Dependence of wheat callus growth,differentiation and mineral content on carbohydrate supply

The effects of different carbon sources on the growth, differentiation and mineral content of wheat callus were investigated. Callus originating from immature embryos showed optimal growth produced the highest ratio of shoots when it was cultured on the medium containing 0.058 M sucrose. Higher carbohydrate concentrations reduced both shoot formation and growth. On the other hand, when sucrose concentration was less than 0.029 M neither differentiation nor greening was observed. Mannitol had a stimulating effect on shoot formation when the medium containing 0.029 M sucrose was supplemented by mannitol to get the final concentration of 0.058 M. The respiration rate increased along with increasing concentration of sucrose and glucose, and reached a maximum in the case of sucrose at the concentration of 0.263 M. On the addition of different concentrations of mannitol to a 0.058 M sucrose medium the respiration remained essentially unchanged. The mineral content of the tissue cultures also depended on the carbohydrate concentration. The water content decreased with increasing carbohydrate concentrations and among carbohydrates examined, sucrose was the most effective. The nitrogen and potassium contents of the calli reached their maximum values at 0.117 M–0.175 M carbohydrate content. The highest phosphorus contents were detected at 0.350 M–0.468 M carbohydrates. Phosphorus proved to be the most sensitive to osmotic changes.     

Regeneration of plants from long-term cultures of Oryza sativa L.

Root and embryo derived callus tissues of rice grown on sucrose alone as carbon source lost their ability to organise shoots by 75 and 100 days in culture respectively. Along with 2% sucrose, either 3% sorbitol or 3% mannitol was found to be necessary in the growth medium for the callus to regenerate whole plants over a period of 1400 days without any decline in the shoot forming ability. Our results indicated that incorporation of sorbitol or mannitol in the callus proliferating medium provides long-term totipotent rice cultures with a high frequency (50–60%) of shoot differentiation.     

Carbohydrate Availability and Shoot Formation in Tobacco Callus Cultures

Tobacco callus grown on a shoot-forming medium containing sorbitol or no carbon source survived, but did not produce shoots. Transfer of tissue from a sucrose medium to carbohydrate-deficient media and vice versa suggested that the growth of the tissue was a function of a total period in contact with available carbohydrate. Both starch and free sugars in the tissue were utilized during shoot initiation. Furthermore, it appeared that the continuous availability of carbohydrate was required for shoot primordium growth and/or their development into leafy vegetative shoots in dark-grown cultures.     

Sucrose-Modulated Morphogenesis in Anagallis arvensis L.

Sucrose was found to have a modulating effect on the morphogenesisof Anagallis arvensis L. leaves cultured in a Murashige-Skoogmedium. Root formation and growth seem to be more independentthan other morphogenetic expressions. Roots formed without exogenoussugars at 25°C but sucrose seemed to be necessary at 32and 35°C. Sucrose at 3% improved shoot formation at 25°Cand had an inhibitory effect at 6%concentration and 35°C.Shoot growth (internode length) is inhibited by sucrose concentrationshigher than 3%. Sucrose could also replace light irradiancein regulating shoot and leaf growth. A higher sucrose concentration,than that required for roots and shoots formation, is necessaryfor flower and fruit formation, but sucrose could not replacethe photoperiod requirement for flowering in culture medium. (Received June 17, 1985; Accepted December 24, 1985)     

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmoticstress, created by high agarose or carbohydrate concentrations,on the regeneration of fertile plants from protoplast-denvedcolonies of several indica (IR43, Jaya, Pusa Basmati 1) andjaponica (Taipei 309) rice varieties. Observations of the culturesdeveloped on media containing one of these carbohydrates (cellobiose,fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose),each at 88 mM, indicated that maltose was the preferential carbonsource for the proliferation of embryogenic callus and shootregeneration. Maltose-containing medium induced shoot formationin 24–66% of the protoplast-derived tissues, dependingupon the rice variety, compared to shoot regeneration from 4–32%of the tissues in sucrose-supplemented medium. Media containing288 mM maltose or an equimolar combination of 88 mM maltoseand 200 mM mannitol, caused water loss from calli and promotedthe growth of embryogenic calli. These calli formed shoots withgreater frequencies when subsequently transferred to shoot regenerationmedium with 88 mM maltose. A medium containing 88 mM maltoseand semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarosehad a similar beneficial effect on the growth of embryogeniccalli and simultaneously supported high-frequency (48–55%)shoot formation. The optimum shoot regeneration frequencies(60–78%) were obtained when protoplast-derived colonieswere serially cultured on to shoot regeneration medium containing1.0% (w/v) agarose for 4 weeks, followed by a 2-week cultureperiod on the same medium with 0.5% (w/v) agarose. Plants regeneratedon medium containing maltose and/or 1.0% (w/v) agarose werephenotypically normal and fertile. Key words: Carbohydrates, Oryza sativa L, indica and japonica rice, osmotic stress, plant regeneration, protoplast-derived colonies     

