Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.)

To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole-acetic acid - BA 6-benzyladeninepurine - S.E.M. standard error of mean     

Plant regeneration from tissue cultures of Pokkali rice is promoted by optimizing callus to medium volume ratio and by a medium-conditioning factor produced by embryogenic callus

The frequency of plant regeneration from seed-derived Pokkali rice callus has been substantially increased. Four conclusions were drawn from the study: (1) Non-embryogenic callus consisting of elongated, highly-vacuolated cells did not produce regenerated plants. Embryogenic callus consisting of small, non-vacuolated cells produced somatic embryos and regenerated plants. (2) The numbers of plants could be markedly increased by optimizing a medium for embryogenic callus production and a second medium for plant regeneration from embryogenic callus. (3) The optimization of callus to medium volume ratio of 6.5 mg embryogenic callus per 1.0 ml of medium significantly increase plant production on regeneration medium. (4) A further significant increase was obtained by using regeneration medium previously conditioned for one or two weeks by optimal amounts of embryogenic callus. At present, the callus derived from a single seed in six months could theoretically be used in the seventh month to produce 127500 plants.This research was supported by the Agency for International Development under Contract No. AID/DSAN-C-0273     

籼型杂交稻亲本成熟胚愈伤组织再生体系的建立

本文给出了6个籼型杂交稻亲本成熟胚愈伤组织再生体系建立优化措施。采用6个有重要育种价值的杂交籼稻亲本成熟胚盾片诱导愈伤组织作为分化再生的外植体,通过调节2,4-D浓度、培养基成分、接种方式、激素组合和愈伤组织分化途径,建立适合籼稻遗传转化的再生体系。结果表明,MB培养基是较为合适的愈伤组织诱导培养基类型,6个品种在MB培养基上的愈伤组织诱导率均在60％-80％之间。半粒米的接种方式能够明显提高愈伤组织诱导率,提高幅度达到28．2％。通过调节2,4-D浓度和激素组合,愈伤组织诱导率最高可达到97．9％,两步分化法和适当干燥处理能够提高愈伤组织的分化效率,6品种愈伤组织分化率均在50％-90％之间。初步建立了6个杂交籼稻亲本品种成熟胚愈伤组织的再生体系,为以后遗传转化奠定基础。     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

In Vitro Selection for Salt Tolerance in Rice

In six cultivars of rice (Oryza sativa L.), Pusa Basmati 1, Basmati 370, Type III, Pant Dhan 4, CSR 10 and Pokkali, embryogenic callus growth, plant regeneration, and proline and total protein contents were studied under salt stress (on agar solidified media containing 0, 0.5, 1.0, 1.5 and 2.0 % NaCl). Four weeks after inoculation the callus fresh mass decreased with increasing salt concentration in all the six cultivars. The regeneration frequency in salt stressed callus was also lower as compared to control. 15 d and 30 d after inoculation proline content increased several fold whereas total protein content decreased markedly with increase in salt concentration.     

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.     

Genotype x culture media interaction effects on regeneration response of three indica rice cultivars

Interactive effects of genotypes with callus induction and regeneration media combinations on green plantlet regeneration response were studied for three indica rice (Oryza sativa L.) cultivars, IR-72, IR-54 and Karnal Local. Isolated mature-embryos were used to derive scutellar callus and fifteen media combinations involving MS, N6, R2, SK1 and some modifications were tested. Regeneration percentage as well as the shoot-bud induction frequency were influenced by genotype, callus induction medium, regeneration medium, interaction between genotype and the two media (callus induction and regeneration) as well the interaction between the callus induction medium and regeneration medium. Basal media combination of SK1m (callusing) and MS (regeneration) was found to be the best for cv. Karnal Local in which regeneration frequency of 88% and shoot-bud induction of 233% was observed. In IR-72, the highest regeneration frequency of 47.5% and shoot-bud induction frequency of 77% was obtained on MS-MS combination. In IR-54, highest regeneration frequency (25%) was recorded on MMS(N)-MMS(N) combination, whereas, highest frequency of shoot-bud induction (50%) was observed on MMS(S)-MS combination. Although genotype and the composition of the callus induction basal medium were the major determinants of regeneration response, an overall analysis of variation also revealed a significant interaction between the media used for de-differentiation (callusing) and re-differentiation (plantlet regeneration). This revised version was published online in June 2006 with corrections to the Cover Date.     

