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Somatic embryogenesis in leaf callus from cauliflower (Brassica oleracea var. Botrytis)
Plant Science Letter~, II (1978) 311--316 © Elsevier/North-HollandScientificPublishersLtd.
311
SOMATIC EMBRYOGENESIS IN LEAF CALLUS FROM CAULIFLOWER (BRASSICA OLERACEA VAR. BOTR YTIS)
L.K. PAREEK and N. CHANDRA Department of Botany, University of Rajasthan, Jaipur 302004 (India) (Received June 20th, 1977) (Accepted October 19th, 1977)
SUMMARY Embryogenesis was induced in leaf callus of cauliflower (Brassica oleracea vat. botrytis) maintained on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA, 1.0 mg 1-1 ) and kinetin (0.5 mg 1-I ). The callus first developed meristematic nodules on the surface of which embryoids were initiated superficially. The callus masses when transferred to the same medium with a lower concentration of IAA (0.1--0.01 mg 1-1 ) developed a much larger number of embryoids, which presumably developed from single superficial cells. All the stages of embryoid formation viz. globular, heart-shaped and torpedo-shaped were observed. A number of abnormalities were also noted. Precocious proliferation of superficial cells of the embryoids resulted in accessory embryoid development. Some of the embryoids showed a reversed polarity with respect to the tissue of origin. The origin, development and organisation of induced embryoids is discussed.
INTRODUCTION Embryoid formation from callus cellswas firstobserved in the carrot [1,2]. Since then the phenomenon of somatic embryogenesis has been demonstrated in tissue and cell cultures of various plants. However, only the carrot system has been studied extensively [3--5]. This work has demonstrated, beyond any doubt, the totipotency of somatic cellsin culture. It is well k n o w n that carrot cells grown on an auxin-containing m e d i u m can be manipulated to foster the development of embryos by transferring them to an auxin-free m e d i u m [3,6]. However, there are no comparable studies on other systems where embryoid formation has been demonstrated [7,8], Abbreviations: IAA, indole-3-acetic acid;MS, Murashige and Skoog.
312 Somatic embryogenesis (in culture) is of considerable interest from several points of view, viz., (i) to understand the physical and chemical requirements for e m b r y o induction and development, (ii) to study the biochemistry underlying this process, (iii) to raise plants which breed true to the maternal parent, and (iv) to obtain virus-free plants from infected plants. In carrot suspension cultures embryoids arise from superficial cells of multiceUular embryonic masses and t h e y presumably originate from single cells [9]. In our studies on morphogenesis in callus cultures of a locally cultivated variety of cauliflower (Brassica oleracea var. botrytis) embryogenesis was induced in MS m e d i u m [10] supplemented with kinetin and IAA. Although bud formation has been reported in tissue cultures of several species of Brassica, including B. oleracea var. botrytis, there is no report in the literature of e m b r y o formation in this variety [11]. Mostly the regeneration of plants in tissue cultures of different cruciferous plants is through bud formation [12--14]. However, haploid e m b r y o formation from microspores has been obtained in Brassica napus [15] and in B. campestris [16]. In the present paper the origin, development and organisation of induced somatic embryoids is described. MATERIALS AND METHODS
The seeds of a local cultivar of cauliflower (Brassica oleracea var. botrytis) obtained from the Government agricultural farm, Durgapura, India, were sown in pots. The y o u n g and expanding leaves were used as explants. Leaves were first washed with 10% solution o f a detergent and cut into small pieces which were t h e n surface-sterilized in a 5% solution of sodium hypochlorite for about 20 rain and washed thoroughly with several rinses o f sterile distilled water. Several cuts were made in the pieces of lamina before planting t h e m on MS agar m e d i u m [10]. The cultures were raised in 125 ml erlenmeyer flasks (Coming) and incubated in a growth r o o m maintained at 26 + 2 ° C and in weak light. For histological preparations the material was e m b e d d e d in wax following customary procedures of dehydration and clearing in an ethanolxylol series. Sections were cut at 10--12 ~m thickness, stained with safranin and fast green and m o u n t e d in DPX. Camera lucida diagrams were drawn from dissections and t e m p o r a r y m o u n t s o f embryoids. RESULTS AND DISCUSSION
