Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

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     

Genetic analysis of plant regeneration in rice (Oryza sativa L.)

Summary A diallel study involving reciprocal crosses of four genotypes (IR8, 36, 54, and 64) was carried out to understand the genetic mechanism of plant regeneration from immature embryo-calli in rice. Regeneration frequency (% of calli that produced plants) varied from a high of 86% for IR54 to a low of 0% for IR36, while regenerated plants per embryo numbered from 0 to 7 when these same IR lines and the F₁ hybrids were compared. Combining ability analysis revealed that both nuclear (with both additive and dominant effects) and cytoplasmic genes are important in controlling regeneration in rice. Parental lines and F1 hybrids with high ability to regenerate were identified.     

Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus

Summary To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.     

Application of activated charcoal and nanocarbon to callus induction and plant regeneration in aromatic rice (Oryza sativa L.)

The investigations of nanotechnology with the application on agricultural products also have been few reported, especially the plant regeneration. The effects of activated charcoal and nanocarbon on the callus induction and plant regeneration of aromatic rice were studied. Activated charcoal was added into the callus induction and regeneration medium. The presence of activated charcoal in the callus induction medium (100–500 mg L^?1), activated charcoal significantly reduced the percentage of the callus induction and biomass accumulation (fresh weight, dry weight and size). Whereas, the regeneration medium supplemented with 100 mg L^?1 of activated charcoal showed the highest percentage of plant regeneration (61.90%) and the ratio of the number of seedlings to the number of regenerated calli (RSR; 3.06) that derived from the callus induction medium (without activated charcoal). Moreover, the induced calli derived from the callus induction medium supplemented with nanocarbon at 5 mg L^?1 showed the highest percentage of callus induction (94.70%), the percentage of green spots (95.83%), the percentage of plant regeneration (60.42%) and the RSR (3.12) when transferred the calli into the regeneration medium (without nanocarbon). After that, nanocarbon was also added into the regeneration medium. The percentage of green spots (96.08%), the percentage of plant regeneration (62.75%) and the RSR (3.16) obtained from the regeneration medium supplemented with 20 mg L^?1 of nanocarbon showed the highest values. This experiment showed that the optimum concentration of activated charcoal and nanocarbon had potential to enhance the callus induction and plant regeneration frequencies in tissue culture medium of aromatic rice.     

Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L.)

Genetic variation of nine upland and four lowland rice cultivars (Oryza sativa L.) was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). Forty-two random primers were used to amplify DNA segments and 260 PCR products were obtained. The results of agarosegel electrophoretic analysis of these PCR products indicated that 208 (80%) were polymorphic. All 42 primers used in this experiment were amplified and typically generated one-to-four major bands. Only two primers showed no polymorphisms. In general, a higher level of polymorphism was found between japonica and indica subspecies while fewer polymorphisms were found between upland and lowland cultivars within the indica subspecies. A dendrogram that shows the genetic distances of 13 rice cultivars was constructed based on their DNA polymorphisms. Classification of rice cultivars based on the results from the RAPD analysis was identical to the previous classification based on isozyme analysis. This study demonstrated that RAPD analysis is a useful tool in determining the genetic relationships among rice cultivars.     

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L⁻¹) and NAA (2 mg L⁻¹) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L⁻¹), BAP, NAA and 0.5 mg L⁻¹ of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L⁻¹), Kin (1.5 mg L⁻¹), NAA (0.5 mg L⁻¹) and TDZ (0.5 mg L⁻¹) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, naphthaleneacetic acid; Kin, kinetin; MS, Murashige and Skoog; BAP, benzylaminopurine; TDZ, thidiazuron     

A low-temperature method for maintaining plant regeneration activity in embryogenic callus of rice (Oryza sativa L.)

 A method was developed to maintain plant regeneration activity of rice cells (Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5  °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5  °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. Received: 7 January 1999 / Revision received: 28 April 1999 / Accepted: 26 May 1999     

Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.)

