Organogenesis and Plantlet Formation from Organ- and Seedling-Derived Calli of Rice (Oryza sativa)

Callus was induced in different somatic organs of Oryza sativa L. Specific minimum 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations in the medium were necessary for the induction of callus from different organs while high levels of 2,4-D (6–10 mg/l) induced callus formation in each organ tested. The optimum 2,4-D concentration for callus induction and growth for root-derived calli was 2 mg/l and for leaf-derived 6 mg/l. Root and shoot organogenesis were induced in both root- and leaf-derived calli by sub-culturing to a medium lacking 2,4-D. Root organogenesis occurred at a higher frequency than shoot organogenesis. Shoot organogenesis rarely occurred in calli without differentiated roots. Increased age of callus cultures almost completely inhibited shoot development. The addition of the cytokinin 6-γ,γ-dimethylallyl-amino purine partially restored the potential for shoot organogenesis. Whole plants were easily recovered from the calli and grown to maturity with some plants exhibiting phenotypic abnormalities.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

Isolation and culture of protoplasts ofCapsicum annuum L. and their regeneration into plants floweringin vitro

Summary Axenic shoot cultures ofCapsicum annuum cv.California Wonder were used as the source for isolation of protoplasts from mesophyll cells. Protoplasts underwent sustained mitotic activity and proliferated to form callus masses on NT or DPD medium enriched with 2,4-D, NAA and BAP each at 1 mg/l level. The callus could be differentiated into whole plants on the differentiation media and plants floweredin vitro under long day conditions.     

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.)

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3–4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2–4 weeks before normal growth in the green house.Abbreviations BA 6-Benzyl-adenine - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - mB5 medium modified B5 medium - WPM Woody Plant medium     

Alcohol dehydrogenase (EC 1.1.1.1) isozymes as markers at 2,4-dichlorophenoxyacetic acid × kinetin combinations in callus cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH; EC 1.1.1.1) isozymes were investigated in tissue ofCereus peruvianus cultured in different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Five ADH isozymes were detected in starch gel and showed different patterns in seeds, seedlings, calli cultured at 32 and 22°C, and plants regenerated from calli cultured in three 2,4-D and kinetin combinations. Four phenotypes formed by different combinations of ADH-2, ADH-3, ADH-4, and ADH-5 were detected in calli cultured at 32°C and in plants regenerated from calli. ADH-1 isozyme was detected only in calli subcultured for 1 or 2 weeks at 22°C and was indicated as a marker of stress conditions that affect the growth ofC. peruvianus callus tissues in culture. ADH phenotypes with either a higher or a lower number of isozymes were detected in different proportions in the callus tissues cultured in media containing different 2,4-D and kinetin ratios. ADH isozyme patterns were found to be sensitive markers at the highest kinetin concentration or at high kinetin/2,4-D ratios. The results indicate a high correlation between the ADH isozyme patterns and the capacity for regeneration. Thus, ADH isozymes are indicated as good biochemical markers and as a powerful tool for monitoring studies ofC. peruvianus callus cultures.This research was supported by the CNPq.     

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     

Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis

Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DPX dibutylphthalate xylol - MS Murashige and Skoog (1962) basal medium - NAA 1-Naphthaleneacetic acid - PAA Phenylacetic acid     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Synchronized shoot regeneration of rice (Oryza sativa L.) calli on solid medium by adjustment of intracellular 2,4-dichlorophenoxyacetic acid concentration

The present research aimed to establish conditions for synchronized plantlet regeneration from rice callus based on a quantitative analysis of the relationship between intracellular 2,4-dichlorophenoxyacetic acid (2,4-D) concentration and shoot regeneration rate. To prepare the rice calli with different intracellular 2,4-D concentrations prior to regeneration, callus precultures were carried out in medium containing 4 mg/l 2,4-D and in 2,4-D-free medium for predetermined periods. As the critical intracellular 2,4-D concentration of the calli precultured in 2,4-D-free medium was too low to analyze precisely by conventional analytical methods, it was estimated using a kinetic model which described the behavior of 2,4-D by taking its uptake, metabolism and/or inactivation rates during the callus preculture into consideration. An experimental relationship between intracellular 2,4-D concentration and regeneration rate of rice calli revealed that the intracellular 2,4-D concentration should be controlled as low as 2.6×10^–2μg/g fresh weight to reach the same synchronization in shoot regeneration as seen with rice seed germination. This condition was realized by feeding sugar into the 2,4-D-free medium after 4 days preculture when the carbon source was exhausted. Received: 29 June 1998 / Revision received: 24 September 1998 / Accepted: 27 October 1998     

The Effect of High-Concentration Auxin and Plant Regeneration in the Immature Embryo Culture of Soybean

Using immature embryos of soybean as explants, green structures and somatic embryoids were able to be induced on higher auxin-containing media. Genotypes, developmental degree of the embryos, origin of the explants and medium compositions all affected the occurrence of the structures and calli. After the green structures were transferred to high 2,4-D containing medium (30 mg/l) calli were reinduced. These calli were maintained on the same medium without being subcultured for 2 months and then transferred to lower hormone-containing media. After 2 weeks, a great number of new green structures in the same shapes were induced. It was shown that high level of 2,4-D played a unique role in lasting the morphogenesis ability of the cultures. When the green structure were cultured on low hormone-containing media they developed new leaves and formed leaf clusters while the apical did not develop. In order to stimulate the apical development the medium containing 2 mg/l GA3 and 0.1 mg/l IBA was used and some plantlets were obtained. The different effects of NAA and 2,4-D on the explants and calli were studied. Calli induced from the cotyledon of immature seeds (416 mm) had a regeneration ability stronger than that from the seedlings. The calli induced by use of the medium containing high concentration of 2,4-D (5–30 mg/l) have higher potentialities in producing green structures. In contrast, the calli induced by high concentration of NAA (10 mg/l) were highly root-morphogenetic. The explants and the calli cultured on the medium containing 5 mg/l 2,4-D could be maintained for a long term without being subcultured frequently.     

