Long-Term Maintenance of Callus Cultures from Immature Embryo of Sorghum bicolor (original) (raw)
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Improved Culture Media for Embryogenic Callus Generation in Sorghum [Sorghum bicolor (L.) Moench]
Phyton, 2019
Many attempts on optimization of sorghum [Sorghum bicolor (L.) Moench] tissue culture induction media have been made, but the culture system remains with some bottlenecks compared to that of other crops. This study aimed at assessing the suitability of various induction media to produce embryogenic callus (yellow and friable) with high induction rates and reduced phenolic exudation. The six culture medium modifications: 3 based on Murashige and Skoog (MS) medium and one each based on Chu N6, Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes. Although there was a genotype influence on the attainment of a yellow callus, friability of the callus was determined to be dependent on the culture medium and not the genotype. Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite® as the gelling agent modified with 1.0 g/l KH2PO4, 1.0 g/l L-proline, 1.0 g/l L-asparagine and 0.16 mg/l CuSO4•5H2O (type E) was found to be the most effective resulting in about 60% yellow coloured callus induction with 25% friability. Addition of CuSO4•5H2O, KH2PO4, L-proline and L-asparagine significantly reduced the phenolic production. Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds. The half strength MS medium was also observed to suppress phenolic production. Medium 190-2 produced the highest regeneration frequency (40%) among the 3-regeneration media tested. The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.
Improved plant regeneration in callus cultures of Sorghum bicolor (L.) Moench
In Vitro Cellular & Developmental Biology - Plant
Sorghum bicolor is a recalcitrant species for tissue culture regeneration and genetic transformation. Browning of explants is one of the factors limiting organ and tissue cultures. To overcome this, callus tissue was initiated from the shoot tips of in vitro germinating seeds (S. bicolor cv. Róna 1), and then cultured on modified MS media (Murashige and Skoog in Physiol Plant 15:473-497, 1962). In the first experiment, we tested callus induction on several media supplemented with casein hydrolysate, polyvinylpyrrolidone, honey, and sucrose. The best callus induction was recorded for the medium with honey and sucrose (80.0%) and for control medium (79.8%). Shoot regeneration was tested on the MS medium with 6-benzylaminopurine (BAP) supplemented with honey and sucrose at a 1:1 ratio (by weight) or with sucrose only. The highest percentage of calluses regenerating shoots was noted for those induced on the medium with sucrose and honey-approx. four times higher when compared to the control. Rooted plantlets were acclimatized with a 92% survival rate. In the second experiment, we analyzed culture responses to various ways of honey application to the induction media: honey (autoclaved or filtered) in presence or absence of sucrose. Supplementation of the medium with fructose, glucose, and maltose at a proportion typical for honey was also investigated. The explant and callus survival rates were similar to those of the honey-sucrose combination in the first experiment. Only presence of both sucrose and honey in the induction medium improved the total regeneration rate to 37.9% over the control (18.8%). Sucrose and honey appear to act synergistically for shoot regeneration in callus cultures of sorghum.
Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol in cereals. Aiming this, in the present study, we have accomplished efficient plant regeneration using mature embryos as a source material in Sorghum bicolor (L.) Moench. Although immature inflorescence and immature embryos are best explant sources for in vitro culture in Sorghum, however they are available only for a limited period in a year. Mature embryos have always been ideal for in vitro studies for the reason that they can be handled easily over other explants and available throughout the year. Mature embryo explants of Sorghum bicolor genotypes viz. IS 3566, SPV 475, CSV13, CSV15, CSV112, IS 348 were cultured on MS medium for efficient callus induction and subsequent plant regeneration. The response of different combination and concentrations of plant growth regulators were compared, and factors affecting the mature embryo tissue culture response were studied in this manuscript. Significant genotypic differentiation was detected in embryogenic callus induction and plantlet regeneration. Genotype IS 3566 showed better tissue culture response than the other genotypes. Efficient embryogenic callus induction was achieved with 2mg l-1 2, 4,5- Trichlorophenoxyacetic acid (2,4,5-T) and multiple shoot induction was achieved by manipulation of 6-benzyl adenine (BAP), Thidiazuron (TDZ), and Indole-3-acetic acid (IAA) in the culture medium. Keywords: Sorghum bicolor, embryos, 2, 4,5-T, embryogenic callus, BAP, TDZ, callus regeneration
AFRICAN JOURNAL OF BIOTECHNOLOGY
Optimization of tissue culture conditions for Sorghum bicolor L. through somatic embryogenesis from immature embryos is important for the genetic manipulation and improvement of this agronomically valuable crop. In an attempt to develop a successfully reproducible in vitro regeneration protocol for a group of diverse sorghum genotypes, 10 sorghum lines including locally adapted and commercially important elite genotypes were assessed for their regeneration potential on different culture media–containing adequate growth regulators combinations. The maximum response of embryogenic callus induction was obtained from explants cultured on Murashige and Skoog (MS) medium supplemented with 1.5 mgL-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.7 mgL-1 L-proline. The addition of kinetin to the MS-based culture media had a negative effect on the formation of embryogenic calli. The results reveal that embryogenic callus formation and regeneration were highly genotype dependent. The line LG...
