Frequency Of Embryogenic Callus Research Articles

Transcriptomic and physiological analyses reveal the acquisition of somatic embryogenesis potential in Agapanthus praecox

Somatic embryogenesis (SE) is an ideal cell model for plant cell totipotency study. The poor frequency of embryogenic callus (EC) induction limits the application of SE in many plants, such as Agapanthus praecox. Herein, we performed transcriptomic and physiological analyses with different callus (Ca) differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the physiological and molecular characteristics of the acquisition of embryogenic potential in A. praecox. A number of differentially expressed genes (DEGs) were significantly related to plant hormonal signaling transduction, starch and sucrose metabolism, and reactive oxygen species (ROS) responses. Compared to Ca, DEGs including heat shock protein, isoamylase, histone H4, alpha-1,2-mannosyltransferase, and sucrose synthase substantially changed expression levels in EC formation. Corresponding to DEGs, physiological indicators including indole-3-acetic acid, cytokinins, brassinosteroids, ethylene, abscisic acid, ROS, hydrogen peroxide, superoxide dismutase, catalase, starch and sucrose may be responsible for the acquisition of embryogenic potential in A. praecox. Moreover, plant growth regulators, carbon source combination, osmotic regulating substance, and DNA methylation inhibitors in the culture medium significantly affected the acquisition of SE potential. Altogether, our results suggested that plant hormone signal regulation, starch and sucrose metabolism, ROS scavenging, chromatin accessibility and DNA methylation jointly contributed to the activation of embryogenic ability in A. praecox.

Integrated Proteomic and Metabolomic Analyses Provide Insights Into Acquisition of Embryogenic Ability in Agapanthus praecox.

Somatic embryogenesis (SE) is an ideal model for plant cell totipotency. Transition from somatic cells to embryogenic cells is the key to SE. The poor frequency of embryogenic callus (EC) induction has limited the application of SE in many plants, such as Agapanthus praecox. We performed large-scale, quantitative proteomic and metabolomic analyses with different callus differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the morphological, physiological, and molecular characteristics of the acquisition of embryogenic ability in A. praecox. Integrated proteomic and metabolomic analyses suggested that callus differentiation direction was potentially regulated by pathways related to carbohydrate and energy metabolism (fatty acid metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, pentose and glucuronate interconversions, starch and sucrose metabolism, galactose metabolism, carbon fixation pathways in prokaryotes, carbohydrate digestion and absorption, and fructose and mannose metabolism), chromatin accessibility and DNA methylation, reactive oxygen species responses and resistance (ascorbate and aldarate metabolism), and plant hormonal signaling. As a validation, we found that carbon source combination and plant hormone regulation in the culture medium significantly affected the acquisition of embryogenic ability, thereby inducing EC. Interestingly, plant hormonal signaling-related genes showed different expression patterns significantly when callus cultured with different carbon sources. Thus, our results suggested that energy supply and hormone signal transduction seemed to cooperatively contribute to the activation of embryogenic ability. Altogether, this study revealed valuable information regarding the molecular and biochemical changes that occurred during EC induction and provided valuable foundation for comprehensive understanding of the mechanisms associated with SE and organogenesis in A. praecox.

Callogenesis and somatic embryogenesis of <i>Oryza sativa</i> L. (cv. MARDI Siraj 297) under the influence of 2, 4-dichlorophenoxyacetic acid and kinetin

