Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro plant regeneration from leaf-derived callus in ginger (Zingiber officinale Rosc.)

Excised tissues from young leaves of ginger cv. Maran were cultured on revised Murashige and Skoog medium supplemented with various concentrations of growth regulators. The presence of 2, 4-D in the culture medium at 9.0–22.6 M resulted in callus growth. Organogenesis and plantlet formation occurred when the concentration of 2,4-D is reduced to 0.9 M and with the addition of 44.4 M BA into the medium. The rate of plant regeneration increased when the growth regulators are completely removed from the culture medium in the subsequent subcultures. The plantlets developed extensive root systems when they were put in MS liquid medium with 5.4 M of NAA. The establishment of these plantlets in soil is about 80%.Abbreviations BA N⁶-benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. ‘Lovely Tokyo’ and P. obconica cv. ‘Aalsmeer Giant White’. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.     

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

Summary We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10⁷ protoplasts.ml^–1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10⁶ protoplasts.ml^–1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.     

Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

Plant regeneration from protoplasts isolated from embryogenic suspension cultured cells of Cinnamomum camphora L.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1±2.1×10⁶/g FW) and viability (91.8±3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl₂·2H₂O, and 0.011% (w/v) NaH₂PO₄·2H₂O. First divisions occurred 7–10 days following culture initiation. The highest division frequency (24.6±2.9%) and plating efficiency (6.88±0.8%) were obtained in liquid medium (MS) supplemented with 30 g l^–1 sucrose, 0.7M glucose, 0.1 mg l^–1 NAA, 1.0 mg l^–1 BA, and 1.0 mg l^–1 GA₃. After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l^–1 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.     

Characterization of polymorphic microsatellite markers,isolated from ginger (Zingiber officinale Rosc.)

The present study reports isolation and characterization of eight polymorphic microsatellite markers for Zingiber officinale Rosc. (Ginger). A total of 34 alleles were detected across the 20 accessions, with an average of 4.3 alleles per locus. Values for observed and expected heterozygosities ranged from 0 to 1.0 and from 0.23 to 0.67, respectively. The heterozygote deficits were observed at three loci. At the significance threshold (P < 0.05) of the eight loci, seven were found to have deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibria were observed between 10 pairs of loci. Our data indicate the existence of moderate level of genetic diversity among the ginger accessions genotyped with eight markers.     

Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Hartog)

Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength of the macroelements, 5 m 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25%. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome complement for wheat (2n = 42).     

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

Plant regeneration from cell suspension-derived protoplasts of Nicotiana africana

Plants have been regenerated from Nicotiana africana Merxm. protoplasts isolated from cell suspensions. Two different sequences of media were assayed, one usually used to regenerate tobacco mesophyll protoplasts (K3,RMO) the other previously recommended for potato mesophyll protoplast regeneration (W-S-S, ST-1, ST-2, S-3). Only the media for potato protoplasts were efficient for African tobacco plant regeneration. The regeneration efficiency was 6.3 plants per 1000 plated protoplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

蔗糖和多效唑对试管生姜形成的影响

用不同浓度的蔗糖、NAA、Ca3(PO4)2、多效唑(Pac)和光照条件对江西兴国九山生姜(ZingiberofficinaleRosc.cv.Jiushan)进行试管姜的诱导,成功诱导出试管姜。结果表明,诱导试管姜的最佳培养基为:MS 6-BA0.2mgL-1 NAA0.5mgL-1 Ca3(PO4)22.5mgL-1 Pac2.5mgL-1 蔗糖8%;适宜浓度的Pac、Ca3(PO4)2、蔗糖有利于试管姜诱导;NAA和6-BA对试管姜的诱导不起促进作用;延长光照时间有利于试管姜膨大。     

Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava

Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×10⁶ protoplasts/g fresh weight). Protoplasts plated at a density of 10⁵–10⁶/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N⁶-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP. Received: 30 August 1996 / Revision received: 9 June 1997 / Accepted: 1 October 1997     

Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens Pall.

A reproducible release of viable protoplasts was obtained from friable calli of Astragalus adsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5 mg/l 2,4-D, 0.5 mg/l BA and 0.5 M glucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems showing a plating efficiency of 0.8±0.5% and 6.5±0.7%, respectively. Upon transfer to Murashige and Skoog (1962) medium (MS) with 1–2 mg/l BA, alone or in combination with NAA or 2,4-D at 0.1 mg/l, the protoplast-derived calli produced complete plantlets through somatic embryogenesis. The maximum percentage of calli producing somatic embryos (52.5± 2.2%) occurred on MS medium containing 0.1 mg/l NAA and 1 mg/l BA, whereas the maximum number of calli regenerating plantlets (64.7±6.2) was obtained on MS medium with 0.1 mg/l NAA and 2 mg/l BA. Received: 25 April 1997 / Revision received: August 1997 / Accepted: 2 September 1997     

Nitrogen nutrition of ginger (Zingiber officinale)

