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Somatic embryogenesis in slash pine (Pinus elliottii) from immature embryos cultured in vitro
Plant Science, 65 (1989) 233-- 241
233
Elsevier Scientific Publ/shers Ireland Ltd.
SOMATIC EMBRYOGENESIS IN SLASH P I N E (PINUS ELLIOTTII) FROM I M M A T U R E EMBRYOS CULTURED IN VITRO
S. MOHAN JAIN*, NIU DONG and R j . NEWTON**
Depa~me#t of Forest So&nee, Tezaa Ay~icultunsl Ezperiment Station, The Tezas A&M University Sy#tem, College Statio~ TX 77843 tTf..S.A.) (Received January 11th, 1989) (Revision received June 28th, 1989) (Aeceptod July 18th, 1989) Embryogenic callus development was initiated in cultured immature embryo explants of four different genotypes of slash pine (P/m~s eUiottii) on several culture media supplemented with auxin and cytokinin. Callus induction, either from the suspensor cells at the base of immature embryos or from the cotyledons of immature embryos, started after 1 - 2 weeks. As the callus started to proliferate, three types of calli were distinguished; white mucilaginous, loose globular, and light green-locee. Histoehemical staining, with acetocarmine, indicated that only white mucilaginous calli were embryogenic. Non~mbryogenic loose globular and light green-loose ralii continued to retain their nature even after several subcultures. The origin of somatic preembryce in P / n ~ elliottii was from single cells of the snspensor cell callus. The developmental stage of the zygotic immature embryoe, genotype and culture medium are critical factors for the production of embryogenic callus. Somatic preembryos are being cultured for further development.
Key Ioo~ls: slash pine; P/nu# eUiottii; somatic embryogenesis; somatic proembryos; single cell origin
Introduction Micropropagation of woody plants can be achieved by induction of adventitious buds, growth of axillary buds or somatic embryogenesis [1--7]. Recent successes in plant regeneration via somatic embryogenesis in Picea abies [2,8--11], Picea glauca [12,13], Picea mm~ana [13], Lariz decidua [14] and Pinus lambertiana [15] have increased the prospects for rapid progress in tree improvement. Somatic embryogenesis in conifers was first reported in Picea abies from immature embryos [16] which lead to further development into plantlets [17,18]. Recently, somatic embryos were readily induced in embryogenic callus from mature embryo explants of Picea abies [2,9,10], Pinus lambertiana [19] and Picea sitchensis [4]. yon Arnold [9] reported induction of embryogenic callus from mature embryos of *Present address: Tea Raseareh Association, Toekali Experiment Station, Jorhat, Assam, 785008 (India) **To whom cerrespendenee should be sent.
Picea abies by varying the composition of the culture medium. However, to this date immature embryos appear to be the most suitable source for initiation of embryogenic callus in both angiosperms [20-22] and gymnosperms
[5]. Embryogenic callus in conifers is white, glossy, translucent and mucilaginous and contains numerous somatic embryos of different sizes which subsequently develop into plantlets. It can be easily distinguished from nonembryogenic callus in Picea abies [2], Pinus taeda [23], Picea mariana and Picea glauca [12,13], and Picea sitchensis [4] and serves as a source from which embryogenic cell suspension cultures for protoplast isolation, regeneration, and gene transfer can be obtained. The regeneration of somatic embryos from protoplasts, derived from embryogenic cell suspension cultures of Pinus taeda [23], Santalum album [24] and Picea glauca [25~28] has been achieved. Kartha et al. [27] regenerated somatic embryos and plantlets from a cryopreserved embryogenic cell suspension of Picea glauco.