<abstract> <p>Callogenesis and embryogenesis are integral parts of many tissue culture procedures for genetic manipulation in rice. However, the efficiency of both processes is largely dependent on the media constituent especially the plant growth regulators (PGRs) due to the genotype-dependent nature of <italic>in vitro</italic> culture protocols. Therefore, this study investigates the effect of two PGRs; 2, 4-dichlorophenoxyacetic acid (2, 4-D) and kinetin (Kin) on callus growth and somatic embryogenesis of an important Malaysian rice cultivar (<italic>Oryza sativa</italic> L. cv. MARDI Siraj 297). Mature rice seeds explants were inoculated in Murashige & Skoog (MS) medium supplemented with different combinations of 2, 4-D (0 to 3.5 mg/L) and Kin (0 to 0.5 mg/L) to induce callogenesis. Parameters for callus growth such as fresh weight (FW), callus induction frequency (CIF), embryogenic callus frequency (ECF), regeneration frequency (RF), number of plantlets per callus (PPC), callus texture and callus color were observed after 35 days of inoculation. The results show that the maximum callus growth was achieved in MS medium supplemented with combination of 2.0 mg/L 2, 4-D and 0.2 mg/L Kin, represented by the highest FW (211 mg), CIF (95%), ECF (90%), RF (100%) and PPC (22 plantlets); along with friable callus texture. Low concentration of 2, 4-D (0 to 0.5 mg/L) in the presence or absence of Kin promotes root growth instead of callus, while high concentrations (above 3.0 mg/L) retard the callus formation. The embryogenic calli from this optimized PGRs combination were successfully formed shoots in MS medium supplemented with 2 mg/L BAP and 1 mg/L NAA, followed by rooting in PGRs-free MS medium. This finding provides an efficient protocol for callogenesis and somatic embryogenesis of MARDI Siraj 297, since this is the first published report regarding somatic embryogenesis induction of this cultivar.</p> </abstract>

Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.)

ABSTRACT In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO4, AgNO3 and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO4, AgNO3 and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO4 or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO4, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO4. In another study, the basal induction and regeneration medium were fortified with AgNO3 or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO3 or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO3 or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.

Polyethylene glycol and chilling overcome Somatic embryogenesis obstacle in Pyrus communis

Abstract The purpose of this investigation is to achieve the procedure of somatic embryogenesis (SE) as an efficient propagation way in four pear cultivars: Natanzi, Ghosi, Williams and Dar Gazi, by focusing on two strategies: One is the application of competent explants, and the other one is stress employment. The results were variable because of cultivars behavior was highly genotype specific. However, the results identified that the highest frequency of embryogenic callus induction was obtained from the endosperm and immature cotyledonary segment explants of P. communis cultivars on Murashige and Skoog medium (MS) containing 8.05 μM 1-naphthaleneacetic acid (NAA), 13.32 μM N6-benzyladenine (BA) supplemented with 250 mg/L glutamine as a reduced nitrogen source and 30 g/L (w/v) sucrose in Ghosi (75.46%). The optimized media were different for each cultivar in the induction phase, namely, Natanzi and Ghosi in MS, Williams in Schenk and Hildebrandt (SH) and Dar Gazi in Nitsch and Nitsch (NN). Then, it was observed that when embryogenic calli were transferred to the media containing 1% (w/v) Polyethylene glycol (PEG), calli of some cultivars further differed into embryos while other cultivars produced in chilling at 4 °C. Also, maturation of embryos occurred at 4 °C for 4 months. Then passages using MS media caused germination of embryos and plantlets were produced. To sum up, the greatest frequency of embryogenic callus (75.46%) occurred in Ghosi, and the highest embryo maturation and germination (77.06% and 37.66%, respectively in mentioned cultivars) were observed in Natanzi.

Somatic Embryogenesis of Immature Cunninghamia lanceolata (Lamb.) Hook Zygotic Embryos

Two efficient somatic embryogenesis systems were developed in Chinese fir, the most important conifer for industrial wood production in China. Three development stages (cleavage polyembryony, dominant embryo, and precotyledon) of immature embryos derived from 25 genotypes of open-pollinated mother trees were used as initial explants. Cleavage polyembryony-stage embryos with a 12.44% induction rate was the most embryogenic response stage. The highest frequency of embryogenic callus (13.86%) induction was obtained from DCR medium supplemented with 1.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.3 mg L−1 kinetin (KN). An average of 53.33 early somatic embryos were produced from approximately 0.2 g (fresh weight) embryogenic callus after 2 weeks of incubation on medium supplemented with 50 μmol L−1 abscisic acid (ABA) and 100 g L−1 polyethylene glycol (PEG) 6000. About 53% dominant embryos have an embryogenic response after a 6-week cultivation on medium supplemented with 1.0–2.0 mg L−1 benzyladenine (BA), 0.2 mg L−1 naphthylacetic acid (NAA) or 2,4-D, and 0.004 mg L−1 thidiazuron (TDZ). After three successive transfer cultures on medium supplemented with 1.5 mg L−1 BA, 0.2 mg L−1 NAA, and 0.004 mg L−1 TDZ, 4.49–16.51% of the embryos developed into somatic embryos.

