Plant regeneration from protoplasts of indica rice: genotypic differences in culture response

Summary Fourteen varieties of indica rice (Oryza saliva L.) were examined for their capacity for plant regeneration from protoplasts using the nurse culture methods developed for japonica rice. Calli induced from germinating seeds were grouped into two types: type I, white and compact; type II, yellow and friable. In four varieties producing type II callus, colony formation (2%–4.5%) and plant regeneration (2%–35%) were observed. The inability to develop suspension cultures was a major obstacle in regenerating plants from protoplasts of the remaining rice varieties studied.     

An assessment of the cultural capabilities of Trifolium repens L. (white clover) and Onobrychis viciifolia Scop. (sainfoin) mesophyll protoplasts

Mesophyll protoplasts isolated from white clover and sainfoin divided to form callus under similar cultural conditions. White clover protoplasts showed varietal differences in their plating efficiency. Sainfoin tissues regenerated readily by forming shoots, but induction of morphogenesis in white clover was only achieved after testing several media and culture sequences. Many of the white clover shoots were abnormal in being fused together to form green plate-like structures, but the latter still developed into plantlets while attached to the parent callus. The ability to isolate, culture, and regenerate mesophyll protoplasts of these two forage legumes is discussed in relation to future attempts to produce somatic hybrids between high tannin containing bloat-safe sainfoin and other major forage legumes such as alfalfa, white clover, and red clover.     

Plant regeneration from protoplasts isolated from suspension cultures of leek (Allium ampeloprasum L.)

A procedure is described for the regeneration of plants from protoplasts of tetraploid leek (Allium ampeloprasum L.), 2n = 4x =32. Regeneration-competent protoplasts could only be obtained from an embryogenic suspension culture that was initiated with friable, embryogenic callus derived from immature embryos. The generally low plating efficiency could be increased by embedding the protoplasts in Ca-alginate, compared to culturing the protoplasts in liquid or agarose-solidified medium. A minimum plating density of 2 × 10⁵ pps/ml was required to obtain microcalli. Upon transfer of the protoplast-derived calli on agarose-solidified BDS medium, morphologically different callus types proliferated. After transfer to regeneration medium, compact or friable calli with an embryogenic appearance produced somatic embryos and plantlets at a frequency of up to 80%. Calli that had been classified as heterogeneous also regenerated shoots, but mainly via organogenesis, at a frequency of 46%. After transfer of shoots to half strength MS medium, healthy, well-rooted plants were obtained, that were successfully transferred to soil. All plants contained the tetraploid DNA level.     

Enhancement of growth and regeneration efficiency from embryogenic callus cultures of Oncidium ‘Gower Ramsey’ by adjusting carbohydrate sources

The embryogenic callus was induced from shoot apex tissues of Oncidium ‘Gower Ramsey’, and the derived callus cultures maintained more than 5 years were viable in growth and exhibited high regeneration capability. Combination levels of exogenous 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ) could stepwise change granular and yellow callus into more friable or compact morphotypes. In the 16-h photoperiod culture, the influences of various carbohydrate sources including sucrose, maltose and trehalose were assessed on formation and development of protocorm-like body (PLB) from the embryogenic callus. Histological observations showed a unicellular origin for these PLBs. The growth of plantlets regenerated on half-strength Murashige and Skoog (MS) medium supplemented with maltose or trehalose was significantly better than those regenerated on sucrose. Approximately, 6000 PLBs could be generated in 2 months from an initial culture of 1 g callus fresh weight, and then more than half of the PLBs developed into plants in 4 months after two subcultures on the medium supplemented with 20 g/l trehalose.     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

A low-temperature method for maintaining plant regeneration activity in embryogenic callus of rice (Oryza sativa L.)

 A method was developed to maintain plant regeneration activity of rice cells (Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5  °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5  °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. Received: 7 January 1999 / Revision received: 28 April 1999 / Accepted: 26 May 1999     

Adventitious shoot regeneration from leaf explants of an African clone of cassava (Manihot esculenta Crantz) and analysis of the conformity of regenerated plants

A protocol for regeneration of adventitious shoots from immature leaf lobes from in vitro plants of cassava has been developed. Induction occurred in the presence of 2,4-D for ten days which was followed by transfer onto the regeneration medium containing 23 μM zeatin. Induction for more than 10 days in 2,4-D resulted in reduced regeneration and delayed shoot formation. A friable callus developed after a longer induction phase. A shorter induction resulted in the formation of foliar structures which did not develop further. Following subculture calluses produced shoots with improved frequency: 1.0 shoot/explant after 4 subcultures and 1.8 shoots/callus after 14 subcultures. When transplanted into soil, the regenerated plants were apparently normal and similar to the stock plants. A sample of 203 regenerants was examined for isozymes and DNA content using flow cytometry. Glutamate oxaloacetate transaminase (GOT) banding pattern was identical to that of the stock plants. Small changes were however detected in the phosphoglucomutase (PGM) pattern for 3.4% of regenerated plants, showing an additional band in the fast migration zone. The DNA content of the regenerated plants was homogeneous and was similar to that of the stock plants. The ploidy level was unchanged (2n = 36 chromosomes). This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytokinin autotrophy and differentiation in tissue cultures of haploid Nicotiana tabacum L.

