Sinapine leakage to detect seed germination potential on a single-seed basis in Brassica has been developed as a rapid test. In this test, sinapine leakage predicts that a seed is non-germinable; however, the major source of errors in this method are false-negative (F–)—i.e., the method predicted a seed was germinable because the seed did not leak, and it did not germinate. The sinapine leakage index (SLI) was used to asses the F– for any seed lot by dividing the number of non-germinable seeds that leaked sinapine by the total number of non-germinable seeds. Seed lots including cabbage, cauliflower, and broccoli (B. oleracea L., Captitata, Botrytis, and Italica groups, respectively) were used to examine the F–. The leakage rate as measured by T50, the time for 50% of heat-killed seeds to leak, was linearly correlated to SLI. Cabbage seeds were viewed by scanning electronic microscopy and leaking non-germinable seeds either had cracks or were shrunken. NaOCl pretreatment has been found to increase the rate of sinapine leakage and SLI. The mode of NaOCl was due to high pH altering the seed coat permeability. Chemical analysis was conducted on isolated seed coats for pectin, tannins, hemicellulose, cellulose, phenolic lignin, and cutin. It was found that the higher SLI (more permeable) lots contained lower amounts of cutin, suggesting that cutin may restrict the diffusion of sinapine through the testa.
P.C. Lee, A.G. Taylor, and T.G. Min
A.R. Kuehnle, T. Fujii, F.C. Chen, A. Alvarez, N. Sugii, R. Fukui, S.L. Aragon, and J.M. Jaynes
Two cultivars of Anthurium andraeanum Hort. hybrids, `Paradise Pink' and `Tropic Flame', were transformed by Agrobacterium to contain gene sequences for Shiva-1, a cecropin-based lytic peptide. The antibacterial gene was driven by a 35-35S cauliflower mosaic viral (35-35S CaMV) promoter and the construct included the secretory signal sequence for pathogenesis-related protein 1b (PR1b). Blight tolerance of regenerated plants was tested by inoculation with a virulent strain of Xanthomonas axonopodis (formerly campestris) pv. dieffenbachiae (Xad) that is bioluminescent to allow detection of symptomless infections in Shiva-1 transformants. Primary regenerants for two Shiva-1 transgenic lines of `Paradise Pink' displayed significantly enhanced tolerance to bacterial blight over blight susceptible `Rudolph' and even the blight tolerant `Kalapana'. Two Shiva-1 transgenic lines of `Tropic Flame' showed no improved resistance when compared to the control at the mean percent leaf infection level. One Shiva-1 transgenic line of `Tropic Flame' was unexpectedly more susceptible to blight than the nontransgenic control. Low expression of Shiva-1 observed in this line is hypothesized to be the cause of its increased susceptibility to Xad.
H.C. Wien and R.J. Sloan
The growth processes of most horticultural crops are too slow to be visually interesting to students. Time lapse photography has been used for years to speed up the action and make plants “come alive.” With the advent of video technology, time lapse techniques have become convenient, easy, and affordable. The system which we have found satisfactory consists of a time lapse video cassette recorder, linked by optical fiber cable to a closed circuit color video camera in a ventilated housing. Typically, the camera has been set up in a greenhouse compartment, monitoring growth processes of vegetable crops, and linked by cable to the VCR in an office 80 m away. Equipment costs with one camera are less than $3000. Two cameras can be set up to do comparative growth studies, with two images side-by-side, using a screen splitter. Costs of the latter system is about $4500. Growth processes such as cabbage head formation, curd growth in cauliflower, and weed-crop competition of mustard and peas have been the subjects so far. The technique lends itself to increasing the visual impact of teaching, and gaining a better understanding of plant growth processes in research.
Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown
Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.
Guo-qing Song, Hideo Honda, and Ken-ichi Yamaguchi
Leaves are usually the target tissue for expressing transgenes conferring resistances to herbicides, pests, and diseases. To achieve leaf-specific expression, a light-harvest chlorophyll a/b binding protein (CAB) of photosystem-II (CAB2) promoter (CAB2-p) from rice (Oryza sativa L.) and the cauliflower mosaic virus 35S promoter were fused to the β-glucuronidase (GUS) reporter and subsequently evaluated in transgenic sweetpotato [Ipomoea batatas L. (Lam.)]. The 35S promoter-directed GUS activities varied from 46.0 to 61.2 nmol 4-methyl-umbelliferyl-β-D-glucuronide (4-MU) per minute per milligram of protein in leaf, stem, primary, and storage roots. In contrast, the CAB2-p directed an uneven distribution of GUS activities (4-MU at 1.1 to 12.6 nmol·min−1·mg−1 protein); GUS activity in mature leaves was ≈12-fold as high as that in storage roots. In addition, GUS assay in leaf tissues revealed that CAB2-p enabled a developmentally controlled and light-regulated GUS expression. These results indicate that the rice CAB2-p could be used to drive leaf-specific expression of linked genes in sweetpotato.
T.K. Hartz and F.J. Costa
The production of cool-season vegetable crops in California's coastal valleys is characterized by high N input (typically 200–300 kg·ha–1 per crop), with two crops per year the norm. N. removal in harvested biomass seldom exceeds 100 kg·ha–1, suggesting a high degree of inefficiency in N management. A project was conducted on a commercial farm in Santa Maria to document the utility of intensive monitoring of soil and plant N status on improving N management. Eight fields were monitored through successive cropping cycles. Slow-release N fertilizer was applied preplant at 110–250 kg·ha–1 in subplots in each field to provide a reference of known N sufficiency against which to compare field productivity; these reference plots also received the same in-season fertilizer N applied in the balance of the field. N monitoring techniques included: in situ and controlled-environment soil incubation to estimate net N mineralization, soil NO3-N analysis by a “quick test” technique using colormetric test strips, and petiole sap analysis by NO3-N selective electrode. It was consistently demonstrated that, for lettuce, cauliflower, and broccoli, maximum crop productivity was obtained with seasonal N applications 50–100 kg N/ha less than the industry norm and that fertilizer cost savings more than offset the cost of crop and soil monitoring.