Elevated carbon supply altered hypericin and hyperforin contents of St. John's wort (Hypericum perforatum L. cv `New Stem') grown in bioreactors

For the high frequency selection of salt-tolerant doubled haploids (DHs) using rice anther culture, the efficiency of anther culture was investigated with different genotype, media condition and NaCl concentrations. The six F₁ hybrids obtained by backcross or three-way cross between indica and japonica differed in salt tolerance. The efficiencies of callus induction and plant regeneration was decreased by NaCl concentration and salt tolerance of donor variety, and those in japonicas were higher than those in indicas. The percentages of callus induction in Gyehwa 5 (japonica, tolerant) and IR61633-B-2-2-1 (japonica, sensitive) were 21.1 and 13.5% on agar medium containing 0.3% NaCl, respectively. The plant regeneration of callus derived from anther floating culture in liquid media was less than that on solid medium. In four F₁ hybrids, the frequencies of high salt-tolerant DHs were 21.4 and 8.9% in 0.3% NaCl medium and the control, respectively. The high frequency of salt-tolerant DHs could be selected in the callus induction medium (0.3% NaCl) and in the combinations crossed with salt-tolerant japonica as the third parent.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of a low glutelin mutant of ‘Koshihikari’ rice variety using the mutated-acetolactate synthase gene derived from rice genome as a selectable marker

We have developed an efficient rice transformation system that uses only rice genome-derived components. The transgenic ‘Koshihikari’ rice, low-glutelin mutant a123, is capable of accumulating large amounts of bioactive peptides in the endosperm. Agrobacterium-mediated transformation using the mutated-acetolactate synthase (mALS) gene expressed under the control of the callus-specific promoter (CSP) as a selectable marker was used to introduce GFP and an anti-hypertensive hexapeptide into ‘Koshihikari’ a123. The CSP:mALS gene cassette confers pyrimidinyl carboxy herbicide resistance to transgenic rice callus, but is not expressed in regenerated plants. Transformation efficiency of transgenic rice line a123 was improved from about 10% to about 30% by modifying callus induction, callus selection and regeneration media conventionally used for rice tissue culture. An erratum to this article can be found at     

Callus induction and plant regeneration of U.S. rice genotypes as affected by medium constituents

Summary This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorbitol or 4% sucrose alone, and 0.5 to 4 mg·L⁻¹ (2.26 to 18.10 μM) 2,4-D with or without 0.5mg·L⁻¹) (2.32 μM) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg·L⁻¹ (9.29 μM) kinetin combined with 0 or 0.1 mg·L⁻¹ (0.54 μM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration.     

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.Abbreviations PA Polyamines - PCA Perchloric acid - PUT Putrescine - SPD Spermidine - SPM Spermine     

The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary Callus induction and later plant regeneration were studied in four widely grown garlic (Allium sativum L.) cultivars from Europe. Root segments from in vitro plantlets were used as starting material. In addition to cultivar effects, the effects of auxin and cytokinin levels and the position of the segments on the root were studied. There were no statistically significant differences among cultivars for the number of root segments that induced callus in the two series of experiments. The average induction frequency was 34.7% in the first series of experiments. Callus induction on apical root segments was significantly higher compared to callus induction on non-apical root segments in the second series of experiments. Two months after callus induction, callus lines were transferred to a regeneration medium consisting of Murashige and Skoog basal medium supplemented with 30gl⁻¹ sucrose and 1 mgl⁻¹ (4.6μM) kinetin. Calluses derived from different experiments were quite uniform with respect to their regeneration potential. Also it was found that our regeneration system was cultivar-independent. The average shoot regeneration frequency was 17.9% in the first series of experiments. Highly significant differences were found in the frequency of shoot regeneration among different callus induction treatments. When the cytokinin 6-(γ,γ-dimethylallylamino)purine (0.1mgl⁻¹∶0.5 μM) was present during callus induction, shoot regeneration ranged from 30.10 to 47.60%. Shoot regeneration from callus induced on non-apical segments was higher, although not significant, compared to callus induction from apical root segments in the second series of experiments. All in all, an efficient callus induction and plant regeneration system was developed from both apical and non-apical segments taken along the entire length of the roots. This system has potential to be used for garlic transformation.     

