Rice Regeneration Research Articles

Effect of oxygen-enriched aeration on regeneration of rice (Oryza sativa L.) cell culture

The effect of the oxygen concentration in the aeration gas on regeneration from rice cells in bioreactor cultures was investigated. The efficiency of regeneration in cultures aerated with over 40% oxygen was higher than that in a flask culture. In the case of a culture in which the dissolved oxygen(DO) was saturated by aeration with air, the efficiency of regeneration was less than the half that of cultures aerated with 40% oxygen. In cultures with the DO levels controlled at 8,10 and 12 mg/ℓ, the efficiency of regeneration was highest at 12 mg/ℓ. In the oxygenenriched cultures, although cell aggregation was observed and the color of plantlets was relatively pale, more than 90% of them grew into healthy plants.

Effect of oxygen-enriched aeration on regeneration of rice ( Oryza sativa L.) cell culture

Effect of oxygen-enriched aeration on regeneration of rice ( Oryza sativa L.) cell culture

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

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

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.

Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

In order to construct transgenic rice plant with an introduced oryzacystatin (OC)-β-glucuronidase (GUS) fusion gene, we first introduced it into rice protoplasts by electroporation, together with a marker gene conferring hygromycinresistance (pUC-HPH). In a transient assay using the transfected protoplasts, both OC and GUS activities were detected. The GUS activity was higher when the OC-GUS fusion protein was expressed than when only a single GUS protein was expressed. Next, to isolate stable transformants, hygromycin-resistant calli were selected. Forty one out of 116 hygromycin-resistant calli expressed a 2.2 kb mRNA transcribed from the chimeric gene and their extracts exhibited the activities of both OC and GUS. Finally, the transgenic calli were regenerated into rice plants whose tissues (leaves, roots and seeds) exhibited GUS activity probably derived from the fusion protein.

Effect of intracellular 2,4-D concentration on plantlet regeneration of rice ( Oryza sativa L.) callus

Effect of intracellular 2,4-D concentration on plantlet regeneration of rice ( Oryza sativa L.) callus

Effect of intracellular 2,4-D concentration on plantlet regeneration of rice (Oryza sauva L.) callus

Intracellular 2,4-dichlorophenoxyacetic acid (2,4-D) concentration profiles in rice (Oryza sativa L.) callus were measured for various subculture periods. 2,4-D was taken up into the callus by uphill transportation from the medium. The intracellular 2,4-D taken up was metabolized gradually during cultivation. The intracellular 2,4-D concentration markedly affected the redifferentiation process after subculture in such a way that the higher its concentration was, the more strongly the redifferentiation process was suppressed, and the lesser was the browning of the callus.

Partial desiccation of mature embryo-derived calli, a simple treatment that dramatically enhances the regeneration ability of indica rice

Regeneration of indica rice varieties remains a limiting factor for researchers undertaking rice Iransformation experiments. As reported for japonica rice and other crops, partial desiccation of indica rice calli dramatically promotes organogenesis and leads to high regeneration ability. We are now able to obtain 66.5%, 61.1% and 73.7% of calli that regenerate plants for the indica varieties TN1, IR72 and IR64 whereas in non desiccated controls only 30.0%, 15.5% and 18.7% of calli regenerated, respectively. Plants obtained were phenotypically normal and 50% were highly fertile. Partial desiccation is a reliable and simple method for improving indica rice regeneration. It also shortens the time of in vitro culture.

Oryzacystatin exogenously introduced into protoplasts and regeneration of transgenic rice.

Oryzacystatin (OC) is a proteinaceous cysteine proteinase inhibitor involved in the biodefense of rice seeds. To create transgenic rice plants with increased OC activity, we introduced an OC expressing vector into rice protoplasts and obtained transformed calli. The expression vector contained a bacterial inaA DNA fragment in the 3'-noncoding region as a tag to distinguish the introduced DNA from the intrinsic OC gene. The OC vector and a selection marker gene conferring hygromycin resistance were used together to transfect into rice protoplasts. A number of hygromycin-resistant calli were obtained and studied by polymerase chain reaction and genomic Southern blotting to find if the exogenous OC gene had been integrated. The calli were studied by northern blotting as well to examine mRNA expression. The results showed that integration and expression of the introduced OC gene occurred in 51% and 27%, respectively, of 156 subcultures from 15 hygromycin-resistant calli. As a final step, transgenic rice plants were regenerated from the calli expressing OC. Leaves and seeds from the plants had higher OC activities than those from nontransgenic plants.

Protoclonal variation of plant regeneration in rice

Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants.

Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL(-1) and 26 mgL(-1) ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.

Diallel analysis of callus growth and plant regeneration in rice seed-callus.

