Highest Callus Induction Research Articles

Screening Wheat Genotypes for High Callus Induction and Regeneration Capability from Immature Embryo Cultures

Selecting the explant genotypes is crucial step in in vitro culture and Agrobacterium-mediated transformation system due to its host range specificity. Immature embryos of five winter and three spring wheat (Triticum aestivum L) cultivars were evaluated for tissue culture response in three callus initiation media. MS medium containing 2,4-0 (2 mg ml-1) plus B5 vitamins (MSB5), MS medium containing 2,4-0 (1 mg ml-1) with no vitamins (MS1GC) or MS medium containing picloram (2.2 mg ml-1) and 2,4-0 (0.5 mg ml-1) plus MS vitamins (CM4C) were used for callus initiation. Percentage of callus induction varied widely with the genotype and initiation medium used, with values ranging from 5.7% to 100%. Embryogenic capacity of genotypes was evaluated by number of somatic embryos formed from cultured immature embryos. Bob White (spring) and NE92458 (winter) were equal and most embryogenic; Pronghorn and 2137 (both winter) were the poorest. CM4C medium was found to be the best medium for initiating embryogenic callus among three culture media tested. A standard regeneration procedure was used. The genotypes with the highest regeneration efficiencies were Bob White, Fielder and NE92458, (1.8, 1.4 and 1.6 plantslexplant, respectively).

Anther culture response in indica rice and variations in major agronomic characters among the androclones of a scented cultivar, Karnal local

-1 YE. However, coconut water (5 and 10% concentrations from Pink Dwarf variety) encouraged green plantlet regeneration only in Karnal local 95. High callus induction was observed with synthetic hormones in comparison to the control set. However, green plantlet regeneration was observed when callus induction medium (CIM) was supplemented with 2, 4-D and IBA in Taraori Basmati and Karnal local 95. Among the varieties Pusa Basmati and Karnal local 95 showed better callus inductions on N6 supplemented with 6% maltose. Maximum callus induction was observed in Pusa Basmati and Karnal local 95 when 6% sucrose or maltose were used as carbon source. Total plantlet regeneration was cent percent in Taraori Basmati when CIM was supplemented with maltose (6%). Maximum green plants were obtained when CIM was added with maltose (6%) for Karnal local 95. Plantlet regeneration in the present study was found to be very low. Androgenic plantlets derived from the scented indica rice, Karnal local 95, were evaluated under field condition to assess variability among segregating A2 generation. Overall mean values in respect of some major agronomic characters viz. plant height, panicle length, number of filled grains per panicle, spikelet sterility (%) and grain yield per plant were reduced except number of panicles per plant. High CV was observed for filled grains per panicle, grains yield per plant and number of panicles per plant suggesting the existence of high variation among the androclones for those characters. Positive selection deems to be improvising those characters. The frequency distribution for number of panicles per plant and panicle length varied largely among the androclones in both direction of the parental mean. Whereas, all the androclones were found to be shorter than the parent and low yielder too. This study elucidates that the genetic modulation through exploitation of androclonal variation is a feasible proposition in scented indica rice.

Effect of genotype on callus induction and plant regeneration from leaf explants of sugarcane ( Saccharum sp.)

Nine sugarcane genotypes (CP59-73, CP63-588, CP80-314, SP71-1081, F160, L62-96, CP70-321, CP57- 614 and Clone III) were evaluated for their callus induction capacity, embryogenic callus production and plant regeneration ability. Leaf cylinders were used as explants using Murashige and Skoog (MS) based medium supplemented with 3 mg l-1 2,-4 dichlorophenoxyacetic acid. Plant regeneration was accomplished on hormone free modified MS medium supplemented with casein hydrolyzate. The genotypes tested showed high callus induction percentage (69 to 95%) and high embryogenic callus percentage (60 to 100%). These genotypes also showed excellent regeneration capacities, with regeneration percentages ranged between 88 and 100%. Significant differences were observed between genotypes for callus induction capacity, embryogenic response and plant regeneration ability indicating that these criteria are genotype dependent. Plant regeneration ability is highly correlated with embryogenic callus production. The in vitro regenerated plants were successfully rooted and well acclimatised in growth cabinet conditions.

Factors Affecting the Anther Culture of Wheat (Triticum Aestivum L.)