Studies on Cell Suspension Culture and Plant Regeneration in Rice

The present paper reports the establishment of rice cell suspension culture system, including callus induction and proliferation, isolation of single cells and small aggregates, cell suspension culture and callus re-formation, as well as regeneration of plantlets. The results have been obtained as follows: 1. The compositions of the different media used for callus induction, callus proliferation, cell suspension and plant regeneration are summarized in Table 1.2. Two kinds of disifectants, mercuric chloride and sodium hypochlorite, were used for surface sterilization of brown rice. The percentage of callus formation and callus yields were much higher when sodium hypochlorite was used (Fig. 3). We suggest that the disinfactant is one of the important factors that affect callus formed at the initial stage has an influence upon subsequent isolation of cells and suspension culture and even plant regeneration. 3. Table 3 shows that addition of yeast extract to the medium improves callus yield greatly and the efficiency of callus formation to a lesser extent. 4. Both medium Ⅱ (modified B5 medium) and N6 medium were suitable for cell suspension culture, but medium II was more effective for cell growth and callus re-formation (Fig. 4 and Table 4). 5. Effect of 2, 4-D on cell growth was tested at the concentration range among 0, 10-6, 10-5, 10-4 to 10-3 M. The results indicated that 10-5 M of 2,4-D was most effective for induction of rice callus. It has also been found that absence of 2,4-D increased callus re-formation in suspension culture, but no plant regeneration was observed. 6. By using 7% sucrose in differentiation medium, for all the three varieties, the plant regeneration frequency was raised up to 3 or 4 times than those of the 3% ones (Table 6). Occurrence of albino plants is often reported as one of the problems in rice anther culture. It is, however, no problem in seed-derived rice cell culture.     

Effects of different gelling agents on the different stages of rice regeneration in two rice cultivars

Plant tissue culture technology offers a solution for meeting the increasing commercial demand on economically important plants such as rice, a widespread dietary staple. However, significant genotype-specific morphogenetic responses constitute a considerable on rice regeneration in plant biotechnology contexts. Aside from genotype dependency, the components of the nutrient media including gelling agents have an important impact on regeneration efficiency. The current study explores the effect of different gelling agents on various stages of rice regeneration in two Egyptian rice cultivars-Sakha104 and Giza178. Media solidified with varying concentrations of a variety of gelling agents (agar, bacto agar, gelrite and phytagel) were tested for their impact on the frequency of callus induction, shoot regeneration and rooting. The results indicated gellan gum (gelrite and phytagel) was superior to agar products (agar and bacto agar) for callus induction. By contrast, no significant differences were found between different gelling agents for shoot regeneration. Gellan gum and media solidified with bacto agar were found to lead to significantly higher root regeneration than agar. The Sakha104 cultivar showed better responses than Giza 178 for callus induction and similar performance to the Giza 178 cultivar for root regeneration irrespective of the gelling agent. This work provides insights into the impact of different gelling agents on the morphogenetic response of two rice cultivars and can be used to help maximize the frequency of rice regeneration.     

Improvement of plant regeneration from long-term cultured calluses of Taipei 309, a model rice variety in in vitro studies

An efficient protocol was developed for regeneration of plants from long-term cultured calluses, which originated from mature seeds of a model rice variety Taipei 309 and were maintained by subculture for at least 6 months. The calluses were precultured for 4 weeks on a medium containing 8.88 μmol 6-benzyladenine, 5.37 μmol α-naphthaleneacetic acid and various concentrations of abscisic acid, which converted the calluses to a state more responsive to the subsequent culture conditions for plant regeneration. Supplementation of 8.69 mmol proline in the preculture medium increased the growth rate of the callus masses by 50% and resulted in the regeneration of 60% more plants. A more pronounced effect was observed after raising the 6-benzyladenine concentration to 55.48 μmol in the preculture medium, which promoted the development of adventitious buds on the calluses and led to the regeneration of some 30% more plants of better quality. Results indicate that manipulation of medium supplements and growth regulators leads to efficient plant regeneration in long-term callus cultures of rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

The impact of selection parameters on the phenotype and genotype of transgenic rice callus and plants

Transgenic rice embryogenic callus and plants were recovered from experiments involving electric discharge particle acceleration (Accell^TM technology). Critical parameters influencing successful delivery and stable integration of exogenous DNA into organized rice tissue had been identified previously. We report here on the effects of one selective agent (hygromycin B) on the phenotype and genotype of recovered callus and plants. The nature, timing and culture practices appeared to be more critical for the successful recovery of transgenic callus and plants than the level of selection used. By utilizing the procedures described in this report, transformation frequencies well in excess of 10% were obtained routinely in all varieties of rice tested. The combination of Accell^TM technology with a selectable and prolific regeneration culture system resulted in the development of a variety-independent and highly efficient method for the routine introduction of any gene into any rice variety.     

