Development of an Efficient Regeneration System via Somatic Embryogenesis Obtained from Mature Embryos in Some Grain and Silage Sorghum Cultivars (original) (raw)
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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.
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...
Efficient plant regeneration from shoot apices of sorghum
An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm -3 each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm -3 kinetin.
American Journal of Plant Sciences, 2014
In this study, an optimum protocol for shoot formation through somatic embryogenesis using mature embryo explants was developed. Calli were initiated on Murashige and Skoog (MS) media supplemented with varying concentration of 2,4-Dichlorophenoxyl acetic acid (2,4-D) ranging from 1.5 mg/l-4.0 mg/l alone or in combination with 0.5 mg/l Kinetin (KN). Significance difference (p < 0.05) was observed among the different concentrations of hormone used for callus induction. The highest percentage callus formation was obtained from the media fortified with 4 mg/l 2, 4-D for mature embryo obtained from imbibed seed while for the preconditioned mature embryo, the media supplemented with 2 m/l 2,4-D + 0.5 mg/l kinetin (KN) recorded more percentage callus formation compared to what was obtained from the media supplemented with 2.5 mg/l2,4-D + 0.5 mg/l. More percentage shoot formation was obtained from the media supplemented with 1 mg/l 6-benzylaminopurine (BA). Average number of shoot per callus was also more in the media fortified with 1 mg/l BA (2.25) but this was not significantly different from what was obtained from the media fortified with 2 mg/l BA+ 0.1 mg/l 2,4-D at 5% level of significance.
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.
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.
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
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.
Long-Term Maintenance of Callus Cultures from Immature Embryo of Sorghum bicolor
A protocol was developed for long-term maintenance of callus cultures, induced from immature embryo of Sorghum bicolor (L.) Moench. Maintenance of callus cultures for prolonged duration in a regenerable state is one of the most important requirements for using in vitro techniques for plant improvements. In the present study calli were maintained satisfactorily on MS medium over 57 weeks and plantlets regenerated from them on transfer to light. Immature embryo was used as source material for callus initiation. The callus cultures were cultured on MS media with 1.5 mg/L of 2,4-D (2,4-dichlorophenoxy acetic acid), 10mg/L silver nitrate (AgNo ), 400mg/L casein hydrolysate (CH), 200mg/L L-proline and L-asparagine. 3 After callus initiation cultures were maintained in dark conditions and subcultured for every 3 week intervals. Greening of callus and regeneration of shoot-buds from callus occurred on transfer to MS media with 2mg/L BAP + TDZ (Thidiazuron). These shoots grew further in media with both, BAP and TDZ, Rooting of shoots occurred on media with NAA, the rooted plantlets could be transferred to autoclaved soil. Genetically uniform plantlets regenerated continuously from established callus upto 57 weeks old; thereby helping in multiplication and to facilitate the year round availability of explants and/or somatic embryos for Sorghum tissue culture and transformation.