Suppression of the cysteine protease, aleurain, delays floret senescence in Brassica oleracea.

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.

Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica).

An improved broccoli transformation system was developed by optimising several factors that affect the rate of effective Agrobacterium-mediated transformation. Leaf explants of cultivar Shogun were co-cultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II (NOS-NPTII-NOS) gene for kanamycin resistance and a ß-glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated including combinations of arginine, mannopine, acetosyringone and the use of feeder cell layers. The new protocol includes the use of 200 µM acetosyringone in LB medium for bacterial growth, the use of a Brassica campestris feeder cell layer, 10 mM mannopine and 50 µM acetosyringone in the co-cultivation medium and 1 mM arginine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary experiments transforming broccoli with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene from pTOM13.

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation.

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment.

Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.

Hairy roots of Brassica napus: II. Glutamine synthetase overexpression alters ammonia assimilation and the response to phosphinothricin.

Hairy roots of Brassica napus (rape cv. Giant) have been produced that contain the cytosolic glutamine synthetase (GS) gene from Glycine max (soybean). Leaf explants were cocultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pLN16. This vector was constructed by inserting a soybean cytosolic GS cDNA into the multiple cloning site of pGA643, placing it under the control of the CaMV promoter. In addition, the T-DNA region of pLN16 contained a NPTII gene for selection of transformed cells. Transgenic hairy roots grew prolifically on hormone-free media containing a selective level of kanamycin. Southern and northern analyses confirmed the presence of soybean GS DNA and transcripts, respectively. These transformed hairy roots also have a greater abundance of the GS polypeptide, approximately 3-6 fold greater GS activity and lower levels of endogenous ammonia. Hairy roots provide a useful system for studying responses to phosphinothricin (PPT). Hairy roots grown in media containing PPT had lower GS activity, greater ammonia accumulation and slower growth than controls. The presence of the soybean GS gene in the hairy roots reduced these PPT-induced effects and resulted in higher GS activity, lower ammonia levels and faster growth than in PPT-treated controls. Greater tolerance of PPT was also seen in shoots regenerated from the hairy roots displaying elevated levels of GS activity.

Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris.

Protoplast fusion was used to combine the cytoplasmic traits of atrazine resistance and male sterility in Brassica oleracea var. italica (broccoli). Leaf protoplasts from broccoli with the petaloid B. nigra type of cytoplasmic male sterility were fused with hypocotyl protoplasts from an atrazine-resistant biotype of B. campestris var. oleifera cv Candle (oilseed rape). A total of 19 colonies regenerated shoots, all of which were broccolilike in phenotype, i.e., lacked trichomes. Four shoots, all from one colony, were atrazine resistant, surviving and growing in the presence of 25 µM atrazine. A leaf piece assay also confirmed that they were atrazine resistant. Molecular analysis showed that they contain chloroplasts from the atrazine-resistant B. campestris parent and mitochondria from the B. nigra parent. No recombination or rearrangement of the mitochondrial genomes in the fusion products was detected. These four plants and their progeny all showed the petaloid B. nigra type of male sterility.