Charles F. Forney and Michael A. Jordan
Methanethiol (MT) is a volatile compound responsible for the strong off-odor that is evolved when fresh broccoli is held under anaerobic atmospheres. Inductive atmospheres can develop in modified-atmosphere packages, resulting in reduced quality. To determine if related vegetables are capable of producing MT, 12 different vegetables from the genus Brassica were cut into ready-to-eat forms. Fifty-gram samples of these cut vegetables were sealed in 500-ml glass jars and flushed with N2. After flushing, jars were held for 24 h at 20C in the dark. Headspace samples from the jars then were analyzed for MT and other volatiles using a GC-MS> The concentration of MT was greatest in jars containing broccoli florets. Broccoli flower buds removed from florets produced 40 times more MT than peduncle and stem tissues (38.3 vs. 0.87 mmol·m–3). Headspace concentration of MT (mmol·m–3) in jars containing these different vegetables was: broccoli florets, 22.7; pak choi leaf blades, 17.8; savoy cabbage, 12.4; broccoflower, 7.5; green storage cabbage, 5.2; red cabbage, 2.7; kale, 0.81; Brussels sprouts, 0.36; pak choi petioles, 0.28; rutabaga root, 0.26; cauliflower florets, 0.18; Chinese cabbage, 0.03; and kohlrabi tubers, 0.02. In addition to MT, ethanol, dimethyl disulfide, and dimethyl trisulfide were detected in the headspace over each of the 12 vegetables. The contribution of these induced compounds to off-odor development in packaged, precut vegetables will be discussed.
Manjul Dutt, Zhijian T. Li, Sadanand Dhekney, and Dennis J. Gray
Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.
Jessica L. Boldt, James E. Barrett, and David G. Clark
Petunia × hybrida `Electric Purple' plants, genetically transformed (Selecta Klemm Co.) via Agrobacterium tumefaciens to constitutively express the Cauliflower Mosaic Virus 35S promoter (CaMV35S) fused to two separate Arabidopsis c-repeat binding factor cDNAs (CBF3 & CBF4), were utilized to evaluate water relations. Non-stressed plants followed a classical stomatal conductance pattern, with maximum conductance between 1000 hr and 1400 hr. CBF3 and CBF4 plants showed an increase in transpiration rates and a decrease in stomatal resistance at 1230 hr, compared to `Electric Purple'. Transpiration rates (per unit leaf area) were similar in CBF3 and `Electric Purple' plants, but CBF4 plants were 12% less than `Electric Purple'. Xylem water potentials at visible wilt were between –1.4 and –1.5 MPa and there were no significant differences between line or irrigation treatment. A fourth experiment observed differential plant responses to stress cycles. Under non-stress irrigation conditions, CBF4 plants showed an increase in stomatal resistance and a decrease in transpiration rate compared to `Electric Purple' plants. There were no differences in the xylem water potential at visible wilt for the first and third stress cycles, but, for the second cycle, xylem water potentials at wilt were –1.9, –1.7 and –1.4 Mpa for CBF4, `Electric Purple' and CBF3 plants, respectively. CBF3 and CBF4 plants showed small differences in performance as compared to `Electric Purple' and under mild stress conditions as imposed in these experiments apparent heterologous overexpression of the Arabidopsis CBF3 & 4 transgenes may not be sufficient for conferring drought tolerance in petunia.
Samuel Salazar-García and Carol J. Lovatt
Avocado trees (Persea americana Mill.) bearing a heavy crop produce a light “off” bloom the next spring. This results in a light crop and a subsequent intense “on” bloom the year after. The objective of the study was to quantify the effects of GA3 canopy sprays applied to `Hass' avocado trees during the months preceding an “off” or “on” bloom on inflorescence and vegetative shoot number and yield. The experiment was initiated approximately seven months before an anticipated “off” bloom in an attempt to increase flowering intensity and yield. GA3 (25 or 100 mg·L-1) was applied to separate sets of trees in September (early stage of inflorescence initiation), November (early stage of inflorescence development), January (initial development of the perianth of terminal flowers), March (cauliflower stage of inflorescence development; only 25 mg·L-1), or monthly from September through January (only 25 mg·L-1). Control trees did not receive any treatment. GA3 (100 mg·L-1) applied in September reduced inflorescence number in both years, but not yield. GA3 (25 or 100 mg·L-1) applied in November before the “on” bloom reduced inflorescence number with a concomitant increase in vegetative shoot number and 47% yield reduction compared to control trees. This treatment might provide avocado growers with a tool to break the alternate bearing cycle by reducing yield in an expected “on” crop year to achieve a higher yield the following year. GA3 (25 mg·L-1) applied in November or January stimulated early development of the vegetative shoot of indeterminate inflorescences. January and March applications did not affect the number of flowering or vegetative shoots produced either year. GA3 (25 mg·L-1) applied in March at the start of an “off” bloom increased 2-fold the production of commercially valuable fruit (213 to 269 g per fruit) compared to the control.