Initiation o f callus and development o f embryoids Callus formation was initiated within a week o f placing leaf segments on MS m e d i u m supplemented with IAA (1.0 mg 1-1 ) and kinetin (0.5 mg 1-1 ). Callus was f o r m e d m o r e profusely at the cut ends. Sections of material at this stage showed that the mesophyll cells situated towards the cut ends o f the explants were dividing. The callus cells f o r m e d initially were green with a large n u m b e r o f chloroplasts. Later the n u m b e r o f chloroplasts declined and starch grains appeared. After 4 weeks the callus grew into a yellowish-green mass with nodules on the surface. When subcultured the nodules separated and developed
313 a
b
c
d f
Fig. 1. Camera lucida drawings of embryoids from the leaf callus of cauliflower (Brasslca oleracea var botrytis) Abbreviations: Cam, cambium-like layer; rc, root cap; sus, suspensor. Magnifications: a--p × 50, q---w × 150, x, y × 450. (a--h) Embryoids arranged in sequential order of development from early globular (a) to a typical dicotyledonous embryoid (h). Note 3 cotyledons in "f" and 4 in "g". (i) An embryoid without any differentiation of co{yledons and shoot apex. (j,k) Embryoids with well developed shoot pole and ill-defined root pole. (l,m) Embryoids with "membranous" and lobed shoot pole. (n,o,p) Accessory embryoid formation on the surface of embryoids. (q) Section of a nodule showing early stages of embryoid development. (r--u) Sections of globular embryoids with (u) and without suspensor. (v) Median longitudinal section of a typical dicotyledonous embryoid with an accessory embryoid. (w) Median longitudinal section through a mature embryoid with two shoot apices surrounded by their respective leaf primordia. (x) A portion of the median longitudinal section passing through the shoot apex of an embryoid. Note the highly enlarged cells and a cambium-llke layer. (y) A portion of the median longitudinal section of the root pole. Note the highly enlarged root cap cells.
314 secondary and tertiary nodules on their surfaces. These nodules consisted of very compactly arranged small cells. On the surface of these nodules, embryoids were initiated superficially (Fig. lq). Some of the structures arising on the surface resembled root primordia. These callus masses when transferred to the same medium with a lower concentration of auxin (0.1--0.01 mg 1-1 IAA) developed a much larger number of embryoids. However, the differentiation of embryoids and growth of callus was poor if grown on a completely auxinfree medium. This callus with nodules and embryoids was maintained for about 3 years by sub-culturing at monthly intervals. The embryos when transferred to basal MS medium developed into plantlets. The embryoids presumably developed from single superficial cells on the nodules of callus. The initial cells divided several times without any appreciable cell enlargement (Fig. 1 q). All stages of embryogenesis from early globular to heart-shaped, torpedo-shaped and typical dicotyledonous embryoids were observed (Figs. 1 a--h, q--w). Pro-embryo like structures were quite common. In many cases a 3 or 4-celled suspensor was observed (Fig. 1 u). However, the embryoids were not as well organized as the normal zygotic embryos. They nevertheless resembled them in essential details. The young globular embryoids varied widely in shape from spherical to ellipsoidal (Fig. 1 r - u ) . Accessory embryoids ~vere formed on the surface of the developing embryoids by precocious proliferation of surface cells {Fig. 1 m--p, v). Thus embryoids of the second and third order were seen developing on a single embryoid (Fig. 1 p). The accessory embryoids were easily separable from the parent embryoid. It seemed as if all the cells of an embryoid were totipotent, capable of giving rise to embryoids. Some of the embryoids had their radicles directed outwards i.e. away from the parent tissue and thus showed a reversed polarity.
