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  • Author or Editor: K. C. Sink x
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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.

Open Access
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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.

Open Access

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.

Open Access

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.

Open Access

Abstract

Rapid in vitro propagation of Phlox subulata L. was achieved by inducing shoot explants, 3.0 to 5.0 mm, to proliferate axillary buds on a basal medium containing Murashige and Skoog (M&S) salts, Nitsch Vitamins and supplemented with 3.5 × 10 3 mg/titer gibberellic acid (GA3), 5.0 mg/liter benzylamino purine (BA), and 40 mg/liter adenine sulfate. GA3 was essential for axillary bud elongation. The proliferating shoots were rooted on a medium con-sisting of M&S salts and Vitamins plus 0.01 to 0.5 mg/liter α-naphthaleneacetic acid (NAA). P. paniculata L. was propagated in vitro by culturing internode stem sections from actively growing shoots on Linsmaier and Skoog (L&S) medium containing L&S salts and vitamins, 10 mg/liter BA, 0.1 mg/liter NAA and 40 mg/liter adenine sulfate. About 65 shoots arose on each stem section and they rooted on L&S plus 1.0 mg/liter NAA. Root initiation was stimu-lated in both Phlox spp. by incubating the cultures at 30°C for 1 week.

Open Access

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 PlantsAdvances and Potential from the first international symposium held in this area of plant biology, at Guelph, Ontario in 1974.

Open Access
Authors: and

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).

Free access

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).

Open Access

Abstract

An investigation of free amino acids, phenols, and indoles from various plant parts of double (DD and Dd) and single (dd) flowered petunia plants was made to determine if a relationship exists between these substances and flower genotype. There was no difference between genotypes in total free amino acid concentration from shoot tips or anthers. Flower buds with the DD genotype and leaves with the Dd genotype were significantly different in total amino acid concentration from similar tissue of the other genotypes. TLC analysis of amino acid extracts from shoot tips, buds, or leaves failed to resolve any qualitative differences between the 3 genotypes. Anthers directly squashed on TLC plates and developed in several solvent systems did not resolve any differences between genotypes. TLC analyses of phenols and indoles indicated they were very similar for the 3 genotypes.

Open Access

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.

Open Access