EN

Picrorhiza kurroa Royle ex. Benth is an endangered and important medicinal plant of alpine Himalayas. Developing an efficient protocol for its mass multiplication is essential to meet the requirements of pharmaceutical industries and also in conservation of this plant under its natural habitat. Present study was undertaken to develop a protocol for in vitro mass multiplication of P. kurroa. Result of study revealed that highest frequency of shoot regeneration was achieved on Murashige and Skoog's basal medium supplemented with 1.0mg/l BAP, 0.5 mg/l Kn and 1.0mg/l GA3, while the best rooting was observed in MS medium supplemented with 2.5 mg/l IBA. MS medium supplemented with 3.0 mg/l 2,4-D resulted in highest frequency of embryogenic callus. Callus inoculated on MS media supplemented with BAP and IAA resulted in both shoot and root formation while the callus on MS media supplemented with NAA and IBA resulted only root formation. The somatic embryos were established from callus on MS medium supplemented 2.5 mg/l 2,4- D after four weeks. MS medium containing 1.0 mg/l BAP and 1.0 mg/l GA3 resulted into shoots from well developed somatic embryos. This protocol will provide a system for the germplasm conservation in P. kurroa by multiplication and regeneration of true to type plants.

Plant Regeneration of Iris germanica L. from Shoot Apexes via an Improved Somatic Embryogenesis Protocol

An improved three-stage protocol for plant regeneration via somatic embryogenesis of the horticulturally important plant Iris germanica L. was developed using shoot apex segments as explants. At the first stage of the experiment, 60% of callus was obtained from shoot apex segments of I. germanica on Murashige and Skoog’s (MS) medium supplemented with 4.52 μm 2,4-dichloropheoxyacetic acid (2,4-D) and 0.44 μm 6-benzyladenine (6-BA). When nonembryogenic calli were subcultured on MS medium with 11.31 μm 2,4-D and 0.44 μm 6-BA, maximum frequency of embryogenic callus (66.0%) was obtained. At the second stage, the treatment of 9% (w/v) sucrose resulted in the optimum somatic embryo (SE) formation (70.0%). More than 90.0% of SEs germinated with bipolar structure and regenerated into plantlets on plant growth regulator-(PGR)free MS medium during the third stage. Regenerated plantlets were successfully acclimatized in greenhouse environment with little somaclonal variation. Histological study showed that somatic embryogenesis stages were asynchronous and SEs developed from the surface and inner tissue of embryogenic calli.

Embryogenic cultures of lily (Lilium spp.): optimising callus initiation, maintenance, and plantlet regeneration

SummaryTo facilitate genetic improvement in lily (Lilium spp.), the efficiency of regeneration from callus cultures induced from a range of explants was optimised for three important cultivars: Oriental hybrid lily ‘Sorbonne’, Oriental hybrid lily ‘Bernini’, and Oriental × Trumpet hybrid lily ‘Robina’. Filaments, pedicels, styles, ovaries, and anthers were used as explant material to identify and optimise the parameters that influence somatic embryogenesis. The results showed that embryogenic competence was dependent on cultivar, explant type, and developmental stage. The highest numbers of embryogenic cultures were induced from filaments of ‘Sorbonne’ (22.8%), ‘Bernini’ (16.5%), and ‘Robina’ (17.8%). The youngest filaments (developmental Stage I) gave the highest embryogenic response in each genotype, showing a three-to-four-fold increase compared with Stage V explants. Of the three regions of the filament used as explants, only the basal region produced embryogenic cultures. The highest proliferation frequency of embryogenic callus was obtained on 1.0× Murashige and Skoog (MS) medium supplemented with 2.0 mg l–1 picloram (PIC; 4-amino-3,5,6-trichloropicolinic acid) and 60 g l–1 sucrose. These cultures also produced the highest numbers of regenerated plantlets when transferred to plant growth regulator (PGR)-free 1.0× MS medium supplemented with 1.0 g l–1 activated charcoal (AC) and 30 g l–1 sucrose. Histological and scanning electron microscopic (SEM) observations of the various developmental stages of somatic embryo development showed the four typical stages: globular, heart-shaped, torpedo, and cotyledonary.