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
234 Recently, a luciferase reporter gene has been successfully introduced into protoplasts of Pinus taeda and Pseudotsuga menziesii by electroporation [5]. Somatic embryoids can be used for artificial seed production [28,29] and cryopreservation [30]. Since somatic embryos arise from a single cell [31,32], the regenerated plants are uniform and have less chromosomal variation [33,34]. Therefore, somatic embryos may be used for the clonal propagation. The purpose of this study was to induce somatic embryogenesis from immature zygotic embryo explants of slash pine (Pinus eUiottii). This is the first report of the induction of somatic embryogenesis in Pinus elliotti£ Materials and Methods
Plant materials Green seed cones of 4 different genotypes ($5PC7, D12PCT, A1PC1, and $4PC3) of slash pine (Pinus eUiottii) were collected every week in the morning hours (09.00--11.00 h) during the zygotic embryo maturation period (June 3 r d - J u l y 29th, 1988). The trees were located in a Texas Forest Service orchard near Alto, Texas. All cones were stored in plastic bags at 4 °C for a few days to 3 months. The developmental stage of embryos was checked after every collection. Cones were thoroughly washed with 75% (v/v) ethanol and seeds were removed from the cones with a sharp scalpel. Seeds were surface sterilized in 10% (v/v) chlorox for 15 min and washed 3 times in sterile distilled water. Immature embryos were aseptically dissected from the seeds under a binocular microscope and placed horizontally on a solidified culture medium in petri dishes. Culture conditio,ts Several basal culture media were tested, either at full or half strength (Table I), for the induction of embryogenic callus from immature embryo explants. The pH of the medium was adjusted to 5.7 with 1 N KOH or HC1 before autoclaving at 120 °C and solidified with 0.3--
0.4°/0 gelrite. Filter-sterilized 1 ILM ABA (abscisic acid) was added to the cooled medium after autoclaving. For somatic embryo development, embryogenic callus was cultured on WPMG medium [35], containing: 5 /~I 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 ttM BAP (NB-benzyladenine); 1 tam 2,4-D and 0.5 ttM BAP; 1 tam ABA. Some of the embryogenic callus was transferred to WPMGN and DCRGN solid media (Table I). Fifteen immature embryo explants from different developmental stages were cultured in 100 x 20 mm petri dishes, and at least 50 embryos per treatment were used. The petri dishes were sealed with parafilm and incubated at 25°C in the dark. The tissues were subcultured on fresh medium after 10-- 12 days.
Histochemical staining The embryogenic nature of callus was determined by staining with 20/0 (w/v) acetocarmine [23]; the preparations were examined under a light microscope and photographed. Results and Discussion
Pinus eUiottii cones were collected once a week, starting from June 3rd, 1988. Inspection of cones collected on June 17th, 1988 demonstrated that fertilization had occurred. Developing immature embryos were isolated from cones collected on June 17th, 1988 through July 29th, 1988, and were cultured on several basal culture media (Table I). Most of the immature seeds contained several embryos, but as they matured, one continued to develop further while the others remained arrested. These results are due to polyembryogeny, a well known phenomenon in conifers which is due to fertilization of several archegonia. Similar observations were recorded in immature embryos of Pinus taeda and Pinus virginiana (personal observation). The size of the embryos differed among seeds within the same green cone of each genotype. Embryo length varied from less than 1--3 mm in cones collected on July 15th, 1988. Cones collected earlier than
235 Table I.
Basal media tested for the induction of somatic embryogenesis from immature embryos of Pinus elliotti~ BSO, DLbuthionine sulfoxime; NAA, a-naphthaleneaceti¢ acid; IBA, indole-3-butyric acid.
Medium
Hormone composition
ReL
DCRG WPMG" DCRGN WPMGN NSIII MNCI MSII(li2b}
20 ~ 2,4-D + 5 ~ BAP + 250 mg/l glutamine 20 ~uM2,4-D + 5 pM BAP + 250 mg[l glutamine 1 ~M NAA + 1 ~M ABA + 0.1 ~ BSO 1 ILMNAA + 1 ~uMABA + 0.1 pM BSO 10 ~aM2,4-D + 5 ~M BAP 20 ~M 2,4-D + 2.5 ~M BAP + 2.5 pM kinetin 50 ~ 2,4-D + 20 ~M BAP + 20 ~M kinetin + 450 mg/l glutamine 10 ~ 2,4-D ÷ 5 ~M IBA 20 I~I 2,4-D + 5 ~M BAP
Gupta and Durzan [15] Lloyd and McCown [35] Gupta and Durzan [15] Gupta and Durzan [15] Jain et al. [2] See Table II of present paper Gupta and Durzan [23]
MS(1/2b}" NS(1]2b)b
Murashige and Skoog [40] Lu and Thorpe [12]
"WPM and MS powder media were purchased from Carolina Biological Supply Company, NC, U.S.A. bComposition of modified sugar solution in NS(1/2b) medium: D-glucose, 180 mg/l; L-maltose, 360 mg/1; L-mannose, 150 rag/I; D-xylose, 150 rag/l; L-galactose, 180 rag/l; D-arabinose, 150 rag/l; L-fructose, 180 rag/1.