Explants from the apical region (10 cm from the tip) of haploid Nicotiana tabacum cv. Wisconsin-38 were cultured on media with and without kinetin. Cell lines were selected in the dark and in the light. Cytokinins were extracted from the apical region of haploid plants and from callus tissues after 84 days of growth (third transfer culture). Chlorophyll was extracted from callus grown under light after 21 days of growth at each of the four cell line selection steps. Kinetin (+) cell lines and cytokinin autotrophic tissues grown in the light showed a compact growth pattern. Microscopic examination of these callus showed the presence of large numbers of nodules consisting of tracheary elements, parenchymatic cells, sieve elements and meristematic cells. Cytokinin-autotrophic callus grown in the dark showed an irregular growth pattern presenting regions of compact tissue and friable tissue. The compact tissue contained large amounts of nodules similar to those of kinetin (+) tissues and of cytokinin autotrophic tissues grown in the light. Extraction of the compact and the friable callus components showed high cytokinin activity in the compact region and low activity in the friable portion. It is suggested that cytokinin synthesis is related to the differentiation of the nodular structures. The amount of chlorophyll increased during the process of cytokinin autotrophic cell line selection.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

美味猕猴桃子叶愈伤组织的原生质体培养和再生植株

从B_5和NN-69培养基(含1mg/L 2,4-D)上分别选出美味猕猴桃子叶愈伤组织系A_(11)B_2和A_(16)N_1。在B_5原生质体培养基中,A_(11)B_2的原生质体再生细胞形成小细胞团;在NN-69原生质体培养基中,A_(16)N_1的原生质体再生细胞能持续分裂形成愈伤组织。经过分步诱导再生,获得A_(16)N_1原生质体再生植株。     

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

Summary The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice. The cell suspension in the AA medium that contained 4 amino acids as the sole nitrogen source was friable, finely dispersed, and readily released a large number of protoplasts. These protoplasts, subsequently cultured in NO₃ medium that contained nitrate as the sole nitrogen source, formed compact calli. The compact calli produced green plants with a frequency of 24%. Out of 15 flowering plants, 4 were haploids, the others were diploids which showed a uniform morphology but varied in seed fertility from 95 to 0%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Somatic embryogenesis in Asparagus officinalis can be an in vitro selection process leading to habituated and 2,4-D dependent embryogenic lines

Long-term embryogenic lines were repeatedly obtained from nine asparagus (Asparagus officinalis L.) genotypes by the selection of rare events, which consisted of the emergence of either a few somatic embryos or an embryogenic callus from a restricted area of a primary callus. In the first case, somatic embryos emerged from 1 % of calli induced with naphtaleneacetic acid and transferred to a medium without auxin. Isolated and subcultured on hormone free medium, these embryos developed habituated embryogenic lines (H lines) growing by adventive embryogenesis. In the second case, 3 % of primary calli developed then subcultured on 2,4-dichlorophenoxyacetic acid (2,4-D) produced a new type of friable and yellowish-white callus, constituted of clusters of globular somatic embryos which can be continuously maintained on 2,4-D (2,4-D lines). Among 2,4-D lines, two types were identified by subculturing them on hormone–free medium. Half of the 2,4-D lines were habituated and half were 2,4-D dependent. Most plants regenerated from H lines exhibited a strong increase in embryogenic capacity compared to control plants, unlike plants regenerated from the 2,4-D dependent lines. This increased embryogenic capacity was transmitted to the progeny as a monogenic dominant trait. H lines would therefore be issued from mutation(s) occurring in vitro, conferring both the embryogenic and habituated phenotypes. On the contrary, in the 2,4-D dependent lines, the embryogenic processes appeared to remain under exogenous auxin control and no evidence of a mutational origin could be inferred from the behaviour of regenerated plants.     

Induction of morphogenically competent callus and suspension cell cultures from leaf explants and mature seeds ofDigitaria sanguinalis

Summary Digitaria sanguinalis (crabgrass) has recently been introduced as a high quality forage crop. We report here a tissue culture system showing a high level of regeneration developed to aid in a breeding program. Two morphologically distinct types of callus, compact opaque and friable translucent, were induced from leaf blade explants and mature seeds when cultured on MS medium containing 0.9 μM 2,4-dichlorophenoxyacetic acid. Proline (25 mM) inhibited induction of callus but was required for continued maintenance. Plants were readily regenerated from the compact opaque callus. Selectively subcultured friable translucent callus continued to produce colony sectors of the morphogenically competent compact opaque callus when transferred to regeneration medium. Suspension cell cultures derived from callus or directly from leaf blade explants also produced regenerable callus.     