The organization of embryoids A prominent feature of the embryoids was a very well developed root part and a lesser developed shoot pole (Fig. 1 v). In some cases the shoot pole was an expanded membranous structure differentiated into two well
315
M a n y abnormal features were observed in embryoid morphology. S o m e of the embryos had well developed cotyledons and an ill developed root pole (Fig. i j,k), while others had a very well defined root pole and illdefined shoot pole with no visible cotyledonary primordia (Fig. 1 i). Embryoids with three or four cotyledons were c o m m o n (Fig. 1 f,g).In fact all stages from a well developed normal embryo to conditions where the embryoid showed a well defined shoot part and an illdefined root part or vice versa were observed. In some cases the embryos showed two shoot apices instead of one (Fig. 1 w). Our observations on the development and histology of embryoids are comparable to the recent studies on Ranunculus sceleratusand Atropa belladonna [7,8,17]. Konar et al. [8] have reported root-like structures with collars in cell cultures of Atropa belladonna. Their diagrams and description of such structures suggest a similarity to embryoids with reversed polarity observed in the present study. Another important question is whether the embryoids arise from single cells or from a group of cells.Our observations indicate that they arise from single cells present on the surface of nodules. The embryogenic initialcells undergo several divisions without any appreciable cell enlargement. Similar observations have been made in carrot cells [5,9]. However, multicellular origin of embryoids has been reported in tissue cultures of carrot [4,18]. Sussex [18] maintained that the unicellular zygote was an unusual m o d e of organogenesis in higher plant development. According to him "the requirement that the zygote be unicellular is a genetic restriction imposed by the special conditions of fertilization and fusion of gametes from two different parents. The usual pattern of plant organogenesis is from a group of cells to function co-ordinately in development." Co-ordinate function of several cells has been experimentally demonstrated in the origin of leaves and lateral roots [19,20]. Therefore, Sussex concluded that the multicellular origin of embryos observed by him [18] and by Halperin and Wetherell [4] was in accord with the origin of other organs. However, Street and Withers [5] while discussing the anatomy of embryogenesis in cell culutres write, "to be properly described as embryogenesis we n o w propose that the developmental process should be shown to produce a perfect embryo frown a single cell, all the derivatives from which become part of a structure which achieves bipolarity (the emergence of a root and shoot pole) at as early a stage as occurs in zygotic embryogenesis." The present authors feel that the evidence for the "single cell origin of embryoids" in cell cultures is only circumstantial as e m b r y o i d formation has n o t been d e m o n s t r a t e d directly from a single cell. Even in studies [21] where e m b r y o formation has been traced from a single isolated cell in culture, a small multicellular mass is formed first which later embarks u p o n e m b r y o formation. J o n e s [22] studied the factors influencing embryogenesis in carrot cultures and arrived at a similar conclusion. According to him "there is still no direct evidence for the aesthetically satisfying transition o f single free vacuolated cells embarking on a developmental sequence analogous to embryogenesis in the zygote." However, a good example of single cell origin of an embryoid is that
316 f r o m t h e m i c r o s p o r e s w h i c h are h i g h l y d i f f e r e n t i a t e d cells m u c h d i f f e r e n t f r o m a s o m a t i c cell in c u l t u r e . ACKNOWLEDGEMENTS T h e a u t h o r s are g r a t e f u l t o P r o f e s s o r H . E . S t r e e t f o r r e a d i n g t h e m a n u s c r i p t T h a n k s are also d u e t o P r o f e s s o r B. Tiagi a n d Dr. D. Singh o f t h e D e p a r t m e n t o f B o t a n y , U n i v e r s i t y o f R a j a s t h a n , J a i p u r , I n d i a f o r l a b o r a t o r y facilities. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
F.C. Steward, Am. J. Bot., 45 (1958) 709. J. Reinert, Planta, 53 (1959) 318. W. Halperin, Am. J. Bot., 53 (1966) 443. W. Halperin and D.F. Wetherell, Am. J. Bot., 51 (1964) 274. H.E. Street and L.E. Withers, The anatomy of embryogenesis in culture, in H.E. Street (Ed.) Tissue Culture and Plant Science, Academic Press, London, 1974, pp. 71--100. S.M. Smith and H.E. Street, Ann. Bot., 38 (1974) 223. R.N. Konar and K. Natraja, Phytomorphology, 15 (1965) 132. R.N. Konar, E. Thomas and H.E. Street, Ann. Bot., 36 (i972) 249. A.A. McWiUiam, S.M. Smith and H.E. Street, Ann. Bot., 38 (1974) 243. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. S.M. Barnocelli, M. Buiatti and A. Bennici, Z. Pflanzenz~ichtg., 70 (1973) 99. J. Lustinec and J. Horak, Experientia, 26 (1970) 919. V. Clare and H.A. Collin. Ann. Bot., 38 (1974) 1067. Y.P.S. Bajaj and P. Nietsch, J. Exp. Bot., 26 (1975) 883. E. Thomas and G. Wenzel, Z. Pflanzenz~ichtg., 74 (1974) 77, W.A. Keller, T. Rajhathy and J. Lacapra, Can. J. Genet. Cytol., 17 (1975) 655. E. Thomas, R.N. Konar and H.E. Street, J. Cell. Sci., 11 (1972) 95. I.M. Sussex, Phytomorphology, 22 (1972) 50. D.E. Foard, Can. J. Bot., 49 (1971) 1601. D.E. Foard, A.H. Haber and T.N. Fishman, Am. J. Bot., 52 (1965) 580. D. Backs-Hiisemann and J. Reinert, Protoplasma, 70 (1970) 49. L.H. Jones, Ann. Bot., 38 (1974) 1077.