ABSTRACTThe limitations of conventional cereal breeding programmes require simple and rapid new breeding technologies such as doubled-haploid production. One of the various techniques used to obtain in vitro haploids of wheat (Triticum aestivum L.) is anther culture. In this study the effects of cultivar, pre-treatment of spikes, callus induction media and carbon sources added into induction medium on anther culture response of Turkish wheats were investigated. Anthers from 5 commercial cultivars (Basri Bey, Golia, Marmara 86, Gönen and Pehlivan) grown in the field were excised and cultured on 2 nutrient media (N6 and Potato). Three cold pre-treatments (1, 2 and 3 weeks at +4 °C) and 2 carbon sources (sucrose and maltose) were tested. Hexaploid wheat cultivars showed low response to current anther culture technique. The 2-week-pre-treatment of spikes resulted in the highest callus induction whereas no callus was obtained from 3-week-pre-treatment. No significant differences was observed between the cultivars. Callus formation within cultivars varied between 3.0 and 7.0 per 1000 anthers plated. Basri Bey produced the highest callus formation among the cultivars used. Sub cultures of calluses on a regeneration media caused green zones. Callus induction medium containing sucrose caused a significant increase in callus production.

Callus induction and plant regeneration via anther culture in mutant tomato (Lycopersicon esculentum Mill.) lines with anther abnormalities

Anthers from two tomato mutants, solanifolia (sf) and trifoliate (tf), characterized by anther abnormalities, 4 male sterile tomato mutants (ms 1526, ms 30, ms 32, and ms 33), and their isogenic fertile lines were cultured in vitro, using three modifications of MS media. The frequency of callus induction in the mutant anthers was significantly higher than in the wild type ones in all genotypes, a result independent of the medium. Pseudofruits surrounded by well-developed calli appeared from the anthers of San Marzano ms 15, Van’s Early ms 26, San Marzano ms 30, and VF 11 Pearson ms 33. The higher callus induction in the mutant anthers correlated with higher frequency of organogenesis. The fact that, in addition to the ms-mutants, solanifolia and trifoliate also affected callus frequency, suggested that in tomato, process/es leading to, or resulting from anther abnormalities might be favorable for enhancing anther callus induction and subsequent regeneration.

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.

Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)

SUMMARY Axenic cultures of Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand (Gracilariales, Rhodophyta) were established in ASP12-NTA solid medium (0.4% agar and 1.0% sucrose) supplemented with plant growth regulators to evaluate the effects on apical callus formation and plant regeneration. Indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA) were added individually or in combinations (IAA : BA) over a range of concentrations from 0.5 to 5 mg L−1. Growth of apical and intercalary segments was stimulated by high concentrations of 2,4-D (5 mg L−1) and a high IAA to BA ratio (IAA : BA = 5:1 mg L−1) respectively. Apical calluses were originated from divisions of apical and cortical cells located at apical regions of thallus segments and lateral branches. Low concentration of IAA (0.5 mg L−1) or a high IAA to BA ratio (IAA : BA = 5:1 mg L−1) were the optimal treatments for inducing apical callus formation in apical segments, while high concentration of IAA (5 mg L−1) stimulated the highest callus induction rate in intercalary segments. Conversely, equal parts IAA and BA (IAA : BA = 1:1 mg L−1) and low concentration of 2,4-D (0.5 mg L−1) stimulated growth of apical calluses from apical and intercalary segments, respectively. Two processes of regeneration were observed: direct regeneration (upright axis originated from cells of proximal region of intercalary segments) and indirect regeneration (adventitious plantlet originated from cells of apical calluses). Direct regeneration was promoted significantly by treatment with a low IAA to BA ratio (IAA : BA= 1:5 mg L−1), and treatments with IAA (0.5 mgL−1) or 2,4-D (0.5 or 5 mg L−1) significantly stimulated the elongation of upright axis. Plant growth regulators are essential to inducing indirect regeneration, and a high concentration of IAA (5 mg L−1) and BA (5 mg L−1) were the optimal treatments for inducing the regeneration of plantlets from apical calluses in apical and intercalary segments, respectively. Regenerating plantlets grew into plants morphologically similar to those formed from germinating spores, and became fertile after 6 weeks. The results suggest that auxins and cytokinins are involved in developmental regulatory processes in G. tenuifrons. The regeneration process from calluses in species of Gracilariales was observed for the first time in the present study. The culture system described for G. tenuifrons could be useful for micropropagation and for biotechnological applications in agarophytic algae.

Combining Ability of Callus Induction and Plant Regeneration in Sorghum Anther Culture

Fifteen F1 hybrids derived from a six-parent diallel mating set (without reciprocal) of sorghum genotypes were used to investigate the combining ability for callus induction and plant regeneration. Anthers at a young stage were collected from the parents and hybrids, which were grown in a greenhouse, and cultured on a modified Murashige and Skoog medium containing 1.0 mg L−1 IAA, 2.5 mg L−1 2,4-D and 2.0 mg L−1 kinetin. Calli of 2 to 5 mm in diameter were transferred to a regeneration medium containing 2.5 mg L−1 kinetin and 3.0 mg L−1 IAA for plant regeneration. The general combining ability (GCA) accounted for a larger proportion than the specific combining ability (SCA) for callus induction (179.83, mean square for GCA vs. 31.75, mean square for SCA) and for plant regeneration (1225.73, for GCA vs. 410.37, for SCA). Some parents and hybrids showed high callus induction ability accompanied with high green-plant regeneration ability. Thus, improvement of the rate of green-plant regeneration in anther culture through crossing and selection may be possible.