Summary Two fertilizer experiments were conducted in the field at Beerwah, South-East Queensland. In the first experiment leaf nitrogen concentrations, and the yield of ginger shoots and rhizomes at early and late harvests increased both with the total amount of nitrogen applied up to the highest level studied (336 kg N/ha as ammonium nitrate) and with the number of applications making up the total. At all levels of nitrogen application the apparent recovery of fertilizer nitrogen increased in the order 1 application <2 applications <4 applications. At 33.6 kg N/ha there appeared to be no advantage in dividing the total N applied into more than 4 applications but the data suggested higher recoveries of nitrogen with 8 applications at 112 kg N/ha and 336 kg N/ha. In the second experiment, ammonium nitrate, urea, and ammonium sulphate were found to be equally effective as nitrogen fertilizers for ginger when applied at equal rates of nitrogen per hectare. However, in terms of cost effectiveness they rated in the order urea > ammonium nitrate > ammonium sulphate.All three nitrogen sources acidified the soil, the decrease in soil pH during the growing season increasing with increasing rate of application. In Experiment 1 split applications, which increased the recovery of applied nitrogen in the crop, also increased the extent of acidification. In Experiment 2 ammonium sulphate tended to be more strongly acidifying than the other fertilizers but the difference was statistically significant only at the highest rate of nitrogen application. Because of the strong effects of nitrogen supply on both yield and soil pH, the highest yields were associated with end-of-season pH values below 5.0.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

Plant regeneration from meristem-derived callus protoplasts of apple (Malus3domestica cv. `Fuji')

A procedure has been established for regeneration from meristem-derived callus protoplasts of scion cultivars of apple that have been difficult to regenerate from leaf protoplasts. Calli were induced from the meristem of apples, Malus×domestica cvs `Fuji' and `Jonagold' and Malus prunifolia var `ringo Asami Mo84-A', cultured on MS medium (2 mg/l 2,4-D, 1 mg/l BA, 0.8% agar) and subcultured in a liquid medium. The ability to regenerate plants from suspension calli was studied under eight different combinations with respect to IAA, ABA, and TDZ concentrations. With the materials studied here, two combinations, one with 0.1 mg/l IAA, 0.1 mg/l ABA, and 2.0 mg/l TDZ and another with 0.1 mg/l IAA, 1.0 mg/l ABA, and 2.0 mg/l TDZ, were effective for plant regeneration. Protoplasts were isolated from the above suspension cultures and then cultured in KM8P medium containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5 mM, pH 5.7). Shoot formation of protoplast-derived calli was studied in the above-mentioned regeneration media. The high concentration of Gelrite (0.5% and 0.7%) was also shown to be important for shoot formation of protoplast-derived calli. Shoot primordia were formed in the medium containing IAA (0.1 mg/l), ABA (1.0 mg/l), and TDZ (2.0 mg/l). Ultimately, five regenerants of `Fuji' protoplasts were obtained from 200 protoplast-derived calli. Received: 19 June 1998 / Revision received: 9 October 1998 / Accepted: 27 October 1998     

In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc.)

Rhizome buds, excised from threeCurcuma spp., and ginger, inoculated aseptically on MS medium with varying levels of BAP and kinetin, produced multiple shoots. For shoot multiplication, a concentration of 3.0 mg/l BAP was found to be optimum for all the species.In vitro plants were successfully established in the field and were morphologically uniform. A simple method to extend the subculture interval was used and its relevance to germplasm conservation is discussed.Abbreviations BAP 6-benzylaminopurine - kinetin 6-furfurylaminopurine - MS Murashige and Skoog (1962)     

Plant regeneration from in vitro-cultured seedling leaf protoplasts of Actinidia eriantha Benth

Newly expanded in vitro leaves of Actinidia eriantha were used for protoplast isolation. Protoplasts were cultured in liquid MS medium (lacking NH₄NO₃) supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.4 M glucose. The plating efficiency after 3 weeks of culture was 19.4%, and calli were recovered without addition of fresh medium. These calli regenerated shoots on transfer to MS medium containing 2.28 μM zeatin and 0.57 μM IAA (indole-3-acetic acid). Regenerated shoots were rooted by immersion in 20 ppm IBA (indole-3-butyric acid) solution before culturing on half-strength MS medium lacking growth regulators. Somaclonal variation, in terms of chromosome number and nuclei per cell of protoplast-derived plants, was estimated. Received: 15 March 1997 / Revision received: 27 January 1998 / Accepted: 7 March 1998     

Metabolic profiling and phylogenetic analysis of medicinal Zingiber species: Tools for authentication of ginger (Zingiber officinale Rosc)

Phylogenetic analysis and metabolic profiling were used to investigate the diversity of plant material within the ginger species and between ginger and closely related species in the genus Zingiber (Zingiberaceae). In addition, anti-inflammatory data were obtained for the investigated species. Phylogenetic analysis demonstrated that all Zingiber officinale samples from different geographical origins were genetically indistinguishable. In contrast, other Zingiber species were significantly divergent, allowing all species to be clearly distinguished using this analysis. In the metabolic profiling analysis, the Z. officinale samples derived from different origins showed no qualitative differences in major volatile compounds, although they did show some significant quantitative differences in non-volatile composition, particularly regarding the content of [6]-, [8]-, and [10]-gingerols, the most active anti-inflammatory components in this species. The differences in gingerol content were verified by HPLC. The metabolic profiles of other Zingiber species were very different, both qualitatively and quantitatively, when compared to Z. officinale and to each other. Comparative DNA sequence/chemotaxonomic phylogenetic trees showed that the chemical characters of the investigated species were able to generate essentially the same phylogenetic relationships as the DNA sequences. This supports the contention that chemical characters can be used effectively to identify relationships between plant species. Anti-inflammatory in vitro assays to evaluate the ability of all extracts from the Zingiber species examined to inhibit LPS-induced PGE(2) and TNF-alpha production suggested that bioactivity may not be easily predicted by either phylogenetic analysis or gross metabolic profiling. Therefore, identification and quantification of the actual bioactive compounds are required to guarantee the bioactivity of a particular Zingiber sample even after performing authentication by molecular and/or chemical markers.