The use of flax (Linum usitatissimum) as a model system for studies on organogenesis in vitro: the effect of different carbohydrates

Three carbohydrate sources, maltose, sucrose and cellobiose, were compared for the respective effects on regeneration from internodal sections of flax. It was found that different patterns of morphogenesis were attributable to the type of carbohydrate and its concentration. Mean shoot regeneration was highest on a medium containing 87 mM maltose with a rate of 2.4 shoots per explant and root formation was stimulated to the greatest degree on a medium with 87 mM sucrose. This medium also resulted in the highest tissue dry weight.     

Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro

Efficacy of phloroglucinol in promoting growth and development of in vitro-derived shoot tips was studied in six potato (Solanum tuberosum L.) genotypes. Different concentrations of phloroglucinol (0, 0.08, 0.4, 0.8, 1.2 and 1.6 mM) were tested in combination with either 0.1 or 0.2 M sucrose in shoot tip proliferation medium based on MS (Murashige and Skoog, 1962) medium supplemented with 5.8 μM GA₃ (gibberellic acid), 1.1 μM BA (N⁶-benzyladenine) and 8.39 μM D-calcium pantothenate. Phloroglucinol fostered multiple shoot formation, promoted axillary shoot proliferation in terms of shoot tip fresh weight and shoot length, and stimulated root formation on the shoot tips. There was significant phloroglucinol × sucrose interaction for number of shoots developed per shoot tip, shoot tip fresh weight and number of roots induced per shoot tip. The beneficial effect of phloroglucinol on shoot tip survival was conspicuous only in genotypes that showed poor survival in the control proliferation medium. There were significant differences in response between the two sucrose levels with regard to shoot tip fresh weight and number of roots per shoot tip. Phloroglucinol in combination with 0.2 M sucrose induced maximum number of roots per shoot tip. Optimum shoot tip growth was fostered in medium containing 0.8 mM phloroglucinol and 0.2 M sucrose. High frequency multiple shoot formation in this medium ensures a faster rate of potato shoot tip multiplication within a limited time and space. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

Sucrose level influences micropropagation and gene delivery into leaves from in vitro propagated highbush blueberry shoots

In an effort to increase in vitro blueberry (Vaccinium corymbosum L.) shoot production without negatively impacting subsequent genetic engineering experiments, studies were conducted to examine the effects of sucrose concentration in the propagation medium on shoot proliferation and on the transfer of an intron-containing -glucuronidase (GUS) gene into leaf explants from the propagated shoots. Numbers of axillary shoots >0.5 cm in length did not significantly increase for `Bluecrop' when sucrose levels were increased from 15 mM to either 29, 44 or 58 mM. The number of axillary shoots increased significantly for Duke ' and `Georgiagem' when sucrose concentrations were increased from 15 to 44 mM, and from 15 to 58 mM, respectively. Four-days of cocultivation with Agrobacterium tumefaciens strain EHA105 yielded highest GUS-expressing leaf zones on leaf explants from shoots cultured on either 15 or 29 mM sucrose. The number of GUS-expressing leaf zones was significantly lower on leaf explants derived from shoots grown on 58 mM sucrose than from those grown on 15 mM sucrose for all three cultivars, and was significantly lower on 44 mM compared to 15 mM for cultivars Duke and Georgiagem. These studies indicate shoot pretreatment conditions for optimizing subsequent blueberry genetic engineering experiments. Thus, a blueberry shoot proliferation medium containing 15–29 mM sucrose is recommended for explants later used for genetic transformation.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.Abbreviations MS(2%) Murashige-Skoog medium containing 2 % sucrose - MS(3%) Murashige-Skoog medium containing 3 % sucrose - 1/2MS half strength Murashige-Skoog medium containing 2% sucrose - B5 Gamborg B5 medium containing 2% sucrose - WP Woody Plant medium containing 2% sucrose     