Plant regeneration from leaf sheath cultures of some rabi sorghum cultivars

High frequency callus induction was obtained from young leaf sheath segments of four rabi type sorghum cultivars viz., M 35-1, SPV-86, A-1 and GRS-1 on Murashige and Skoog (MS) medium supplemented with 2 mgl − 1 of 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was unorganised in the beginning, but became highly organised and nodular within 2–3 months. On MS medium without 2,4-D, plantlets occurred at high frequency through somatic embryogenesis. Addition of 0.5 mgl − 1 of 6-benzyl aminopurine improved the formation of plants Genotypic influence with regard to the frequency of callus Induction, type of callus, pigment production and regeneration of plantlets were observed.

Embryogenesis and plant regeneration in anther culture of sunflower (Helianthus annuus L.)

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N6-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants.

Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%.

Effects of light regimes on anther culture response in bread wheat

This experiment was initiated to further test the effects of light regimes during callus induction and plant regeneration on anther culture response of spring wheat (Triticum aestivum L.). Spring wheat cultivars 'Edwall' and 'WA 7176' with high callus induction from anther culture but low green plant production were used. Different gro-lux light and dark regimes during callus induction, and gro-lux light and fluorescent light regimes during plant regeneration were used. Callus induction decreased significantly at relatively high light intensity (315 µmol m−2 s−1) applied at any period of culture when compared to continuous dark. Light regimes used continuously and from the 15th to the last day of callus induction also had a significant negative effect on plant regeneration compared to continuous dark and light application in the first half of callus induction. During plant regeneration, '15 day dark + 7 day gro-lux light' significantly increased plant regeneration compared to both 'gro-lux' and 'fluorescent light' regimes. Light regimes during both callus induction and plant regeneration and their interaction effects were found to be highly significant on green plant proportion and green plant yield. 'Continuous light' application during callus induction increased green plant proportion more than other applications in contrast to its negative effect on plant regeneration. During plant regeneration, '15 day dark + 7 day gro-lux light' had the higher green plant proportion compared to only 'fluorescent light' and only 'gro-lux light'. The highest green plant yields were obtained from '15 day dark + 7 day gro-lux light' during plant regeneration in combination with either 'continuous dark' or 'continuous light' regimes during callus induction.

Regeneration of anther-derived plants of Avena sterilis

Anther culture response of accessions of several hexaploid Avena species was studied with respect to requirement for auxin. The highest callus induction frequency was achieved withA. sativa L. cv. Stout (17.3%). The three most responsive genotypes, two of A. sativa and one of A. nuda L., had two-fold higher anther culture responses on growth regulator -free medium than on a medium containing 2,4-d. The A. sterilis L. accession CAV 2648 was the only genotype which consistently produced white, embryogenic structures (on solid MS+ 10% sucrose) and did so irrespective of the presence of 2,4-d. When transferred onto medium with lower sucrose concentration and an auxin transport inhibitor (TIBA), three green (two diploid [2t n=6t x=42] and one haploid [1t n=3t x=21]) and two albino plantlets were regenerated. The diploid regenerants set seed in the greenhouse. This first report of plants recovered from anther culture in the wild oat A. sterilis may provide an avenue to understand better and possibly overcome problems associated with androgenesis in cultivated oat.

Effects of Growth Regulators and Genotypes on Callus and Embryoid Induction from Maize Anther Culture *

Fifty genotypes and ten growth-regulator combinations were used in two experiments (I and II) to investigate genotype and hormonal effects on production of callus and embryoids via anther culture in maize (Zea mays L.). Hormonal effects across all genotypes were not significant in either experiment. However, highest callus-induction frequencies in both experiments occurred on YP basal medium plus 2,4-D at 2.0 mg 1−1, and kinetin at 1.5 mg 1−1 indicating that this combination was more effective than others. Genotypic differences in callus or embryoid induction across all media were significant. The most responsive genotypes in experiment I were single-cross hybrids Yuanwu × 592 and K727 × K305, which produced 18.3 and 6.7 % calli, respectively, with their appropriate media. The most responsive entry in experiment II was CIMMYT Pool 29, which produced 15.0 % calli on appropriate medium and an average of 10.0 % calli across 10 media. Twenty-three plantlets was regenerated from this study. Most of them developed embryogenically.

