An aleurain-like protein, BoCP5, is up-regulated during harvest-induced senescence in broccoli floret and leaf tissue. BoCP5 is most closely related to an Arabidopsis protein (91%, AAF43041) and has 71% identity to barley aleurain (P05167). The mRNA for this gene accumulates within 6 h after harvest in broccoli florets, and its expression is reduced in tissue that has been held in senescence-delaying treatments (e.g. water, sucrose feeding, controlled atmosphere). The gene is also expressed in leaves during aging-related and harvest-induced senescence. Analysis of protein bands that cross-react with antibodies raised to the bacterial BoCP5 fusion protein, revealed prominent immunoreactive bands at ca. 26, 28, 31, and 38 kD in floret tissue. The 31 kD band was absent in protein extracts from leaf tissue. Agrobacterium-mediated transformation was used to produce transgenic broccoli plants with down-regulated BoCP5. A reduction in the postharvest expression of BoCP5 in floret tissue was achieved for four transgenic lines in the current study. In three of these lines postharvest floret senescence (yellowing) was delayed, and florets contained significantly greater chlorophyll levels during postharvest storage at 20 degrees C than wild-type plants. Line 4 showed the greatest down-regulation of BoCP5, and in this line postharvest protease activity remained at pre-harvest levels, and the yield of soluble proteins extracted from florets after harvest was significantly greater than that of wild-type tissue.

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We report on the production and selection of transgenic Brassica oleracea var. Italica lines with a downregulated soluble acid invertase (BoINV2). Explants of broccoli (cv. Triathlon) were transformed with an antisense construct of BoINV2 under the control of an Asparagus officinalis‐derived harvest‐induced promoter using Agrobacterium tumefaciens‐mediated transformation. BoINV2 is upregulated in wild‐type broccoli floret tissue after harvest. Transgenic broccoli lines showed reduced BoINV2 mRNA accumulation immediately after harvest compared with wild‐type. Downregulation of BoINV2 had no significant impact on the expression of a second broccoli acid invertase gene (BoINV1), but plants with downregulated BoINV2 also had lower expression of a senescence‐associated cysteine protease (BoCP5) compared with wild‐type. The total soluble sugar levels in floret tissue of antisense BoINV2 lines were greater than wild‐type tissue after harvest (up to 1.5 times higher). Soluble protein content of wild‐type tissue decreased from 48 h after harvest with an increase in protease activity. In comparison, two antisense BoINV2 lines retained at‐harvest levels of soluble protein until 72 and 96 h after harvest and had lower postharvest endoprotease activity compared with wild‐type. Antisense BoINV2 lines also had a slower rate of floret sepal chlorosis after harvest compared with wild‐type.

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of microcolonies with plating efficiencies 3-10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.