Kao Medium Research Articles

English

A protocol for the isolation and regeneration of protoplasts from leaf explant of Rhyncholaelia digbyana is presented. The protoplasts were isolated using hemicellulase enzymes at 1.5, 2.25, and 3% (w/v), pectinase at 0.5 and 0.75% (w/v) and cellulase at 1 and 2% (w/v). Protoplast counting was carried out with a Neubauer camera and an optical microscope at 40X, and viability was determined with Evans blue dye at 0.025% (w/v). The protoplasts were cultivated following the standard plate method, using the K&M media with 0.06 M of saccharose, 2.3% of Gelrite and plant growth regulators. It produced a yield of 386250±1875 protoplasts/g of tissue using an enzymatic combination of 1.5% (w/v) of hemicellulase, 0.5% (w/v) of pectinase and 1% (w/v) of cellulase with an incubation time of 4 h. The colonies were observed after two months of culture and the highest number of colonies (1.66±0.50) was obtained when the protoplasts were cultured in Kao medium with 4.53 µM of 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.912 µM of Zeatin and 3.10 µM of 6-Benzylaminopurine (BA). The nuclear DNA content estimated by flow cytometry for R. digbyana was 27.39±3.8 pg of DNA equivalent to 26.79×109 pb and the number of mitotic chromosomes counted was 2n=40.   Key words: Isolation, in vitro culture, plant regeneration, flow cytometry.

Establishment of Protoplast Culture of Solanum sisymbriifolium

The optimal culture conditions were studied for plant regeneration from mesophyll protoplasts of Solanum sisymbrizfolium. Aseptic seedlings ofS. sisymbrizfolium were used as a material for protoplast culture. Many viable protoplasts were isolated by incubating leaf slices in an enzyme solution containing 0.250/0 Meiserase and 0.050/0 Macerozyme for 16 h at 25 'C wlthout shaking. Protoplast density of 5.0 X 10*/ml in Kao medium containing 5.0mg/1 NM, 1.0mgfl 2,4-D and 1.0mg/1 BA was optimal for colony formation. Most colonies formed when protoplasts were cultured at 25 'C after initial culture at 30'C for one week. On the MS agar medium with 1.0mg/1 zeatin, 38.50/0 of protoplast~lerived calli differentiated shoots. These shoots rooted on 1/2 MS medium with 5.0mg/1 sucrose and 2.5g/1 gellan gum, and developed into whole plants.

EFFICIENT CALLUS INDUCTION AND PLANT REGENERATION VIA SOMATIC EMBRYOGENESIS FROM IMMATURE LEAF-DERIVED PROTOPLASTS OF GROUNDNUT (ARACHIS HYPOGAEA L.)

High yields of protoplasts were obtained from immature leaves of aseptically grown plants ofArachis hypogaeausing an enzyme solution containing cellulase 2.0% (w/v) and Macerozyme 1.0% (w/v) in 0.6 M mannitol. Isolated protoplasts were cultured in Kao's medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and mini calli in 4 weeks. After 4 weeks, protoplast colonies were transferred to the Murashige and Skoog (MS) medium supplemented with a-naphthalene acetic acid (NAA) and BAP. Colonies proliferated into actively growing calli. Further attempts to regenerate plants from such calli were not successful. However, protoclones differentiated roots on the same medium. Alternative methods for plant regeneration from protoplast derived callus cultures were tried through somatic embryogenesis. Protoplast-derived calli treated with 2,4-D and BAP formed somatic embryos. Somatic embryogenesis began in the proembryo stage and proceeded from globular to dicotyledonary stage. Embryos were then transferred onto hormone-free MS medium for germination. Five to ten percent of these embryoids germinated and grew to plantlets. Regenerated plants were transferred to plastic cups and grown to maturity.

Somatic embryogenesis and plant regeneration inMedicago suffruticosa

A successful procedure has been designed for the regeneration of plantlets from leaf sections of the self-pollinating species,Medicago suffruticosa. Callus growth was promoted by a 4-week culture period on liquid Kao's medium containing 4.9 μM benzyladenine and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-d), followed by a 4-day treatment in which the benzyladenine was elevated to 44.4 μM. Shoots/plantlets were observed after 3–4 weeks culture on growth regulator-free agar-solidified medium. Under these conditions, the regeneration frequency from callus was 18% and a histological study showed that this regeneration was through somatic embryogenesis. The growth regulator treatment, with a relatively high concentration of growth regulators (44.4 μM benzyladenine) for a short time period (4 days), is important for inhibiting polyphenol compounds and for stimulating callus growth and plant regeneration.

