scholarly journals Teknik Isolasi dan Kultur Protoplas Tanaman Padi

2016 ◽  
Vol 3 (2) ◽  
pp. 60 ◽  
Author(s):  
Deden Sukmadjaja ◽  
Novianti Sunarlim ◽  
Endang G. Lestari ◽  
Ika Roostika ◽  
Tintin Suharlini
Keyword(s):  
Room Temperature ◽  
Protoplast Culture ◽  
Somatic Hybridization ◽  
Sucrose Solution ◽  
Protoplast Isolation ◽  
Protoplast Regeneration ◽  
Isolation And Purification ◽  
Enzyme Solution ◽  
Wild Rice Species ◽  
Rice Genotypes

<p>Protoplast<br />fusion or somatic hybridization technology is an alternative<br />technology for production hybrids of plants that are difficult<br />to be produced by conventional methods due to their sexual<br />incompatibility. An experiment was conducted to develop<br />techniques for isolation, purification, and culture of rice<br />protoplasts of cultivar IR64 and a wild rice species (Oryza<br />officinalis). Optimization of protoplast isolation and purification<br />methods from both rice genotypes were successfully<br />done. The highest protoplast density was obtained by<br />digesting embryonic callus or stems of young seedling in an<br />enzyme solution containing of 2% cellulose, 0.1% pectolyase,<br />0.5% macerozyme, 0.5% driselase, 5 mM ES, and 13% mannitol<br />in CPW solution. The protoplast digestion was done for<br />three hours by soaking in the enzyme solution followed by<br />shaking at 50 rpm under a room temperature. Purification of<br />the protoplasts were done by separating them from plant<br />debris using a 25% sucrose solution. Protoplast regeneration<br />was not successful using although different media compositions<br />and conditions. Growth process from cell division to<br />cell aggregate was only successful on IR64 protoplast culture<br />on a medium that contained AgNO3.</p>

scholarly journals Effect of Enzyme Treatments on Protoplast Isolation from Leaves of Vetiver (Vetiveria spp.)

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Sumana Leaungthitikanchana ◽  
Khachapohn Thongdonyod ◽  
Nootjaree Singphan
Keyword(s):  
Cell Wall ◽  
Protoplast Fusion ◽  
Incubation Time ◽  
Protoplast Culture ◽  
Protoplast Isolation ◽  
Fusion Technology ◽  
Vetiveria Zizanioides ◽  
Enzyme Solution ◽  
Protoplast Yield ◽  
High Viability

Protoplast isolation is a first and important step for establishing a new plant with desired traits through protoplast fusion technology. This experiments were conducted to evaluate various concentration of enzymes and incubation time on protoplast yield and viability in two vetiver ecotypes, Kamphaeng Phet 2 (Vetiveria zizanioides Nash) and Prachuap Khiri Khan (V. nemoralis A.Camus). The results revealed that protoplast yields were significantly affected by different enzyme treatments. The highest protoplast yield (6.12x105 protoplasts/ml) and high viability (98.61%) in Kamphaeng Phet 2 was obtained through the process of cell wall digestion when treated with enzyme solution containing 0.5% (w/v) cellulase onozuka R-10 and 0.5% (w/v) macerozyme R-10 in combination. While, the optimal enzyme solution for protoplast isolation from leaves of Prachuap Khiri Khan was the combination of 1.0% (w/v) cellulase onozuka R-10 and 0.4% (w/v) macerozyme R-10, resulting in the highest yield (6.80x105 protoplasts/ml) and viability (96.56%) of protoplasts. Meanwhile, incubation time of 24 h with the optimal enzyme solution resulted in the highest protoplast yields of both ecotypes. Our findings have the potential to generate an efficient protocol to isolate the protoplast from leaves of vetiver which can be used for further research studies in protoplast culture and fusion for vetiver improvement. Keywords: Cellulase onozuka R-10, Macerozyme R-10, Protoplast isolation, Vetiver

scholarly journals Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

2021 ◽  
Vol 12 ◽  
Author(s):  
Xueyuan Li ◽  
Sjur Sandgrind ◽  
Oliver Moss ◽  
Rui Guan ◽  
Emelie Ivarson ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Shoot Regeneration ◽  
Gene Editing ◽  
Protoplast Culture ◽  
Callus Formation ◽  
Protoplast Regeneration ◽  
Induction Medium ◽  
Shoot Induction ◽  
Brassica Napus L ◽  
High Concentrations

