Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.     

Somatic cell genetic studies inBrassica species

In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.     

In vitro plantlet formation by organogenesis in E. camaldulensis and by somatic embryogenesis in Eucalyptus citriodora

Adventitious shoots were formed through callus on leaf explants of Eucalyptus camaldulensis Dehnh. (River red gum) taken from shoot cultures of mature trees. Callus formed in dark on a medium containing 1 g/l casein hydrolysate, 3 mg/l 1-naphthaleneacetic acid, 0.1 mg/l 6-benzyladenine and 50 g/l sucrose. Shoot initiation occurred in 4 weeks on calli shifted to light on a regeneration medium containing 10% coconut milk, 0.5 mg/l 6-benzyladenine and 20 g/l sucrose. Rooting occured in dark on a liquid medium containing 4 mg/l 1-naphthaleneacetic acid. Zygotic embryos of Eucalyptus citriodora Hook f. (Lemon scented gum) cultured in dark on a medium containing 3 mg/l 1-naphthaleneacetic acid and 50 g/l sucrose formed somatic embryoids which grew to normal plantlets on the same regeneration medium used for organogenesis.Abbreviations BAP 6-benzyladenine - CH Casein hydrolysate - CM Coconut Milk - NAA 1-naphthaleneacetic acid NCL Communication no. 4162     

Factors influencing induction,propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborg's B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of gelling agents on plaunotol accumulation in callus cultures ofCroton sublyratus Kurz

Callus cultures established from the leaves ofCroton sublyratus Kurz (Euphorbiaceae) contained little or no plaunotol on various media with different hormone combinations. Plaunotol accumulation was observed in calli which were cultured in media with increasing concentrations of gelling agents, especially gellan gum and agarose. The accumulation was observed within three weeks after transfer to the medium and was accompanied by chlorophyll increase, tracheid development and slow growth. Light and increasing concentration of gelling agents were observed to be indispensable for plaunotol accumulation in the callus.Abbreviations MS Murashige and Skoog (1962) basal medium - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine     

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.     

Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media

A comparison of semi-solid vs. liquid embryo proliferation media was made using two Gossypium hirsutum L. genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from mature, flowering plants were cultured on two modified Murashige and Skoog media. Medium 1 included 4.0 mg l^-1 NAA and 1.0 mg l^-1 kinetin; medium 2 contained 0.1 mg l^-1 2,4-D and 0.1 mg l^-1 kinetin. After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and the other on semi-solid (0.2% Gelrite) proliferation medium. Composition of proliferation medium was identical to that of initiation medium, except no growth regulators were added. Embryos were counted after eight weeks. The percentage of explants forming callus was influenced by genotype/initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid), and an initiation medium x genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3 embryos/culture) than on semi-solid proliferation medium (134.6 embryos/culture).Abbreviations NAA naphtaleneacetic acid - 2,4-D 2,4-D dichlorophenoxyacetic acid     

Nodular somatic embryogenesis and frond regeneration in duckweed,Lemna gibba G3

Duckweed(Lemna gibba) is a useful model system for elucidating plant development, but the techniques needed for regenerating fronds from calli are not yet well established. This study examined the effects of auxin, sucrose, and gelling agents on callus and frond formation inL. gibba G3. After three weeks of culturing on a solid medium, two types of calli were observed: watery, pale-green, and undifferentiated; or white, compact calli that were organized into nodules and which resembled somatic embryogenie calli. Homogeneous callus lines were produced through selective subculture. To induce nodular calli, auxin (2,4-D) was absolutely required, with an effective concentration of 5 to 20 μM; induction was found to be possible with up to a maximum concentration of 4.4%. The calli were then maintained on a medium with a reduced 2,4-D concentration (1 μM), and were transferred every three weeks. Optimal callus induction and growth were obtained by using 3% sucrose with a combination of 0.15% Gelrite and 0.4% agar. Fronds, however, could be regenerated only on distilled water solidified with a combination of 0.4% agar and 0.15% Gelrite. On this medium, 87% of the callus expiants regenerated into fronds after four weeks of culture. These new fronds were morphologically normal but small, approximately 15 to 20% of the size of stock fronds. Continued culture of these fronds in an SH medium produced normal duckweeds, and histological examination of the cultures revealed several distinct types of callus nodules. Nonetheless, because zygotic embryogenesis inL. gibba does not produce distinct bipolar structures, the developmental pathway of frond regeneration from these nodular cultures remains unknown.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

Tissue Culture of Cassava on Chemically Defined Media

Defined media that promote the initiation and undifferentiated growth of callus derived from stem explants of four cultivars of cassava, Manihot esculenta Crantz, are described. Growth rates and yields of cassava callus after 4 weeks of culture are shown to be comparable to those of callus of Nicotiana tabacum L. cv. Wisconsin No. 38. Nitrogen sources of ammonium nitrate or of ammonium chloride plus succinate supported growth of all four cultivars. Sucrose was superior to glucose as a carbon source. The cassava cultivars differed in their response to increasing concentrations of sucrose between 0.5% (w/v) and 3%, two of them increasing in dry matter with increasing sucrose concentrations of up to 3%. When cultured in the light on defined media that contained higher ratios of cytokinin to auxin, callus of the latter two cultivars turned green. Roots but not shoots differentiated from the callus of all cultivars. The influence of hormone concentrations, sucrose level, and nitrogen source on greening and root formation is summarized.     

