Callus Induction Potential Research Articles

GWAS determined genetic loci associated with callus induction in oil palm tissue culture.

GWAS identified six loci at 25kb downstream of WAK2, a crucial gene for cell wall and callus formation, enabling development of a SNP marker for enhanced callus induction potential. Efficient callus induction is vital for successful oil palm tissue culture, yet identifying genomic loci and markers for early detection of genotypes with high potential of callus induction remains unclear. In this study, immature male inflorescences from 198 oil palm accessions (dura, tenera and pisifera) were used as explants for tissue culture. Callus induction rates were collected at one-, two- and three-months after inoculation (C1, C2 and C3) as phenotypes. Resequencing generated 11,475,258 high quality single nucleotide polymorphisms (SNPs) as genotypes. GWAS was then performed, and correlation analysis revealed a positive association of C1 with both C2 (R = 0.81) and C3 (R = 0.50), indicating that C1 could be used as the major phenotype for callus induction rate. Therefore, only significant SNPs (P ≤ 0.05) in C1 were identified to develop markers for screening individuals with high potential of callus induction. Among 21 significant SNPs in C1, LD block analysis revealed six SNPs on chromosome 12 (Chr12) potentially linked to callus formation. Subsequently, 13 SNP markers were identified from these loci and electrophoresis results showed that marker C-12 at locus Chr12_12704856 can be used effectively to distinguish the GG allele, which showed the highest probability (69%) of callus induction. Furthermore, a rapid SNP variant detection method without electrophoresis was established via qPCR-based melting curve analysis. Our findings facilitated marker-assisted selection for specific palms with high potential of callus induction using immature male inflorescence as explant, aiding ortet palm selection in oil palm tissue culture.

Effects of various plant growth regulators upon callus induction potential using leaf explants of Clinacanthus nutans (Sabah Snake grass)

Effects of various plant growth regulators upon callus induction potential using leaf explants of Clinacanthus nutans (Sabah Snake grass)

Response of Different Explants for Callus Induction in Cucumber

In vitro regeneration of cucumber is relatively difficult for genetic improvement. In this regard, different concentrations of growth regulators and three types of explants (cotyledon, hypocotyl and leaf disc) were investigated for their efficiency on callus induction potential. Among different explants explored for callus induction with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), leaf disc responded earlier (4.67 days) and showed higher percentage of callus induction (91.50%) with 2 mg/l 2,4-D supplemented Murashige and Skoog (MS) media. The same concentration of 2,4-D resulted in the maximum callus fresh (0.56 g) and dry weight (0.39 g) from leaf disc explant. Then the callus was transferred to untreated, 2.0 mg/l BAP + 0.2 mg/l NAA + 1.0 mg/l Kn, 2.0 mg/l BAP + 1.0 mg/l NAA + 1.0 mg/l Kn and 2.0 mg/l BAP + 1.5 mg/l NAA + 1.0 mg/l Kn fortified MS medium. After transferring the callus of different explants to shoot regeneration media containing different concentrations of 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and Kinetin (Kn), only cotyledon callus started to regenerate shoot. The combination of BAP (2 mg/l) + NAA (0.2 mg/l) + Kn (1 mg/l) showed highest shoot regeneration percentage (67.77%) and the maximum number of shoots (5.12) per explant were recorded in the treatment combination of 2 mg/l BAP + 0.2 mg/l NAA + 1 mg/l Kn. These results provided a basis for the optimization of the callus induction protocol of cucumber for genetic transformation.

Surface sterilization and callus induction potential of various indica rice (Oryza sativa L.) Varieties