July 15th, 1988, contained mostly developed immature embryos with suspensor cells. Embryos varying in length responded differently in the production of embryogenic callus. Immature embryo explants isolated from cones collected during June 1 7 t h - J u l y 8th, 1988, did not produce callus on any of the media tested (Table I). This was probably because the immature embryos were either not in the right stage of development or were desiccated during isolation from the cones. The length of these immature embryos was less than 1 mm and in most cases only suspensor cells were visible. It appeared that zygotic immature embryos, with the attached suspensor cells, were essential for callus production. After 1 week, proembryos were seen in cultured immature embryo explants; however, they did not divide further to form callus and eventually turned brown. Suspensor cells began to turn 'rubbery' after 1 week. After 1 - 2 weeks, callus development had begun in the immature embryo explants (July 15th-29th, 1988) of four different genotypes ($5PC7, D12PC7, A1PC1 and $4PC3) on WPMG, MNCI (Table II), DCRG and MSII(1/2b) media containing both auxin and cytokinin (Fig. 1). As the callus started to proliferate, three types of calli could be distinguished: white mucilaginous (Fig. 2), loose globular, and light
green-loose, Jain et al. [2] also observed these three types of calli in cultured Picea abies mature embryos. Similarly, Hakman et al. [16] recovered three types of tissues from immature embryo explants of Picea abies - a bright green callus that consisted of rounded small cells, a green compact tissue often found to be covered with needles and bud-like structures, and a translucent and friable white callus with organized structures. White mucilaginous callus was initiated from 6% and 20/0 of the $5PC7 immature embryos on WPMG and MNCI media, respectively, and from 20/0, 20/0 and 4% of the D12PCT, A1PC1 and $4PC3 embryo explants on DCRG, WPMG and MNCI media, respectively. Loose globular and light green-loose calli developed from embryos ( 2 - 3 mm long) in all four genotypes on all tested media. Well developed embryos with cotyledons ( 3 - 5 mm long) started to swell after 1 week on all media tested (Table I), and they became 'sticky' and 'rubbery'. Most of the swollen embryo explants did not produce any callus, even after several subcultures onto the fresh media. Embryogenic callus induction in Pinus taeda and Pinus virginiana was not observed in any of the media tested (Table I) (personal observation). White mucilaginous callus was translucent, loose and glossy and seemed to develop from
236 Table II. Compositionof the new medium(MNCI). Constituents Cone. (mg/l) Macroelements NH,NOs KNOs MgSOc7H20 KHzPO, CaCl,.2H,0 Ca(NOs).4HzO KC1
400 2000 320 170 170 550 750
Microelements KI H~BO~ MnSOcHzO ZnSO¢7H20 NarMoO,2HsO CuSO,.SHtO CoC1~.6H20
0.83 3 10 3 0.25 0.025 0.025
FeSO¢7HzO NazEDTA
27.8 37.3
Vitamins Nicotinic aci Pyridoxine- HCI Thiamine-- HCI Lysine Sugars D-Xylose ~Glucose ~Arabinose L.Maltose L.Galactose L.Fruetose L~Mannose ~Suerose Amino acids l~Glutamine L-Alanine L-Cysteine-- HCI L-Arginine L~Leucine I~Phenylalanine L~Tyrosine Glyeine Myo-inositol Casein hydrolysate Gelrite
0.5 0.5 5.0 100 150 180 150 360 180 180 150 30 000
200 0.05 0.02 0.01 0.01 0.01 0.01 2 200 500 4000
the suspensor cells at the base of the developing embryo head, about I mm or less in length. Loose globular and light green-loose calli formed from the cotyledons as well as from the hypocotyl of the explants. Nagmani et al. [32] demonstrated, in Picea glauca and Picea abies, that callus arising from the radicle of the zygotic embryo is non~embryogenic while that from the hypocotyl is embryogenic. Staining with acetocarmine indicated that the loose globular and light green-loose calli were non-embryogenic. Similar results were obtained with mature embryo explants of Picea abies [2]. White mucilaginous callus was embryogenic, staining red with acetocarmine. Cells of this callus were long and narrow. In conifers, the morphology of embryogenic callus is white mucilaginous, glossy and translucent, containing numerous somatic embryos of different sizes, which later develop into plantlets [4,13,23]. Loose globular and light green-loose calli were subcultured several times after 1 0 - 1 2 days on the fresh culture media. Some of the calli were subcultured on WPMG and DCRG media containing 10 ~M 2,4-D and 5 I~M BAP; 5 2,4-D and 1/aM BAP; I pM 2,4-D and 0.5 pM BAP, and on WPMGN and DCRGN media. After 4 - 5 weeks, these calli remained nonembryogenic in nature. Similar results were observed in both Pinus taeda and Pinus virgin~aria (pers. observation). The formation of three types of calli under identical culture conditions demonstrated the presence of different cell populations in the explants which respond differentially to exogenous plant growth regulators [36]. It appears that only certain types of cells respond by the formation of embryogenic callus under appropriate culture conditions. The high concentrations of auxin and cytokinins in MSII(1/2b) (Table I) were harmful to the immature embryo explants and caused them to turn brown. Low concentrations of growth hormones were optimal for embryogenic callus production. Similar responses were seen in both Pinus virginiana and Pinus taeda except formation of embryogenic callus (personal observation).
237
Fig. 1. Im'tiation of embryogenie callus from immature embryo explants of genotype $5PC7 after 10 days on WPMG medium (bar ffi 25 mm). Fig. 2. White, mucilaginous embryogenic callus developed from immature embryos of genotype $5PC7 after 30 days on WPMG medium (bar = 25 ram).
