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Improved in vitro clonal propagation methods are valuable tools for nurseries and growers, and are essential for manipulation and improvement of tree fruit germplasm using the tools and techniques of biotechnology. We have developed a rapid shoot multiplication procedure for clonal propagation of apple, Malus ×domestica cv. Gale Gala and pear, Pyrus communis L. cv. Bartlett. Rapid clonal multiplication was achieved after the following series of steps: pre-conditioning of micropropagated shoots, sectioning pre-treated stems into thin slices, placing slices onto shoot induction medium and incubating directly under cool-white fluorescent lights or after a brief dark incubation. Multiple induction of shoots recovered from stem slice explants within three weeks of culture. A maximum of 37% of cultured apple stem slices, and 97% of pear stem slices, showed induction of shoots. More shoots were recovered on phytagel solidified shoot induction medium than on agar. Cultured stem slices of both apple and pear showed maximum recovery of shoots from shoot induction medium supplemented with thidiazuron (TDZ) compared to medium supplemented with BAP and kinetin. Under ideal conditions, pear stems generated four times the shoots as the same quantity or length of apple shoots. Micropropagated shoots were rooted and transferred to the greenhouse and field nursery for further evaluation. Chemical names used: N-phenyl-N′-1,2,3-thidiazol-5-ylurea (thidiazuron or TDZ); 6-benzylaminopurine (BAP).

Haploids are useful in basic studies on intergenomic relationships, in molecular studies, and in practical breeding. Haploid production in durum wheat has had limited success. The objective of this study was to develop an efficient method of durum haploid production via maize pollination. Pollination of seven agronomically superior durum wheat (Triticum turgidum L., 2 n= 4x = 28, AABB) cultivars [Durox, Langdon (LDN), Lloyd, Medora, Monroe, Renville, and Vic] and three important cytogenetic stocks [LDN 5D(5B), LDN Ph1 ph1b, and Cappelli ph1c ph1c with pollen from three maize (Zea mays L.) cultivars resulted in haploid embryos. In vitro culture of these embryos produced haploid green seedlings. Post-pollination treatment with 3 mg L -1 2,4-D and 120-180 mg L -1 AgNO 3 gave the best yield of embryos, whereas 3 mg L -1 2,4-D plus 120 mg L -1 AgNO 3 promoted the conversion of embryos into plantlets. We produced a total of 142 mature, green haploid seedlings which included haploids with and without the homoeologous pairing suppresser gene, Ph1. Clear genotypic differences in haploid production were observed, Medora with 3 mg L -1 2,4-D + 180 mg L -1 AgNO 3 being the highest yielder. Renville proved to be more consistent yielder of haploid embryos as well as seedlings, over all treatments. Among the three Langdon genotypes - Langdon Ph1 ph1b, Langdon 5D(5B) substitution line, and normal Langdon - the substitution line gave the best response. It appears, therefore, that the substitution of chromosome 5D for 5B confers on durum higher ability to produce haploids.

The objective of this study was to detect the presence of alien chromatin in intergeneric hybrids of durum wheat (Triticum turgidum, 2n=4x=28; AABB genomes) with the perennial grass Thinopyrum junceiforme (2n=4x=28; J1J1J2J2) using RAPD markers. The first step was to identify amplification of species-specific DNA markers in the parental grass species and durum wheat cultivars. Initially, the genomic DNA of five grass species (Thinopyrum junceiforme, Th. bessarabicum, Lophopyrum elongatum, Leymus karataviensis and Elytrigia pycnantha) and selected durum cultivars (‘Langdon’, ‘Durox’, ‘Lloyd’, ‘Monroe’, and ‘Medora’) was screened with 40 oligonucleotide primers (nano-mers). Three oligonucleotides that amplified DNA fragments specific to a grass species or to a durum cultivar were identified. Primer PR21 amplified DNA fragments specific to each of the five durum cultivars, and primers PR22 and PR23 amplified fragments specific to each of the grass species. Intergeneric hybrids between the durum cultivars ‘Langdon’, ‘Lloyd’ and ‘Durox’ and Th. junceiforme, and their backcross (BC) progeny were screened with all 40 primers. Six primers amplified parent-specific DNA fragments in the F1 hybrids and their BC1 progeny. Three primers, PR22, PR23 and PR41, that amplified Th. junceiforme DNA fragments in both F1 and BC1 were further analyzed. The presence of an amplified 1.7-kb Th. junceiforme DNA fragment in the F1 hybrids and BC1 progeny was confirmed using Southern analysis by hybridization with both Th. junceiforme genomic DNA and Th. junceiforme DNA amplified with primer PR41. With the exception of line BC1F2 no. 5, five selfed progeny of BC1 and a BC2 of line 3 (BC1F2 no. 3בLloyd’) from a cross of ‘Lloyd’×Th. junceiforme showed the presence of the 1.7-kb DNA fragment. All selfed BC1 and BC2 lines retained the 600-bp fragment that was confirmed after hybridization with Th. junceiforme DNA amplified with primer PR22. Other experiments using RFLP markers also showed the presence of up to seven Th. junceiforme DNA fragments in the F1 hybrids and their BC progeny after hybridization with Th. junceiforme DNA amplified with primer PR41. These studies show the usefulness of molecular markers in detecting alien chromatin/DNA fragments in intergeneric hybrids with durum wheat.