The present study was conducted to standardize the efficient and reliable surface sterilization and callus induction protocol for 22 rice (Oryza sativa L.) varieties developed by Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, Maharashtra (India) using different combinations of various plant growth regulators. The dehusked mature rice seed was used as explant. Prior to callus induction, surface sterilization protocol was standardized using 70% ethanol and different concentrations of mercuric chloride (HgCl2). The results revealed that surface sterilization treatment with 70% ethanol for 1 min, followed by 1.0% HgCl2 for 10 min and then washing the seeds 4-5 times was most effective in establishing maximum aseptic cultures (86.67%). Callus induction potential differed significantly among the rice varieties for the concentrations of plant growth regulators in MS medium. Rice cv. ‘Ratnagiri-711’ followed by cv. ‘Karjat-8’ were most superior in callus induction and proliferation potential with the respective callus induction frequency of 87.56 and 87.0%, and callus weight of 269.37 and 235.20 mg after 45 days of inoculation. The supplementation of 2,4-D @ 1.0 to 2.0 mg L-1 along with 0.5 mg L-1 BAP in MS medium was found optimum for embryogenic callus formation. All the calli formed on MS medium supplemented with combinations of plant growth regulators were yellowish-white in colour but with different textures as compact, soft, friable that were embryogenic and mucilaginous that were non-embryogenic.

Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice.

DH (Doubled haploid) is the immortal mapping population and an outcome of single meiotic cycle, contributed from male partner. An improved procedure was developed for high frequency androgenesis in japonica genotypes, K-332 and GS-88 and their F1s. A total of 207 fertile, green, di-haploid plants were generated from K-332 × GS-88 hybrids using the improved anther culture protocol. The investigation was carried out to evaluate callus induction potential and regeneration response for the genotypes and the derived F1s on N6 media and modified N6 media (N6M). Whereas, N6 failed to induce callusing, agarose solidified N6M media supplemented with 4% maltose, growth regulators; NAA (2 mg/l), 2, 4-D (0.5 mg/l), Kinetin (0.5 mg/l), and silver nitrate induced high calli percentage of 27.6% in F1s, 9.5% and 6.7% in GS-88 and K-332 respectively. Murashige and Skoog (MS) media supplemented with 3% sucrose, and the hormonal combination BAP (2 mg/l), Kinetin (1 mg/l) and NAA (1 mg/l) induced high green shoot regeneration rates (0–60.0%). The effect of cold pre-treatment at 4°C and the stage of anther collection and their interaction was studied. The effect of cold pre-treatment (CP) of collected boots at 4°C (for CP2: 2, CP4: 4, CP6: 6 and CP8: 8 days) at different stages of panicle emergence (BES4-6: 4–6, BES7-10: 7–10, BES11-13: 11–13, BES>13: more than 13 inches was worked out in relation to the effect on response of calli induction, albino regeneration, green plant regeneration and number of shoots/green calli. CP referred to the number of days for which the collected boots were incubated before they were inoculated. BES was the length (inches) between flag leaf and penultimate leaf at the time of boot collection. We concluded that CP6 and BES7-10 showed better response to callus proliferation and regeneration of plantlets across genotypes. The appropriate pre-treatment, stage of anther collection and favourable media composition resulted in high calli induction and green plant regeneration rates in recalcitrant japonica genotypes. The modified N6 media resulted into efficient callus induction and is expected to be useful for studies which aim at rapid generation of mapping populations for genetic studies.

The establishment of callus and cell suspension cultures of Hylocereus costaricensis for the production of betalain pigments with antioxidant potential

Hylocereus costaricensis, commonly known as red-purple dragon fruit or pitaya, is a climbing vine cactus that is currently widely grown as a fruit crop and has gained popularity as a potential source of both natural colourants and antioxidants contributed by betalain compounds. Betalains comprised of red-purple betacyanins and yellow betaxanthins. This study aimed to establish callus and cell suspension cultures of H. costaricensis from in vitro germinated seedlings and to evaluate their betalain content and antioxidant profiles. Epicotyl and cotyledon segments of 60-day-old axenic seedlings were examined for their callus induction potential on half-strength Murashige and Skoog (MS) medium supplemented with various concentrations of naphthaleneacetic acid (NAA) and Thidiazuron (TDZ) under different illumination (16-h photoperiod and darkness). Different combinations of picloram and 2,4-dichlorophenoxyacetic acid (2,4-D) were subsequently tested for the induction of friable callus. The betalains, total phenolic content, total flavonoid content and antioxidant activity DFRSA in cell suspension cultures were quantified using spectrophotometric analysis. Generally, cotyledon explants cultured under 16-h photoperiod was optimum for callus induction. Compact callus with roots was formed on medium supplemented with NAA while granular callus was formed in TDZ supplemented medium. Among the 36 treatments tested, supplementation of 10 μM picloram induced friable callus with average fresh mass of 0.817 g (10.2-fold as compared to the control treatment). Picloram at 10 μM concentration was also found to be effective for callus proliferation and initiation of cell suspension culture. Yellow and red callus were produced and proliferated on the same medium through repetitive clonal selection. The higher concentration of sucrose was more effective for cell mass accumulation while lower sucrose concentration stimulated the accumulation of betalain compounds and antioxidants in the cell suspension cultures of H. costaricensis. This study indicates that callus and cell suspension culture could be used as an efficient system for sustainable production of betalain compounds which can contribute to functional food colouring and pharmaceutical industries.