Plant tissue culture studies in Sorghum bicolor: immature embryo explants as the source material
Sorghum is a wonder crop from physiological point of view. It is the most important cereal crop, after rice and wheat. The number of reports describing the use of transgenic Sorghum for basic studies in Biotechnology is still limited when compared to other crops. In one hand, the success of the transformation techniques is mainly dependent upon the availability of optimal protocols for highly efficient tissue culture techniques. On the other hand, regeneration in Sorghum is difficult. Hence, in this study an efficient and reproducible method for in vitro development of embryogenic callus and regeneration in Sorghum bicolor was developed. Immature embryo explants of Sorghum bicolor were cultured on MS nutrient medium supplemented with different concentrations and combinations of auxins and cytokinins. Embryogenic callus was initiated on MS medium supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l Kinetin (KN). Addition of KN to MS medium containing 2,4-D resulted in a significant enhancement on embryogenesis of the embryos. Amino acids also supported an improved frequency of embryogenesis. Therefore induction of a high frequency of somatic embryogenesis in immature embryos on MS medium is possible. Development of embryogenic callus, induction of somatic embryo and subsequent shoot regeneration was proficient at a concentration of 2 mg/l BAP. The regenerated shoots readily rooted on half strength MS medium supplemented with 1 mg/l naphthalene acetic acid (NAA). The regenerated plantlets were successfully transferred to soil and subsequently plants produced seeds. There was no difference between the acclimatized plants in comparison with in vivo plants in the respect of morphological characters
Adventitious shoot regeneration from immature embryos of sorghum
Plant cell, Tissue and organ culture, 2002
Eleven genotypes of sorghum were examined for their response in tissue culture, and the tissue culture system was optimized. The cultures were initiated from immature embryos taken approximately two weeks after flowering. The response of immature embryos varied with the genotype. 'C. Kafir' and 'PE932 025' showed the highest frequency of callus induction and regenerable callus formation under appropriate culture conditions. Regeneration occurred at high frequencies when cytokinins (kinetin or 6-benzyladenine) had been added in the callus induction medium, followed by regeneration medium devoid of growth regulators. The addition of proline and polyvinylpyrrolidone also enhanced shoot formation, but the addition of cytokinins to regeneration media did not improve shoot formation. On the revised culture medium, plants were regenerated from up to 100% of sorghum immature embryos.
Regeneration of sorghum from shoot tip cultures and field performance of the progeny1
Plant Cell Tissue and Organ Culture, 2000
A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg l−1) and kinetin (0.1 mg l −1) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about 5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l−1) and indole-3-acetic acid (0.5 mg l −1) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation over other protocols using different explants are discussed.
Novel explant for somatic embryogenesis in Sorghum bicolor (L.) Mohen (Versión electrónica)
2015
This work was carried out with the objective to form somatic embryos of sorghum, cv. 'CIAP 132R-05' starting from callus obtained from sections of in vitro shoots. For the formation of callus, different concentrations of 2,4-D were studied as well as three concentrations of ascorbic acid to eliminate the phenolic oxidation. To increase the percentage of callus formation with embryogenic structures, different segments of the shoots were used. For the formation of somatic embryos, different concentrations of 2,4-D; 6-BAP and L-Proline were added to the culture media. The greatest callus formation (50%) was obtained in the culture medium with 18.1 µM of 2,4-D. When 50 mg l-1 of ascorbic acid was added to the culture medium, the percentage of callus formation increased to 67.5%, and was couple with absence of oxidation of the medium and the explant. The frequency of callus formation with embryogenic structures increased to 95% with the use of segment 1 of the shoot sections in v...
Enhanced shoot regeneration in tissue culture studies of Sorghum bicolor
The effect of combination of benzyl aminopurine (BAP), thidiazuron (TDZ) and indole acetic acid (IAA) was studied on in vitro shoot proliferation from immature embryo explants of Sorghum bicolor (L.) Moench, an economically important cereal. Proliferation of multiple shoots was achieved on MS medium supplemented with 1.5 mgL-1 BAP + 1.5 mgL-1 TDZ + 1.0 mgL-1 IAA + 1000 mgL-1 L-asparagine, L-proline, L-glutamine and serine. Upto 72 plantlets per explant were obtained in the present study. Response of six varieties; IS 3566, SPV 475, CSV 13, CSV 15, CSV 112 and IS 348 to multiple shoot formation was studied. The maximum number of shoots were observed in the variety IS 3566. The in vitro proliferated and elongated shoots were transferred individually to a root induction medium containing 2% sucrose + 1.0 mgL-1 NAA and within 21 days 162 roots per culture which were produced from multiple shoots. The regenerated plantlets were transferred to 1:1 soil and vermiculite mixture and acclimatized with 60 % survival rate. Fully acclimatized plants were grown in garden soil in greenhouse and their morphological and physiological parameters as similar with seedlings.
Applied Ecology and Environmental Research, 2019
An effective regeneration system was developed from embryonic callus that was formed by using mature embryos in 6 sorghum cultivars (Gözde 80, Greengo, Leoti, Beydarı, Aldarı and Akdarı). Different auxin (2,4-D and 2,4,5-T) and cytokinin (zeatin and kinetin) combinations on somatic embryogenesis were studied. The highest embryonic callus in all cultivars was derived from cultures in MS medium containing 1 mg/l 2,4-D. The transfer of embryonic callus obtained from medium containing 2,4,5-T + kinetin to the shooting medium (1 or 2 mg/l BA +1.5 mg/l TDZ +1 mg/l IAA) and subsequently rooting medium (½ MS with 1 mg/l NAA) resulted in a higher shooting and rooting. Different concentrations of BA in the shooting medium did not affect shoot formation. Akdarı and Greengo cultivars produced better callus induction and regeneration than the other cultivars as grain and silage types, respectively. Rooting and surviving rates varied between 10.55-68.37% depending on the growth regulators used at the beginning of culture. Growth and survival rates were increased in plants transferred from high-shoot-rate cultivars to the rooting medium.