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

Polymorphisms and evolutionary history of retrotransposon insertions in rice promoters

Genome ◽  
2011 ◽  
Vol 54 (8) ◽  
pp. 629-638 ◽  
Author(s):  
Z. Xu ◽  
S. Rafi ◽  
W. Ramakrishna
Keyword(s):  
Oryza Sativa ◽  
Wild Rice ◽  
Evolutionary Dynamics ◽  
Genomic Sequence ◽  
Promoter Regions ◽  
Higher Plant ◽  
Wild Rice Species ◽  
History Of ◽  
Aa Genome ◽  
Rice Genotypes

Retrotransposons are ubiquitous in higher plant genomes. The presence or absence of retrotransposons in whole genome and high throughput genomic sequence (HTGS) from cultivated and wild rice was investigated to understand the organization and evolution of retrotransposon insertions in promoter regions. Approximately half of the Oryza sativa subsp. japonica ‘Nipponbare’ promoters with retrotransposons conserved in Oryza sativa subsp. indica ‘93-11’ and four wild rice species showed higher sequence conservation in retrotransposon than nonretrotransposon regions. We further investigated, in detail, the evolutionary dynamics of five retrotransposons in the promoter regions of 95 rice genotypes. Our data suggest that four of five insertions (Rp2–Rp5) occurred in the ancestor of AA genome, while the other insertion (Rp1) predates the ancestral divergence of Oryza officinalis (CC genome). Four retrotransposons (Rp2–Rp5) were present in 52% (Rp2), 29% (Rp3), 53% (Rp4), and 43% (Rp5) of the rice genotypes with AA genome type, and the fifth retrotransposon (Rp1) was present in 95% of the rice genotypes with AA, BBCC, or CC genome types. Furthermore, most of these retrotransposons were found to evolve slower than flanking promoter regions, suggesting a role in promoter function for regulating downstream genes.

scholarly journals ISOLATION AND CULTURE OF Celosia cristata L. CELL SUSPENSION PROTOPLASTS

2003 ◽  
Vol 9 (1) ◽  
pp. 1-6
Author(s):  
Retno Mastuti ◽  
Hiroshi Miyake ◽  
Takeshi Taniguchi ◽  
Yoji Takeoka
Keyword(s):  
Plant Regeneration ◽  
Cell Suspension ◽  
Protoplast Culture ◽  
Somatic Hybridization ◽  
Culture Media ◽  
Developmental Competence ◽  
Gelling Agent ◽  
Ms Medium ◽  
L Cell ◽  
Celosia Cristata

Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolated from 3- to 9-d old cultures in enzyme solution containing 2 percent (w/v) Cellulase YC and 0.5 percent (w/v) Macerozyme R-10 which was dissolved in washing solution (0.4 M mannitol and 10 mM CaCl2) at pH 5.6 for 3 hours. The highest number of viable protoplasts was released from 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examined with four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2 percent agarose significantly enhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery callus when they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regeneration from the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in this study may facilitate the establishment of somatic hybridization using C. cristata as one parent.

scholarly journals Effect of Osmo Dehydration on Quality Attributes of Bilimbi (Averroha bilimbi) Fruits

2022 ◽  
Author(s):  
G.S. Aparna ◽  
P.R. Geetha Lekshmi ◽  
C. Mini
Keyword(s):  
Mass Transfer ◽  
Water Loss ◽  
Weight Reduction ◽  
Room Temperature ◽  
Sucrose Solution ◽  
Quality Attributes ◽  
Hot Water ◽  
Solid Gain ◽  
Sensory Score ◽  
Osmotic Treatment

Background: Bilimbi is a profusely bearing tree and majority of fruits produced are wasted due to lack of proper preservation methods. Osmo-dehydration studies on quality attributes of bilimbi (Averroha bilimbi) was conducted with the objective to standardize the process variables for osmodehydrated bilimbi and to assess the retention of bioactive compounds. Methods: Harvested mature bilimbi fruits of uniform size were washed, surface dried, pricked and blanched in hot water for one minute. Blanched fruits were subjected to osmotic treatment, with sucrose solution of 40, 60 and 80°B for 60, 120 and 180 minutes. The osmodehydrated bilimbi fruits were analyzed for mass transfer, biochemical and sensory qualities. Best treatments were stored for four months in the room temperature. Result: Mass transfer characters viz., solid gain, water loss, percentage weight reduction, yield and biochemical parameters such as reducing sugar and total sugar increased with increase in osmotic concentration and immersion time whereas free acids, ascorbic acid and antioxidant activity were decreased. The osmotic treatment of 80°B for 180 minutes recorded the highest value for solid gain (5.10%), water loss (16.72%), weight reduction (22.57%), ratio of water loss to solid gain (3.25%) and yield (21.13%) which exhibited superior sensory scores for taste (8.43), flavor (8.27), texture (8.46) and overall acceptability (8.43). The best three treatments selected based on sensory analysis were subjected to storage stability studies under room temperature. Osmodehydrated bilimbi obtained highest sensory score at the end of storage.

