Search

Mature and efficient tissue culture systems are already available for most japonica rice varieties (Oryza sativa ssp. geng). However, it remains challenging to regenerate the majority of indica rice varieties (Oryza sativa ssp. xian). In this study, quantitative trait loci (QTLs) associated with rice callus regeneration ability were identified based on the plant regeneration rate (PRR) and total green plant rate (TGPR) of the 93-11 × Nip recombinant inbred line population. Significant positive correlations were found between PRR and TGPR. A total of three QTLs (one for PRR and two for TGPR) were identified. qPRR3 (located on chromosome 3) was detected for both traits, which could explain 13.40% and 17.07% of the phenotypic variations of PRR and TGPR, respectively. Subsequently, the effect of qPRR3 on callus regeneration ability was validated by cryptographically tagged near-isogenic lines (NILs), and the QTL was narrowed to an interval of approximately 160 kb. The anatomical structure observation of the regenerated callus of the NILs revealed that qPRR3 can improve the callus regeneration ability by promoting the regeneration of shoots. [ABSTRACT FROM AUTHOR]

Key message: The Agrobacterium-mediated transformation system of three elite indica rice varieties was explored and two varieties yielded positive transgenic plants.Fourteen elite indica rice varieties with different genotypes were selected for callus induction, and varieties NY49, KW and CL38 were chosen for the establishment of the Agrobacterium transformation system because of their excellent tissue culture characteristics. The callus induction frequency was the highest when the concentration of 2,4-Diphenoxyacetic acid (2,4-D) was respectively at 2.5 mg/L, 3.0 mg/L and 2.8 mg/L for NY49, KW and CL38 after optimizing the tissue culture conditions. Moreover, medium types of CIM1, CIM8 and CIM7 were most suitable for the varieties NY49, KW and CL38, respectively, with high callus induction frequency. The regeneration frequency of CL38 and NY49 was as high as 73.33% and 50.00%, respectively, in MS medium containing 2 mg/L 6-benzyladenine (BAP), 2 mg/L kinetin (KT) and 0.5 mg/L naphthylacetic acid (NAA). Finally, transgenic seedlings of CL38, NY49 and NIP were obtained with infection of Agrobacterium EHA105 carrying the pYLCRISPR/Cas9PUB-H vector. The successful editing frequency detecting by sequencing in CL38 and NIP were 62.50% and 77.78%, respectively. The establishment of the transformation system provides a useful tool for gene function study and gene editing breeding in indica rice cultivars.The Agrobacterium-mediated transformation system of three elite indica rice varieties was explored and two varieties yielded positive transgenic plants.Fourteen elite indica rice varieties with different genotypes were selected for callus induction, and varieties NY49, KW and CL38 were chosen for the establishment of the Agrobacterium transformation system because of their excellent tissue culture characteristics. The callus induction frequency was the highest when the concentration of 2,4-Diphenoxyacetic acid (2,4-D) was respectively at 2.5 mg/L, 3.0 mg/L and 2.8 mg/L for NY49, KW and CL38 after optimizing the tissue culture conditions. Moreover, medium types of CIM1, CIM8 and CIM7 were most suitable for the varieties NY49, KW and CL38, respectively, with high callus induction frequency. The regeneration frequency of CL38 and NY49 was as high as 73.33% and 50.00%, respectively, in MS medium containing 2 mg/L 6-benzyladenine (BAP), 2 mg/L kinetin (KT) and 0.5 mg/L naphthylacetic acid (NAA). Finally, transgenic seedlings of CL38, NY49 and NIP were obtained with infection of Agrobacterium EHA105 carrying the pYLCRISPR/Cas9PUB-H vector. The successful editing frequency detecting by sequencing in CL38 and NIP were 62.50% and 77.78%, respectively. The establishment of the transformation system provides a useful tool for gene function study and gene editing breeding in indica rice cultivars. [ABSTRACT FROM AUTHOR]

