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.     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

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     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium × formolongi by utilizing meristematic nodular cell clumps

Plant regeneration from protoplasts of Lilium × formolongi cv. Azusa was achieved by utilizing suspension cultures of meristematic nodular cell clumps with a high plant regeneration ability. Creamy-white calli with embryogenic potential were initially induced from the seeds on Murashige and Skoog (MS) medium containing 4.1 μM picloram. The calli were then transferred into a liquid medium of the same composition, in which they turned into compact cell clumps which consisted of meristematic nodules. Protoplasts were readily isolated from these meristematic nodular cell clumps. Colonies were successfully formed from the protoplasts by embedding in 0.1% gellan gum-solidified MS medium containing 4.1 μM picloram and 0.5 M glucose. They regenerated shoots and roots on MS medium containing 2.2 μM benzylaminopurine (BAP). The plants thus obtained produced flowers with normal fertile pollen 8 months after successful transfer into soil. These plants had normal chromosome numbers (2n = 24) but had shorter leaves than original plants. They set seeds after as well as cross pollination.     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

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     

Regeneration of fertile Arachis paraguariensis plants from callus and suspension cultures

Highly morphogenic callus cultures were isolated from stamens of a wild peanut species, Arachis paraguariensis. These cultures were initiated on modified N6 medium containing 0.2 mg1l^-1 4amino-3,5,6-trichloro-picolinic acid (picloram) and 0.5 mg l^-1 6-benzylaminopurine (BAP) and were maintained on modified N6 medium with 0.008 mg l^-1 picloram and 0.25 mg l^-1 BAP. Buds formed on the calli growing on the maintenance medium developed into shoots when they were transferred to a MS salts based medium with no hormones. The cultures could also be maintained as a suspension culture in N6 liquid medium. When cell clumps larger tham 840 m were collected from the suspension culture and transferred to MS medium without hormones, they formed shoots in liquid culture. Root formation rarely occurred in agar or liquid cultures. Therefore, grafting to stems of rooted seedlings was used to obtain plants from regenerated shoots. Eight out of 50 field grown plants produced viable seed.     

Comparison of 2,4-D and picloram for selection of long-term totipotent green callus cultures of sugarcane

Long-term regeneration of sugarcane (Saccharum spp. hybrid and Saccharum spontaneum L.) callus cultures was achieved by selection of green callus on MS agar medium containing 0.5 mgl^-1 picloram or 2,4-D. Newly initiated sugarcane callus cultures were a complex mixture of different tissue types including white, nonregenerative and green, regenerative tissues. The proportion of the tissue types changed as a function of time in culture, genotype, and amount and kind of auxin. Green callus on picloram media always regenerated green plants. Nine hybrids and ten wild relatives of sugarcane produced green calli on picloram media whereas only three hybrids were grown as green calli on 2,4-D media in long-term culture. Green calli were inoculated into liquid MS medium with 0.5 mgl^-1 picloram for suspension culture. These cultures were totipotent after 19 months. For routine culture, we initiated callus cultures on modified MS medium with 3 mgl^-1 2,4-D, then in two to three weeks we subcultured callus on MS medium with 0.5 mgl^-1 picloram and selected for green callus. Green calli regenerated large numbers of green plants after more than four years.     

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

Plant regeneration from leaf protoplasts of <Emphasis Type="Italic">Solanum virginianum</Emphasis> L. (<Emphasis Type="Italic">Solanaceae</Emphasis>)

Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 10⁶/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.     

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     

Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 10⁵ protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 μmol m⁻² S⁻¹ of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.     

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 protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 10⁶ g^-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.     

Fertile indica and japonica rice plants regenerated from protoplasts isolated from embryogenic haploid suspension cultures

Summary A system to regenerate fertile rice (Oryza sativa L.) plants (both indica and japonica varieties) from protoplasts isolated from anther-derived embryogenic haploid suspension cultures has been established. Green plants were regenerated from protoplast-derived cell clusters five months after suspension culture initiation. Protoplast yields and subsequent growth of the protoplast-derived microcalli were enhanced by transferring suspension cells into AA medium (Muller et al. 1978) three to four days prior to protoplast isolation. Protoplasts were cultured initially in Kao medium (Kao et al. 1977) and in association with nurse cells for four weeks. Protoplast-derived microcalli were transferred onto N6 (Chu et al. 1975) or MS (Murashige and Skoog 1962) media for callus proliferation. Callus growth was more rapid and the calli were more enbryogenic when grown on N6 medium. The 2,4-D concentration used to develop the suspension culture was important. Cell cultures grown in medium containing 0.5 mg/l 2,4-D released protoplasts whose plating efficiency was higher than for protoplasts obtained from suspension cultures grown in 2.0 mg/l 2,4-D. However, suspension cells grown in 2.0 mg/l 2,4-D were superior with regard to the ability of protoplast-derived calli to regenerate green plants. Amongst several hormone treatments evaluated, a combination of 0.5 mg/l NAA + 5.0 mg/l BAP resulted in the largest number of green plants regenerated. There were no significant differences between BAP or kinetin regarding total number of plants regenerated. More than 200 green plants have been produced form six independently initiated suspension cell lines. The number of regenerated plants per 10⁶ protoplats plated anged from 0.4 to 20.0, and the average seed fertility of single panicles of these R_O plants was about 40%.     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.)

A procedure is described to regenerate plants from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Somatic embryogenic calli were induced from ginger shoot tips on solid MS medium with half the concentration of NH₄NO₃ and supplemented with 1.0 mg l⁻¹ 2,4-Dichloroacetic acid (2, 4-D) and 0.2 mg l⁻¹ Kin. Rapid-growing and well-dispersed suspension cultures were established by subculturing the embryogenic calli in the same liquid medium. Protoplasts were isolated from embryogenic suspensions with an enzyme solution composed of 4.0 mg l⁻¹ cellulase, 1.0 mg l⁻¹ macerozyme, 0.1 mg l⁻¹ pectolyase, 11% mannitol, 0.5% CaCl₂ and 0.1% 2-(N-morpholino) ethane sulphonic acid (MES) for 12–14 h at 27°C with a yield of 6.27 × 10⁶ protoplasts g⁻¹ fresh weight. The protoplasts were cultured initially in liquid MS medium with 1.0 mg l⁻¹ 2, 4-D and 0.2 mg l⁻¹ Kin. Then the protoplast-derived calli (1.5 cm²) were transferred to a basal MS medium containing 0.2 mg l⁻¹ 2, 4-D, 5.0 mg l⁻¹ benzyladenine (BA), 3% sucrose and 0.7% agar. The white somatic embryos were transferred to MS medium lacking growth regulators for shoot development. Shoots developed into complete plantlets on a solid MS medium supplemented with 2.0 mg l⁻¹ BA and 0.6 mg l⁻¹ α-Naphthaleneacetic acid (NAA). In addition, the effects of AgNO₃, activated charcoal (AC) and ascorbic acid (AA) on browning of protoplast-derived calli are discussed.