85 SYNERGISTIC EFFECT OF DEHYDRATION AND LOW TEMPERATURE ON THE SURVIVAL OF PANGASIUS CATFISH (PANGASIANODON HYPOPHTHALMUS) EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 190
Author(s):  
X. N. Bui ◽  
T. H. Nguyen ◽  
T. N. Nguyen ◽  
V. H. Nguyen ◽  
T. T. Nguyen ◽  
...  
Keyword(s):  
Low Temperature ◽  
Synergistic Effect ◽  
Room Temperature ◽  
Sucrose Solution ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Water Tank ◽  
Group 4 ◽  
Group 3 ◽  
Group 2

Embryo cryopreservation is an important need for industrial catfish production, as well as for biodiversity conservation. However, due to the limit of membrane permeability and dehydration in fish embryos, no successful cryopreservation method has been reported. We present herein results of the effect of dehydration and low temperature on the survival of catfish embryos, to understand their cryobiological behaviour and develop an efficient freeze method for this species. Embryos at blastocyst stage were collected at 10 h after insemination and kept in a water tank with an air pump. Embryos with somites and optic cups clearly distinguished were selected and treated according to 4 treatment groups: group 1, control group: embryos were kept in water at room temperature for 2 h; group 2, treated for dehydration: embryos were kept in 1 M or 2 M sucrose solution for 2 h at room temperature; group 3, treated for low temperature: embryos were kept in water for 2 h at 0°C; and group 4, treated for dehydration and low temperature: embryos were kept in 1 M or 2 M sucrose solution for 2 h at 0°C. After treatments, all embryos were washed twice in water and incubated in separated water tanks for 2 days. The survival of embryo was evaluated by the rate of intact and hatched embryos during the first day after treatment. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. Our results showed that in the control group, all of the embryos were intact and hatched. In the group 2, the survival rate of embryos treated with 1 M sucrose was not different compared with the control group. However, in this group, no hatched embryos were obtained when treated with 2 M sucrose (Table 1). Also, no hatched embryos were observed when they were kept at 0°C without dehydration (group 3). A significant improving hatching rate was obtained in group 4 for embryos treated at 0°C and dehydrated in 2 M sucrose (90 v. 0% in group 3; P < 0.05). In conclusion, the combined treatment of dehydration (in 2 M sucrose concentration) and low temperature (at 0°C) can result in synergistic effect that are able to protect embryos from damages caused by treatment with either low temperature or dehydration conditions. Table 1.Treated catfish embryos This work was supported by a project from MARDVN.

On the X-Ray Inactivation at Various Temperatures of Trypsin in Dry State and Aqueous Solution

1968 ◽  
Vol 23 (7) ◽  
pp. 962-968 ◽  
Author(s):  
Øyvind Oksmo ◽  
Tor Brustad
Keyword(s):  
Aqueous Solution ◽  
Indirect Effect ◽  
Room Temperature ◽  
Sample Temperature ◽  
Dilute Solution ◽  
X Ray ◽  
Enzyme Solution

The X-ray sensitivity of trypsin has been studied both in dry state and in dilute solution (0.1 mg/ml) of 10-3 N HCl, as a function of the sample temperature during irradiation. Oxygen present during irradiation acts as a sensitizer when the exposure is carried out in frozen aqueous solution as well as in dry state, but it acts as a protector when the enzyme solution is exposed at room temperature. There is a considerable indirect effect when trypsin is exposed in frozen aqueous solution, above 200 °K, and even at 77 °K a small indirect effect is apparent.When trypsin is exposed in 10-3 N HCl at room temperature about 85 per cent of the indirect effect is eliminated when the solution is saturated with O2 , (known as an effective scavenger for e ⊖ aq and H) and most of the remaining indirect effect is eliminated if the enzyme solution also contains 100 mM C2H3OH (known as an effective OH scavenger).

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