Regeneration and characterization of somatic hybrids between Brassica napus and Diplotaxis harra

Stem cortex protoplasts of Brassica napus, inactivated with iodoacetate, were fused with cell suspension - derived protoplasts of Diplotaxis harra by polyethylene glycol (PEG) treatment. PEG at 15–18% (w/v) induced 5–9% heterofusions. The hybrid callus selection was based on morphology: D. harra calli were yellow and very fine, while B. napus protoplasts typically produced green, well defined calli. A number of green, large calli were selected after fusion experiments and flowering plants were regenerated from two of these callus lines. The morphological traits as well as cytological and biochemical analysis of four isoenzymes confirmed the hybrid nature of the regenerants.     

Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting

The experimental conditions for an efficient and reproducible enrichment of fusion products by flow cytometry, using protoplasts of different Brassica species as hybridization material, have been investigated. The heterokaryons were identified by the endogenous chlorophyll autofluorescencence of mesophyll protoplasts of one parent and the fluorescense of exogenously supplied carboxyfluorescin to the hypocotyl protoplasts of the other parent. By using a low head drive frequency (11 kHz), a large nozzle (110 μm) and a low nozzle pressure (30–35 kPa) good survival of the protoplasts was obtained after sorting. Heterokaryons were sorted using these parameters and on average 80% of the protoplasts were fusion products as judged by microscopy. They were cultured in small volumes, 150 μl, and started to divide after 3–5 days and regenerated calli easily. Isozyme analysis of the calli confirmed that 81% had the pattern typical for a hybrrid. Differentiation into shoots have been obtained from some of the hybrid calli; these shoots were also confirmed to be true hybrids.     

Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.)

Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readilyisolated by soaking chopped leaf tissue in distilled water for 16 h prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N₆ medium and and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots.     

Studies on the Somaclonal Variation of Regenerated Plants from Protoplasts of Actinidia deliciosa

Protoplasts were isolated from leaf, petiole and stem segment-derived calli induced from one pistillate plant of Actinidia deliciosa line No. 26. Regenerated plantlets were obtained from protoplasts of leaf-derived and stem segment-derived calli, while only calli regenerated from protoplasts of petiole-derived calli. Seventy-six plants from protoplasts of leaf-derived calli and 21 plants from that of stem segment-derived calli survived after transplanting or grafting during 1987–1989. One staminate plant and two pistillate plants bloomed in May, 1991 and fruited soon afterwards. In all of those three plants regenerated from protoplasts of leafderived calli, sex differentiation occurred from somatic cells of Actinidia was verified. Somaclonal variation on leaf shape and plant morphology was obviously appeared. Chromosome number identified from 16 plants varied from 116 to 180.     

A rapid procedure for plant regeneration from protoplasts isolated from suspension cultures and leaf mesophyll cells of wild Solanum species and Lycopersicon pennellii

A rapid procedure for protoplast isolation, culture and plant regeneration has been developed for two Solanum species (S. lycoperisicoides and S. verrucosum) and Lycopersicon pennellii. Freshly isolated protoplasts were initially cultured in liquid Solanum Culture Medium (SCM), containing 2,4-dichlorophenoxy acetic acid (2,4-D). Subsequent dilution with fresh culture medium without auxins appeared to be essential to obtain rapid regeneration medium later on. The resulting micro calli were first grown in a culture medium containing 0.5 mg/l 6-BAP and 0.05 mg/l NAA and 0.2 M mannitol and 7.3 mM sucrose to induce greening, at a lower osmolarity (300 mOsm · kg⁻¹). Then, the green micro calli were transferred to shoot induction medium, containing 2 mg/l zeatin, 0.1 mg/l IAA and 2% sucrose (150 mOsm · kg⁻¹). In this way plants could be regenerated from leaf mesophyll protoplasts and suspension cell-derived protoplasts of L. pennellii and S. lycopersicoides within 2 months. Shoot regeneration from leaf mesophyll protoplasts of the two lines of S. verrucosum could be obtained 3 months after protoplast isolation.     

A method for production of interspecific hybrids within Brassiceae via somatic hybridization,using resynthesis of Brassica napus as a model

A general method for the production of somatic hybrids within Brassiceae has been developed using mesophyll protoplasts from one parent and hypocotyl protoplasts from the other. Fusion products were easily identified by their intermediate phenotype between the parental protoplasts. They could be isolated 24 h after fusion with micropipettes using a micromanipulator. At that time their frequency was about 15%. They were cultured in small volumes, 10 μl, and a plating efficiency of 14% was obtained. Hybrid calli were obtained from the fusion products, which was confirmed by isozyme analysis. Ploidy level of one hybrid shoot was determined by flow cytometric DNA analysis.     