Evaluation of the Genotype-dependency of the Leafbased Regeneration and Transformation System in Maize

Tissue culture and genetic transformation in maize are very laborious. The existing regeneration methods, which mainly use immature embryos as starting material, are highly genotype-dependent. Leaf segments can be used as an alternative explant source to produce embryogenic calli. Although a reliable leafbased regeneration and transformation system has been recently reported for maize, however, the genotype-dependency of this method has not been described yet. To this end, we evaluated the production of embryogenic calli from young leaves of several maize genotypes. The results showed that, overall callus induction potential as well as embryogenic callus induction rate is significantly different among the tested genotypes, demonstrating the genotypedependency of this system. However, induced embryogenic calli from different genotypes remained their embryogenic capability during several callus multiplication rounds. In addition, embryogenic calli showed high potential for biolistic-based genetic transformation, as revealed by transient GUS expression.
 Plant Tissue Cult. & Biotech. 28(2): 261-268, 2018 (December)

Effect of growth hormones in induction of callus, antioxidants, and antibacterial activity in Nerium odorum

Nerium odorum is an imperative species with worldwide therapeutic and commercial uses. N. odorum has been considered as a potentially important plant for industrial and pharmacological applications. Callus induction potential of N. odorum was assessed from leaf explants cultured on Murashige and Skoog’s medium using different plant hormonal treatments. A range of different concentrations of 6-benzylaminopurine, 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and kinetin (0.5–5.0 mg/L) have been incorporated in the culture medium to investigate the biomass, polyphenols production and oxidizing and antibacterial activity of the callus. The results showed highest callus induction at 2.0 mg/L in growth hormone combination of IAA and 2,4-D. The total phenolic content was 92.14 mg GA/g dry weight (DW). In addition, the 2,2-diphenyl-1- picrylhydrazyl activity and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) activities were 46.7 μg/mL DW and 19.9 μg/mLDW, respectively. Interestingly, the explants produced hard calli using combination of IAA and 2,4- D presented a higher phenolic content and antioxidant capacity when compared to individual growth hormones of either IAA or 2,4-D. Altogether, these results demonstrate the extraordinary effect of different growth hormones on polyphenols production, antioxidant, and antibacterial activities in N. odorum.

Study on Callus Induction System of 4 Genotype of Napiergrass (Pennisetum purpureum )

The aim of this study was to produce callus induction potential of 4 napiergrass (Pennisetum purpureum) genotypes (Dwarf Late, Hybrid, Merkeron and Wrukwona). Callus was induced from shoot apices of shoot tillers on MS media containing 2,4-D and BAP. On the MS medium containing 2 mg L–1 2,4-D and 0.5 mg L–1 BAP all genotypes could produce embryogenic calli, with different rate of growth. The best genotype for producing embryogenic calli was dwarf napiergrass in 60 day culture. These genotypes would be usefull for tissue cultured based research and for napiergrass improvement program, particularly in genetic transformation. Culturing shoot apices on MS medium containing 2 mgL–1 2,4-D and 0.5 mgL–1 BAP was more suitable than on MS medium containing 0.5 mgL–1 2,4-D. In the subculture with similar medium composition, proliferation occured poorly on dwarf napiergrass, whereas none happened on the three other genotypes. On the hormon-free medium, all genotypes germinated in different rates. This research pointed out that dwarf napiergrass gave the best response toward induction medium. However, its proliferation and regeneration needed to be optimized in order to obtain more obvious data. This genotype would be usefull for tissue culture based research and for napiergrass improvement program, particularly in genetic transformation.