311
SOMATIC EMBRYOGENESIS IN LEAF CALLUS FROM CAULIFLOWER (BRASSICA OLERACEA VAR. BOTR YTIS)
L.K. PAREEK and N. CHANDRA Department of Botany, University of Rajasthan, Jaipur 302004 (India) (Received June 20th, 1977) (Accepted October 19th, 1977)
SUMMARY Embryogenesis was induced in leaf callus of cauliflower (Brassica oleracea vat. botrytis) maintained on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA, 1.0 mg 1-1 ) and kinetin (0.5 mg 1-I ). The callus first developed meristematic nodules on the surface of which embryoids were initiated superficially. The callus masses when transferred to the same medium with a lower concentration of IAA (0.1--0.01 mg 1-1 ) developed a much larger number of embryoids, which presumably developed from single superficial cells. All the stages of embryoid formation viz. globular, heart-shaped and torpedo-shaped were observed. A number of abnormalities were also noted. Precocious proliferation of superficial cells of the embryoids resulted in accessory embryoid development. Some of the embryoids showed a reversed polarity with respect to the tissue of origin. The origin, development and organisation of induced embryoids is discussed.
INTRODUCTION Embryoid formation from callus cellswas firstobserved in the carrot [1,2]. Since then the phenomenon of somatic embryogenesis has been demonstrated in tissue and cell cultures of various plants. However, only the carrot system has been studied extensively [3--5]. This work has demonstrated, beyond any doubt, the totipotency of somatic cellsin culture. It is well k n o w n that carrot cells grown on an auxin-containing m e d i u m can be manipulated to foster the development of embryos by transferring them to an auxin-free m e d i u m [3,6]. However, there are no comparable studies on other systems where embryoid formation has been demonstrated [7,8], Abbreviations: IAA, indole-3-acetic acid;MS, Murashige and Skoog.
312 Somatic embryogenesis (in culture) is of considerable interest from several points of view, viz., (i) to understand the physical and chemical requirements for e m b r y o induction and development, (ii) to study the biochemistry underlying this process, (iii) to raise plants which breed true to the maternal parent, and (iv) to obtain virus-free plants from infected plants. In carrot suspension cultures embryoids arise from superficial cells of multiceUular embryonic masses and t h e y presumably originate from single cells [9]. In our studies on morphogenesis in callus cultures of a locally cultivated variety of cauliflower (Brassica oleracea var. botrytis) embryogenesis was induced in MS m e d i u m [10] supplemented with kinetin and IAA. Although bud formation has been reported in tissue cultures of several species of Brassica, including B. oleracea var. botrytis, there is no report in the literature of e m b r y o formation in this variety [11]. Mostly the regeneration of plants in tissue cultures of different cruciferous plants is through bud formation [12--14]. However, haploid e m b r y o formation from microspores has been obtained in Brassica napus [15] and in B. campestris [16]. In the present paper the origin, development and organisation of induced somatic embryoids is described. MATERIALS AND METHODS
The seeds of a local cultivar of cauliflower (Brassica oleracea var. botrytis) obtained from the Government agricultural farm, Durgapura, India, were sown in pots. The y o u n g and expanding leaves were used as explants. Leaves were first washed with 10% solution o f a detergent and cut into small pieces which were t h e n surface-sterilized in a 5% solution of sodium hypochlorite for about 20 rain and washed thoroughly with several rinses o f sterile distilled water. Several cuts were made in the pieces of lamina before planting t h e m on MS agar m e d i u m [10]. The cultures were raised in 125 ml erlenmeyer flasks (Coming) and incubated in a growth r o o m maintained at 26 + 2 ° C and in weak light. For histological preparations the material was e m b e d d e d in wax following customary procedures of dehydration and clearing in an ethanolxylol series. Sections were cut at 10--12 ~m thickness, stained with safranin and fast green and m o u n t e d in DPX. Camera lucida diagrams were drawn from dissections and t e m p o r a r y m o u n t s o f embryoids. RESULTS AND DISCUSSION