Plant regeneration from cultured protoplasts of a glutinous rice

Young embryos of rice (Oryza sativa L. subsp, japonicavar. Guo-xiang No. 1) were cultured on MS agar medium(2,4-D 2 mg/l). Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l ). After selection, the small, grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l, 6-BA 0.2 mg/l)~1 with agarose block culture method. The protoplasts grew, divided and formed calli. After inducing differentiation, the regenerated mature plants were obtained.     

Regeneration of plantlets from mature embryo calli of Western Ghats land race cultivar of rice, Oryza sativa L

The Malnad region located in the Western Ghats of Karnataka is known for the cultivation of indigenous rain fed land race cultivar of rice. The present study was to investigate the callogenic and caulogenic potentialities of the two indigenous rice cultivar namely Karimundaga and Kanadatumba using dehusked mature embryo explants. For callus and shoot bud differentiation, the explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-D (1-3 mg/L), IAA (1-2 mg/L), Kn (1-4 mg/L) and BAP (1-4 mg/L). The morphogenic potentialities of the two rice cultivar differed in texture of callus. In both the cultivar callogenic frequency was optimized at 1 mg/L 2,4-D concentration, it was 94% in Karimundaga and 58% in Kanadatumba. Supplementation of IAA either alone (1-2 mg/L) or in combination with Kn or BAP at 1 to 4 mg/L concentration of each induces shoot bud differentiation from the calli. In the cultivar Karimundaga caulogenic frequency was highest (10.60 +/- 2.55) at 1.0 mg/L IAA and 4.0 mg/L BAP concentration. While in the cultivar Kanadatumba highest number of shoot buds (7.90 +/- 2.69) was differentiated at 1.0 mg/L IAA and 4.0 mg/L Kn concentration. The calli derived regenerants were successfully acclimatized in the greenhouse and agro-morphological variations were evaluated. The growth characteristics and yield related parameters exhibited by in vitro plants were lower than the in vivo plants.     

Isozyme modifications during morphogenesis of callus from barley and wheat

Callus cultures fron non-organogenic, young and one-year old, and morphogenic calli were used to assess the value of isozymes analysis for the prediction of morphogenic capacity by studying esterase, peroxidase and acid phosphatase. Basic isozyme patterns of each enzyme for the callus were retained in all the callus stages and in the callus which has differentiated into shoots. With the development of shoot and/or root some conspicuous isozymes appeared for esterase and acid phosphatase and some disappeared for peroxidase. As the isozyme changes became apparent only after shoot or root initiation these enzymes could not be used as markers to distinguish between morphogenic and non-morphogenic calli.     

Plant Regeneration from Protoplasts of Suspension Cells of Saposhnilkovia divaricata (Turcz.) Schischk

Embryogenic calli were produced from the segments of the young roots, hypocotyls or petioles of test-tube seedlings on MS agar medium containing 1 mg/1 2,4-D. When shaken in the MS liquid medium, the calli formed cell suspension with many embryogenic cell clumps.Using the enzyme mixture: Onozuka R-10 1.5%+MacerozymeR-10 0.3%+Snailase 0.5%+CaCl2 5 mmol/l + Mannitol 0.6 mol/1 (pH=5.8), protoplasts were obtained from the cell clumps which had been subcultured for three to' seven days. When cultivated, the protoplasts grew and began to divide after four days, and formed cell clumps about l—2 mm within fifty days. Protoplast-derived calli were formed from the cell clumps on the MS agar medium with 0.5 mg/l 2,4-D. When transferred onto the MS agar medium containing 0.1 mg/1 6-BA or 0.1 mg/1 2,4-D and 0.5 mg/1 6-BA, the calli differentiated into embryoids. On the MS agar medium without phytohormone, the embryoids grew into plantlets.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Leaf callus induction and suspension culture establishment in lychee ( Litchi chinensis Sonn.) cv. Huaizhi

This study reports a protocol for leaf callus induction and suspension culture establishment in lychee cv. Huaizhi. The results showed that 12-day-old leaf explants cultured under a photoperiod of 16/8 h with their adaxial side touching the medium were the optimum conditions for leaf callus induction. Globular embryos were formed when the induced calli were kept on the callus induction medium without 2,4-D for 24 weeks. Friable calli were induced after 2–3 subcultures at 4 weeks intervals on the Murashige and Skoog medium supplemented with 3 mg/L IAA and 2 mg/L BAP. Suspension culture was established when these friable calli were subcultured six times in liquid callus induction medium.     

Regeneration responses of callus from different expiants and changes in isozymes during morphogenesis in wheat

Immature embryo and root meristem expiants of wheat were cultured on modified medium of Murashige and Skoog and Gamborg’s medium supplemented with 2,4-dichlorophenoxy acetic acid. Morphogeriic callus cultures were obtained from both the expiants. The frequency of shoot formation varied from 22% to 48% from callus obtained from embryos while only root formation could be induced from root meristem expiants. Cultures from young and old non-differentiating calli, and calli with shoot and/or root formation at different intervals were analysed for isozymes of esterase, peroxidase and acid phosphatase for studying the morphogenic capacity. With the development of shoot and/or root from callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root/shoot development rather than in the induction of morphogenesis in callus. Basic isozyme pattern of each enzyme for the callus was retained in all the callus stages.     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.