甘蔗幼叶切段培养中的体细胞胚胎发生

本文从形态学和组织学方面研究了甘蔗幼叶胚性愈伤组织发生及体细胞胚胎的形成过程。甘蔗幼叶片切段培养于含2.4—D1.5mg/1的MS培养基上,4—6天后切段开始形成愈伤组织,约10天后愈伤组织表面出现白色颗粒状结构。将含有白色颗粒状结构的愈伤组织转移至不含激素的培养基中,7—10天后可见有小植株长出。组织学和形态学观察表明,甘蔗离体再生植株是通过体细胞胚胎发生途径。     

Sugarbeet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.): shoot regeneration from callus and callus protoplasts

The successful application of recombinant DNA technology for crop plants requires efficient regeneration systems. A detailed study on the regeneration potential of callus and callus-derived protoplasts of a recalcitrant species, sugarbeet, was performed. A reproducible and highly efficient method for induction of regenerable friable callus was established from etiolated hypocotyl explants. A reduced sucrose concentration proved beneficial. Successful shoot regeneration could be demonstrated in 10 out of 12 tested lines. Seed germination, followed by callus induction and shoot regeneration required only a single culture medium. Additionally, the regeneration capacity of roots and root-derived callus was demonstrated. Highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants. To our knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet.     

Regeneration of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethnobotanical herb

A procedure was developed for plant regeneration of Hybanthus enneaspermus, a rare ethnobotanical herb from the Deccan peninsula in India, through seed-derived callus. Seeds demonstrated a high induction frequency (69.4±2.8%) and a high yield (364.4±2.5 mg) of light-yellow friable callus on Murashige and Skoog's (MS) medium containing 2.6 μm NAA and 2.2 μm BA within 4 weeks of incubation. After 1 year of subculture, yellow friable and light-green compact calli types were established from initial light-yellow friable callus. Shoot differentiation was achieved from light-green compact callus, but not from yellow friable callus. Shoot differentiation resulted when light-green compact callus was transferred to MS medium supplemented with 8.8 μm BA and 2.6 μm NAA; the highest percentage of calli forming shoots (66.6±4.8%) and the highest number of shoots (8.9±0.3) were achieved in this medium. Differentiated shoot buds elongated to 4–5 cm within 4 weeks. The addition of casein hydrolysate (500 mg/l) and more potassium phosphate (1.86 mm) to the culture medium enhanced shoot differentiation. Rooting was achieved on the shoots using half-strength MS medium containing 4.8 μm IBA. About 70% of the plants were established in pots containing pure garden soil after 2 weeks of hardening. The regenerated plants were morphologically uniform and exhibited normal seed set. Received: 23 July 1998 / Revision received: 18 November 1998 / Accepted: 26 November 1998     

Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria

 Stem segments of seedlings from two Alstroemeria breeding lines, cultured on media supplemented with 4 mg/l 2,4-dichlorophenoxyacetic acid and 0.5–1.0 mg/l 6-benzylaminopurine (BA), initiated soft callus, which became compact after subculture on a medium with only 0.5 mg/l BA. Friable embryogenic calli were initiated from compact callus on a medium supplemented with 10 mg/l picloram. Proembryos developed from friable embryogenic calli via embryos into plants after subculture on medium supplemented with 0.1 mg/l BA. The proembryos formed friable embryogenic calli again after culture on medium supplemented with 10 mg/l picloram. The total time needed to regenerate a complete plantlet from friable callus was approximately 6 months. This system for the production of embryogenic material is considered to have valuable applications for genetic transformation in Alstroemeria. Received: 22 April 1999 / Revision received: 16 July 1999 · Accepted: 20 July 1999     

Production of asymmetric hybrids between Arabidopsis thaliana and Brassica napus utilizing an efficient protoplast culture system

Application of the protoplast culture method developed for Brassica protoplasts to protoplasts of Arabidopsis thaliana has increased the opportunities for interspecific hybridizations involving Arabidopsis. A more-efficient and much-simpler method was established compared to the earlier-reported protocol developed for A. thaliana protoplasts in which alginate beads were utilized. Mesophyll protoplasts of A. thaliana (ecotypes 'Landsberg erecta' and 'Wassilewskija') were cultured in the modified 8p liquid medium, which had been developed for Brassica protoplasts. For comparison, protoplasts were cultured in sodium alginate beads supplied with B5 medium according to the protocol for A. thaliana. The protoplasts divided with high frequencies in the 8p medium, and calli proliferated more rapidly than in the sodium alginate beads. High frequencies of shoot differentiation and regeneration were observed in calli of both ecotypes, from about 30% in the ecotype 'Wassilewskija' to about 60% for 'Landsberg erecta'. The more-rapidly the calli developed, the higher the regeneration frequencies were. Asymmetric hybrids between A. thaliana and Brassica napus were obtained by treating the protoplasts of A. thaliana with iodoacetamide (IOA) and B. napus protoplasts with UV-irradiation before fusion with polyethylene glycol (PEG). By using the culture procedure developed for Brassica protoplasts, calli developed and plants were regenerated. Although most of the plants regenerated after cell fusion were A. thaliana-like and were judged to be escapes from IOA treatment, more than ten plants showed hybrid features of both morphological and molecular characters. Among the hybrids that have flowered so far, both male-fertile and male-sterile plants have been obtained. Back-crossings to A. thaliana are now in progress as is morphological and molecular characterization of the plants.     

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