Initiation o f callus and development o f embryoids Callus formation was initiated within a week o f placing leaf segments on MS m e d i u m supplemented with IAA (1.0 mg 1-1 ) and kinetin (0.5 mg 1-1 ). Callus was f o r m e d m o r e profusely at the cut ends. Sections of material at this stage showed that the mesophyll cells situated towards the cut ends o f the explants were dividing. The callus cells f o r m e d initially were green with a large n u m b e r o f chloroplasts. Later the n u m b e r o f chloroplasts declined and starch grains appeared. After 4 weeks the callus grew into a yellowish-green mass with nodules on the surface. When subcultured the nodules separated and developed
313 a
b
c
d f
Fig. 1. Camera lucida drawings of embryoids from the leaf callus of cauliflower (Brasslca oleracea var botrytis) Abbreviations: Cam, cambium-like layer; rc, root cap; sus, suspensor. Magnifications: a--p × 50, q---w × 150, x, y × 450. (a--h) Embryoids arranged in sequential order of development from early globular (a) to a typical dicotyledonous embryoid (h). Note 3 cotyledons in "f" and 4 in "g". (i) An embryoid without any differentiation of co{yledons and shoot apex. (j,k) Embryoids with well developed shoot pole and ill-defined root pole. (l,m) Embryoids with "membranous" and lobed shoot pole. (n,o,p) Accessory embryoid formation on the surface of embryoids. (q) Section of a nodule showing early stages of embryoid development. (r--u) Sections of globular embryoids with (u) and without suspensor. (v) Median longitudinal section of a typical dicotyledonous embryoid with an accessory embryoid. (w) Median longitudinal section through a mature embryoid with two shoot apices surrounded by their respective leaf primordia. (x) A portion of the median longitudinal section passing through the shoot apex of an embryoid. Note the highly enlarged cells and a cambium-llke layer. (y) A portion of the median longitudinal section of the root pole. Note the highly enlarged root cap cells.
314 secondary and tertiary nodules on their surfaces. These nodules consisted of very compactly arranged small cells. On the surface of these nodules, embryoids were initiated superficially (Fig. lq). Some of the structures arising on the surface resembled root primordia. These callus masses when transferred to the same medium with a lower concentration of auxin (0.1--0.01 mg 1-1 IAA) developed a much larger number of embryoids. However, the differentiation of embryoids and growth of callus was poor if grown on a completely auxinfree medium. This callus with nodules and embryoids was maintained for about 3 years by sub-culturing at monthly intervals. The embryos when transferred to basal MS medium developed into plantlets. The embryoids presumably developed from single superficial cells on the nodules of callus. The initial cells divided several times without any appreciable cell enlargement (Fig. 1 q). All stages of embryogenesis from early globular to heart-shaped, torpedo-shaped and typical dicotyledonous embryoids were observed (Figs. 1 a--h, q--w). Pro-embryo like structures were quite common. In many cases a 3 or 4-celled suspensor was observed (Fig. 1 u). However, the embryoids were not as well organized as the normal zygotic embryos. They nevertheless resembled them in essential details. The young globular embryoids varied widely in shape from spherical to ellipsoidal (Fig. 1 r - u ) . Accessory embryoids ~vere formed on the surface of the developing embryoids by precocious proliferation of surface cells {Fig. 1 m--p, v). Thus embryoids of the second and third order were seen developing on a single embryoid (Fig. 1 p). The accessory embryoids were easily separable from the parent embryoid. It seemed as if all the cells of an embryoid were totipotent, capable of giving rise to embryoids. Some of the embryoids had their radicles directed outwards i.e. away from the parent tissue and thus showed a reversed polarity.