Formation of proembryos Two-celled somatic proembryos were formed by a quantal or unequal cell division in an embryogenic cell (Fig. 3). This type of cell division gave rise to a distal, small semicircular embryonal initial cell with dense cytoplasmic contents, and a large proximal suspensor initital cell. Similar results were obtained by Nagmani et al. [32] in Picea abies and Picea glauca. They have further suggested that the origin of somatic embryos in Picea abies and Picea glauca can be traced to single cells and that
these cells do not undergo any early free nuclear divisions that are characteristic of early zygotic embryogenesis in Picea smithiana and Abies p/ndrow [37]. Instead, the single cells divided by quantal cell division that produced two unequal daughter cells with an unequal distribution of cytoplasmic contents, thus forming the embryonal and suspensor initials [32]. Embryonal initial cells divided further into 2 cells (Fig. 4) and subsequently divided into several cells, resulting in an early somatic proembryo (Figs. 5 and 6) with a suspensor ini-
Fig. 3. A two-celled somatic proembryo with embryonal and suspensor initial cells of genotype $5PC7 after 30 days on WPMG medium (bar = 0.45 mm). Fig, 4. Division of an embryonal initial cell of genotype $5PC7 after 35 days on WPMG medium (bar = 0.45 ram).
238
Figs. 5--6. Development of somatic proembryo with several cells of genotype $5PC7 after 40 days on WPMG medium (bar = 0.45mm).
tial cell. However, there was no further indication of division of suspensor initial cells.
Effect of genotypes and media Out of four genotypes tested, $5PC7 responded best with 60/0 of the immature embryo explants producing embryogenic callus on WPMG medium. Other genotypes (D12PC7, A1PC1 and $4PC3) responded in lower frequency. But $4PC3 genotype was more effective (4%) than $5PC7 (20/0) on MNCI medium in forming the embryogenic callus from immature embryos compared to $4PC3 (0%). The genotype, A1PC1 responded with 2% on WPMG medium. These results indicate that WPMG and MNCI media appear to be suitable for the initiation of somatic embryogenesis from immature embryos of $5PC7, $4PC3 and AIPC1 genotypes of Pinus eUiotti. Culture medium affects the behavior of genotypes for somatic embyrogenesis. There may be a genetic control of somatic embryogenesis, possibly by one or only a few genes, which are expressed under certain culture conditions. Genes that control culture characteristics for somatic embryogenesis and their location within the genome are yet to be identified [2]. The frequency of embryogenic callus formation is rather low in all the genotypes tested.
Further modifications in the culture medium may be necessary for improving the efficiency of immature embryo explants for the production of embryogenic callus. Jain et al. [2] suggested that genotype, controlled pollination and pre-conditioning of the explants are of tremendous importance in the initiation and enhancement of somatic embryogenesis. The evaluation of different genotypes, as well as the effect of the growth conditions is important for the efficient formation of embryogenic callus [13]. However, Mathias and Simpson [38] suggested that the genotype may be a more significant factor affecting culture response than the medium. Our results indicate that the developmental stage of the zygotic immature embryos, genotype and culture medium are important factors for the production of somatic embryogenic callus.
Development of somatic embryos For the development of somatic embryos, embryogenic callus was subcultured on WPMGN and WPMG medium containing 1 ABA, 5 / ~ I 2,4-D and 1 / ~ I BAP and 1/~M 2,4-D and 0.5 ~M BAP. Embryogenic callus was subcultured on the fresh medium after 10-- 12 days and kept in the dark. After 3 weeks, embryogenic callus did not indicate the formation of
239
7--9. 1.8 ram).
A small somatic proembryo head with suspensor cells of genotype $4PC3 after 30 days on MNCI medium (bar =
Fig. 10. Two somatic proembryo6 developed on the same suspensor cells in opposite directions (genotype $4PC3 after 30 days on MNCI medium) (bar ffi 1.8 ram).
an embryonal mass of cells in any of the media tested. Histochemical staining with acetocarmine showed that most of the embryonal cells did not develop into somatic embryos. However, in some cases, the development of somatic proembryos with suspensor cells was observed (Figs. 7-9). The development of somatic proembryos, with two embryo heads on opposite sides of the same suspensor cells, was observed (Fig. 10). Hakman et al. [39] also observed two somatic embryos sharing a common suspensor in Picea glauca. We are further investigating the formation of somatic embryos.
and C.R. McKinley of the Texas Forest Service for providing the plant material for this study and Ms. L. McGee for typing the manuscript. This research was supported by the Texas Agriculture Experiment Station ERA Program. TAES Technical Article No. TA24223. References 1
2
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Acknowledgements The authors thank Drs. J.P. van Buijtenen
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Elsevier Scientific Publ/shers Ireland Ltd.
