High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea ) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).     

Asymmetric somatic hybridization between Triticum aestivum L. and Haynaldia villosa Schur

Suspension cell-derived protoplasts of wheat, inactivated with different concentrations (0-2.5mol/L) of IOA, were fused by PEG method with the Haynaldia villosa protoplasts which originated from the calli 4-5d after subculture and were irradiated with 60Co-γ ray. Cell colonies, calli or regenerated plants were obtained from different combinations of fusion. The calli and plants were verified to be hybrids by chromosome counting, isozyme analysis and morphological inspection.     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

Construction of the cybrid transplastomic Brassica napus plants containing Lesquerella fendleri chloroplasts

Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.     

Somatic hybridization between the zinc accumulator Thlaspi caerulescens and Brassica napus

Somatic hybrids between the zinc hyperaccumulator Thlaspi caerulescens and Brassica napus were produced by electrofusion of protoplasts isolated from each species. Optimization of electrofusion parameters yielded interspecies heteroplasmic fusion rates of up to 13%. Hybrids were selected by screening the growing calli for Zn tolerance. In addition, a second novel selection technique was developed based on the observation that a high proportion of hybrid microcalli grown in liquid media did not adhere to the wall of the culture vessel, while microcalli derived from parental cells did. Seventeen from a total of 64 regenerated plants were conclusively verified as hybrids by AFLP DNA analysis. The hybrid plants were grown in soil for up to 4 months, and at least five flowered. Several of these hybrids survived when grown on high-zinc media.These hybridsaccumulated levels of zinc and cadmium that would have been toxic for B. napus. The data indicate that transfer of the trait for metal hyperaccumulation in plants is possible through somatic hybridization. Received: 1 December 1998 / Accepted: 30 January 1999     

A procedure for regenerating Japonica and Indica varieties of rice from protoplasts

Fertile rice plants have been regenerated from protoplasts of two japonica rice varieties (Radon and Baldo) using a protocol initially developed for plant regeneration from protoplasts of an indica rice. Embryogenic calli were developed from immature embryos of Radon and Baldo rice on a callus induction medium, and then used to establish cell suspensions. Protoplasts were isolated from the cell suspensions, and cultured on a Millipore filter placed on a Kao/agarose medium that contained cell clusters from suspensions of IR52 or IR45. The protoplasts grew vigorously on Kao medium and developed into embryogenic calli within two to three weeks. Somatic embryo development occurred during a subsequent transfer of the calli to an LS medium for two to three weeks. The calli were then transferred to MS or N6 plant regeneration medium, and within one to three weeks, plants regenerated from 21 to 32% of the Radon calli, and 33 to 35% of the Baldo calli. Based upon these results and the previous success in regenerating an indica variety from protoplasts, this procedure has great promise for regenerating a range of rice varieties, and probably for regeneration of other monocotyledonous plants from protoplasts     

Nurse culture of low numbers of Medicago and Nicotiana protoplasts using calcium alginate beads

Protoplasts of two species, lucerne and tobacco, were cultured in semi-solid droplets of calcium alginate as a means of nurse culturing very low numbers of protoplasts. It was shown that increasing autoclave times decreased the gelling capacity of the alginic acid. A convenient measure of viscosity is described to allow appropriate adjustment of the alginate solution. Tobacco protoplasts are shown to be more sensitive to higher alginate concentrations than lucerne, however beads with a final alginate concentration of approximately 1.5% were suitable for both species. Agitation of the beads in liquid medium was needed for optimum division frequencies. The volume of liquid medium affected the culture response. Interestingly, the local cell density (bead cell density) was shown to be more influential than the total cell density. Nurse beads with higher densities of protoplasts of the same species were visually marked with activated charcoal. Experiments were performed to determine whether nursing was effective with calcium alginate encapsulation and to what extent the cell densities could be lowered. When there were no nurse beads, divisions effectively ceased at 10⁴ per ml with lucerne and 10³ per ml with tobacco. In the presence of nurse beads, protoplasts in the test beads grew at high frequency down to the lowest densities tested, namely 50 per ml for tobacco. With these methods transformed lucerne protoplasts from electroporation experiments and somatic hybrids have been recovered and plants regenerated with much greater efficiency that was hitherto possible.     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Regeneration of Plants from Protoplasts of Arabidopsis thaliana L. cv. Columbia (C24), Via Direct Embryogenesis

This report describes a protocol for the regeneration of fertileplants from mesophyll protoplasts of Arabidopsis thaliana raceColumbia (C24). Regeneration was rapid and reproducible. Theprotocol is especially novel in that a large proportion of regeneratingprotoplasts regenerated via direct somatic embryogenesis. Protoplastsisolated from in vitro-grown plants entered sustained divisionafter 3–5 d in culture medium and over a period of severaldays 6–22% of protoplasts underwent at least one celldivision. Approximately 2–16% of these protoplasts continuedto divide and after 3 weeks in culture had formed macroscopiccolonies, of which 70–80% were regular embryo-like structures.Four weeksafter release from the alginate culture matrix andtransfer to solid medium in the light, 68–88% of thesestructures had produced well-developed shoots. Shoots couldbe maintained in culture or established in peat blocks. Theregenerated plants were fertile. Key words: Arabidopsis thaliana, protoplast, regeneration, embryogenesis, dicamba     

Comparison of Flowering Time Genes in Brassica Rapa, B. Napus and Arabidopsis Thaliana

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.     

