Manabu Hagimori

Production and characterization of somatic hybrids between the Japanese radish and cauliflower

SummarySomatic hybrids between the Japanese radish and cauliflower (Brassica oleracea) were produced by protoplast electrofusion in order to introduce clubroot disease resistance in the Japanese radish (Raphanus sativus) into Brassica crops. After electrofusion of iodoacetamide-treated cauliflower protoplasts with untreated radish ones, culture was performed under conditions, that allowed only cauliflower protoplasts to regenerate. Out of 40 regenerated plants, 37 were morphologically of a hybrid type and 3 of a cauliflower type. On the basis of isozyme and RFLP analysis, all of the hybrid-type plants tested proved to be true hybrids. Of the 10 true hybrids tested, 9 were found to contain chloroplasts similar to those found in the Japanese radish, while only 1 contained those of the cauliflower. Using two mitochondrial genes as probes, we were able to show that 3 hybrids contained mitochondria of the Japanese radish, with some modification, while 7 hybrids had either parental or new patterns. All of the hybrid-type plants showed resistance to clubroot disease as high as that found in the Japanese radish. Some hybrids were self-fertile. All of the self-fertile hybrids were found to contain 36 chromosomes, indicating that they were amphidiploids. In addition, a few seeds were obtained from a backcross of the self-fertile hybrids to both parents.

Recovering vitrified carnation (Dianthus caryophyllus L.) shoots using Bacto-Peptone and its subfractions.

Vitrified shoots regenerated from carnation petals (Dianthus caryophyllus L. cv. Scania) were recovered by culturing them in a medium containing 3.0 g/l Bacto-Peptone. Wax structures not found on vitrified shoots developed on the abaxial surface of leaves of recovered shoots and on those of normal leaves. Recovered shoots were rooted and successfully acclimatized while vitrified shoots could not survive the acclimatization process. The Bacto-Peptone solution was fractionated and the efficiency of each fraction for the recovery of vitrification was examined. Only basic, non high molecular fractions whose molecular weight was less than 10,000 were effective.

Studies on the production of Digitalis cardenolides by plant tissue culture I. Determination of digitoxin and digoxin contents in first and second passage calli and organ redifferentiating calli of several Digitalis species by radioimmunoassay

With first and second passage calli induced from seedlings of Digitalis purpurea, D. lanata, D. lutea, D. mertonensis, D. ambigua and D. ferruginea Gigantea and those induced from leaf discs of D. purpurea and D. lanata, the contents of digitoxin and digoxin equivalents were assayed and compared with the contents involved in the inocula. Although the total contents of digitoxin and digoxin equivalents in the first passage calli induced from seedlings varied between zero and nine times as high as in the original seedlings, those in the second passage calli were almost undetectable. The total contents of digitoxin equivalents in the first passage calli induced from leaf discs of D. purpurea were approximately equal to those in the original leaf discs, but those in the second passage calli were less than those in the inocula. In the first passage calli induced from leaf discs of D. lanata, the total contents of digitoxin equivalents decreased but those of digoxin equivalents slightly increased. However, in the second passage calli, the amounts of both cardenolides decreased. Root-forming calli accumulated no more digitoxin nor digoxin equivalents than completely dedifferentiated calli. However, shoot-forming calli accumulated considerable amounts of cardenolides, which were assayed as digitoxin and digoxin equivalents by radioimmunoassay.

Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwacks medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally.

RAPD-based method for cultivar-identification of calla lily (Zantedeschia spp.)

A RAPD-based identification system for calla lily cultivars (Zantedeschia spp.) was constructed after screening 60 arbitrary 10-mer primers. Using this method, two or three sequential PCR reactions enable clear identification of 12 cultivars within several days.

Nurse culture of Japanese radish (Raphanus sativus L.) mesophyll protoplasts

Abstract Mesophyll protoplasts of 11 cultivars of Japanese radish ( Raphanus sativus L.) were cultured. Using protoplasts or cultured cells of different species as nurse, the efficiency and reproducibility of colony formation were improved and protoplasts of all cultivars grew into colonies. Cells of Nicotiana, Brassica, Daucus, Lactuca and Asparagus were effective as nurses, while those of Oryza were not. N. tabacum protoplasts just isolated and those cultured for more than 50 days showed equal effectiveness as nurses. The number of nurse cells correlated with the efficiency of colony formation of the nursed protoplasts: generally highest efficiency was achieved when the numbers of nurse cells and the nursed protoplasts were nearly equal. Although cell division and colony formation were improved by the nurse culture method, viability and cell wall synthesis were unaffected. Conditioned medium showed less promotive effect than nurse culture.

Production of somatic hybrids between Nicotiana benthamiana and N. tabacum and their resistance to aphids

Abstract In order to introduce aphid resistance into tobacco, somatic hybrids between tobacco and aphid resistant Nicotiana benthamiana were produced. The hybrids which were proved to be true hybrids on the basis of electrophoretic analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and restriction fragment length polymorphism (RFLP) analysis showed significant resistance to aphids. In the leaf surface lipids of the hybrids, lipids specific to N. benthamiana were detected with those specific to tobacco. Five out of 22 of the hybrids were fertile in self-pollination.

Calcium chloride as a major component contributing to aphid resistance ofNicotiana benthamiana

Substances with antiaphid activity were extracted from the leaf surface of aphid-resistantN. benthamiana and purified. Sugar esters and diacyl glycerols were isolated from the chloroform extract, but only diacyl glycerols showed significant activity. However, the major activity was found in the water extract rather than in the chloroform extract. From the water extract calcium chloride was isolated as the most abundant active substance. Only calcium chloride showed significant activity among several calcium salts and chlorides of several metals that are abundant in plants. Calcium contents per unit area of leaf surface ofN. benthamiana and aphid-resistantN. gossei were almost equal to each other and 10–100 times higher than that of aphid-susceptibleN. tabacum.