Enhancement of callus induction and its regeneration efficiency through in vitro techniques has been optimized for 2 abiotic stresses (salt and air desiccation) using 3 rice genotypes viz. BR10, BRRI dhan32 and BRRI dhan47. The highest frequency of callus induction was obtained for BRRI dhan32 (64.44%) in MS medium supplemented with 2, 4-D (2.5 mgL⁻¹) and Kin (1.0 mgL⁻¹). Different concentrations of NaCl (2.9, 5.9, 8.8 and 11.7 gL⁻¹) were used and its effect was recorded on the basis of viability of calli (VC), relative growth rate (RGR), tolerance index (TI) and relative water content (RWC). It was observed that in all cases BRRI dhan47 showed highest performance on tolerance to VC (45.33%), RGR (1.03%), TI (0.20%) and RWC (10.23%) with 11.7 gL⁻¹ NaCl. Plant regeneration capability was recorded after partial air desiccation pretreatment to calli for 15, 30, 45 and 60 h. In this case BRRI dhan32 gave maximum number of regeneration (76.19%) when 4 weeks old calli were desiccated for 45 h. It was observed that air desiccation was 2-3 folds more effective for enhancing green plantlet regeneration compared to controls. Furthermore, desiccated calli also showed the better capability to survive in NaCl induced abiotic stress; and gave 1.9 fold (88.80%) increased regeneration in 11.7 gL⁻¹ salt level for BRRI dhan47. Analysis of variance (ANOVA) showed that the genotypes, air desiccation and NaCl had significant effect on plant regeneration at P < 0.01.     

Plantlet regeneration from glume calli of maize (Zea mays L.)

Summary Totipotent callus cultures were established from anther-free glumes of Sweet corn, Seed corn, DHM 103 and DHM 101 on MS medium supplemented with 1–2 mg/l 2,4-D. The callusing response of the glumes was tested on six different media. Glumes at the uninucleate stage of pollen development callused with a high frequency compared to other stages. Organogenesis was observed in 40% of the cultures on media devoid of hormones. A total of 76 plantlets were regenerated on medium with 0.5–1.0 mg/l of both IAA and kinetin. Cytological observations in root tips indicated a diploid chromosome number (2n=20).     

Effect of culture methods on the regeneration of albino rice (Oryza sativa L.) plantlets

In our study, we investigated the effects of regeneration conditions on both green and albino rice plants (Oryza sativa L.). The regeneration frequency of an albino cell line was compared to a normal cell line obtained from mature seed under two kinds of culture conditions; namely, the static culture on semi-solid regeneration medium and the suspension culture in liquid regeneration medium. The albino cell line, from which only albino plantlets were regenerated, was induced from the albino leaf segments. There were no significant differences in the regeneration frequencies between normal and albino calli on the semisolid regeneration medium. On the other hand, the frequency of regeneration of albino calli was significantly lower than that of the control specifically in the liquid regeneration medium.     

A comparison of methods for callus culture and plant regeneration of RD25 rice (Oryza sativa L.) in two laboratories

While methodology is transferable from one laboratory to another, an exact transfer does not usually occur and even a nearly exact transfer of methods does not always result in repeatable data. Researchers should not expect that an effort to duplicate a published procedure will necessarily lead to identical results.In attempting to transfer rice tissue culture methods between laboratories in Fort Collins, Colorado, USA and Bangkok, Thailand, we discovered that a combination of the methods of each laboratory produced the best results in term of callus productions and plant regeneration. In the experiments reported here, the type of culture vessel used and the geographical location were also important variables.Supported by the USAID/Cooperative Agreement No DAN-4137-A-00-4053-00.     