SOMATIC EMBRYOGENESIS IN SLASH P I N E (PINUS ELLIOTTII) FROM I M M A T U R E EMBRYOS CULTURED IN VITRO
S. MOHAN JAIN*, NIU DONG and R j . NEWTON**
Depa~me#t of Forest So&nee, Tezaa Ay~icultunsl Ezperiment Station, The Tezas A&M University Sy#tem, College Statio~ TX 77843 tTf..S.A.) (Received January 11th, 1989) (Revision received June 28th, 1989) (Aeceptod July 18th, 1989) Embryogenic callus development was initiated in cultured immature embryo explants of four different genotypes of slash pine (P/m~s eUiottii) on several culture media supplemented with auxin and cytokinin. Callus induction, either from the suspensor cells at the base of immature embryos or from the cotyledons of immature embryos, started after 1 - 2 weeks. As the callus started to proliferate, three types of calli were distinguished; white mucilaginous, loose globular, and light green-locee. Histoehemical staining, with acetocarmine, indicated that only white mucilaginous calli were embryogenic. Non~mbryogenic loose globular and light green-loose ralii continued to retain their nature even after several subcultures. The origin of somatic preembryce in P / n ~ elliottii was from single cells of the snspensor cell callus. The developmental stage of the zygotic immature embryoe, genotype and culture medium are critical factors for the production of embryogenic callus. Somatic preembryos are being cultured for further development.
Key Ioo~ls: slash pine; P/nu# eUiottii; somatic embryogenesis; somatic proembryos; single cell origin
Introduction Micropropagation of woody plants can be achieved by induction of adventitious buds, growth of axillary buds or somatic embryogenesis [1--7]. Recent successes in plant regeneration via somatic embryogenesis in Picea abies [2,8--11], Picea glauca [12,13], Picea mm~ana [13], Lariz decidua [14] and Pinus lambertiana [15] have increased the prospects for rapid progress in tree improvement. Somatic embryogenesis in conifers was first reported in Picea abies from immature embryos [16] which lead to further development into plantlets [17,18]. Recently, somatic embryos were readily induced in embryogenic callus from mature embryo explants of Picea abies [2,9,10], Pinus lambertiana [19] and Picea sitchensis [4]. yon Arnold [9] reported induction of embryogenic callus from mature embryos of *Present address: Tea Raseareh Association, Toekali Experiment Station, Jorhat, Assam, 785008 (India) **To whom cerrespendenee should be sent.
Picea abies by varying the composition of the culture medium. However, to this date immature embryos appear to be the most suitable source for initiation of embryogenic callus in both angiosperms [20-22] and gymnosperms
[5]. Embryogenic callus in conifers is white, glossy, translucent and mucilaginous and contains numerous somatic embryos of different sizes which subsequently develop into plantlets. It can be easily distinguished from nonembryogenic callus in Picea abies [2], Pinus taeda [23], Picea mariana and Picea glauca [12,13], and Picea sitchensis [4] and serves as a source from which embryogenic cell suspension cultures for protoplast isolation, regeneration, and gene transfer can be obtained. The regeneration of somatic embryos from protoplasts, derived from embryogenic cell suspension cultures of Pinus taeda [23], Santalum album [24] and Picea glauca [25~28] has been achieved. Kartha et al. [27] regenerated somatic embryos and plantlets from a cryopreserved embryogenic cell suspension of Picea glauco.
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
234 Recently, a luciferase reporter gene has been successfully introduced into protoplasts of Pinus taeda and Pseudotsuga menziesii by electroporation [5]. Somatic embryoids can be used for artificial seed production [28,29] and cryopreservation [30]. Since somatic embryos arise from a single cell [31,32], the regenerated plants are uniform and have less chromosomal variation [33,34]. Therefore, somatic embryos may be used for the clonal propagation. The purpose of this study was to induce somatic embryogenesis from immature zygotic embryo explants of slash pine (Pinus eUiottii). This is the first report of the induction of somatic embryogenesis in Pinus elliotti£ Materials and Methods
Plant materials Green seed cones of 4 different genotypes ($5PC7, D12PCT, A1PC1, and $4PC3) of slash pine (Pinus eUiottii) were collected every week in the morning hours (09.00--11.00 h) during the zygotic embryo maturation period (June 3 r d - J u l y 29th, 1988). The trees were located in a Texas Forest Service orchard near Alto, Texas. All cones were stored in plastic bags at 4 °C for a few days to 3 months. The developmental stage of embryos was checked after every collection. Cones were thoroughly washed with 75% (v/v) ethanol and seeds were removed from the cones with a sharp scalpel. Seeds were surface sterilized in 10% (v/v) chlorox for 15 min and washed 3 times in sterile distilled water. Immature embryos were aseptically dissected from the seeds under a binocular microscope and placed horizontally on a solidified culture medium in petri dishes. Culture conditio,ts Several basal culture media were tested, either at full or half strength (Table I), for the induction of embryogenic callus from immature embryo explants. The pH of the medium was adjusted to 5.7 with 1 N KOH or HC1 before autoclaving at 120 °C and solidified with 0.3--
0.4°/0 gelrite. Filter-sterilized 1 ILM ABA (abscisic acid) was added to the cooled medium after autoclaving. For somatic embryo development, embryogenic callus was cultured on WPMG medium [35], containing: 5 /~I 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 ttM BAP (NB-benzyladenine); 1 tam 2,4-D and 0.5 ttM BAP; 1 tam ABA. Some of the embryogenic callus was transferred to WPMGN and DCRGN solid media (Table I). Fifteen immature embryo explants from different developmental stages were cultured in 100 x 20 mm petri dishes, and at least 50 embryos per treatment were used. The petri dishes were sealed with parafilm and incubated at 25°C in the dark. The tissues were subcultured on fresh medium after 10-- 12 days.