Immature embryos (stage I) and cotyledonary somatic embryos (stage III) of black spruve [Picea mariana (Mill) B.S.P.] were bombarded with tungsten particles coated with a gene construct containing the fusion of gus::nptll. GUS (β-glucuronidase) activity was monitored histochemically with X-gluc giving a blue colour where transient gene expression was detected in the bombarded tissues. A high transient expression of gus was observed in stage I embryo cultures 2 d after bombardment (202 GUS foci per 300 g tissue)

Observations were made on the maturation of stamens and ovaries from cultured maize (Zea mays L.) ear inflorescences. Immature ears (5.1–10.0 mm long) of maize were cultured in kinetin medium to study microsporogenesis and pollen maturation in developing stamens. Male spikelets developed on ears cultured in kinetin medium. Meiosis-I began by 7 days of culture in the developing anthers and the mature tri-nucleate pollen grains were developed by 20 days of culture. Further, kinetin was required in the culture medium for at least initial 5 days to obtain the microspores in differentiated stamens.

The data indicate that partial hepatectomy results in a decrease in degradation of targeted DNA. This appears to be due to an inhibition of the endocytotic pathway shortly after hepatectomy and is associated with the accumulation of targeted DNA within a population of light endosomal vesicles. It is likely that these vesicles serve as a reservoir from which targeted DNA gradually escapes and can be found in the nucleus. The DNA targeted to liver is capable of expressing the marker gene, and the DNA in the vesicles is transfection competent, suggesting that a substantial portion is intact. Overall, the receptor-mediated delivery system is highly efficient in transporting foreign DNA to liver cells. Partial hepatectomy and the ensuing cellular events provide a means of inhibiting the degradative portion of the endocytotic pathway. Pharmacological agents that can mimic the cellular processes that occur after partial hepatectomy may be useful in increasing the duration of foreign gene expression without the trauma of surgery.

Asialoglycoproteins (ASG) are internalized by hepatocytes by ASG receptor (ASGR)-mediated endocytosis. We have shown previously that when a plasmid DNA, pAlb(9-12)CAT (expressing chloramphenicol acetyltransferase driven by an albumin promoter enhancer), was complexed with an ASG-polylysine conjugate and injected intravenously in rats, 80% of the DNA was internalized by the liver. In normal recipient rats, over 95% of the internalized DNA was degraded in 4 h; the plasmid was undetectable after 48 h. In contrast, when 66% hepatectomy was performed 20 min after DNA administration, the internalized DNA persisted for several weeks in cytoplasmic vesicles (Chowdhury, N. R., Wu, C. H., Wu, B. Y., Yerneni, P. C., Bommineni, V. R., and Chowdhury, J. R. (1993) J. Biol. Chem. 268, 11265-11271). Since microtubules are required for the translocation of ligand-containing endosomes to lysosomes, the site of ligand degradation, we hypothesized that persistence of the endocytosed DNA might be related to changes in microtubular structure and function. To test this hypothesis, we examined hepatocellular microtubules by immunofluorescence confocal microscopy. Liver from untreated rats or sham-operated controls showed a network of fibrillar microtubules throughout the cytoplasm. The extent of the microtubular network was substantially reduced 3-6 h after 66% hepatectomy. By 24 h, microtubules had regenerated. Intraportal infusion of cycloheximide (250 mg/kg body weight) 15 min before 66% hepatectomy, prevented microtubular disruption, indicating that protein synthesis is required for this process. Immunotransblot analysis showed that hepatic alpha-tubulin concentration remained unchanged through microtubular disassembly and subsequent reassembly, which is consistent with conservation and reutilization of tubulin released by depolymerization of microtubules.

