuc. = nucellus. c 50 cultures per treatment for Shamouti, and 25 cultures for Valencia and Marsh.
No f u r t h e r d e v e l o p m e n t occurred on t he original n u t r i e n t m e d i u m after 12 weeks of culture, a n d t h e size of t h e e m b r y o i d s r e m a i n e d 1-2 m m for S h a m o u t i a n d 3-6 m m for Marsh Seedless an d Valencia. T h e y were, therefore, t r a n s p l a n t e d s e p a r a t e l y to m e d i a eontailfing B M ddifferent levels of k i n e t i n a n d I A A , or coconut milk, for Valencia an d Marsh, while S a m o u t i e m b r y o id s were t r a n s p l a n t e d to only one m e d i u m due to t h e low n u m b e r of e m b r y o id s available. Th e d e v e l o p m e n t of t h e cultures on fresh n u t r i e n t m e d i a was r ap i d (Table 2). I n m a n y cases more t h a n one p l a n t d ev el o p ed per culture. Their av er ag e n u m b e r was highest on t h e lower k i n et i n -b higher I A A concentrations for Valencia a n d on CCM for Marsh. M a n y cultures of Marsh an d S h a m o u t i a n d some of Valencia f o r m e d new em b r y o i d s f r o m existing ones. I n t h e S h a m o u t i cultures t her e was no stem elongation,
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J. Kochba, P. Spiegel-Roy and H. Safran:
II
III
1V
Fig. 1. Micrographs of Citrus ovules. Ovules at explanting; no differentiation of embryos visible. I - I I Shamouti orange (4 weeks after anthesis); I I I Valencia orange (6 weeks after anthesis); I V Marsh Seedless grapefruit (8 weeks after anthesis)
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241
Fig. 2. ~r of Citrus ovules. Ovules 10 weeks after anthesis. Nueellar embryos evident. I Valencia orange; I I Marsh Seedless grapefruit
and only few roots were formed. More Marsh than Valencia embryoids formed elongated stems and roots. Several combinations of kinetin -IAA or CCM stimulated plant development more than did ME. The lower level of kinetin combined with the higher level of IAA favoured root formation in both eultivars, but inhibited stem elongation. On most media, more cultures had roots than elongated stems, but the higher level of kinetin ,-~ the lower level of IAA or CCM was better than the other kinetin/IAA ratios for the development of both organs. Cultures in which stems did not elongate showed a "rosette" type of growth with short internodes and many small, thick leaves (Fig. 3). Newly formed embryoids of all three cultivars were planted on media containing GAs, IAA or kinetin (Table 3). Both GAs and IAA greatly improved rooting of Shamouti embryoids, while Valencia embryoids rooted best with the addition of GAs and Marsh with addition of IAA. More stems from all eultivars elongated on GAa than on any other medium. A higher percentage of Marsh and Valencia than of Shamouti eultnres yielded fully developed plants which could be transferred to the greenhouse for further growth.
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J. Kochba, P. Spiegel-Roy and H. Safran:
Table 2. Developmenta of transplanted embryoids on media containing different levels of kinetin and IAA or coconut milk (CCM) b Addition to basal medium c
Developing plantlets per culture Avg. No.
Cultures forming new embryoids (%)
0.1 mg/1 Kinetin + 1.0 rag/1 IAA
Shamouti 1.08
31
0
3
1.0 mg/1 Kinetin + 1.0 rag/1 IAA 1.0 rag/1 K i n e t i n + 0.1 rag/1 IAA 0.1 mg/1 Kinetin + 0.1 rag/1 IAA 0.1 rag/1 Kinetin + 1.0 rag/1 IAA CCM 5 % ME 500 rag/1
Valencia 1.04 1.16 1.24 1.84 1.24 1.11
8 12 8 12 4 26
0 12 0 8 20 0
19 16 44 52 44 42
32 32 52 42 26 20
20 32 16 4 26 10
48 42 36 71 26 10
1.0 rag/1 Kinetin ~- 1.0 rag/1 IAA 1.0 mg/1 Kinetin + 0.1 rag/1 IAA 0.1 mg/1 Kinetin ~- 0.1 rag/1 IAA 0.1 mg/1 Kinetin-t- 1.0 rag/1 IAA CCM 5% ME 500 mg/1
Marsh leedless 1.40 1.53 1.44 1.63 2.11 1.00
Cultures with stems (%)
roots (%)
a 40 days after transplanting. b 40-45 cultures per treatment. c Murashige's citrus medium.
Discussion Our objective in raising in vitro nucellar seedlings from Shamouti, Valencia a n d Marsh Seedless was to exploit the earliest stages of e m b r y o formation for use in m u t a t i o n research, with the u l t i m a t e aim of producing solid m u t a n t s [9]. The stage of e m b r y o d e v e l o p m e n t in the explants was therefore crucial since nucellar p l a n t p r o d u c t i o n from already existing proembryos i n the explants would be of little use. We were able to show t h a t no embryonic d e v e l o p m e n t had started i n the explants from 1- to 8-week-old fruitlets. I n i t i a t i o n of nucellar embryo f o r m a t i o n
seems to differ with cultivar. While Button and Bornman [2], and Bitters st al. [I] obtained results similar to ours, Singh [7] found globular a d v e n t i v e embryos in 55-day-old ovules of Citrus reticulata cv. N a g p u r i a n d R a n g a n et al. [4] detected no nucellar embryos in seeds from 4-month-old i m m a t u r e fruits of the sour orange (Citrus aurantium) while a zygotic embryo was present. No zygotic e m b r y o could be detected i n our explants or in later stages of seed development which we examined.