In vitro cormlet development in Crocus sativus

An improved protocol for generation of viable cormlets from tissue culture derived shoots of saffron has been developed. Multiple shoots were generated from apical buds, small corms and in vitro developed single shoots. Bunches of two to three shoots when cultured on half strength Murashige and Skoog (MS) medium containing 3 mg dm⁻³ benzyladenine (BA) and 80 g dm⁻³ sucrose developed 1.89 cormlets per shoot bunch with an average fresh mass of 1.18 g. It took nine months from culture of apical buds to the harvest of cormlets but under field conditions 22 months. Sucrose appeared to be essential for cormlet induction as no cormlets were developed in the medium devoid of sucrose and only 0.29 per shoot in medium containing mannitol. In vitro derived cormlets sprouted from apical and axillary buds on MS medium containing 12 mg dm⁻³ BA, 3 mg dm⁻³ indolebutyric acid and 30 g dm⁻³ sucrose. Daughter cormlet formation from in vitro derived cormlets was also observed.     

Morphogenesis in hypocotyl cultures of Digitalis obscura: Influence of carbohydrate levels and sources

Hypocotyl explants of Digitalis obscura L. were grown on Murashige and Skoog medium supplemented with 0.57 μM IAA and 4.40 μM BA. The effects of sucrose, maltose, glucose, galactose or mannitol on their growth and bud formation were investigated. None of the carbohydrate sources tested was superior to sucrose, and best results were obtained with 2.0% (w/v) of this disaccharide. Although mannitol did not support morphogenesis, it had a promotive effect on bud formation when added to 1.0 or 1.5% sucrose-supplied media to give the molar sucrose equivalent to 2.0%. The inhibitory effect of high sucrose concentrations could be duplicated by substituting mannitol for sucrose on a molar basis. Our data suggest a dual role of sucrose as osmotic and energy source in D. obscura hypocotyl cultures.     

In vitro corm production of Gladiolus dalenii and G. tristis

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l^-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l^-1 Gelrite^®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l^-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.     

Effects of salt formulations, carbon sources, cytokinins, and auxin on shoot organogenesis from cotyledons of Pinus pinea L.

Adventitious shoots were induced on cotyledons of Pinus pinea. Among seven salt formulations, three carbon sources (sucrose, glucose and fructose), and two cytokinins [6-benzyladenine (BA) and thidiazuron (TDZ)] with and without [-naphthaleneacetic acid (NAA)], cotyledons grown on 1/2 MS medium containing sucrose and 30 M BA produced the highest frequency of shoot organogenesis (>90%) and mean number of shoots/explant (>40% of cotyledons produced over 20 shoots/cotyledon). As for the site of shoot organogenesis along the cotyledonary explant, the highest number of shoots per explant was observed along the basal segment of the cotyledon (distal to the hypocotyl) over all cytokinin and auxin treatments tested. Shoots were elongated on a growth regulator-free medium containing activated charcoal.     

In Vitro Regeneration from Leaf Explants of Pineapple (Ananas comosus L, Merr)

Multiple shoots were regenerated from leaf explants obtained from in vitro grown shoot cultures of pineapple. Each leaf was horizontally cut into three pieces (～ 0.5 cm, basal, middle and tip) and cultured onto MS basal medium supplemented with 2% sucrose and various growth regulators.The explant containing the basal part of the leaf gave rise to tiny protuberances which grew into shoots.The highest number of shoots were obtained on MS basal medium supplemented with 2,4-D (0.90 µM) and 2iP (0.98 pM).These shoots were subcultured ontowhite’s basal medium supplemented with 1% sucrose, NAA (0.54 µM) and IBA (1.97 µM). Plantlets produced in vitro were transferred to paper cups containing autoclaved soil or Soilrite, hardened in the greenhouse and established in soil.The protocol provides an easy propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Uptake of mannitol from the media by in vitro grown plants