Isolation and culture of barley megasporocyte protoplasts

An efficient technique has been developed for the isolation of barley megasporocyte protoplasts at early meiotic prophase. Ovules were dissected out of ovaries under aseptic conditions, subjected to a brief enzymatic digestion, and then transferred to a modified Kao medium with 90 g/l sucrose and 20 mM CaCl2. A small incision was made with a scalpel through the softened epidermal cell layer of the nucellus and the megasporocyte could then be liberated into the medium by applying gentle pressure on the nucellus. The megasporocyte appeared to be completely devoid of a wall and changed its in situ pyriform shape to completely spherical when extruded into the medium. Four to nine protoplasts could typically be isolated per spike. Protoplasts cultured in medium degenerated after a few days. Viability was dramatically improved if protoplasts were co-cultivated with barley microspores undergoing microspore embryogenesis. More than half of the protoplasts were still alive after 6 days of culture, and in some cases they survived more than 12 days of culture. Fluorescence microscopy of the cultured protoplasts stained with 4′,6-diamidino-2-phenylindole (DAPI) or aniline blue revealed that the protoplasts remained uninuclear and reformed their callose wall.

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

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 10(6) protoplats plated anged from 0.4 to 20.0, and the average seed fertility of single panicles of these RO plants was about 40%.

Effect of Karyotype on Somatic Embryogenesis from Immature Triticale (x Triticosecale Wittmack) Embryos

To assess the tissue-culture response of different karyotypes of spring triticales, immature embryo explants of 20 secondary triticales with a complete karyotype (complete rye genome with or without a 6D/6A substitution) or substituted karyotype (incomplete rye genome with a 2D/2R substitution), two primary triticales with octoploid and two with hexaploid karyotypes, were cultured on MS and on Kao medium supplemented with 2,4-D. Complete triticales produced significantly more embryogenic callus than substituted types. Complete 6D/6A karyotypes showed the highest embryogenic response. Octoploid primary triticales showed high and uniform production of embryogenic callus on both media. These findings complement earlier results on the agronomic and quality differences between the karyotypes.

High Variation in Cardenolide Content of Plants Regenerated from Protoplasts of the Embryogenic Cell Strain VII of Digitalis lanata

Summary Protoplasts prepared from cells of the embryogenic D. lanata cell strain VII were purified by flotation and cultivated in a modified Kao medium with 1 μmol L -1 2,4-D, 5μmol L -1 NAA and 2.5 μmol L -1 zeatin solidified with Sea Plaque agarose. In a first step, small proembryonic cell colonies were formed. Later on, globular and bipolar somatic embryos were regenerated in a medium with 5 μmol L -1 BA and 0.05μmol L -1 NAA. Plantlets were obtained from the bipolar embryo structures in the absence of plant growth regulators on the surface of perlite wetted with nutrient solution. A few plantlets could be adapted to greenhouse conditions and to the open ground. A fraction of the cell colonies regenerated from individual protoplasts formed cardenolides if cultivated on the surface of solidified Nm I containing 5 μmol L -1 2,4-D and 0.1 μmol L -1 kinetin. The percentage of these colonies was higher if compared with that of the mother culture under the same conditions. The regenerated plants showed greater variability relative to the cardenolide content than seed-born plants, clone plants or plants regenerated directly from cell colonies of strain VII.

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

Rapid plant regeneration through organogenesis and somatic embryogenesis from cultured protoplasts of Brassica juncea

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×104 ml-1 in Kao's medium supplemented with 1.0 mgl-12,4-D, 0.1 mgl-1 NAA and 0.5 mgl-1 zeatin riboside. After three days of culture in darkness at 25±1°C, cultures were transferred to light (70 μEm-2s-1) in a 16/8 h ligo ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl-1 2,4-dichlorophenoxyacetic acid and 1.0 mgl-1 benzyladenine. On the fifteenth day, microcalli were plated on K3 medium gelled with 0.5% agarose (Type 1, low EEO, Sigma). After a further period of two weeks, transfers were made to specific media to achieve either organogenesis or somatic embryogenesis. Time taken from plating protoplasts to obtaining plantlets is 8–10 weeks. Using this procedure, several hundred regenerated plants have been hardened in a growth chamber and transferred to soil.