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Fig. 3. Development of transplanted Citrus embryoids into plants in culture. Valencia orange, BSI-~-1rag/1 GA3. I Normal stem elongation and root growth; I I l~osette shoot growth and normal root development Nucellar embryo development in the cultivars examined by us clearly preceded zygotic embryo development. As no zygotic embryo was present, we could use whole nucelli as well as ovules for culture. Shamouti ovules, especially, had to be cultured at an early stage of development because of the rapid degeneration of ovules. Great difficulty was encountered in extracting nucelli from the very small ovules. Probably because of the damage caused to the exceedingly small nucellar explants during extraction, ovules yielded more successful cultures. Although nucelli gave better results than ovules for Valencia and Marsh, the use of entire ovules can be more rewarding due to comparative ease of extraction. Rangan et al. [5] and Bitters et al. [1] had no difficulty in raising entire plants of Citrus on the same medium on which nucellar embryo development was initiated. In the cultivars used in our experiments, fm-ther development did not occur on the same nutrient medium, but we were able to overcome this by the addition of growth substances, namely, kinetin, IAA or GA s. I t is well known that kinetin-auxin ratios can affect organ formation in tobacco callus [8]. The stimulating effect
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J. Kochba, P. Spiegel-Roy and H. Salran:
Table 3. Root and stem formation a in embryoids on medium containing GA3 as compared to additions of kinetin or IAAb Addition to BM c
Cultures with elongated
developed
stems
roots
(%)
(%)
Shamouti
GA3 1 rag/1 IAA 0.1 rag/1
9 6
36 34
Valencia
GA3 1 rag/1 Kinetin 0.1 mg/l IAA 0.1 rag/1
30 21 14
59 25 25
Marsh
GA~ I mg/l Kinetin 0.1 mg/1 IAA 0.1 mg/1
38 5 22
38 34 41
a 42 days after transplantation. b 34-40 cultures per treatment. e Murashige's citrus medium.
of GA a on rooting of C i t r u s embryoids was first reported b y B u t t o n a n d B o r n m a n [2] for W a s h i n g t o n Navel. We f o u n d the same to hold true for S h a m o u t i a n d Valencia. Moreover, a d d i t i o n of GA s s t i m u l a t e d stem elongation in the cultures of all cultivars. This resulted in a higher percentage of p l a n t s from i n - v i t r o cultures successfully established in the greenhouse. The use of our technique, n a m e l y the culture of plants from nueellar a n d ovular explants, allows i n d u c t i o n of m u t a t i o n s i n cells, giving subsequently rise to entire plants. Contribution from the Volcani Center, Agricultural Research Organization, Bet Dagan, Israel, 1972 Series, No. 2057-E.
References 1. Bitters, W.P., Murashige, T., Rangan, T. S., Nauer, E.: Investigations on established virus-free citrus plants through tissue culture. Calif. Citrus Nurserymen's Soc. 9, 27-30 (1970). 2. Button, J., Bornman, C. It. : Development of nucellar plants from unpollinated and unfertilized ovules of the Washington ~avel orange in vitro. J. S. Afr. Bot. 37, 127-134 (1971).
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3. Murashige, T., Tucker, D. P. H.: Growth factor requirement of Citrus tissue culture. Proc. 1st Int. Citrus Syrup. vol. 3, p. 1155-1161, H. D. Chapman, ed. Univ. Calif. Riverside: Publ. Dept. 1969. 4. Rangan, T. S., Murashige, T., Bitters, W.B.: I n vitro initiation of nucellar embryos in monoembryonie Citrus. Hort. Sci. 3, 226-227 (1968). 5. Rangan, T. S., Murashige, T., Bitters, W. B. : I n vitro studies of zygotic and nucellar embryogenesis in Citrus. Proe. 1st Int. Citrus Syrup. vol. 1, p. 226-229, H. D. Chapman, ed. Univ. Calif. Riverside: PuN. Dept. 1969. 6. Sass, J. E.: Botanical mierotechnique. Ames: Iowa State Univ. Press i958. 7. Singh, V.P.: Raising nucellar seedlings of some Rutaceae in vitro. In: Plant tissue and organ culture, Int. Syrup. Delhi, India, p. 275 277, P. Maheshwari and N. Ranga Swamy, eds. Univ. Delhi: Int. Soc. Plant Morphologists 1963. 8. Skoog, F., Miller, C. 0. : Regulation of growth and organ formation in plant tissues. Symp. Soe. exp. Biol. 11, 118-131 (1957). 9. Swaminathan, M.S.: The discovery of induced mutations. In: Manual on mutation breeding, p. 131-134. Int. Atomic Energy Agency, Teeh. iRep. Ser. No 119, Vienna 1970. P. Spiegel-Roy Inst. of I-Iortieulture J. Kochba Dept. of Fruit Breeding H. Safran Dept. of Citrieulture The Voleani Center Agricultural Research Organization P.O.B. 6 Bet Dagan, Israel
