The apical meristems of Calanthe orchid embryos were exposed to 1 mg/ml pBI-121 DNA in an electric field. pBI-121 contains the GUS marker gene glucoronidase under the control of the 35 S cauliflower mosaic virus promoter. A pipette containing 0.3% agarose and acetate buffer containing the DNA was placed on one end of the embryo; while the opposite end was in contact with a pipette containing only buffer and agarose. Uptake of the DNA into the meristem was monitored by 4′6-diamidino-2-phenylindole (DAPI) fluorescence. Optimal uptake occurred after 10 min of electrophoresis at 10 volts and 0.5 milliamps. Under these conditions, 55% of the embryos survived the treatment and 57% of those which survived were transformed as measured by GUS-positive staining. Leaves from 6 month old plants which developed from the transformed embryos expressed specific patterns of GUS staining.
Shin Je Kim, Kyung-Hee Paek, and Byung-Dong Kim
A cDNA clone of cucumber mosaic virus (CMV) 117 N-satellite RNA driven by the cauliflower mosaic virus (CaMV) 35S transcript promoter, was stably integrated into the genome of Petunia hybrida `Bluepicoti' tissues by Agrobacterium tumefaciens Ti plasmid-mediated transformation. Transgenic plants producing CMV satellite RNA showed delayed disease development when inoculated with CMV-Y, a helper virus for the I17N-satellite RNA. Furthermore, transgenic petunia plants showed delayed disease development against tobacco mosaic virus (TMV), a tobamovirus not related to CMV. Northern blot analysis revealed that large amounts of unit length satellite RNA (335 bp) were produced in CMV-infected transgenic petunia plants; whereas, mainly transcripts driven by the CaMV 35S promoter (approximately 1 kb) were produced in TMV-infected transgenic plants. SDS-PAGE and Western blotting showed that symptom reduction was correlated with a reduction in the amount of viral coat protein in transgenic plants.
Craig S. Charron and Carl E. Sams
There has been significant interest in the glucosinolate-myrosinase system in plants of the Brassicaceae due to accumulating evidence that some glucosinolate degradation products are anticarcinogenic and/or suppressive to plant pathogens. Because glucosinolate hydrolysis is catalyzed by endogenous myrosinase, characterization of myrosinase activity is important for elucidating the potential bioactivity of crop glucosinolates. We measured the specific activity in citrate-phosphate buffer extracts across the pH range 4.5–6.5 of two cultivars each of five Brassica groups grown during two fall and two spring seasons. Specific activity in two kale cultivars was highly variable, but tended to have highest activity from pH 5.0–6.0. In both cauliflower cultivars from Fall 2000, Fall 2001, and Spring 2002, optimal pH was around pH 6.0. In Spring 2000, however, specific activity was highest at pH 5.0. Maximum specific activity in both cabbage cultivars occurred in the pH range 5.5–6.0 in Fall 2000, Fall 2001, and Spring 2002. In Spring 2000, specific activity in `Red Acre' cabbage was uniform across the range pH 4.5–5.5 and maximum specific activity was at pH 5.0 for `Early Round Dutch' cabbage. Both brussels sprouts cultivars had pH maxima around pH 5.5–6.0 and significantly lower activity at pH 4.5. Specific activity in broccoli was much like that of cauliflower in that highest activity occurred around pH 5.5–6.0 in Fall 2000, Fall 2001, and Spring 2002, but in Spring 2000, maximum activity was at pH 5.0. These results indicate that in most cases, pH optima were in the range 5.5–6.0, but varied somewhat with season and genotype.