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 1·10(6) to 15·10(6) 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, N(6)-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.

Chromosome Irregularities in Wheat and Triticale Plants Regenerated from Leaf Base Callus

AbstractBasal leaf sections of Triticum aestivum L., cv. ‘Chinese Spring’, and XTriticosecale Wittmack cv.‘Welsh’were cultured on Kao's medium with 2 mg/l of 2,4‐D. Callus was induced on the leaf sections at frequencies of 64 and 63 %, respectively, and plant‐lets regenerated at 18 and 31 %, respectively. Compared to the controls, the regenerated plants showed higher frequencies of univalent chromosomes at meiosis and low frequencies of trivalents, and quadrivalents; indicating that the callus phase had induced low frequencies of chromosome irregularity.

Study of factors affecting the culture of Brassica napus L. and B. juncea Coss. mesophyll protoplasts

Various factors affecting the culture of Brassica napus and B. juncea mesophyll protoplasts were examined in order to develop suitable culture media for these species. The basic components (salts and vitamins) of culture media K3 and Kao best supported cell division and colony development in protoplast culture of both species. The addition of casamino acids to Kao's medium resulted in colony browning in B. napus genotypes. B. napus protoplasts grew well with glucose as the osmotic stabilizer, whereas B. juncea protoplasts responded better to sucrose. High NAA and low 2,4-D combinations were effective in stimulating colony growth. Colony development was rapid for a range of genotypes cultured with these recommendations in these media and plant regeneration was obtained from protoplast-derived calli in both species.

Plant regeneration from cotyledon protoplasts of glycine canescens and G. clandestina

Protoplasts isolated enzymatically from cotyledons of wild Glycine species were cultured in agarose-solidified droplets of Kao protoplast culture medium. Incubation of droplets in liquid Kao medium mixed with a B5-based formulation resulted in protoplast-derived colonies. Subculture of tissues with liquid SC2 medium containing B5 salts and vitamins, 3% sucrose, 1.1 mg 1 −1 BAP and 0.005 mg 1 −1 IBA, followed by transfer to agar solidified SC2 medium, induced shoots in two accessions, G. clandestina G1231 and G. canescens G1171. Shoots elongated on agar B5-based medium with 0.2 mg 1 −1 BAP and 0.005 mg 1 −1 IBA(SC6), and rooted on hormone-free, half-strength semi-solid B5 medium.

Chromosome instability in somaclones of a Triticum crassum × Hordeum vulgare hybrid

Immature inflorescence culture and subsequent plant regeneration was practiced for four successive cycles using a Triticum crassum × Hordeum vulgare hybrid cultured on Kao's medium supplemented with 2,4-dichlorophenoxyacetic acid (5 mg/mL). In one line, chromosomal mixoploidy was observed among both mitotic and meiotic cells. Variation in chromosome number of 20 to 98 was observed in mitotic and 14 to 68 among meiocytes in the first cycle regenerants. The range in chromosome number decreased in subsequent regeneration cycles. Fragmented chromosomes were observed at low frequencies in both groups of cells. The high frequency of univalents at meiosis was attributed to possible elimination of chromosomes carrying meiotic pairing control genes.Key words: somaclonal variation, hybrids (intergeneric), tissue culture, chromosomal mixoploidy.

Cytogenetics of tissue culture regenerated hybrids of Triticum tauschii × Secale cereale

Immature inflorescences of Triticum tauschii (Coss.) Schmal. × Secale cereale L. cv. 'Puma' hybrids were induced to callus on Kao's medium supplemented with 5 mg/L 2-4-dichlorophenoxyacetic acid (2.4-D). Plantlets with diploid and tetraploid chromosome numbers were subsequently regenerated from the callus. Chromosome pairing in the diploid regenerants and tetraploid amphiploids was incomplete and the plants were sterile.Key words: Triticum, Secale, chromosome pairing, callus, tissue culture.

Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L.

A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.