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l−1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l−1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l−1 glycine, 690 mg l−1 proline, and 1.0 g l−1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l−1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l−1 6-benzylaminopurine; addition of 2.0 mg l−1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l−1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.

English

  Genetic transformation is considered as one of the most promising options for improvement of crop traits. Current transformation methods for sweetpotato depend on plant regeneration through organogenesis or somatic embryogenesis. Somatic embryogenesis and plant regeneration at a high frequency has been restricted to a few sweetpotato varieties. Three auxins namely:  2,4-dichlorophenoxyacetic acid (2,4-D), 4-fluoroamphetamine (4-FA) and 4,5-trichlorophenoxyacetic acid (2,4,5-T)were investigated in this study for enhancing somatic embryogenesis from various plant organs of recalcitrant African sweetpotato cultivars. 2,4-D was found to be the best (p ≤ 0.05) for induction of embryogenic callus. Cultivar Bwanjule had the highest (20.2%) embryogenic callus frequency among the five African cultivars tested. The highest number of plants in this study was regenerated from the non-African cultivar variety Jonathan on media supplemented with 0.2 mg Zeatin. The emergence of roots from callus of recalcitrant Ugandan cultivars and the comparable high embryogenic responses in this work demonstrate the potential for regenerating plants from African cultivars that have not been regenerated before. The regeneration of roots in this work could be useful for the initiation of root cultures. The most important application of this work is in genetic transformation of sweet potato, particularly for improvement of resistance to weevils.   Key words: Embryogenesis, plant growth regulators, plant regeneration, Ipomoea batatas

Regeneration from embryogenic callus and suspension cultures of the wild medicinal plant Cymbopogon schoenanthus

Somatic embryogenesis and plant regeneration from both callus and suspension cultures of the wild medicinal plant Cymbopogon schoenanthus subsp. proximus has been achieved. The species is rare and confined in its distribution to Africa. A range (0.5 to 8 mg/l) of 2,4-dichlorophenoxyacetic acid (2,4D) for the induction of embryogenic callus from seed cultures were used. Results show that 1.0 and 4.0 mg/l 2,4-D gave a 90 to 100% frequency of embryogenic callus containing mature embryos within three months of culture. Testing the effect of phosphorus and nitrogen concentrations on somatic embryogenesis from callus cultures showed that high phosphorus and low nitrogen concentrations enhanced embryo induction. Low NH4NO3 enhanced growth and maturation of somatic embryos, while low KNO3 enhanced germination and shoot production. Suspension cultures were initiated from embryogenic callus on 0.5, 1.0 and 2.0 mg/l 2,4-D, then plated on 3 different combinations of 2,4-D and 6-benzyl adenine (BA). Mature embryos were highest on both 0.5 and 1.0 mg/l 2,4-D (X = 15.8, 17.3), while shoot germination was enhanced by using BA in the regeneration media. The effect of high phosphorus concentration in the culture media was significant on both embryo induction and early maturation (X = 41). Lower ammonium nitrate concentrations enhanced growth and maturation of embryos. Both embryo maturation and the germination of shoots after 6 and 8 months of plating were best on low ammonium nitrate (X = 36.8 and 27.6 shoots, respectively). The presented embryogenic system will be of value for the clonal propagation, ex situ conservation and production of bioactive compounds from this threatened plant species.

In vitro regeneration of selected commercial Tanzanian open pollinated maize varieties

Four Tanzanian open pollinated maize varieties namely; Kito, Situka M-1, Staha and TMV-1 were regenerated in vitro using immature zygotic embryos as ex-plants. Callus induction was achieved using Murashige and Skoog (MS) basal medium supplemented with 1, 1.5, 2 or 2.5 mg/l of 2, 4-D. Callus induction was significantly affected by the genotype of the varieties. Among the varieties tested, Kito, Situka M-1 and Staha had significantly higher callus induction frequency of 84.4, 92.7 and 88.7%, respectively compared to TMV-1 (43.3%). Embryogenic callus induction percentage was significantly influenced by the genotype, 2, 4-D concentrations and their interaction effect implying differential response of the genotypes to 2, 4-D concentrations. Among the genotypes tested, Kito gave significantly (p<0.05) the highest embryogenic callus frequency of 69.5%. Great variations were observed among the genotypes with regard to regeneration frequencies. Staha was comparatively the most regenerable variety with RF of 32.6% than even Kito which had the highest callus induction and embryogenic callus formation. MS medium amended with 1 mg/l 2, 4-D was observed to be the optimum formulation for the induction of embryogenic calli for genotypes tested in this study. Using this regeneration system Situka M-1, Staha and TMV-1 can now be improved against various production constraints through genetic engineering. Key words : 2, 4-dichlorophexyacetic acid, embryogenic callus, immature zygotic embryos, regeneration frequency, somatic embryos, Tanzanian open pollinated maize.