Selection of stable mutants from cultured rice anthers treated with ethyl methane sulfonic acid

To increase the frequency of stable mutants from cultured anthers of rice, the effects of EMS treatment on callus induction, plant regeneration and mutant induction were investigated according to the timing of treatment after anther inoculation on the medium. The frequency of callus induction was highest in anthers treated with 0.5% EMS 10 days after culture. Anthers treated directly at the initiation of culture exhibited a very low callus induction level, and the such calluses exhibited a poor plant regeneration capacity. The frequency of regeneration of green plants was significantly decreased by EMS treatments immediately after anther inoculation as compared with control. The frequencies of stable mutants were 20.7% and 12.0% in EMS treatments at 10 and 20 days, but unstable mutants were 43.1% and 52.6%, respectively. A total of 14 stable mutants, semidwarf mutants (4 lines), grain-shape mutants (2 lines) and glabrous mutants (8 lines) were selected from doubled haploid lines of the A₂ generation. The frequencies of callus induction, green plant regeneration and stable mutants were maximal in anthers treated with 0.5% EMS 10 days after culture.     

佳禾早占成熟种胚高效率组织培养方法的研究（简报）

In order to improve the embryogenic callus induction rate and the regeneration rate of JiaHe-ZaoZhan rice, the influence of different factors were investigated, media with different hormones were used. Induction medium was supplemented with 1.5 mg/L 2,4-D + 3 mg/L NAA + 0.1 mg/L KT + 1 mg/L phytic acid + 20 mg/L PAA. Embryogenic call were treated under the condition of 25 degrees C before transferring to regeneration medium, the regeneration medium contained 0.5 mg/L 6-BA + 3 mg/L NAA + 0.5 mg/L KT + 1 mg/L phytic acid. The experiment results indicated that the hormone treatments had certain effects on the callus induction. Under the optimal medium, culture condition and the hormone treatments, the embryogenic callus induction could reach over 95%, and dry treatment of embryogenic callus had been found to increase the frequency of plant regeneration, significantly the plant regeneration rate could reach over 80%. Transplanted into pots, the young plants grew well. Then a experimental system with stability and high regenerating efficiency has been established for the mature seeds of rice (JiaHe-ZaoZhan).     

Operational optimization of a turbine blade reactor using macroporous support and its application to rice callus regeneration

Optimal operation condition was investigated for immobilized rice callus culture using a turbine blade reactor (TBR²) with polyurethane foam supports. By using polyurethane foam block as immobilization support, the inhibition of cell growth at a high agitation speed was avoided because the hydrodynamic stress against immobilized cell was probably reduced. Experimental results in each operational condition were assessed by means of rice callus growth, immobilization ratio in TBR and those regeneration frequencies in regeneration culture using solid medium. Concerning with pore size of polyurethane foam and support size, three-millimeter cube support of polyurethane foam with an average pore size of 1.3 mm was the most suitable support. The maximum immobilization ratio was 50% under 5% support volume by volume of growth medium. For improving the immobilization ratio of rice callus in the TBR, the optimum TBR operation and modification were investigated further. By repeating a periodic operation 3 times (agitating at 300 rpm for 5 min and then 50 rpm for 2 min, and then 200 rpm of constant agitation speed during the remaining time), almost all supports could entrap rice callus and homogeneous immobilization was attained. The immobilization ratio was improved as compared with that using a constant operation at 200 rpm. Next, the TBR was modified by setting an air sparger inside the stainless mesh cylinder. In the modified TBR, the floating support by air bubbles was reduced, and the immobilization ratio increased further and reached 86.3% when we increased the support volume to 15% under periodic operation on a daily basis. The regeneration frequency of immobilized callus was also slightly increased by periodic operation and modification of the TBR.     

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Summary Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.