A novel method for regenerating plants from mesophyll protoplasts of Arabidopsis thaliana line WS

A procedure has been developed for the successful regeneration of plants from mesophyll protoplasts of Arabidopsis thaliana line WS. The protocol is an improved version of that of Damm and Willmitzer (1988). The main changes in original procedure are as follows:

1. A mixture of Cellulase Y-C (0.5%) and Pectolyase Y-23 (0.05%) is used for the isolation of protoplasts. Use of these enzymes reduces the incubation time to 50 min.
2. α-Naphthaleneacetic acid is used as the auxin throughout cultures of protoplasts and calli.
3. Protoplasts and calli are incubated under dim white light (0.8–8 μW/cm²) during culture.

With these modifications, we were able consistently to obtain regenerated shoots from about 70% of calli that had been transferred to shoot-forming medium even though the plating efficiency was rather low (about 0.5–1.5%).     

Effects of Alteria alternata f.sp. lycopersici toxins at different levels of tomato plant cell development

Since the host-specific toxins of Alternaria alternata f. sp. lycopersici play an important role in pathogenesis, they potentially could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Prerequisite for this is that sensitivity to the Alternaria alternata f.sp. lycopersici pathotoxins is manifest at the cellular level. To gain insight into cellular effects of AAL-toxins and into the mechanisms of plant insensitivity to AAL-toxins, effects of AAL-toxins on leaves, leaf discs, roots, calli, suspension cells, minicalli and protoplasts of susceptible and resistant tomato genotypes were studied. In leaves of susceptible genotypes, toxins cause severe necrosis, while in leaves of resistant genotypes necrosis was never observed. Inhibition effects of toxins were observed at all other levels in susceptible and resistant genotypes: toxins inhibited shoot induction on leaf discs, root growth and growth of calli, suspension cells and protoplasts. This indicates a cellular site for AAL-toxins. Differences in sensitivity to AAL-toxins between susceptible and resistant genotypes were observed in leaves and roots, but were not observed during shoot induction on leaf discs, in calli, suspension cells and protoplasts. However, differences in sensitivity to AAL-toxins in roots were at least 20 times less than in leaves. Therefore insensitivity seems related to a higher level of tomato plant differentiation and is most pronounced in leaves.     

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     

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     

Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum

Summary Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved growth in the presence of kanamycin.     

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.     

Stably transformed callus of wheat by electroporation-induced direct gene transfer

Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.     

新疆杨愈伤组织原生质体的游离与纯化

目的:以愈伤组织为材料,研究新疆杨原生质体的游离、纯化。方法:以新疆杨愈伤组织为材料,采用简单试验设计和方差分析方法,对新疆杨原生质体游离的影响因素进行研究,并利用二乙酸荧光素染色法观察原生质体活力。结果:适宜新疆杨愈伤组织原生质体游离的较适宜条件是:CPW+2.0%纤维素酶R-10+1.0%离析酶R-10+1.0%果胶酶Y-23+0.6 mol/L甘露醇,酶解温度27℃,酶解时间8 h。在此条件下,原生质体产量达8.5×106个/(g.FW),活力达83.6%。原生质体纯化可采用蔗糖等密度离心法,较适蔗糖浓度为30%。结论:研究筛选出的酶解因素组合与等密度离心条件较适宜新疆杨愈伤组织原生质体的游离和纯化。     

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.     

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt

Summary Green mesophyll protoplasts of the dihaploid potato line 1982 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 679 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%–4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.     

Plant regeneration from protoplasts of the monocotyledonous Haworthia magnifica v. Poelln.

Calli were initiated from flower buds, gynoecia and inflorescence segments of Haworthia magnifica v. Poelln. and subcultured on solid medium. Two liquid culture steps were necessary to prepare the calli for the isolation of protoplasts capable of sustained cell divisions. Plants were regenerated from protoplast-derived calli. The influence of both the osmolality of the culture media and exudates on the viability of protoplasts and protoplast-derived cell colonies is briefly discussed.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

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

Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa…     

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR⁺) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR⁺ calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5–9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1–25.8% of the tomato genome. Thus, DNA contained in 3.8–13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.