Regenerability of elite tropical maize (Zea mays L.) inbred lines using immature zygotic embryo explants

Five elite tropical maize inbred lines; CML395, CML443, CML442, MAS [MSR/312]-117-2-2-1-B-5-B) and CML216 as a control, were evaluated for their regenerability making use of calli derived from immature zygotic embryos. Murashige and Skoog basal salts supplemented with 1.0, 1.5, 2.0 and 2.5 mg/L 2,4dichlorophenoxyacetic acid were used to induce callus. Callus induction frequency and formation of embryogenic callus varied significantly (p<0.01) depending on genotype and level of 2,4dichlorophenoxyacetic acid. Shoot regeneration efficiency also differed significantly (p<0.01) depending on genotype. Significantly (p<0.05) higher callus induction and frequency of embryogenic callus were obtained at 1 mg/L 2,4-dichlorophenoxyacetic acid, indicating this as the optimal level for regenerating these inbred lines. CML395 and CML442 revealed significantly (p<0.05) higher callus induction and embryogenic callus frequency compared to CML443 and MAS [MSR/312]-117-2-2-1-B-5B), while they were at par with the control inbred line CML216. Plants were regenerated from all the inbred lines except CML443 and were successfully acclimatized and grown to maturity. CML395 was the best regenerable line with significantly (p<0.05) higher regeneration efficiency of 109.3%. It was concluded that CML395, CML216 and CML442 can be used in in vitro genetic transformation.

Plant regeneration via somatic embryogenesis from root explants ofDaucus carota L., subsp.halophilus

A system for induction of callus and plant regeneration via somatic embryogenesis from root explants of Daucus carota L. sub sp. halophilus was evaluated. The influence of plant growth regulators (PGRs) including 2,4dichlorophenoxyacetic acid (0.25–2.50 mg/L−1 2,4-D) on callus induction from root explants of in vitro plantlets was studied. 2,4-dichlorophenoxyacetic acid (2,4-D), and Kinetin were used for somatic embryogenesis. The results showed that the highest (100%) callus induction on Murashige and Skoog (MS) medium supplemented with 2,4-D (0.75 mg/L−1) and highest rate (100%) of callus multiplication in MS + 2,4-D (1.5 mg/L−1) + NAA (0.50 mg/L−1)was observed. The highest induction frequency of embryogenic callus emerged on Murashige and Skoog (MS) medium supplemented with MS + 2,4-D (1.5 mg/L−1) + Kn (0.25 mg/L−1). Mean of 4 somatic embryos per embryogenic callus were obtained and approximately 90% of them developed into plantlets. The plant regeneration system established in this study will facilitate mass propagation and may be applied to culture of the roots of wild Daucus carota. [Medicinal Plants 2012; 4(2): 00–00]

