Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis).

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2-2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60-80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2-5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100-150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.

Vital fluorescent staining technique for microspores of Brassica napus.

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.

Microspore culture in brassica.

Pioneering research in Brassica microspore culture (1,2,3)rapidly led to the realization that microspores provide a powerful alternative to protoplast culture as a single-celled culture method in plants. These two single-celled systems are fundamentally different, both in tissue origin and in genetic variability. The microspore system improves significantly on the protoplast system by virtually eliminating the large somaclonal variation associated with protoplast selection, by utilizing a true haploid cell system, and by resulting in a more synchronized embryo development that facilitates accurate mutation and selection methods. Most critical, however, are the observations that plant regeneration frequencies in excess of 80% can be readily obtained and that the entire sequence from microspore isolation to plantlet development may take place in as little as 4 wk (4).

Microspore mutagenesis and selection: Canola plants with field tolerance to the imidazolinones.

In vitro microspore mutagenesis and selection was used to produce five fertile double-haploid imidazolinone-tolerant canola plants. The S2 plants of three of the mutants were resistant to at least the field-recommended levels of Assert and Pursuit. One mutant was tolerant to between five and ten times the field-recommended rates of Pursuit and Scepter. Two semi-dominant mutants, representing two unlinked genes, were combined to produce an F1 hybrid which was superior in imidazolinone tolerance to either of the heterozygous mutants alone. Evaluation of the mutants under field conditions indicated that this hybrid and the original homozygous mutants could tolerate at least two times the field-recommended rates of Assert. The field results indicated the mutants were unaffected in seed yield, maturity, quality and disease tolerance. These genes represent a potentially valuable new herbicide resistance system for canola, which has little effect on yield, quality or maturity. The mutants could be used to provide tolerance to several imidazolinones including Scepter, Pursuit and Assert.

Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium tumefaciens.

Microspore-derived embryos of Brassica napus were transformed using the disarmed octopine-producing LBA4404 strain of Agrobacterium tumefaciens containing the binary vector pBin19. Octopine-producing strains have previously been reported to be ineffective in transforming Brassica. Four actively growing yellow/ green sectors were selected from the embryos on 50 mg/l kanamycin and plants regenerated. Analysis for NPT-II activity in these young plants initially indicated no expression of the bacterial NPT-II gene. The plants were nevertheless grown to maturity, selfed and S1 seed was collected. Three of the S1 plants produced microspores which were from 4 to 20 times more tolerant to kanamycin than the original parent. Southern analysis revealed that one plant (EC-1) had a single site of insertion and the other two plants (EC-2 and EC-6) had two sites of insertion with sequence homology to the bacterial NPT-II gene. Microspores from the EC-2 and EC-6 transgenics produced embryos on approximately five times the level of kanamycin tolerated by microspores from untransformed plants, while the EC-1 transgenic produced microspores with more than 20 times the tolerance to kanamycin. Analysis of S1 progeny of the EC-1 transgenic indicated that 100% of the progeny exhibited the trait through both Southern analysis and by expressing tolerance to kanamycin in microspore-derived embryos.

Plant development from isolated microspores of Zea mays L.

This paper reports the development of plants from mechanically isolated microspores of corn (Zea mays). Large populations of corn microspores were isolated using technology previously developed for rapeseed. Embryos and callus were developed from microspores in the late uninucleate stage. Scutellar-type embryos developed after two weeks and these could be transferred and germinated on a hormone free medium. However, the large majority of plants recovered from embryos developed only upon transfer to a corn embryogenic callus medium. These embryos produced shoots through organogenesis, and subsequently could be induced to form roots. Plants were developed from these colonies and grown in the greenhouse. The frequency of mature plants developed from the embryos was approximately 5 %. Non embryogenic callus which developed from some microspores have thus far either failed to develop or have developed only roots. Seed set has been obtained on some of the regenerated plants.

The characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection of microspores and protoplasts.

Brassica napus L.(cv Topas) plants tolerant to chlorsulfuron (CS) were isolated after selection experiments utilizing microspores and haploid protoplasts. The first microspore-derived plant (M-37,) was CS tolerant, haploid and sterile. Normal plant morphology and fertility was restored after colchicine doubling. A CS tolerant plant was also selected from protoplasts (P-26) isolated from microspore-derived embryo tissue and grown on medium containing CS. P-26 was aneuploid, CS tolerant and had very low fertility. The two selected lines produced selfed progeny which were tolerant to from 10-100 times the CS levels of the corresponding Topas plants. Microspores and protoplasts derived from the selfed plants were also CS tolerant. The segregation pattern for CS tolerance from reciprocally crossed progeny of M-37 and Topas was consistent with a semi-dominant nuclear mode of inheritance. Biochemical analysis of the two mutants indicated that the microspore-derived mutant and F1 crosses contained an altered acetohydroxyacid synthase (AHAS) enzyme, while the AHAS activity of the protoplast mutant was similar to Topas. Selfed seed from the M-37 plants have provided tolerance to CS in both greenhouse and field tests. S1 plants from a second microspore selected mutant (M-42) have tolerated 30 g/ha of CS in greenhouse tests. The two single-celled selection systems are discussed and the microspore selection system highlighted as a new method for in vitro selection.

Efficient isolation of microspores and the production of microspore-derived embryos from Brassica napus.

Mechanical isolation of Brassica napus microspores from whole buds with a micro-blender enabled the rapid isolation of large numbers of microspores free from tissue and cellular debris. The procedure resulted in savings of time and labor and could be used independent of the size and number of buds to be examined. Between 700 and 1000 embryos per bud were obtained. Compared to anther removal there was no difference in embryo yields or quality. Microspore isolation under cool conditions and overnight incubation prior to plating improved the frequency of embryogenesis. Over 75% of the embryos developed into normal torpedo-stage structures if the medium was replenished during culture and the embryos placed on a gyratory shaker. Over 80% of the torpedo-shaped embryos would ultimately develop into plants. The implications of these techniques to genetic and physiological studies are discussed.