Chromosome doubling and aneuploidy in anther-derived plants from two winter wheat lines

Anther-derived, doubled haploid populations were obtained from two hard red winter wheats, 'Centurk' and NB88. Spontaneous doubling frequency, efficiency of colchicine treatment, and vernalization requirement were evaluated within each population. In cytological evaluation among the regenerates, haploids, diploids, haploid aneuploids, and diploid aneuploids were observed. Most regenerates were either haploid or diploid. The frequency of anthers producing at least one haploid, diploid, or aneuploid was the same for both genotypes. The regenerates from the same anther had the same ploidy level 83 % of the time, suggesting the callus was usually derived from one microspore. Over 60% of the anthers produced at least one diploid plant. The high frequency of spontaneous doubling suggests that chromosome doubling by colchicine treatment could be eliminated. Ninety-eight percent of the colchicine-treated 'Centurk' plants produced seed, while only 43% of the NB88 colchicine-treated plants produced seed. Anther culture did not replace the vernalization requirement. Vernalization was required for uniform flowering. Results indicate that it is feasible to use 'Centurk', NB88, and other genotypes with high callus induction, plantlet regeneration, and colchicine response to efficiently produce doubled haploids.Key words: wheat, Triticum aestivum L., anther culture, doubled haploid, chromosome doubling, aneuploidy, vernalization.

Induction of Haploid Callus from Anthers of Four Species of Arabidopsis

Summary Cultured anthers of four different Arabidopsis species are shown to give rise to haploid callus. Anthers of several geographic races of A. thaliana all gave high callus induction frequencies (43–87 %). The frequency of diploids among such callus was low (0–5 %) for all species. Aneuploidy was observed in primary anther callus of all four species as well as in diploid callus originating from germinating seedlings. The degree of aneuploidy did not appear to be related to hormonal content of the cultural medium but was very closely and negatively correlated with the chromosome number of the source tissue (r = -0.9).

Studies on in vitro response to callus induction and gene transfer technique of five high yielding indica rice varieties.

Due to growing population, there is an increasing demand of rice production but the productivity of rice is lessened day by day. To overcome this problem various biotechnological tools can be used for developing various rice varieties. However, the lack of a simple and efficient protocol for callus induction, embryogenic callus formation and quick plant regeneration in this cereal crop. In this study embryogenic calli from mature seeds of five indica rice varieties viz. Binadhan-5, Binadhan-6, BRRI dhan-48, BRRI dhan-58 and IR-64were observed that is done in four different concentration of growth regulators (2,4-D). The highest callus induction were observed in media containing 2 mg/L 2,4-D. Among those varieties BRRI dhan-48 showed highest rate of callus induction. In this experiment Agrobacterium mediated gene transfer is also tried although plants were found nut need to be tested by molecular methods and tested the desired characters for selecting those plantlets as transgenic plant. This study will be useful for selecting suitable callus induction medium for callus induction in future.

&lt;i&gt;In vitro&lt;/i&gt; regeneration performance of &lt;i&gt;Corchorus olitorius&lt;/i&gt;

The experiment was conducted during May to December 2008 in the Biotechnology Laboratory of Bangladesh Agricultural University, Mymensingh to observe the callus induction, regeneration potentiality and to establish a suitable in vitro plantlet regeneration protocol of Corchorus olitorius. MS medium supplemented with different phytohormone concentrations and combinations were used to observe the callus induction, shoot regeneration and root formation ability of the cotyledon with attached petiole derived explant of three genotypes viz. O-9897, O-72 and OM-1. The highest callus induction (92.85%) was observed in O-9897 followed by O-72 (82.14%) in the MS media supplemented with 2.5 mg/L BAP + 0.5 mg/L IAA. Genotype O-9897 in MS media supplemented with 2.5 mg/L BAP + 0.5 mg/L IAA produced the highest percentage of shoot regenerants (83.33%) followed by O-72 (75.00%) in the media supplemented with 2.5 mg/L BAP + 0.5 mg/L IAA. The root formation from regenerants was the best on halfstrength of MS media supplemented with 0.6 mg/L IBA in genotype O-9897 (45.00%). The in vitro regenerated plantlets from the genotypes O-9897 could be established in the field. Therefore, the genotypes O-9897 of C. olitorius in MS media supplemented with 2.5 mg/L BAP + 0.5 mg/L IAA could be used for callus induction and shoot regeneration. Keywords: Regeneration; Phytohormone; Corchorus olitorius DOI: 10.3329/jbau.v8i1.6390J. Bangladesh Agril. Univ. 8(1): 1-6, 2010