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Abstract
Most horticultural plant breeders probably are skeptical of the “potential for crop improvement by genetic manipulation through the use of protoplasts,” which has been professed optimistically in review papers and at genetic engineering conferences and meetings (45). This attitude may be warranted to some degree (43). Certainly, the ability to manipulate quantitatively inherited traits will be very difficult to achieve. Exogenous DNA may be introduced into protoplasts, but the subsequent integration, phenotypic expression, and sexual transmission of the new DNA are prerequisite to any possible exploitation by plant breeders. Significant advances have been and are being made presently, however, toward the realization of somatic cell genetic manipulations by the use of protoplasts. These efforts should increase genetic diversity of available germplasm and provide more efficient means for handling specific traits at various steps in plant breeding.
The histology and morphology of developing asparagus Asparagus officinalis L.) somatic embryos arising in callus cultures were examined and contrasted with that documented for zygotic embryos. Histological sections of lateral bud-derived callus cultured for 2 weeks on embryo induction medium consisting of Murashige and Skoog salts and vitamins (MS) with 1.5 mg NAA/liter and 0.1 mg kinetin/liter indicated the formation of distinct groups of embryogenic cells. At 4 weeks, the callus was comprised of embryos in the early and late globular stages and a few bipolar embryos. Within 2 weeks on embryo development medium consisting of MS with 0.05 mg NAA/liter and 0.1 mg kinetin/liter, the globular embryos developed a bipolar shape having an expanded upper region that formed the cotyledon and a smaller region that formed the radicle. Within 4 to 6 weeks on this latter medium, each mature bipolar embryo was opaque and had a large cotyledon, a distinct shoot apex at the cotyledon-hypocotyl junction, and vascular connections between the radicle, shoot apex, and cotyledon. Many mature somatic embryos resembled the asparagus zygotic embryos in having a crescent shape, whereas others had a short but wide cotyledon. Both somatic embryo types converted to plantlets at equal rates. Chemical names used: N- (2-furanylmethyl)-1 H -purin-6-amine (kinetin); 1-naphthaleneacetic acid (NAA).
Abstract
Callus cells were used as the cell source for isolating protoplasts of Salpiglossis sinuata L. Callus was initiated on the cut edges of leaf sections cultured on Uchimiya and Murashige (UM) medium under 24 μEm-2s-1 at 30°C. Friable callus from subcultures on UM was subjected to an enzymatic solution of 1% Driselase, 0.75% Pectinase, 2% Cellulase R-10, and 8% mannitol in CPW salts, and incubated at 50 rpm, for 4-5 hr to release protoplasts. Following washing, counting, and dilution, protoplasts were plated in liquid Murashige and Skoog medium (MS) modified by deleting the ammonium ions and adding (mg/liter): 250 L-glutamine, 0.1 L-serine, 2.0 thiamine, 0.5 indoleacetic acid (1AA), 1.0 2,4-D, 0.5 6-benzylamino purine (BA) and 9% mannitol. Within 2-5 days, cell wall synthesis and the first cell division occurred. Green-yellowish colonies, 2 to 3 mm in diameter, formed in 2.5 months and were transferred to MS + 1.0e mg/liter N6-isopentenyladenine (2iP), where shoot primordia were evident within 21 days. After full development and elongation of shoots, they were dipped in 1000 ppm indolebutyric acid (1BA) and placed in MS + 0.001 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) to initiate roots. Rooting was carried out at 22±2°C under 80µm-2s-1 Cool White fluorescent tubes. Nine of 10 regenerated plants examined cytologically were 2n = 44, normal for the species, and 1 plant was 2n =88.
Outstanding asparagus crowns were identified in established Michigan asparagus fields, MSU germplasm, variety trials, or were provided by commercial sources. The single-crown selections were micropropagated to provide cloned plants for the trials. Field trials consisting of four replications of 12 plants each were established at two locations. Crowns were planted 8 inches deep and spaced 18 inches apart in rows 4.5 or 5 feet apart. Five, 37, and 25 selections were planted in 1998, 1989, and 1991, respectively. Plots were not harvested until 2 years after planting, when they had partial harvests of six pickings. In the third and following years, plots received full harvests of 20 to 25 pickings. In the third full harvest at the Hart location, clones Hart-2 and Hart-3 yielded 6989 and 6875 lb/A, respectively, and were significantly more productive than Syn4-56, which had 3720 lb/A. At Benton Harbor, Hart-4 produced 4184 lb/A, significantly higher than the Syn4-56 yield of 3088 lb/A at that location. These significant differences were not observed until the second full harvest.
Abstract
Specific auxin-containing formulations which contained primarily indolebutyric acid (IBA), and/or napthaleneacetic acid (NAA) were most effective in promoting rooting of terminal stem cuttings of poinsettia (Euphorbia pulcherrima Willd.).
