Immature zygotic embryo explants (isolated or with intact megagametophytes) from 10 loblolly pine (Pinustaeda L.) clones (7-34, 7-56, 11-9, 11-16, 11-25, 10-1003, 10-1007, 10-1011, 10-1018, and 10-1019) were surveyed for their potential to form embryogenic tissue from the suspensor region of zygotic embryos. After over 14 000 explants were cultured, embryogenic cultures were initiated from explants of 8 of the 10 clones; only explants from clones 11-25 and 10-1019 were not responsive. Embryogenic tissue was initiated from zygotic embryos with intact megagametophytes on MSG basal medium with no exogenous plant growth regulators or with 2–5 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0–1 mg/L N6-benzyladenine (BA). The highest initiation frequency (5%) was obtained from isolated zygotic embryos of clone 7-34 less than 0.5 mm in length just prior to cotyledon primordia development on DCR basal medium with 3 mg/L 2,4-D and 0.5 mg/L BA. Two types of embryogenic cultures were maintained on medium with 2,4-D and BA: (i) those that contained pre-embryonal masses of cells interspersed with unaggregated suspensorlike cells, but which rarely contained well-formed somatic embryos, and (ii) those that frequently contained well-formed somatic embryos. Somatic embryo development from both types of cultures progressed to a precotyledonary stage on medium with 2.6 mg/L abscisic acid.
Megagametophytes were excised from immature ovules of Larixdecidua Mill. Archegonia, proembryos or embryos, and suspensors were removed from these gametophytes and the remaining tissue was then cultured on media with various auxins, cytokinins, and organic nitrogen sources. After 8–9 weeks, a few of the cultures produced callus. When subcultured on hormone-free medium, this callus grew rapidly and produced a large number of embryoids with suspensors. Embryogenesis was intense through six to eight successive transfers and then slowed down in subsequent transfers. Many of the embryoids formed plantlets, the ploidy of which still remains to be determined.
Our progress is reviewed on development of somatic embryogenesis in conifers for mass propagation. A distinct embryogenic callus (EC) phenotype, white, mucilaginous, and rapidly growing, has been initiated on modified MS media with 2,4-D or NAA (2-5 mg/L) and BA(0-1 mg/L) from immature embryos of Norway spruce (Picea abies), white spruce (Picea glauca), loblolly pine (Pinus taeda), pond pine (Pinus serotina), and white pine (Pinus strobus). EC has also been initiated from mature embryos of Norway spruce and maintained as rapidly growing (48 hour doubling) liquid suspensions. Initiation of EC in Picea and Pinus differ markedly in several ways. Precotyledonary embryos were optimal in Pinus and EC originated from the suspensor region. In Picea EC originated from the hypocotyl and cotyledon region of predominantly post-cotyledonary embryos. Biochemically, EC of Picea and Pinus were similar and distinctly different from nonembryogenic callus (NEC) in terms of ethylene evolution rates (EC low and NEC high), level of total reductants, including glutathione (EC low and NEC high), and protein synthesis rates (EC high and NEC low). Conifer somatic embryos contained proplastids closely resembling those found in early zygotic embryos. On proliferation medium in the light, EC was white and maintained the proplastid morphology, whereas, NEC was green and contained mature chloroplasts with grana. These biochemical and ultrastructural differences served to both verify and predict embryogenic potential.With Norway spruce somatic embryos, maturation frequencies as high as 25% have been attained. Germination frequencies as high as 82% (mean 56%) have been obtained. Twenty-nine percent of the somatic embryo plantlets survived transfer to the greenhouse, set a dormant terminal bud, overwintered to -5°C, and renewed vegetative growth synchronously with control seedlings. This is the first report of overwintering and renewed vegetative growth from resting buds of conifer somatic embryo plants.
In the summer of 1985, female gametophytic explants of Larixdecidua were cultured on LM medium containing 2,4-D (2.0 mg/L). In the subsequent growing season (1986), various concentrations of 2,4-D (0, 0.2, 2.0, and 20 mg/L) were used as media supplements and evaluated in material collected weekly from early June to late July. Optimal induction of callus and embryogenesis occurred at 2.0 mg/L 2,4-D and depended on collection date. Two kinds of callus were produced, a white embryogenic callus developed from explants from late June onwards, and a green nonembryogenic callus developed from middle to late July. Various concentrations of sucrose and sorbitol were tested in osmotically equivalent combinations together with the optimal level of 2,4-D (2.0 mg/L). The production of embryogenic callus was best on 3% sucrose and no sorbitol. Embryogenic callus developed from both chalazal and micropylar portions of the excised megagametophyte.
Isolated zygotic embryos and female gametophytes containing zygotic embryos were cultured on MSG and DCR basal media, supplemented with three different carbon sources added individually to the medium at four levels each. The media also contained various levels of 2,4-dichlorophenoxy acetic acid (2,4-D) and N6-benzyladenine (BA). Embryogenic tissue extruded from female gametophytes during 4 weeks in culture on media containing either glucose or maltose or sucrose. Embryogenic tissue initiation was most frequently from explants collected on July 14, 1992, when the zygotic embryos within the female gametophytes were precotyledonary. A total of 33 embryogenic cultures were initiated from 944 explants cultured. One of 192 explants cultured on basal media with no growth regulators produced embryogenic tissue. The embryogenic tissue showed numerous somatic embryos at stages 1 and 2 of development, corresponding to their zygotic embryo counterparts.
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