In vitro androgenesis is an attractive biotechnological technique that provides rapid transformation of heterozygous hybrids into homozygous lines and thus, significantly reducing the duration of breeding cycle. Due to difficulty in generating embryogenic calli, low plant regeneration and frequent albino plant regeneration, the prospective benefits of doubled haploid technology have not been exploited fully in indica rice hybrids. The present study, therefore aims at optimizing a previously standardized in vitro androgenic protocol to improve callus and shoot regeneration efficiency in an indica rice hybrid, KRH 4. Accordingly, the most potential anthers for androgenesis were obtained from boots with flag leaf to penultimate leaf distance of 10-12 cm. In particular, anthers at 4/6th position (ratio of length of stamen to the glume) within a spikelet were shown to have the highest callus induction efficiency. Further, callusing was advanced by 18 days and callus induction was improved by 13 per cent when sucrose was replaced with maltose a carbon source to the previously standardized callus induction media for KRH 4 rice hybrid. Finally, among the optimized callus induction media, T3 media containing N6 basal media supplemented with 2, 4-D (2 mg/L), NAA (1 mg/L), kinetin (0.5 mg/L), maltose (3%) and clerigel (0.2%) and maintained at a pH of 5.8, had the highest callus induction efficiency and also produced more embryonic calli. Similarly, shoot regeneration efficiency of embryonic calli (2 mm) was enhanced by inoculating embryonic calli in R2 regeneration media containing N6 basal media supplemented with kinetin (3 mg/L), NAA (0.1 mg/L), BAP (1 mg/L), sucrose (3%) and clerigel (0.2%) maintained at a pH of 5.8 and kept under absolute dark for 48 hrs followed by transferring them to BOD incubator maintaining 16-h light/8-h dark regime at 27 ± 2 °C. [ABSTRACT FROM AUTHOR]

This study investigated developmental changes in cold stressed microspores of Indica rice variety At 303. After 3 d at 10°C, approximately 76% of microspores were in the late uni-nucleate stage. Even after 5 d, 49% of viable cells remained in the late uni-nucleate stage without advancing to the bi-nucleate stage. In comparison, microspores undergoing normal gametogenesis in planta progressed rapidly from uni-nucleate to bi-nucleate stage eventually forming tri-nucleate pollen during this period. Thus, cold stress prevented normal microspore development and retained cells in the late uni-nucleate stage which is the most favorable stage for in vitro induction of sporophytic development in rice. Uni-nucleate microspores subjected to cold stress showed a characteristic pattern consisting of several minute vacuoles surrounding a centrally positioned nucleus, which can be interpreted as an early indicator of sporophytic determination in Indica rice microspores. During in vitro culture phase, freshly plated yellow anthers became brown. After 4 wk in culture, 51% of the anthers had discolored. Significantly, all yellow anthers contained only non-viable cells whereas 10% of the brown anthers had few viable cells. Some microspores in brown anthers underwent division on callus induction medium. The first division was symmetrical and occurred after 2 wk. The second division occurred after 4 wk and resulted in four-celled structures. Anther-derived callus was either compact or friable. Histo-differentiation occurred mostly from compact callus. Cell clusters, each delimited by a protoderm, were observed in histological sections of callus grown for 2 to 4 wk on regeneration medium. Within a cellular unit, two heterogeneous cell populations were arranged in concentric rings with larger cells in the center and smaller cells towards the periphery. However, an apical-basal polarity that is present in embryo-like structures was not observed. Therefore, it may be surmised that in Indica rice, regeneration from anther-derived callus takes place not by the formation of somatic embryos but by direct organogenesis. [ABSTRACT FROM AUTHOR]

Mature and efficient tissue culture systems are already available for most japonica rice varieties (Oryza sativa ssp. geng). However, it remains challenging to regenerate the majority of indica rice varieties (Oryza sativa ssp. xian). In this study, quantitative trait loci (QTLs) associated with rice callus regeneration ability were identified based on the plant regeneration rate (PRR) and total green plant rate (TGPR) of the 93-11 × Nip recombinant inbred line population. Significant positive correlations were found between PRR and TGPR. A total of three QTLs (one for PRR and two for TGPR) were identified. qPRR3 (located on chromosome 3) was detected for both traits, which could explain 13.40% and 17.07% of the phenotypic variations of PRR and TGPR, respectively. Subsequently, the effect of qPRR3 on callus regeneration ability was validated by cryptographically tagged near-isogenic lines (NILs), and the QTL was narrowed to an interval of approximately 160 kb. The anatomical structure observation of the regenerated callus of the NILs revealed that qPRR3 can improve the callus regeneration ability by promoting the regeneration of shoots.

Metal nanoparticles are widely used in various fields of industry and their impact in the agricultural field is increasing. This research was carried out to investigate the effects of ZnO and TiO2 nanoparticles on callus induction and plant metabolism in indica rice. Indica rice was assessed in plant tissue culture condition to estimate the influence of metal nanoparticles on morphological and physiological factors over 4 weeks. The seeds of Oryza sativa L. var. KDML105 were exposed to 1-3 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) in the callus induction medium. The induced-calli were cultured on the callus induction medium supplemented with 2 mg L-1 of 2,4-D and showed a high increase in size and dry weight contents under the light condition when compared with the control and other treatments. Moreover, this research also investigated the potential impacts of ZnO and TiO2 nanoparticles at 200, 400 and 800 mg L-1 on morphological and metabolic responses of the induced-calli cultured on the callus induction medium supplemented with 2,4-D and each type of metal nanoparticles. At 4 weeks after treatment, the induced-calli were analyzed for plant growth and assays on aspects of their metabolism were undertaken. The induced-calli exposed to 200 mg L-1 of ZnO or TiO2 nanoparticles were found to have significant increase of biomass (size and dry weight of callus), total phenolic compounds and total flavonoid contents in KDML105 variety. The results demonstrated that utilization of ZnO and TiO2 nanoparticles at appropriate concentration could positively induce the production of beneficial secondary metabolites in the induced-calli of indica rice and are worthy of further study for applications in other plant species. [ABSTRACT FROM AUTHOR]

Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue stained 5–8 μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features. Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli. However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological observations where characteristic embryogenic units (EU) were noticed in these genotypes. These meristematic regions displayed high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent nuclei and were located on the callus surface or inside surrounded by parenchymal cells. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the optimum concentrations of 2, 4-D, L-proline and casein hydrolysate (CH) for efficient callus induction and plantlet regeneration from culturing mature embryos of Sangyod, an economically important indica rice variety in Thailand. The highest frequency of callus induction (73.08 ± 2.65 %) was obtained from MS medium supplemented with 2 mg/L 2,4-D, 750 mg/L CH and 200 mg/L L-proline. The combination of 1 mg/L BA, 0.5 mg/L Kn and 0.5 mg/L NAA containing solidified MS medium gave the maximum mean fresh weight of callus (938.9 ± 44 mg), green spot formation (64.17 ± 7.08 %), shoot induction frequency (66.25 ± 6.80 %) and mean number of shoots/explant (6.12 ± 0.36 shoots). The greatest mean number of shoots/explant (14.93 ± 0.97 shoots) and root formation percentage (82.71 ± 3.03 %) was observed in liquified MS medium supplemented with 0.5 mg/L NAA and 1 mg/L BA. [ABSTRACT FROM AUTHOR]

In vitro embryogenic callus is a critical factor for genetic transformation of rice, especially for indica varieties. In this study, we investigated the relationship between polyamines, including putrescine (Put), spermidine (Spd) and spermine (Spm), and callus browning, and we studied the effect of exogenous Put on callus regeneration and on the content of endogenous polyamines. In addition, the expression levels of arginine decarboxylase gene (Adc1) and S-adenosylmethionine decarboxylase gene (Samdc) in embryogenic callus were studied by quantitative Real-time PCR analysis. The results showed that the contents of endogenous Put and Spd in the browning callus were significantly lower than those in normal callus. Exogenous Put could effectively improve the growing state of callus of indica rice and enhance the development of embryogenic callus. The content of endogenous polyamines in embryogenic callus, especially Spd and Spm, was increased after addition of exogenous Put. Additionally, exogenous Put also had an obvious impact on the expression levels of Adc1 but partial effect on the expression levels of Samdc gene. This study could increase the knowledge of both embryogenic callus induction and polyamine catabolism in callus in indica rice. [ABSTRACT FROM AUTHOR]

Salt tolerant crops are essential to bring the currently uncultivable salinity affected areas under cultivation. Here, we report Agrobacterium-mediated transformation of Pisum sativum calcineurin B-like protein ( PsCBL) and P. sativum calcineurin B-like (CBL)-interacting protein kinase ( PsCIPK) genes for development of salt-tolerant rice ( Oryza sativa L.). The four indica rice cultivars used in this study were 'BR 11', 'BRRI dhan 49', 'Paijam', and 'Chinigura', with the maximum callus transformation frequency reaching 66.6 ± 7.2, 5.1 ± 6.2%, 79.1 ± 7.2 and 58.3 ± 7.2%, respectively. Among the transformed calli, the highest rates of regenerated plantlets were 62.9 ± 8.2% for 'BR 11', 73.5 ± 7.8% for 'BRRI dhan 49', and 65.5 ± 7.8% for 'Chinigura'. 'Paijam' calli were recalcitrant to regeneration, which led us to directly transform 'Paijam' seeds without an intervening callus phase. The maximum transformation rate of 'Paijam' plantlets obtained was 46.3 ± 5.3%. Successful transformation events were confirmed through GUS histochemical assays and PCR analyses of putative transgenic plants. The transgenic rice plants 'BR 11', 'BRRI dhan 49', 'Paijam', and 'Chinigura' were able to survive exposure to 150 mM NaCl stress for over 3 wk, while non-transformed control plants became bleached and died within 1 wk for the same treatment. Our results indicated that stable transformation of these rice cultivars either with PsCBL or PsCIPK significantly improved salt tolerance. [ABSTRACT FROM AUTHOR]

Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600 = 1.0) and co-cultured at 26 ± 2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 &mgr;m acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration. [ABSTRACT FROM AUTHOR]