Higher plants grown in vitro are very seldom fully autotrophic. Therefore, such cultures are usually supplied with exogenous sugars. However, at higher sugar concentration a decrease in dry matter accumulation is observed which can be explained by a decrease in osmotic potential of the medium.To test this explanation a series of experiments with mannitol, a sugar alcohol often used for simulation of osmotic stress, were performed with excised wheat embryos, rape seedlings and potato stem segments grown in vitro. As the presence of mannitol in the medium caused a significant decrease in dry matter accumulation, the content of mannitol in the shoot tissues was determined using HPLC analysis to estimate the uptake and transport of mannitol from roots to shoots. Mannitol contents up to 30% of dry weight in wheat and 20% in rape and potato shoots were found, indicating that mannitol is easily taken up by in vitro plants and transported to shoots. There were no large changes in the content of glucose, fructose and sucrose caused by the presence of mannitol in the tissues. These data show that mannitol can not be used as an inert osmoticum in in vitro studies.     

Changes in the starch content during organogenesis in in vitro cultured Begonia rex stem explants

Stem explants, excised from greenhouse-grown Begonia rex plants, were cultured on basal medium (T. Murashige and F. Skoog, Physiol. Plant. 15: 473–497, 1962) contained in sterile Petri dishes. The medium was supplemented with benzyladenine (0.1 mg 1⁻¹) naphthaleneacetic acid (0.01 mg 1⁻¹) and, according to experimental requirements, with either sucrose (3%) or mannitol (3%). Histochemical and biochemical examination of the starch content of the explant was carried out over several days. There was no starch deposition or organogenesis in tissue cultured on mannitol and carbohydrate-free growth medium. The most dramatic finding was the heavy accumulation of starch in tissue cultured on sucrose medium. This copious accumulation preceded any organ formation and was mainly in regions which ultimately gave rise to shoot primordia. The heavy build-up of starch preceding organogenesis was also observed when explants previously cultured on mannitol medium were transferred to medium containing sucrose. During shoot primordia development there was a decrease in the starch content of the cultured tissue indicating the utilization of the polyglucan in the organogenic process.     

In vitro PPFD and media composition affect both in and ex vitro performance of Alstroemeria Butterfly-hybrids

The objective was to reduce in vitro production costs while retaining or improving plant quality, in particular the suitability for pot plant production. Plants were grown at photosynthetic photon flux densities (PPFD) of 0–40 μmol m^-2 s^-1 and sucrose concentrations of 3–7% during the multiplication phase and the effects of sucrose, BA, and NAA during root formation were investigated. Ex vitro growth were tested in both experiments. A small reduction in the rhizome multiplication rate was found with increasing PPFD and sucrose concentration. Increasing sucrose concentration reduced the number of aerial shoots. Aerial shoots were etiolated when cultured in darkness and their number increased with increasing PPFD at 3% sucrose, whereas PPFD did not affect the number of aerial shoots at 5 or 7% sucrose. During the multiplication phase a synergistic promoting effect of PPFD and sucrose was observed on root formation. Root formation after transfer to rooting medium was affected by sucrose and PPFD during the multiplication phase. PPFD did not influence root formation after propagation on 7% sucrose, whereas on 3 or 5 % sucrose root formation was gradually inhibited when PPFD was decreased below 17 μmol m^-2 s^-1. The formation of thick roots was promoted by propagation in light, and not influenced by sucrose concentration. Ex vitro growth was not affected by in vitro conditions, except for 7% sucrose during the multiplication phase that reduced flowering. Root formation on rooting medium was reduced by BA and promoted both by NAA and high levels of sucrose. The root inhibiting effect of BA could not completely be overcome by simultaneous application of NAA and high sucrose concentrations. Thick roots were only produced in the presence of NAA, and not affected by sucrose treatment. Ex vitro flowering was negatively influenced by the presence of BA during root formation and by high levels of sucrose if BA was absent in the rooting medium. High sucrose levels and NAA could partially compensate for the negative effect of BA on flowering. This revised version was published online in June 2006 with corrections to the Cover Date.