Genetic characteristics of callus growth and plant regeneration in rice with seed-derived calli were studied in 6 × 6 diallel crosses using Japonica cultivars. Genetic parameters estimated by Hayman's method showed the high additive gene effects and involvement of two groups of genes for callus growth, which were identified as incomplete dominance. On the other hand, both dominance and additive effects were important for plant regeneration, and further epistatic effects were observed in this character, while the analysis of 5 × 5 subdiallel for plant regeneration showed non-epistatic relation and involvement of 2 groups of genes. Graphical analysis using Vr and Wr for the callus growth showed that Norin 1, Somewake and Daikoku 1 possessed dominant genes which suppress callus growth, while Kuju, Sasanishiki and Murasaki-ine whose callus growth was more vigorous had recessive genes. The frequency distribution of callus growth in F2 generation between Kuju and Somewake showed a segregation which agreed with the expected ratio for a single factor control (3:1). On the other hand, relationship between Vr and Wr in the 5 × 5 subdiallel for plant regeneration showed that Daikoku 1, which had a relatively high capacity for plant regeneration, possessed more dominant genes, while Norin 1 and Kuju, which had lower capacities, possessed recessive genes. The reciprocal F1s between Norin 1 and Somewake showed excellent capacities for plant regeneration (average of reciprocal crosses: 62%). Strategies for genetical improvement of plant regeneration are discussed.

Efficient plant regeneration from rice protoplasts in general medium

We established an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant regeneration frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.

Diallel Analysis of Callus Induction and Green‐Plant Regeneration in Rice Anther Culture

Low efficiencies of callus induction and green‐plant regeneration have limited the application of anther culture in breeding programs. This study was conducted to determine the inheritance of callus induction and plant regeneration in rice (Oryza sativa L.) anther culture. Sixteen genotypes derived from a four‐parent diallel mating set of two Japonica cultivars (Taipei 309 and Taipei 177) and two indica cultivars (IR36 and Basmati 370) were evaluated for their callus induction and plant regeneration abilities. For callus induction, the effects of genotype and genotype ✕ medium interaction were significant (P < 0.05). Similarly, genotype and abscisic acid (ABA) level were found to be significant (P < 0.05) for green‐plant production. The japonica cultivars generally had a higher percent callus induction and plant regeneration, and number of calli producing green plants than did the indica cultivars. Similar modes of inheritance were observed for both characters. Combining ability analysis demonstrated the predominance of additive gene effects in the control of both characters with the japonica cultivars having higher combining ability for green‐plant regeneration. Reciprocal effects were not significant (P > 0.05), suggesting the absence of maternal control on the characters. Further analysis showed gene action to be partially dominant with high response being highly recessive and controlled by a few genes. There were no indications of inter‐allelic interaction.

Regeneration of Indica Rice ( Oryza sativa L.) from Primary Callus Derived from Immature Embryos

Summary To identify important parameters controlling the regeneration of indica rice (Oryza sativa L.) callus, immature embryos of IR54 were plated onto callus induction medium consisting of either MS or N6 basal salts. Various medium supplements were subsequently compared for ability to induce callus growth and plant regeneration. It was found that callus growth and plant regeneration were influenced by organic supplements to the basal media, medium solidifying agents, the number of embryo-derived calli per plate during plant regeneration, and phytohormone concentration during callus induction. However, these factors often influenced regeneration in contrasting ways depending on the basal medium used. For optimum plant regeneration N6 callus induction medium was supplemented with 1 g 1 -1 casein hydrolysate, 0.2 % (w/v) Gelrite, and 0.5 mg -1 -1 2,4-D. Regeneration was accomplished by subculturing one callus per plate to identical medium lacking 2,4-D.

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.

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

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.

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.

Variations in Callus Formation and Plant Regeneration in African Rice ( Oryza glaberrima Steud)

Eighteen cultivars of African (Oryza glaberrima) and two of Asian rice (O. sativa) were assessed for callus growth and plant regeneration. Mature seeds were used for callus initiation on media containing 2.2 mg · l(-1) 2,4-D with or without coconut milk. There were significant differences between the 20 cultivars in the size of calli produced. Addition of coconut milk increased callus size in most cultivars. Plant regeneration abilities by the cultivars were determined on two media. Calli of 16 cultivars were able to regenerate plants. The medium containing 0.2 mg · l(-1) NAA and 11 mg · l(-1) BA was more suitable for plant regeneration than that with 0.1 mg · l(-1) IAA and 0.2 mg · l(-1) kinetin.

High frequency, long term regeneration of rice from callus culture

Totipotency in cereal tissue cultures is often lost soon after callus initiation, but embryogenic cultures (producing embryo-like forms which develop into complete plants) derived from various plant parts provide long term totipotent cultures. Rice ( Oryza sativa vars. Pokkali, IR-36, Mashuri, and Giza-159) calli containing both embryogenic and non-embryogenic regions were initiated from immature embryos and mature seeds cultured on Linsmaier and Skoog (LS) medium, 4% sucrose, 1% agar and variable 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The most callus was initiated on 1.0 mg/l 2,4,5-T or 2,4-D for Pokkali, on 1.0 mg/l 2,4,5-T for Giza-159, on 2.0 mg/l 2,4,5-T for IR-36 and on 1.0 mg/l 2,4-D for Mashuri. There was not as distinct a visual difference between rice embryogenic and non-embryogenic callus as is found in pearl millet, proso millet and oats. Embryogenic rice callus initiates multiple shoots with roots which develop into fertile plants. Non-embryogenic callus initiates occasional shoots without roots. Twenty percent of embryogenic calli but only 6% of non-embryogenic calli initiated shoots after 50 weeks in culture. Embryogenic calli consistently initiated an average of 9 shoots per 10 cc of callus while non-embryogenic calli initiated 0.4 shoots per 10 cc. Differential shoot initiation indicated that visual selection was accurate in distinguishing between the two callus types.