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alflalfa tissues.     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin     

水稻砷污染及其对砷的吸收和代谢机制

水稻是当今世界大部分地区(尤其是东南亚)的主要的粮食作物之一,同时也是砷(As)进入食物链的主要途径之一。日益严重的水稻田As污染,不但影响了稻米的产量和品质,而且通过食物链威胁着人体健康。如何减少水稻地上部(尤其是米粒)As的含量和降低其毒性,及提高水稻As耐性是亟需解决的世界食品安全问题。深入了解水稻对As的吸收、积累和代谢的生理及分子生物学机制是解决水稻As污染的关键途径。综述国内外研究,对今后深入研究提出建议。     

Potassium nutrition of rice (Oryza sativa L.) varieties under NaCl salinity

A salt-tolerant (Pokkali) and a salt-sensitive (IR28) variety of rice (Oryza sativa L.) were grown in a phytotron to investigate the effect of K (0, 25, 50 and 75 mg K kg^–1 soil) application on their salt tolerance. Potassium application significantly increased potential photosynthetic activity (Rfd value), percentage of filled spikelets, yield and K concentration in straw. At the same time, it also significantly reduced Na and Mg concentrations and consequently improved the K/Na, K/Mg and K/Ca ratios. IR28 responded better to K application than Pokkali. Split application of K failed to exert any beneficial effect over basal application.     

Heterosis breeding in rice (Oryza sativa L.)

Summary Studies conducted at the International Rice Research Institute (IRRI) during 1980 and 1981 have shown up to 73% heterosis, 59% heterobeltiosis and 34% standard heterosis for yield in rice. The latter was estimated in comparison to commercial varieties: IR36 and IR42 (yield 4–5 t/ha in wet season trials and 7–8 t/ha in dry season trials). Generally speaking, absolute yield was lower and extent of standard heterosis was higher in wet season than in dry season with some exception. Yields up to 5.9 t/ha (22% standard heterosis) in the wet season and 10.4 t/ha (34% standard heterosis) in the dry season were obtained. Most of the hybrids performed better in some season while some performed better in both seasons. Hybrids showed better lodging resistance although they were 5–10 cm taller. F₁ hybrids had significant positive correlations with the parental traits viz., yield (r = 0.446), tillering (r = 0.746), height (r = 0.810) and flowering (r = 0.843). Selection of parents among elite breeding lines on the basis of their per se yield performance, diverse origin and resistance to insects and diseases should give heterotic combination. Yield advantage of hybrids was due primarily to increase in number of spikelets per unit area even though tiller number was reduced. Grain weight was either the same or slightly higher. High yielding hybrids also showed significant heterosis and heterobeltiosis for total dry matter and harvest index. For commercial utilization of heterosis in rice, effective male sterility and fertility restoration systems are available and up to 45% natural outcrossing on male sterile lines has been observed. Consequently, F₁ rice hybrid have been successfully developed and used in China. Prospects of developing hybrid rice varieties elsewhere appear bright especially in countries that have organized seed production, certification and distribution programs and where hybrid seed can be produced at a reasonable cost.     

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     

Marker-assisted selection for identification of plant regeneration ability of seed-derived calli in rice (Oryza sativa L.)

Quantitative trait loci (QTL), associated with the ability of plant regeneration from seed-derived callus of rice, were mapped using a recombinant inbred (RI) population from Milyang 23/Gihobyeo. Each flanking marker, RZ474 and RZ575, tightly linked to two QTLs (qSGR-3-1 and qSGR-3-2) that are located on chromosome 3 was used in marker-assisted selection (MAS). These markers were tested on IR 36/MG RI036 (F3), Milyang 23/MG RI036 (F3), and forty-one rice cultivars. A restriction fragment length polymorphism (RFLP) marker, RZ575, that is located on chromosome 3 could effectively differentiate lines with high and poor regeneration ability, based on marker genotypes. This marker might be applicable for screening rice germplasms with high regeneration ability. Its introgression into elite lines might also be valuable in breeding programs to develop highly responsive genotypes to tissue culture.