The organization of embryoids A prominent feature of the embryoids was a very well developed root part and a lesser developed shoot pole (Fig. 1 v). In some cases the shoot pole was an expanded membranous structure differentiated into two well
315
M a n y abnormal features were observed in embryoid morphology. S o m e of the embryos had well developed cotyledons and an ill developed root pole (Fig. i j,k), while others had a very well defined root pole and illdefined shoot pole with no visible cotyledonary primordia (Fig. 1 i). Embryoids with three or four cotyledons were c o m m o n (Fig. 1 f,g).In fact all stages from a well developed normal embryo to conditions where the embryoid showed a well defined shoot part and an illdefined root part or vice versa were observed. In some cases the embryos showed two shoot apices instead of one (Fig. 1 w). Our observations on the development and histology of embryoids are comparable to the recent studies on Ranunculus sceleratusand Atropa belladonna [7,8,17]. Konar et al. [8] have reported root-like structures with collars in cell cultures of Atropa belladonna. Their diagrams and description of such structures suggest a similarity to embryoids with reversed polarity observed in the present study. Another important question is whether the embryoids arise from single cells or from a group of cells.Our observations indicate that they arise from single cells present on the surface of nodules. The embryogenic initialcells undergo several divisions without any appreciable cell enlargement. Similar observations have been made in carrot cells [5,9]. However, multicellular origin of embryoids has been reported in tissue cultures of carrot [4,18]. Sussex [18] maintained that the unicellular zygote was an unusual m o d e of organogenesis in higher plant development. According to him "the requirement that the zygote be unicellular is a genetic restriction imposed by the special conditions of fertilization and fusion of gametes from two different parents. The usual pattern of plant organogenesis is from a group of cells to function co-ordinately in development." Co-ordinate function of several cells has been experimentally demonstrated in the origin of leaves and lateral roots [19,20]. Therefore, Sussex concluded that the multicellular origin of embryos observed by him [18] and by Halperin and Wetherell [4] was in accord with the origin of other organs. However, Street and Withers [5] while discussing the anatomy of embryogenesis in cell culutres write, "to be properly described as embryogenesis we n o w propose that the developmental process should be shown to produce a perfect embryo frown a single cell, all the derivatives from which become part of a structure which achieves bipolarity (the emergence of a root and shoot pole) at as early a stage as occurs in zygotic embryogenesis." The present authors feel that the evidence for the "single cell origin of embryoids" in cell cultures is only circumstantial as e m b r y o i d formation has n o t been d e m o n s t r a t e d directly from a single cell. Even in studies [21] where e m b r y o formation has been traced from a single isolated cell in culture, a small multicellular mass is formed first which later embarks u p o n e m b r y o formation. J o n e s [22] studied the factors influencing embryogenesis in carrot cultures and arrived at a similar conclusion. According to him "there is still no direct evidence for the aesthetically satisfying transition o f single free vacuolated cells embarking on a developmental sequence analogous to embryogenesis in the zygote." However, a good example of single cell origin of an embryoid is that
316 f r o m t h e m i c r o s p o r e s w h i c h are h i g h l y d i f f e r e n t i a t e d cells m u c h d i f f e r e n t f r o m a s o m a t i c cell in c u l t u r e . ACKNOWLEDGEMENTS T h e a u t h o r s are g r a t e f u l t o P r o f e s s o r H . E . S t r e e t f o r r e a d i n g t h e m a n u s c r i p t T h a n k s are also d u e t o P r o f e s s o r B. Tiagi a n d Dr. D. Singh o f t h e D e p a r t m e n t o f B o t a n y , U n i v e r s i t y o f R a j a s t h a n , J a i p u r , I n d i a f o r l a b o r a t o r y facilities. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
F.C. Steward, Am. J. Bot., 45 (1958) 709. J. Reinert, Planta, 53 (1959) 318. W. Halperin, Am. J. Bot., 53 (1966) 443. W. Halperin and D.F. Wetherell, Am. J. Bot., 51 (1964) 274. H.E. Street and L.E. Withers, The anatomy of embryogenesis in culture, in H.E. Street (Ed.) Tissue Culture and Plant Science, Academic Press, London, 1974, pp. 71--100. S.M. Smith and H.E. Street, Ann. Bot., 38 (1974) 223. R.N. Konar and K. Natraja, Phytomorphology, 15 (1965) 132. R.N. Konar, E. Thomas and H.E. Street, Ann. Bot., 36 (i972) 249. A.A. McWiUiam, S.M. Smith and H.E. Street, Ann. Bot., 38 (1974) 243. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. S.M. Barnocelli, M. Buiatti and A. Bennici, Z. Pflanzenz~ichtg., 70 (1973) 99. J. Lustinec and J. Horak, Experientia, 26 (1970) 919. V. Clare and H.A. Collin. Ann. Bot., 38 (1974) 1067. Y.P.S. Bajaj and P. Nietsch, J. Exp. Bot., 26 (1975) 883. E. Thomas and G. Wenzel, Z. Pflanzenz~ichtg., 74 (1974) 77, W.A. Keller, T. Rajhathy and J. Lacapra, Can. J. Genet. Cytol., 17 (1975) 655. E. Thomas, R.N. Konar and H.E. Street, J. Cell. Sci., 11 (1972) 95. I.M. Sussex, Phytomorphology, 22 (1972) 50. D.E. Foard, Can. J. Bot., 49 (1971) 1601. D.E. Foard, A.H. Haber and T.N. Fishman, Am. J. Bot., 52 (1965) 580. D. Backs-Hiisemann and J. Reinert, Protoplasma, 70 (1970) 49. L.H. Jones, Ann. Bot., 38 (1974) 1077.