Denise Duclos and Thomas Björkman
The genetic factors that control reproductive development in B. oleracea remain a mystery. Broccoli differs from cauliflower in its floral development stage at harvest. We are studying the role of meristem identity genes (MIGs) in the transition from inflorescence meristem (cauliflower) to floral buds (broccoli). The objectives are to determine stage-specific roles of MIGs during reproductive development and to check whether expression of flowering genes in heading B. oleracea is as predicted by the Arabidopsis flowering model. We tested a model of arrest in B. oleracea that incorporates FUL, a redundant gene of AP1 in controlling inflorescence architecture and floral meristem identity, the meristem gene TFL1, the flowering gene LFY, and AP1/CAL, and genes involved in flower transition. Conclusions. 1) Arrest at the inflorescence meristem stage is highly correlated with a decrease in LFY to TFL1 ratio, given by a decrease in TFL1 expression. 2) Transcription of AP1c is stimulated at the time of floral primordium initiation, suggesting a role in floral transition but not in floral organ specification. Plants recessive for AP1a, AP1c, and CAL formed normal floral buds containing all four whorls of organs, and did not necessarily form curd. We suggest that their ability to flower could be related with the ectopic expression of FUL by affecting TFL1 expression. FUL paralogs were highly expressed at all stages of development of the triple mutant plants. 3) The lack of upregulation in AP1 transcripts at the floral bud stage, and the absence of an A-function mutant phenotype imply that other genes act redundantly with AP1 in the specification of sepal identity and questions the role of AP1a and AP1c as A-function genes in B. oleracea.
Mark W. Farnham and Anthony P. Keinath
Wirestem, caused by Rhizoctonia solani, is a destructive disease of B. oleracea cole crops and is distributed worldwide. Effective means of wirestem control include soil fumigation and soil treatment with pentachloronitrobenzene, which are increasingly expensive and environmentally undesirable. As a consequence, alternative methods of wirestem control are needed. Thus, we conducted controlled-environment and field experiments to develop methodology to study host-plant resistance and possibly biocontrol agents as potential wirestem control alternatives. Seedlings of 12 cultivars (three each of cauliflower, broccoli, cabbage, and collard) at the four- to five-leaf stage were transplanted to trays in a growth room or into field plots and covered with soil infested with R. solani AG-4 sclerotia. Disease progression (percent of plants healthy, diseased, and dead) was observed every 3 days for 2 weeks in the controlled environments and for 3 weeks in field trials. At the end of two studies, plants were dug with roots intact and rated for disease using a 1 to 10 scale. In all trials, percent healthy plants stabilized at about 2 weeks after inoculation. Incidence of wirestem disease varied among experiments ranging from 70% to 100% diseased, dead plants in controlled environments, and from 51% to 88% and 33% 65% in the two respective field studies. Disease rating was always negatively and significantly correlated with percent healthy plants. Although a genotype × environment interaction was observed, some cultivars (i.e., `Snowcone' and `Snowcrown' cauliflowers) were always severely diseased, while others (i.e., `Viking' broccoli and `Blue Max' collard) were consistently among the least diseased.
P.C. Lee, A.G. Taylor, and T.G. Min
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.
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.
Robert L. Geneve and Sharon T. Kester
Early seedling growth rate can be used to estimate seed vigor for small-seeded vegetable and flower seeds. However, hand measurement of small seedlings is tedious and difficult to reproduce among analysts. Computer-aided analysis digital images of seedlings should improve accuracy and reproducibility. A flat-bed scanner fitted with base and top lighting provided high resolution images of even small-seeded species like petunia [Petunia ×hybrida `Blue Picotee' (Hort) Vilm.] and lisianthus [Eustoma grandiflorum `Mariachi Pure White' (Raf.) Shinn]. Uniform lighting was provided and images were captured and analyzed in less than 2 minutes. A clear, cellulose film was used as the germination substrate in petri dish germination assays to facilitate capturing images with a flat-bed scanner. The transparent medium permitted seedlings to be imaged without removal from the petri dish and also allowed for repeated measures of the same seedlings in order to calculate growth rate. Six species evaluated in this study included cauliflower (Brassica oleracea L., var. Botrytis), tomato (Lycopersicon esculentum Mill. `New Yorker'), pepper (Capsicum annuum L. `North Star'), impatiens [Impatiens walleriana Hook. f. `Impact Lavender'], vinca [Catharanthus roseus (L.) G. Don. `Little Bright Eye'], and marigold (Tagetes patula L. `Little Devil Flame'). For germination and early seedling growth, the cellulose film compared favorably with other standard germination media (blue blotter and germination paper) for five of the six species tested. Computer analysis of seedling length was possible for all six species and was statistically similar to hand measurements averaged for three analysts.
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.