Histochemical staining The embryogenic nature of callus was determined by staining with 20/0 (w/v) acetocarmine [23]; the preparations were examined under a light microscope and photographed. Results and Discussion
Pinus eUiottii cones were collected once a week, starting from June 3rd, 1988. Inspection of cones collected on June 17th, 1988 demonstrated that fertilization had occurred. Developing immature embryos were isolated from cones collected on June 17th, 1988 through July 29th, 1988, and were cultured on several basal culture media (Table I). Most of the immature seeds contained several embryos, but as they matured, one continued to develop further while the others remained arrested. These results are due to polyembryogeny, a well known phenomenon in conifers which is due to fertilization of several archegonia. Similar observations were recorded in immature embryos of Pinus taeda and Pinus virginiana (personal observation). The size of the embryos differed among seeds within the same green cone of each genotype. Embryo length varied from less than 1--3 mm in cones collected on July 15th, 1988. Cones collected earlier than
235 Table I.
Basal media tested for the induction of somatic embryogenesis from immature embryos of Pinus elliotti~ BSO, DLbuthionine sulfoxime; NAA, a-naphthaleneaceti¢ acid; IBA, indole-3-butyric acid.
Medium
Hormone composition
ReL
DCRG WPMG" DCRGN WPMGN NSIII MNCI MSII(li2b}
20 ~ 2,4-D + 5 ~ BAP + 250 mg/l glutamine 20 ~uM2,4-D + 5 pM BAP + 250 mg[l glutamine 1 ~M NAA + 1 ~M ABA + 0.1 ~ BSO 1 ILMNAA + 1 ~uMABA + 0.1 pM BSO 10 ~aM2,4-D + 5 ~M BAP 20 ~M 2,4-D + 2.5 ~M BAP + 2.5 pM kinetin 50 ~ 2,4-D + 20 ~M BAP + 20 ~M kinetin + 450 mg/l glutamine 10 ~ 2,4-D ÷ 5 ~M IBA 20 I~I 2,4-D + 5 ~M BAP
Gupta and Durzan [15] Lloyd and McCown [35] Gupta and Durzan [15] Gupta and Durzan [15] Jain et al. [2] See Table II of present paper Gupta and Durzan [23]
MS(1/2b}" NS(1]2b)b
Murashige and Skoog [40] Lu and Thorpe [12]
"WPM and MS powder media were purchased from Carolina Biological Supply Company, NC, U.S.A. bComposition of modified sugar solution in NS(1/2b) medium: D-glucose, 180 mg/l; L-maltose, 360 mg/1; L-mannose, 150 rag/I; D-xylose, 150 rag/l; L-galactose, 180 rag/l; D-arabinose, 150 rag/l; L-fructose, 180 rag/1.