Study on Callus Induction System of 4 Genotype of Napiergrass (Pennisetum purpureum )

The aim of this study was to produce callus induction potential of 4 napiergrass (Pennisetum purpureum) genotypes (Dwarf Late, Hybrid, Merkeron and Wrukwona). Callus was induced from shoot apices of shoot tillers on MS media containing 2,4-D and BAP. On the MS medium containing 2 mg L–1 2,4-D and 0.5 mg L–1 BAP all genotypes could produce embryogenic calli, with different rate of growth. The best genotype for producing embryogenic calli was dwarf napiergrass in 60 day culture. These genotypes would be usefull for tissue cultured based research and for napiergrass improvement program, particularly in genetic transformation. Culturing shoot apices on MS medium containing 2 mgL–1 2,4-D and 0.5 mgL–1 BAP was more suitable than on MS medium containing 0.5 mgL–1 2,4-D. In the subculture with similar medium composition, proliferation occured poorly on dwarf napiergrass, whereas none happened on the three other genotypes. On the hormon-free medium, all genotypes germinated in different rates. This research pointed out that dwarf napiergrass gave the best response toward induction medium. However, its proliferation and regeneration needed to be optimized in order to obtain more obvious data. This genotype would be usefull for tissue culture based research and for napiergrass improvement program, particularly in genetic transformation.

Effects of variations in culture media and hormonal treatments upon callus induction potential in endosperm explant of Barringtonia racemosa L.

ObjectiveTo induce callus from the medicinally valuable species, Barringtonia racemosa L. (B. racemosa) whereby the formation of callus is essential for micropropagation studies and in vitro plant secondary metabolites production. MethodsThe callus induction potential in B. racemosa was assessed from endosperm explant cultured on different culture media and plant hormonal treatments. Lloyd and McCown's woody plant medium and Murashige and Skoog's medium were used in the study as culture media. On the other hand, various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (1.0–2.0 mg/L) and kinetin (0.5–2.5 mg/L) had been incorporated in the culture media to exert the effects of auxin and cytokinin on callus induction. ResultsFrom the present study, it was found that the profuse [(1.681 ± 0.770) g fresh weight, (0.239 ± 0.239) g dry weight] and friable callus formation was optimally produced with desirable morphology and considerable percentage of callus induction (56.70%) in endosperm explants cultured on 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 1.5 mg/L kinetin in Murashige and Skoog's medium. ConclusionsA reliable protocol for inducing callus formation of profuse and friable morphology in endosperm explant of B. racemosa had therefore been successfully established.

Identification of Callus Induction Potential of Nine Different Genotypes of Indica Rice

Callus induction potential of nine economically important indica rice genotypes of Tamil Nadu and neighboring areas was examined on different callus induction medium. Nine genotypes include Improved White Ponni(I W Ponni), BPT-5204, TKM-9, CR1009, ADT-39, ADT-43, KKL(R) -1, CO(R)-48 and Savitri/ Ponmani. These rice genotypes were having distinct useful agronomical trait like resistance to various pest, tolerance to salinity and drought conditions. Most of the indica rice genotypes having good grain quality and nutritional properties are susceptible to pest and diseases causing loss in annual yield. The yield could be enhanced by genetic improvement by rice transformation to enable them to withstand biotic and abiotic stress. First step towards this approach is to produce a pleuripotent cell mass, callus induced from scutellum of mature rice seeds. MS basal medium with various 2, 4-Dichlorophenoxy acetic acid (2, 4-D) concentrations (2mg/L, 2.5mg/L, 3mg/L, 4mg/L and 5 mg/L) was used for these studies. Four indica rice genotypes I. W. Ponni, ADT- 43, BPT-5204 and TKM-9 found to be potent for callus induction from scutellum of mature seed at the concentration of 2mg/l to 5 mg /l 2,4-D. The varieties ADT-39, KKL (1) R, CO(R) 48, CR1009 and Savitri/Ponmani were recalcitrant and no embryogenic calli was obtained on either of the 2, 4-D concentration. 34 % of healthy calli obtained from I W Ponni 20 % of healthy callus induction obtained from BPT-5204 and 10 % of healthy calli from each of the ADT-43 and TKM-9. These indica rice genotypes could be used for various plant science researches.