Creation and analysis of Brassica napus + Arabidopsis thaliana somatic hybrids possessing maize Spm/dSpm heterologous transposable system

Functionally asymmetric somatic hybrids possessing heterologous transposable element Spm/dSpm were obtained following intertribal somatic hybridization between Brassica napus and transgenic Arabidopsis thaliana. Mobile genetic elements actively transposed in the hybrid genomes. Complete elimination of A. thaliana genome was not observed.     

Plant regeneration from leaf protoplasts of Brassica oleracea var. italica CV Green Comet broccoli

A procedure is described for regeneration of plants from leaf protoplasts of the hybrid broccoli cultivar, Green Comet (Brassica oleracea var italica). The totipotency of protoplasts isolated from plants regenerated from hypocotyl explants (GCR) was greater than that of protoplasts from plants grown directly from seed (GC). Using medium B developed by Pelletier et al (1983), division efficiencies greater than 70% were obtained in leaf protoplasts isolated from GCR. Approximately 1% of these protoplasts formed calli on solidified medium; 77% of the calli regenerated shoots. In contrast, protoplasts from seed-grown material showed a lower division efficiency (15–22%) and fewer protoplast-derived calli produced shoots. Some of the 178 protoplast-derived plants grown to maturity had variant phenotypes.Abbreviations NAA napthalene acetic acid - BA 6-benzylaminopurine - MES 1-morpholino-ethane sulfonate This work has been submitted by D. R. in partial fulfillment of the requirement for the Ph.D. degree     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

Plant protoplasts immobilized in calcium alginate: a simple method of preparing fragile cells for transmission electron microscopy

A simple method for electron microscopic preparation of plant protoplasts is described. The main problems in preparing these fragile protoplasts for electron microscopy have been cell collapse due to steep gradients between protoplasts and fixatives and unacceptable loss of material during the many steps of the procedure. These problems may be solved by immobilization of the protoplasts in calcium alginate beads. The free diffusion properties of this gel prevent steep gradients. The beads also simplify handling and prevent loss of material. Protoplasts isolated from hypocotyls of rape, Brassica napus (var. Niklas), have been used as a model system. Transmission electron microscopy of the immobilized protoplasts osmotically stabilized with glucose demonstrated adequate structural and ultrastructural preservation.     

用PEG法把外源基因导入甘蓝型油菜原生质体再生转基因植株

通过PEG处理把外源基因导入甘蓝型油菜原生质体。转化介质中二价阳离子的种类和浓度、携带DNA及PEG溶液的pH值都会影响基因导入效率。以潮霉素抗性和卡那霉素抗性作标记,均成功地筛选到了抗性愈伤组织。相对转化频率分别为1.3%和0.2%。前者明显高于后者。把抗性愈伤组织转到分化培养基上,分化出芽。诱导生根后移栽到土壤中,生长状况良好。酶活性测定和Southern blotting分析表明外源基因已插入到植物细胞基因组中。该遗传转化系统存在的主要问题是抗性愈伤组织分化频率较低。本文对其原因作了初步分析。     

Development of rapeseed with high erucic acid content by asymmetric somatic hybridization between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Crambe abyssinica</Emphasis>

PEG-induced asymmetric somatic hybridization between Brassica napus and Crambe abyssinica was carried out. C. abyssinica is an annual cruciferous oil crop with a high content of erucic acid in the seed oil valuable for technical purposes. UV-irradiated mesophyll protoplasts of C. abyssinica cv 'Carmen' and cv 'Galactica' were fused with hypocotyl protoplasts of different genotypes of B. napus cv 'Maplus' and breeding line '11502'. Shoot regeneration frequency varied between 6.1% and 20.8% among the different doses of UV-irradiation, ranging from 0.05 J/cm(2) to 0.30 J/cm(2). In total, 124 shoots were regenerated, of which 20 asymmetric somatic hybrids were obtained and verified by nuclear DNA content and AFLP analysis. AFLP data showed that some of the characteristic bands from C. abyssinica were present in the hybrids. Cytological analysis of these hybrids showed that 9 out of 20 asymmetric hybrids had 38 chromosomes, the others contained 40-78 chromosomes, having additional chromosomes between 2 and 40 beyond the 38 expected for B. napus. The investigation into the fertility of asymmetric somatic hybrids indicated that the fertility increased with increasing UV-doses ranging from 0.05 J/cm(2) to 0.15 J/cm(2). All of the hybrids were cultured to full maturity, and could be fertilized and set seeds after self-pollination or backcrosses with B. napus. An analysis of fatty acid composition in the seeds was conducted and found to contain significantly greater amounts of erucic acid than B. napus. This study indicates that UV-irradiation could be used as a tool to produce asymmetric somatic hybrids and to promote the fertility of the hybrids.