Feng H. Huang

Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.)

SummaryA culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine.

Polyethylene glycol-promoted development of somatic embryos in loblolly pine (Pinus taeda L.)

SummaryThe effect of polyethylene glycol (PEG) combined with abscisic acid (ABA) and KCl on somatic embryo development in loblolly pine was investigated. Two embryogenic cell lines, which had not produced cotyledonary stage embryos using previously published methods, were employed in this study. As a maturation medium, basal medium was supplemented with 0 to 10% PEG (MW 3350), 10 to 40 mg/l (37.8 to 151.3 µM) ABA, 0 or 10 mM KCl, 1.5 g/l activated charcoal, 30 g/l sucrose, and 6 g/l agar. Without PEG in the maturation medium, most somatic embryos at stage 1 could not mature further. Embryogenic tissues on the maturation medium with 5 to 7.5% PEG consistently produced stage 2 and stage 3 somatic embryos. PEG at 10% significantly decreased the number of stage 2 and 3 embryos compared to PEG at 5 to 7.5% ABA at 40 mg/l (151.3 µM), combined with 7.5% PEG and 10 mM KCl, gave the maximum number of stage 3 embryos in both cell lines. ABA at 10 mg/l (37.8 µM) induced an over-proliferation of embryogenic tissues and generally failed to produce mature embryos. KCl also significantly enhanced initial stage embryo formation and subsequent embryo maturation.

Effect of basal medium, growth regulators and Phytagel concentration on initiation of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L.)

Abstract Low initiation frequency is one of the main barriers in applying somatic embryogenesis to the clonal production of Pinus species. Factors affecting initiation, including basal medium, plant growth regulators, and Phytagel concentration, have been investigated in loblolly pine (Pinus taeda L.). BM1 basal medium proved superior to DCR1 and LP (LP basal salts plus BM1 organic nutrients). No extrusion from megagametophytes was exhibited on LP medium. The combination of 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) resulted in a higher extrusion frequency than that of 11 mg/l 2,4-D, 4.5 mg/l BA and 4.3 mg/l kinetin. Phytagel at 1 g/l resulted in the highest explant browning, but the lowest extrusion frequency, while 4 g/l Phytagel induced some dry embryogenic extrusions. Phytagel at 2 g/l was regarded as the best level for initiation of embryogenic cultures.

Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

SummarySeveral factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory.

Genotype-dependent response of spinach cultivars to in vitro callus induction and plant regeneration

Abstract Callus was induced from leaf explants of four spinach ( Spinacia oleracea L.) cultivars on Murashige-Skoog (MS) medium supplemented with 2 mg 1 −1 6-(furfurylamino)purine (kinetin) and 0.1, 0.5 or 1 mg 1 −1 2,4-dichlorophenoxyacetic acid (2,4-D) either in darkness or exposed to light (50 μE m −2 s −1 ). Callus production was cultivar dependent. Maximum callus proliferation was obtained with 0.5 mg 1 −1 2,4-D, which also gave rise to the highest percentage of shoot formation. Callus induced in darkness exhibited higher regeneration capacity than callus induced in the light. Regeneration capacity of the callus varied among cultivars and was enhanced by the addition of ascorbic acid. Gibberellic acid was required for shoot regeneration. Complete plants were obtained by inducing the shoots to root with 1 mg 1 −1 indole butyric acid (IBA).

In vitro seed production from sex-modified male spinach plants regenerated from callus cultures☆

Abstract Shoots regenerated from leaf-derived callus of spinach (Spinacia oleracea L. cultivar ‘High Pack’) were induced to flower by exposure to 14-h photoperiods (50 μmol m−2 s−1) and day/night temperatures of 20° C 16° C . Leaves from male and female plants served as sources for explants. Shoots regenerated from callus were induced to flower on a hormone-free medium. The majority of regenerated plants exhibited sex corresponding to their respective explant donors; a proportion of regenerants of male origin, however, shifted to monoecious. These sex-modified plantlets developed viable seeds in vitro. This phenomenon can be useful in producing seeds from elite selected male plants.

Micropropagation ofAcacia mearnsii

SummaryA micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 µEm−2·s−1). Multiple shoot formation was promoted by BAP at 2 mg · liter−1 (8.87µM) and higher combined with or without 0.01 mg · liter−1 (0.049µM) IBA. Cytokinins at concentrations of less than 1 mg · liter−1 combined with 0.01 to 0.1 mg · liter−1 auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter−1 BAP and 0.01 mg · liter−1 IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter−1[4.3µM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter−1 (3.22µM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.

In vitro plant regeneration of spinach from mature seed-derived callus

SummaryA system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA3). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.

Micropropagation of Zoysiagrass (Zoysia japonica Steud.)

Zoysiagrass (Zoysia Willd.), chromosome number 20, is a member of the Gramineae family (Forbes 1952) and belongs to the subfamily Eragrostoideae. It originated in tropical eastern Asia and has now been introduced and distributed throughout the warm humid, warm semi-arid, and transitional regions of the world (Hanson et al. 1969; Beard 1973). In the USA, three species of Zoysia are used for turfgrass: Z. japonica Steud., Z. matrella (L.) Merr., and Z. tenuifblia Willd., ex Tring.; respectively, they are known by the common names Japanese lawngrass or Korean lawngrass, Manilagrass, and Mascarenegrass or Korean velvetgrass (Beard 1973).