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)

A reproducible and efficient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied significantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortified with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the maximum number of shoot buds (15.20 ± 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not beneficial for the valepotriate synthesis, as it helped to accumulate significantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries.

Micropropagation of Cold Resistant Blackberry Cultivar 'Gazda'

Blackberries are some of the most consumed berries in the world. Areas planted with blackberry in Europe and especially in Romania are limited mainly due to low frost resistance of traditional varieties. One of the most frost resist ant cultivar, suitable for cold Romanian winters, i s the variety 'Gazda'. Due to several drawbacks of the co nventional propagation of this cultivar when using root cuttings, the efficiency of a micropropagation protocol was tested, in order to obtain high quali ty planting material. For the in vitro culture establishment axillary dormant buds were i ncubated on MS medium supplemented with BAP. Proliferation of the regenerated shoots was tested on MS and DKW media using different concentrations of plant growt h regulators: BAP, 4-CPPU and Zeatin. BAP caused average proliferation rates but also generat ed large masses of basal callus. 4-CPPU was the solely plant hormone that did not generate severe c allus at any concentration and also provided the highest proliferation rate. It was also tested the callus induction potential from in vitro obtained leaves using 15 hormonal variants (5 hormone types in diff erent concentrations). For most variants it was achieved a substantial mass of callus, however no d irect regeneration was obtained. In vitro plantlets were successfully rooted and acclimatized directly ex vitro in Jiffy-7 pellets.

Establishment of an efficient in vitro culture and particle bombardment-mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop

Plants belonging to the genus Miscanthus are considered promising bioenergy crops. Here, we report the establishment of tissue culture system through particle bombardment-mediated transformation in Miscanthus sinensis Anderss. Callus was induced efficiently from mature seeds in a medium containing a combination of a relatively high level of 2,4-dichlorophenoxyacetic acid (5 mg L−1) and a relatively low level of 6-benzyladenine (BA) (0.01 mg L−1). Callus induction potential of 18 accessions of M. sinensis, which were collected from various sites in Japan, was compared. Significant correlation was detected between compact (embryogenic) callus induction frequency and average annual air temperature in collection sites. An accession from Tanegashima Island showed the highest production of compact callus. We found that a higher level of BA causes callus browning; the 2 mg L−1 BA is the optimal concentration for regeneration. Both compact and friable calli were suitable for particle bombardment transformation. Through selection under the presence of 50 mg L−1 hygromycin for 3 weeks and further selection under the presence of 150 mg L−1 for 1 month, hygromycin-resistant calli survived, of which 72.2% had been entirely transformed. Plants were regenerated from calli in the presence of hygromycin; transcripts of the hpt and gfp genes, which were cobombarded to the calli, were detected in the regenerated plants. This is the first report on the establishment of the in vitro culture of M. sinensis using mature seeds, the variation of callus formation among accessions collected from various sites in Japan, and particle bombardment-mediated transformation in the genus Miscanthus.

Identification of Callus Induction Potential of 15 Indonesian Rice Genotypes

The callus induction potential of 15 indica rice genotypes from Indonesia was examined in comparison with that of the japonica rice Nipponbare. Callus was induced from embryos of mature seeds and root segments on MS and CI media. There was genotype × medium × explant interaction for inducing white/cream/yellow callus with an organized structure (callus type I and II) and for callus browning, but not for callus induction ability and diameter of callus. Genotypes significantly differed in inducing high quality of calluses depending on medium and explant used. Four indica types, Fatmawati, Ciapus, BP-23 and BP-360-3, had callus induction-related traits similar to those of Nipponbare. These genotypes would be useful for tissue-culture based research and for crop improvement, particularly for genetic transformation. Culturing seed explant on MS was more suitable for callus induction than either root explant on MS or both explants on CI medium.