July 15th, 1988, contained mostly developed immature embryos with suspensor cells. Embryos varying in length responded differently in the production of embryogenic callus. Immature embryo explants isolated from cones collected during June 1 7 t h - J u l y 8th, 1988, did not produce callus on any of the media tested (Table I). This was probably because the immature embryos were either not in the right stage of development or were desiccated during isolation from the cones. The length of these immature embryos was less than 1 mm and in most cases only suspensor cells were visible. It appeared that zygotic immature embryos, with the attached suspensor cells, were essential for callus production. After 1 week, proembryos were seen in cultured immature embryo explants; however, they did not divide further to form callus and eventually turned brown. Suspensor cells began to turn 'rubbery' after 1 week. After 1 - 2 weeks, callus development had begun in the immature embryo explants (July 15th-29th, 1988) of four different genotypes ($5PC7, D12PC7, A1PC1 and $4PC3) on WPMG, MNCI (Table II), DCRG and MSII(1/2b) media containing both auxin and cytokinin (Fig. 1). As the callus started to proliferate, three types of calli could be distinguished: white mucilaginous (Fig. 2), loose globular, and light
green-loose, Jain et al. [2] also observed these three types of calli in cultured Picea abies mature embryos. Similarly, Hakman et al. [16] recovered three types of tissues from immature embryo explants of Picea abies - a bright green callus that consisted of rounded small cells, a green compact tissue often found to be covered with needles and bud-like structures, and a translucent and friable white callus with organized structures. White mucilaginous callus was initiated from 6% and 20/0 of the $5PC7 immature embryos on WPMG and MNCI media, respectively, and from 20/0, 20/0 and 4% of the D12PCT, A1PC1 and $4PC3 embryo explants on DCRG, WPMG and MNCI media, respectively. Loose globular and light green-loose calli developed from embryos ( 2 - 3 mm long) in all four genotypes on all tested media. Well developed embryos with cotyledons ( 3 - 5 mm long) started to swell after 1 week on all media tested (Table I), and they became 'sticky' and 'rubbery'. Most of the swollen embryo explants did not produce any callus, even after several subcultures onto the fresh media. Embryogenic callus induction in Pinus taeda and Pinus virginiana was not observed in any of the media tested (Table I) (personal observation). White mucilaginous callus was translucent, loose and glossy and seemed to develop from
236 Table II. Compositionof the new medium(MNCI). Constituents Cone. (mg/l) Macroelements NH,NOs KNOs MgSOc7H20 KHzPO, CaCl,.2H,0 Ca(NOs).4HzO KC1
400 2000 320 170 170 550 750
Microelements KI H~BO~ MnSOcHzO ZnSO¢7H20 NarMoO,2HsO CuSO,.SHtO CoC1~.6H20
0.83 3 10 3 0.25 0.025 0.025
FeSO¢7HzO NazEDTA
27.8 37.3
Vitamins Nicotinic aci Pyridoxine- HCI Thiamine-- HCI Lysine Sugars D-Xylose ~Glucose ~Arabinose L.Maltose L.Galactose L.Fruetose L~Mannose ~Suerose Amino acids l~Glutamine L-Alanine L-Cysteine-- HCI L-Arginine L~Leucine I~Phenylalanine L~Tyrosine Glyeine Myo-inositol Casein hydrolysate Gelrite
0.5 0.5 5.0 100 150 180 150 360 180 180 150 30 000
200 0.05 0.02 0.01 0.01 0.01 0.01 2 200 500 4000
the suspensor cells at the base of the developing embryo head, about I mm or less in length. Loose globular and light green-loose calli formed from the cotyledons as well as from the hypocotyl of the explants. Nagmani et al. [32] demonstrated, in Picea glauca and Picea abies, that callus arising from the radicle of the zygotic embryo is non~embryogenic while that from the hypocotyl is embryogenic. Staining with acetocarmine indicated that the loose globular and light green-loose calli were non-embryogenic. Similar results were obtained with mature embryo explants of Picea abies [2]. White mucilaginous callus was embryogenic, staining red with acetocarmine. Cells of this callus were long and narrow. In conifers, the morphology of embryogenic callus is white mucilaginous, glossy and translucent, containing numerous somatic embryos of different sizes, which later develop into plantlets [4,13,23]. Loose globular and light green-loose calli were subcultured several times after 1 0 - 1 2 days on the fresh culture media. Some of the calli were subcultured on WPMG and DCRG media containing 10 ~M 2,4-D and 5 I~M BAP; 5 2,4-D and 1/aM BAP; I pM 2,4-D and 0.5 pM BAP, and on WPMGN and DCRGN media. After 4 - 5 weeks, these calli remained nonembryogenic in nature. Similar results were observed in both Pinus taeda and Pinus virgin~aria (pers. observation). The formation of three types of calli under identical culture conditions demonstrated the presence of different cell populations in the explants which respond differentially to exogenous plant growth regulators [36]. It appears that only certain types of cells respond by the formation of embryogenic callus under appropriate culture conditions. The high concentrations of auxin and cytokinins in MSII(1/2b) (Table I) were harmful to the immature embryo explants and caused them to turn brown. Low concentrations of growth hormones were optimal for embryogenic callus production. Similar responses were seen in both Pinus virginiana and Pinus taeda except formation of embryogenic callus (personal observation).
237
Fig. 1. Im'tiation of embryogenie callus from immature embryo explants of genotype $5PC7 after 10 days on WPMG medium (bar ffi 25 mm). Fig. 2. White, mucilaginous embryogenic callus developed from immature embryos of genotype $5PC7 after 30 days on WPMG medium (bar = 25 ram).
