Jun-ichi Shimizu

Citrus cybrid regeneration following cell fusion between nucellar cells and mesophyll cells

Abstract Through the cell fusion between nucellar callus cells and mesophyll cells in two Citrus combinations, we obtained the regenerated plants resembling mesophyll parent besides the somatic hybrids. In the fusion experiment between sudachi (Citrus sudachi Hort.) and lime (C. aurantifolia Swingle), 4 out of 12 regenerated clones were similar to mesophyll parent (lime) as we already reported. In another experiment of sudachi and lemon (C. limon Burn) 4 out of 6 clones were similar to mesophyll parent (lemon). These regenerated plants had 18 chromosomes, which were equal to diploid parents and showed the same nuclear rDNA fragment patterns as those of mesophyll parents. The composition of the mitochondrial genomes of these regenerated plants was investigated. The mtDNA analysis revealed that all of the clones that resembled mesophyll parents had identical mtDNA fragment patterns to those of the nucellar callus parent, indicating that these regenerated clones resembling mesophyll parents were cybrids. These results suggest that the mitochondria of nucellar cells may play a significant role in Citrus embryogenesis.

Somatic hybridization in Citrus using embryogenic cybrid callus

Abstract Embryogenic callus of a lime-type cybrid possessing the nuclear genome of lime ( Citrus aurantifolia Swing.) and the mitochondrial genome of sudachi ( C. sudachi Hort. ex Shirai) was induced from a somatic embryo which had been obtained through protoplasts fusion between nucellar callus of sudachi and mesophyll of lime [1]. Protoplasts derived from the cybrid callus were fused electrically with mesophyll protoplasts of lemon ( C. limon Burm.). After 5 months, plants were regenerated from eigth fusion-derived cell clones through embryogenesis. The plants showed two types of leaf morphology; thick and broad leaf shape, identical to lemon. Chromosome counts and nuclear and mitochondrial DNA analysis confirmed that the former plants were somatic hybrids which were allotetraploid (4x = 36), possessing the nuclear genomes of both parents and the mitochondrial genome of sudachi. The latter lemon-type plants were novel cybrids possessing 18 chromosomes with the nuclear genome of lemon and the mitochondrial genome of sudachi. Lime-type cybrid plants, identical with one of the parents, were not found in this study. These results indicate that embryogenic cybrid callus lines can be used as fusion material, resulting in further production of somatic hybrid and cybrid plants.

Acid citrus somatic hybrids between sudachi (Citrus sudachi Hort. ex Shirai) and lime (C. aurantifolia Swing.) produced by electrofusion

Abstract Conditions were established for the production of hybrid plants between sudachi embryogenic nucellar suspension culture and line mesophyll protoplasts by electrofusion. Under these conditions, 12 clones out of a total of 40 embryoids obtained were regenerated to complete plants through embryogenesis. Eight of the regenerated clones were shown to be somatic hybrids on the basis of chromosome number and analysis of nuclear ribosomal DNA. The other 4 were recognized to have lime-type characteristics. This electrofusion method considerably simplified protoplast fusion of citrus plants and allowed successful production of acid citrus somatic hybrids.

Cell Regeneration and Division of Grape Mesophyll Protoplasts

Summary Conditions were established that resulted in high yields and consistent division in 10 -15 of the protoplasts from one to 3 month old mature leaf tissues of Vitis vinifera cv. Koshu Sanjaku grown at 25 °C and 80 % relative humidity without direct sunlight in a greenhouse. Enzymes used for protoplast isolation were the combination of 1 % Macerozyme R-10, 0.01 Pectolyase Y-23, 1 % Cellulase Onozuka RS and 0.4 % Dricelase. Use of a higher concentration of MES buffer than 0.1 M was essential to obtain high yields and viabilities of protoplasts. From these protoplasts new cell walls were regenerated within the first few days and 4 to 6 cell-structures developed from one protoplast by cell division after 9 days of culture in Gamborgs BS liquid medium supplemented with 1.0 mg · 1−1 2,4-D, 0.5 mg · 1−1 6-benzyladenine and 0.5 M sorbitol.