Regeneration of plants from callus tissue of the pasture legume Lotononis bainesii

In vitro regeneration of plants from both cotyledon-, and leaf — derived calli of Lotononis bainesii Paker was studied under defined nutritional, hormonal and environmental conditions. Explants from both, cotyledons from seedilings of 4 days old and fully expanded leaves from mature plants, were cultured on MS medium containing 0.8% agar and supplemented with 0.01, 0.1, and 1 mg/1 concentrations of naphthaleneacetic acid (NAA) and 0.1, 1, and 3 mg/1 levels of benzyladenine (BA) in various combinations. Multiple shoot (on an average 4 shoots per callus) regeneration from primary callus occurred within 15 to 35 days of culture in most of the media tested. Although the best medium for shoot regeneration from cotyledon-derived callus contained NAA and BA at 1, and 0.1 mg/1 levels, respectively, maximal shoot regeneration from leaf-derived calli was achieved by using NAA and BA at 0.01 and 0.1 mg/1, respectively. Roots were induced to differentiate by transferring the regenerated shoots onto a medium lacking growth regulators.Supported by a research fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (República Argentina).     

Restoration of regeneration potential of long-term cultures of red fescue (Festuca rubra L.) by elevated sucrose levels

A tissue culture protocol for restoring embryogenic ability and increasing green plant regeneration from long-term callus (5-year old) and suspension cultures of Dawson red fescue (Festuca rubra var trichyoplylla Gaud) was developed. Pretreatment with elevated levels of sucrose over the standard level (60 mM) enhanced regeneration capacity and decreased the number of albino plants. The highest degree of embryogenesis and green shoot number occurred when calli were pre-treated on MS basal medium supplemented with 120 mM sucrose. Mannitol caused callus discoloration and death if added to pre-treatment media at 60, 90, 120, 150 or 180 mM. Cell suspension growth was greatest when 135 mM sucrose was added to the pre-treatment growth media. High concentrations of sucrose (135 and 180 mM) were necessary for plant regeneration from suspension aggregates pretreated with 135 or 180 mM sucrose and then plated on a growth regulator-free regeneration medium composed of half-strength MS salts and B5 vitamins.Journal Paper no. 3032 of the Massachuesetts Agricultural Experiment Station     

Plant regeneration in zigzag clover (Trifolium medium L.)

A study was conducted to regenerate plants from explant tissue and from callus culture of zigzag clover (Trifolium medium). Petiole segments from two strains of zigzag clover were cultured on L2 or in SL2 media. Shoots were regenerated via organogenesis from petiole segments of both strains of zigzag clover. Direct shoot regeneration was noticed as early as eight days after the initiation of cultures. Shoots were also regenerated via somatic embryogenesis from petiole-derived calli of the two strains on L2 and SL2 media. Regenerated plants have normal morphological characteristics.     

Biotechniques for improving acid aluminum tolerance in alfalfa

Alfalfa (Medicago sativa L.), cultivars ARC, Regen Y and Saranac were selected in vitro in a recently developed acid/aluminum toxic media. The new media produced higher initiation rates and higher fresh callus weights than those obtainable with the media described by Meredith and Connor for the selection of aluminum resistant variants in Nicotiana plumbaginofolia. Both rescue and direct initiation yielded adequate amounts of healthy callus for the initiation of embryogenesis. The toxic effect of the acid/aluminum media is expressed in both the percent of explants initiating callus and in the fresh-weights obtained during initiation and two subsequent subcultures.     

Cell regeneration and sustained division of protoplasts from cotton (Gossypium hirsutum L.)

Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbiochem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K₃ media of Kao et al., 1974 with 0.9 M BAP, 5 M IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5×10⁵ protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed.Abbreviations IAA indoleacetic acid - BAP 6-benzyl-adenine Arizona Experimental Station Publication No. 4373     

Anthocyanin production in callus cultures ofDaucus carota as influenced by nutrient stress and osmoticum

Summary Daucus carota callus developed red pigments under the influence of indole-3 acetic acid and kinetin. Maximum yield of anthocyanin at the end of 3 weeks was 5.4% on dry weight basis. The callus subjected to phosphate and nitrate stress produced 7.2% and 8.5% anthocyanin respectively. Feeding of sucrose at 7.5% level resulted in production of 15% anthocyanin. Mannitol as osmoticum had positive influence on anthocyanin production.     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.