바이오에너지용 수수 품종의 재분화율 증진을 위한 배지와 생장조절제 효과

본 연구는 수수의 성숙 종자로부터 캘러스유기 및 재분화 효율 조사하기위하여 수행되었다. 배발생 캘러스 형성을 살펴보기 위해 생장조절제 효과를 조사하였다. 수수 성숙종자로부터 캘러스 유기에 적합한 생장조절제 종류와 농도는 2 mg/L 2,4-D인 것으로 나타났다. 배발생 캘러스의 고효율 빈도는 B5 배지에 2 mg/L 2,4-D을 첨가하였을 때 가장 양호한 것으로 나타났다. 배발생 캘러스로부터 식물체 재분화는 BAP와 IBA를 넣은 MS 배지가 가장 적절한 것으로 나타났다. BAP 농도가 높을수록 신초재분화가 활발하였다. 따라서, 본 연구 결과로부터 수수 기내배양시 신초와 발근 형성에 효과적인 생장조절제 조합은 0.5 <TEX>$mg\;l^{-1}$</TEX> BAP와 0.25 <TEX>$mg\;l^{-1}$</TEX> IBA인 것으로 나타났다. This study was carried out to optimize the embryogenic callus induction and plant regeneration from mature seeds of Sorghum bicolor. The effect of growth regulators was investigated on formation of embryogenic callus. The highest frequency of embryogenic callus was observed when the mature seeds were cultured on B5 medium supplemented with 2 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D). The highest frequency of plant regeneration from embryogenic callus was observed on MS medium with 0.5 <TEX>$mg\;l^{-1}$</TEX> 6-benzyl amino purine (BAP) and 0.25 <TEX>$mg\;l^{-1}$</TEX> indole-3-butyric acid (IBA) to optimize the shoot regeneration. High concentration of BAP (1 <TEX>$mg\;l^{-1}$</TEX>) supplemented with IBA (0.25 <TEX>$mg\;l^{-1}$</TEX>) was effective combination for shoot multiplication. MS medium supplemented with 1 <TEX>$mg\;l^{-1}$</TEX> IBA was found to be the most effective for inducing roots. Normal rooted plantlets were transferred to the greenhouse for hardening with over 90% survival rate. Hence, this reproducible protocol could be useful for mass propagation and genetic transformation of S. bicolor.

English

A system for induction of callus and plant regeneration via&nbsp;somatic embryogenesisfrom root explants of&nbsp;Hevea brasiliensis&nbsp;Muell. Arg. clone&nbsp;Reyan&nbsp;87-6-62 was evaluated. The influence of plant growth regulators (PGRs)&nbsp;including 2,4-dichlorophenoxyacetic acid&nbsp;(2,4-D), 6-benzylaminopurine (6-BA)&nbsp;and&nbsp;kinetin&nbsp;(KT)&nbsp;on callus induction of root explants from&nbsp;in vitro&nbsp;plantlets&nbsp;were studied.&nbsp;The results showed that&nbsp;the highest induction frequency of embryogenic callus emerged on Murashige and Skoog (MS) medium&nbsp;supplemented with 1&nbsp;mg/l&nbsp;KT, 0.2&nbsp;mg/l&nbsp;6-BA without 2,4-D.&nbsp;Mean of 4 somatic embryos per embryogenic callus were obtained and approximately 11.8% of them developed into plantlets. The regenerated plantlets were successfully transplanted to sand bed. The plant regeneration system established in this study will facilitate&nbsp;mass propagation&nbsp;and&nbsp;may be applied to culture the roots of high-yielding&nbsp;rubber trees. &nbsp; Key words:&nbsp;Hevea brasiliensis&nbsp;Muell. Arg.,&nbsp;root explants, callus, somatic embryogenesis, regeneration.

Comparison of in vitro regeneration pathways in Punica granatum L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA3). However, adding 24 μM silver nitrate (AgNO3) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l−1 sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l−1 sucrose.

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm−3 N6-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm−3) and BA or kinetin (1–5 mg dm−3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.

Somatic embryogenesis in apomictBoechera holboellii

In this study we establish an efficient method for the regeneration for Boechera holboellii via somatic embryogenesis. Immature cotyledons from siliques of 4-6 month-old plants were cultured on MS medium supplemented with plant growth regulators (BA - 6-benzylaminopurine; NAA - alpha-naphthaleneacetic acid; TDZ - 1-phelyl-3-(1,2,3-thiadiazol-5-yl) urea: 2,4 D-2,4-dichlorophenoxy-acetic acid). A high frequency of embryogenic callus was produced after two weeks in culture. The somatic embryos were obtained with a frequency of 10% of explants on MS medium supplemented with 1.34 muM NAA + 8.87 muM BA and 2.68 muM NAA + 17.74 muM BA within 3 weeks in culture. The alternative regime of MS medium supplemented with 1.34 muM NAA + 4.44 muM BA produced somatic embryos at a frequency of 38%.