Formation of proembryos Two-celled somatic proembryos were formed by a quantal or unequal cell division in an embryogenic cell (Fig. 3). This type of cell division gave rise to a distal, small semicircular embryonal initial cell with dense cytoplasmic contents, and a large proximal suspensor initital cell. Similar results were obtained by Nagmani et al. [32] in Picea abies and Picea glauca. They have further suggested that the origin of somatic embryos in Picea abies and Picea glauca can be traced to single cells and that
these cells do not undergo any early free nuclear divisions that are characteristic of early zygotic embryogenesis in Picea smithiana and Abies p/ndrow [37]. Instead, the single cells divided by quantal cell division that produced two unequal daughter cells with an unequal distribution of cytoplasmic contents, thus forming the embryonal and suspensor initials [32]. Embryonal initial cells divided further into 2 cells (Fig. 4) and subsequently divided into several cells, resulting in an early somatic proembryo (Figs. 5 and 6) with a suspensor ini-
Fig. 3. A two-celled somatic proembryo with embryonal and suspensor initial cells of genotype $5PC7 after 30 days on WPMG medium (bar = 0.45 mm). Fig, 4. Division of an embryonal initial cell of genotype $5PC7 after 35 days on WPMG medium (bar = 0.45 ram).
238
Figs. 5--6. Development of somatic proembryo with several cells of genotype $5PC7 after 40 days on WPMG medium (bar = 0.45mm).
tial cell. However, there was no further indication of division of suspensor initial cells.
Effect of genotypes and media Out of four genotypes tested, $5PC7 responded best with 60/0 of the immature embryo explants producing embryogenic callus on WPMG medium. Other genotypes (D12PC7, A1PC1 and $4PC3) responded in lower frequency. But $4PC3 genotype was more effective (4%) than $5PC7 (20/0) on MNCI medium in forming the embryogenic callus from immature embryos compared to $4PC3 (0%). The genotype, A1PC1 responded with 2% on WPMG medium. These results indicate that WPMG and MNCI media appear to be suitable for the initiation of somatic embryogenesis from immature embryos of $5PC7, $4PC3 and AIPC1 genotypes of Pinus eUiotti. Culture medium affects the behavior of genotypes for somatic embyrogenesis. There may be a genetic control of somatic embryogenesis, possibly by one or only a few genes, which are expressed under certain culture conditions. Genes that control culture characteristics for somatic embryogenesis and their location within the genome are yet to be identified [2]. The frequency of embryogenic callus formation is rather low in all the genotypes tested.
Further modifications in the culture medium may be necessary for improving the efficiency of immature embryo explants for the production of embryogenic callus. Jain et al. [2] suggested that genotype, controlled pollination and pre-conditioning of the explants are of tremendous importance in the initiation and enhancement of somatic embryogenesis. The evaluation of different genotypes, as well as the effect of the growth conditions is important for the efficient formation of embryogenic callus [13]. However, Mathias and Simpson [38] suggested that the genotype may be a more significant factor affecting culture response than the medium. Our results indicate that the developmental stage of the zygotic immature embryos, genotype and culture medium are important factors for the production of somatic embryogenic callus.
Development of somatic embryos For the development of somatic embryos, embryogenic callus was subcultured on WPMGN and WPMG medium containing 1 ABA, 5 / ~ I 2,4-D and 1 / ~ I BAP and 1/~M 2,4-D and 0.5 ~M BAP. Embryogenic callus was subcultured on the fresh medium after 10-- 12 days and kept in the dark. After 3 weeks, embryogenic callus did not indicate the formation of
239
7--9. 1.8 ram).
A small somatic proembryo head with suspensor cells of genotype $4PC3 after 30 days on MNCI medium (bar =
Fig. 10. Two somatic proembryo6 developed on the same suspensor cells in opposite directions (genotype $4PC3 after 30 days on MNCI medium) (bar ffi 1.8 ram).
an embryonal mass of cells in any of the media tested. Histochemical staining with acetocarmine showed that most of the embryonal cells did not develop into somatic embryos. However, in some cases, the development of somatic proembryos with suspensor cells was observed (Figs. 7-9). The development of somatic proembryos, with two embryo heads on opposite sides of the same suspensor cells, was observed (Fig. 10). Hakman et al. [39] also observed two somatic embryos sharing a common suspensor in Picea glauca. We are further investigating the formation of somatic embryos.
and C.R. McKinley of the Texas Forest Service for providing the plant material for this study and Ms. L. McGee for typing the manuscript. This research was supported by the Texas Agriculture Experiment Station ERA Program. TAES Technical Article No. TA24223. References 1
2
3
Acknowledgements The authors thank Drs. J.P. van Buijtenen
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