Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojecticle bombarded leaf cells.

Young leaf tissue of orchardgrass (Dactylis glomerata L.) was placed on Schenk and Hildebrandt medium containing 30 µM dicamba. Microprojectiles coated with DNA containing the selectable bar gene (Basta® tolerance) and the reporter gene uidA coding for ß-glucuronidase (GUS), both driven by the maize ubiquitin promoter (Ubi1), were propelled into the tissue with a particle inflow gun. Transient GUS expression was observed as blue spots of various sizes on leaf segments. Somatic embryos staining entirely blue were also produced, and embryos germinated on medium containing 3.0 mg 1-1 bialaphos. Leaves of 67 putative transformed plants were painted with 0.1% Basta. Ten showed no reaction, and 6 showed only a localized response. Cultured leaf segments from tolerant plants also produced somatic embryos that expressed GUS. The genetic transformation was confirmed by Southern blot hybridization and PCR analyses of T0 plants and by PCR analyses of somatic embryos produced from T0 plants.

Cryopreservation of Papaver somniferum Cell Suspension Cultures*.

The effect of culture age and various cooling rates on the survival and regrowth of cryopreserved cell cultures of PAPAVER SOMNIFERUM L. is described. Survival and regrowth following cryopreservation, in liquid nitrogen, occurred only with 4 and 5 day old cultures. No regrowth was observed in 3 or 6 day old cultures. Cooling rate had a significant effect on survival following cryopreservation with an optimum cooling rate of 0.5 degrees C min (-1).

AgNO3 increases type II callus production from immature embryos of maize inbred B73 and its derivatives.

Incorporating 10 to 100 µM AgNO3 into Phytagel(™) (0.2%) solidified N6 medium containing 1 mg/L 2,4-D, 100 mg/L casamino acids and 25 mM praline (N6 1-100-25) promoted type II callus production from cultured Zea mays L. immature embryos of FRB73, B73 X A188 and a proprietary B73 BC6 genotype. Under these conditions, approximately 15, 80 and 80% of the respective FRB73, B73 X A188 and B73 BC6 explants produced type II calli after 2 to 3 weeks incubation in the dark at 28 C. In the absence of AgNO3, the type II culture response from B73BC6 immature embryos was 25% on N6 1 100-25 solidified with Phytagel(™) (0.2%) as compared to 0% for that solidified with 0.8% agar. Duncan's medium was tested using 10 to 100 µm AgNO3 and generally promoted type I callus initiation, although up to 6% of the explants produced type II cultures in the presence of 0.2% Phytagel(™). Ethylene emanation rates of up to 370 and 115 nL g-1 h-1 were detected from B73 X A188 immature embryos and calli, respectively, cultured on N6 1-100-25.

High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase.

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.

Effect of ethylene on sanguinarine production from Papaver somniferum cell cultures.

A Papaver somniferum cell line capable of producing sanguinarine equivalent to 3% of cell dry weight was used to determine if ethylene was involved in signalling the biosynthesis of this alkaloid. A 3.3-fold increase in ethylene emanation from these cell suspension cultures was observed 7 h after elicitation with a Botrytis fungal homogenate. The rate of ethylene release then decreased to near zero after 48 h, suggesting that a pulse of ethylene production may be involved in sanguinarine production. However, sanguinarine biosynthesis was not promoted when either the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), or the ethylene releasing agent, 2-chloroethylphosphonic acid (ethephon), was added to the culture. These results strongly suggest that ethylene is not intimately involved in the production of sanguinarine from Papaver somniferum cell cultures or in the transduction of the elicitation event.

Effect of 1-Aminocyclopropane-1-carboxylic acid and aminoethoxyvinylglycine on ethylene emanation and somatic embryogenesis from orchardgrass leaf cultures.

Ethylene emanation rates were assessed from leaf tissues of an embryogenic seed plant (Cycle 0) and regeneration cycle plants selected for enhanced embryogenesis (Cycles I, II and IV). In all experiments, ethylene was assessed from the basal 1 cm portion of the innermost leaf. Ethylene emanation was five-fold higher in Cycle II and Cycle IV plants than in Cycle 0 and nonembryogenic (NE) seed plants. After two days culture on Schenk and Hildebrandt medium containing 30 µM dicamba (SH-30), ethylene emanation from Cycle 0 and Cycle II leaf sections increased by 55-fold. Culture of leaf explants for 30 days on SH-30 containing 1 mM 1-aminocyclopropane-1-carboxylic acid (ACC) reduced the embryogenic response by 99%. Treatment of leaf explants with 1 mM aminoethoxyvinylglycine (AVG) reduced ethylene emanation but did not affect embryogenesis. The data indicate that ethylene mediated by ACC may hinder the embryogenic response from orchardgrass leaf cultures.

Effect of l-aminocyclopropane-l-carboxylic acid, silver nitrate, and norbornadiene on plant regeneration from maize callus cultures.

The effect of the ethylene antagonists norbornadiene and silver nitrate and the ethylene precursor l-aminocyclopropane-l-carboxylic acid (ACC) on Zea mays plant regeneration was studied. A 12-fold increase in plant regeneration, as measured by number of plants obtained per gram fresh weight from callus cultures of maize inbreds Pa91 and H99, was obtained by 250 µM norbornadiene and 100 µM silver nitrate treatments. An increase in amout of nonregenerable tissue and a 68% decrease in plant regeneration were associated with callus treated with 1 mM ACC. Ethylene emanation from 1 mM ACC treated callus reached a maximum of 170 nl g(-1) h(-1) after 3 days compared to 7 nl g(-1) h(-1) for the control. The free proline content was up to 80% lower in 1 mM ACC treated callus grown for 30 days on medium with or without 12 mM proline, respectively, as compared to each control. These studies indicate that ethylene action inhibitors such as norbornadiene and silver nitrate can be used to increase plant regeneration efficiency from maize callus cultures.