The culture of grass inflorescences has been attempted for three different reasons: (a) to induce somatic embryogenesis (Sharma et al. 1984; Vasil 1985; Rhodes et al. 1986; Pareddy and Petolino 1990); (b) to study the factors responsible for spikelet maturation and grain-filling (Donovan and Lee 1977, 1978; Singh and Jenner 1983; Nicholls 1986; Armstrong et al. 1987; Trione and Stockwell 1989); and (c) to study the factors underlying normal development and morphogenesis (Polowick and Greyson 1982; Pareddy and Greyson 1985; Bommineni and Greyson 1987). This last objective, as studied in maize inflorescences, is the focus of the present chapter.

After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85% of the plasmid appeared in the liver, 80% of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4% of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66% hepatectomy 20 min after DNA administration, 20, 9, and 7% of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66% hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.

Young ears of maize were cultured in two different liquid media containing either kinetin (KN) or kinetin + gibberellic acid (KN + GA3) in order to manipulate stamen and gynoecium development. In KN medium, stamens developed and gynoecia aborted in the flowers of the cultured immature ears. In the KN + GA3 medium, however, ovaries with silks developed and stamens aborted. These differential morphological events were recorded with SEM photomicrographs at regular intervals after excision of ear inflorescences. In addition, the mitotic activity in the developing or aborting organs was determined over a 75-h period. It increased from 6% to 14% in developing organs (i.e. stamens in KN medium, and gynoecia in KN + GA3 medium) and gradually decreased to 1% in the degenerating organs (i.e. gynoecia in KN medium, and stamens in KN + GA3 medium) by 45 h of culture. The mitotic activity reached zero in degenerating flower organs by 75 h of culture. Whether these differential sensitivities to the exogenously applied members of these two plant growth regulator classes are unique to our in vitro system or reflect a more general control feature of in vivo inflorescences must await further clarification.

Maize shoot apices (1 to 2mm size) from two sources were used to recover normal plantlets. The first explant source included shoot apices from the embryonic axis of immature embryos, 12–14 days post pollination in the glasshouse (spring) or 15–20 days post pollination in the summer nursery. In most explants, the shoot apical meristem was surrounded by a coleoptile primordium which was removed before culture. The second explant source included shoot apices from the plumules of 72 h imbibed mature kernels. The coleoptile and all other leaf layers (leaf-1 to leaf-3 or 4) of the plumule were removed before culture to expose the apical meristem. Among the genotypes studied, a recovery of 43% (Mo17) to 100% (Oh43) of plantlets was achieved from shoot apices from immature embryo plumules cultured in MS medium. Recovery of 80% of Oh43 plantlets in MS medium and 40% of A188 plantlets from apices of plumules of imbibed (72 h) seeds in MS medium containing 2,4-dichlorophenoxyacetic acid was recorded. The plantlets derived from both explant sources grew normally and produced viable seeds upon pollination.

Immature ear primordia of maize when cultured on a defined liquid medium grew and differentiated in response to two variables: 1) size of initial explants and 2) concentration of kinetin in the medium. Overall growth of primordia, less than 15 mm at explanting, reached an optimum fresh weight and ear length when kinetin was 10-6M. Ears less than 10 mm, in the presence of kinetin, produced many male spikelets with well-developed stamens in both upper and lower flowers. Ears greater than 15 mm, at explanting, produced only female flowers regardless of the kinetin concentration. Different proportions of male and female and bisexual flowers were produced depending upon the initial size of inflorescences and the concentration of kinetin.

In contrast to plant tissue culture procedures which promote callus proliferation, cell suspensions and plant propagation, the culture of flower and inflorescence primordia can be designed to favor normal growth and differentiation. The technique has revealed details of the nutritional requirements for development and the biochemical features of organ maturation and differentiation (Tepfer et al., 1962; Raman and Greyson, 1978; Bilderback, 1971). Prominent in these investigations has been the use of the plant growth regulators frequently associated with sexual differentiation (Galun et al. 1962).