Abstract
The isolation, culture, and plant regeneration of protoplasts derived from callus or suspension cultures of Pentunia alpicola Smith and Downs was established. Protoplasts formed macrocalli, at 85% percent plating efficiency, in Murashige and Skoog (MS) liquid medium containing 2,4–D (1.0 mg·liter–1), NAA (0.5 mg·liter–1), BA (0.5 mg·liter–1), and 20% coconut water. Shoot regeneration from macrocalli occurred at 65% frequency on MS containing zeatin (1.0 mg·liter–1), and shoots were readily rooted on either MS containing NAA (0.01 mg·liter–1) or IBA (1.0 mg·liter–1). However, plantlets failed to grow in artificial planting medium. The results provide the basis for somatic cell genetic studies for a Petunia sp. that has distincitive morphology compared to cultivated petunias. Chemical names used: (2,4-dichlorophenoxy)acetic acid (2,4–D), 1-naphthaleneacetic acid (NAA), N-(phenylmethyl)-1H-purin-6-amine (BA), (E)-2-methyl-4-(1H-purin-6-ylamino)-2-buten-l-ol (zeatin), and 1H-indole-3-butyric acid (IBA).
Abstract
Fresh indoleacetic acid oxidase (IAA-OX) preparations from the shoot tips of double flowered genotypes, DD and Dd, and single, dd, flowered petunias (Petunia hybrida Hort.) generally exhibited no lag-phase and had similar rates of IAA destruction. Water-soluble, heat-stable fractions from the 3 genotypes inhibited the destruction of IAA by horseradish peroxidase (HRP) and the IAA-OX preparations. The inhibitor fractions behaved similar to ferulic acid which induced a 10 to 20 minute lag-phase. In all combinations of inhibitor fraction and IAA-OX preparation or HRP, neither the length of the lag-phase nor the subsequent rate of IAA destruction was correlated with the genotypes investigated.
Abstract
Shoot tips of Nicotiana aiata Link & Otto, N. forgetiana Sander and N. sanderae W. Wats seedlings were established on modified Murashige and Skoog (MS) salts and vitamins medium to provide leaf material as the cell source for protoplasts. Viable protoplasts (8.0 × 105 to 1.5 × 106/ml per g fresh weight) were enzymatically isolated from N. alata in 2.5% Driselase plus 4.0–4.7% mannitol or sorbitol in cell protoplast wash solution (CPW). An enzyme mixture of 1.0% Driselase, 1.0% Macerozyme R-10, 1.0% Cellulase R-10, 0.5% potassium dextran sulfate and 4.0% mannitol in CPW released 4.0 × 105 to 1.0 × 106 and 7.6 × 105 to 8.5 × 105/ml protoplasts per g leaf tissue respectively from N. forgetiana and N. sanderae. Only 4 of 13 tested culture media, also exhibiting species selectivity, promoted sustained cell division of protoplasts to the macroscopic callus stage in 28-35 days. Optimum plating efficiency ranged from 15 to 37% when the protoplasts were cultured at 5.0 × 104/ml in Cool White light. Plating efficiency did not increase when cultures were placed under Gro-Lux light. Macroscopic callus was readily regenerated to shoots in 2 months on MS medium + 1.0 mg/liter zeatin (Z) or MS + 2.0 mg/liter idoleacetic acid (I A A) + 1.0 mg/liter benzy lamino purine (BA). Rhizogenesis occurred in hormone-free MS medium. Regenerated plants flowered in the greenhouse and exhibited minor variation in flower form and pigmentation.
Abstract
Three sets of Petunia hybrida Vilm. lines were used with each set comprised of the 3 genotypes, multiflora (gg), grandiflora (GG), and heterozygote (Gg). Seed germination was consistently high for the hybrid Gg (92%), intermediate for gg (77%) and low for GG (45%). The fresh and dry wt of 28-day-old seedlings was inconsistent but the Gg hybrid was the most vigorous at 49 days followed by the gg and GG genotypes. No differences were observed in N, P, K, Na, Mn, Fe, Cu, Zn, or Al in vegetative leaves of the 3 genotypes. Differences in Ca, Mg, and B occurred, but they were not uniform with respect to genotype or to genotypes within a set. Calcium and Mg were generally highest in gg and lowest in GG. Boron in 1 of 2 experiments showed the same pattern. The physiological roles of the observed differences in elemental composition with respect to chlorophyll composition, sugar metabolism, and vigor as indicated by an increase in fresh and dry wt, in the 3 genotypes are discussed.
Abstract
Research efforts aimed at producing haploid plants by tissue culture of anthers or isolated pollen grains have increased recently. Likewise, the ability of the plant breeder to utilize haploid plants in the breeding program has added a new dimension and possible efficiency to the manner in which horticultural crops can be genetically improved. This review will discuss the recent progress that has been made in the art and science of anther and pollen culture to produce haploid plants and the potential for use by horticultural plant breeders. Previous reviews on this subject have been published by Bottino (12), DeBerg (24), Kimber and Riley (49), Melchers (57, 58), Smith (87), Sunderland (89) and the publication Haploids in Higher Plants – Advances and Potential from the first international symposium held in this area of plant biology, at Guelph, Ontario in 1974.