Highest Callus Induction Frequency Research Articles

Withania somnifera (Ashwagandha): Regeneration through Meristem Culture

High frequency of callus induction was achieved from shoot tip explants (80%) of Withania somnifera (Hindi name-Ashwagandha) as compared to leaf explants (70%), on MS medium supplemented with IAA (56μM) and Kinetin (56μM). When hormone-free MS medium was fortified with vitamins [thiamine. HCI (3μM), nicotinic acid (40.7μM) and pyridoxine. HCI (24.3μM)], it was found suitable for complete plant regeneration through meristem culture.

Rapid and High-Frequency In Vitro Plant Regeneration from Leaflet and Petiole Explants of Groundnut ( Arachis hypogaea L.)

An efficient protocol for regeneration of groundnut plantlets from immature and mature leaflet and petiole explants excised from axenic seedlings has been developed. The highest frequency of callus induction obtained from leaflet explants was on Murashige and Skoog (MS) medium containing 2.0 mg/L of α-naphthalene acetic acid (NAA) and 0.5 mg/L of kinetin combination. MS medium containing different auxins in combination with 6-benzylaminopurine (BAP) induced shoot buds. A BAP (2.0 mg/L) and NAA (0.5mg/L) combination resulted in the highest frequency of shoot-bud regeneration. Subsequent shoot multiplication was obtained on MS medium supplemented with either BAP or kinetin (5.0 mg/L) in combination with NAA (1.0 mg/L). Immature leaflet explants were found to be more responsive to shoot induction than mature leaflet explants. Direct shoot-bud regeneration was also observed from petiole explants on the same regeneration medium used for leaflet callus. Regenerated shoots were rooted on MS medium containing indole-3-butyric acid (2.0 mg/L) and kinetin (0.5 mg/L). Plantlets obtained were successfully established in the field, where they grew to maturity and set viable seeds.

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes.

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture.

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.

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.

Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator combination in japonica rice (Oryza sativa)

The influence of maltose and growth regulators on microspore culture response was investigated in japonica rice. High frequency of callus induction of isolated microspores was obtained with liquid medium containing MS salts, 100 mg l−1 myo-inositol, 1 mg l−1 thiamine-HCl, 500 mg l−1 glutamine, 60 g l−1 maltose, and several growth regulators. The effect of maltose on promoting callus formation was associated with keeping a high proportion of swollen microspores after 5 day preculture and increasing the microspore division rate on the 3rd day after culture initiation. No significant effect of maltose in place of sucrose on plantlet regeneration was seen in regeneration medium. Among the growth regulators tested, the combination of auxin 2,4-dichlorophenoxyacetic acid (1 mg l−1), naphthaleneacetic acid (1 mg l−1), and cytokinin (6-benzyl-aminopurine 1 mg l−1) in the medium proved to be much better for callus formation than in the other media, and the percentage of callusing microspores of that medium reached 0.86%. Indole-3-acetic acid (0.5 mg l−1) and kinetin (2 mg l−1) in regeneration medium were beneficial for green plantlet differentiation. The results also showed that the frequencies of microspores initial division, callus formation and green plant regeneration varied among genotypes no matter what kind of growth regulator and sugar were used. Xiushui 117 was the best variety for callusing followed by 02428 & Taipei 309. Taipei 309 showed a good ability for green plantlet regeneration.

Anther callus induction and green plant regeneration at high frequencies from an interspecific rice hybrid Oryza sativa Linn. x O. rufipogon Griff

Callus induction and green plant regeneration at high frequencies from an interspefic hybrid, Oryza sativa L. x O. rufipogon Griff. has been achieved by simply coordinating the growth regulators in the induction medium. The study was conducted with two different basal media (Potato-2 and N6) and seven different combinations of growth regulators 2,4-D, NAA and kinetin. Synergistic effects of the two auxins in enhanced anther response to callus induction and subsequent green plant regeneration were observed in both media. The highest frequency of callus induction was obtained on Potato-2 medium supplemented with 1 mg/12,4-D, 2 mg/l NAA and 1 mg/l kinetin. The same combination of growth regulators which yielded higher frequencies of callus induction also induced higher mean number of calli per anther. Although the calli formed on N6 medium showed high regenerability, there was a concomitant increase in the number of albinos among the regenerants. The auxins in the induction media had considerable influence on the regeneration capacity of the calli. The regeneration frequencies were higher from calli formed in the presence of both auxins in the induction media. The levels of growth regulator combinations seem to influence the green plant regeneration especially for calli induced on Potato-2 medium. Among the pollen grain derived plants the majority were either haploids or double haploids and very few were chromosomal variants.

Wheat anther culture: effect of genotype and environmental conditions

Twenty-two cultivars and lines of winter and spring wheat (Triticum aestivum L.) were studied, most for the first time, for their anther culture response. The response was genotype dependent. Plants grown in the field gave higher callus induction frequency than those grown in the greenhouse and the controlled environment chamber. Donor plants grown in a season of low drought stress as compared to a season of severe drought stress resulted in a higher frequency of callus induction. Spherical microcalli were observed in two wheat genotypes in some of only those anthers that were placed with only one loculus in contact with the medium. Wheat lines that were more responsive to anther culture were identified.

Induction and growth of callus derived from rachilla explants of young inflorescences of coconut palm

When suitable stages of rachilla tissues derived from coconut palm were selected as an explant source, a higher frequency of callus induction was observed in medium containing activated carbon and 20–30 ppm 2,4-dichlorophenoxyacetic acid (2,4-D).

Germplasm and Physiologic Effects on Induction of High‐Frequency Hormone Autonomous Callus and Subsequent Shoot Regeneration in Sugarbeet1

The availability of adapted germplasm capable of regenerating shoots from callus may affect the speed with which tissue culture‐mediated genetic improvements can be brought to commercial usage. To assess the shoot regeneration capability in Beta vulgaris L., 17 germplasm sources including sugarbeet, table beet, fodder beet and leaf beet were screened for callus‐forming ability and shoot regeneration. Petiole and blade explants from in vitro shoot cultures grown with 6‐benzyladenine (BA) as the only growth regulator were challenged to initiate callus on a Murashige‐Skoog basal medium (MS) with 0.25 or 1.0 mg L−1 BA. Bud regeneration was scored on the callus induction medium or after transfer of primary callus to MS + 1.0 mg L−1 BA + 0.3 mg L−1 3‐indoleacetic acid. Some individual genotypes from each of the 17 germplasm sources produced callus, and bud regeneration from callus was seen in 14 of the 17 germplasm sources, indicating that this ability is widespread in the species. Direct adventitious budding on petioles was also found within all germplasm sources. Use of BA as the sole growth regulator induced buds on callus within the range 0.1 to 3.0 mg L−1. Kinetin and tzeatin alone induced buds, but at the higher concentrations of 30 and 10 mg L−1, respectively. Environmental conditions and leaf part affected callus induction frequency on explants. Leaf blade segments were considerably more responsive than petiole segments. Explants cultured at 31°C initiated callus at least five times more frequently than those at 23°C. Highest frequency of callus induction with one genotype occurred in the dark, and no callusing occurred in 200 μmol m−2 s−1 light from fluorescent lamps. An uncomplicated procedure for shoot regeneration evolved through these experiments, permitting lone explants to initiate callus and subsequently regenerate shoots on MS + 1.0 mg L−1 BA without transfer. This system is applicable to a wtde range of B. vulgaris L. germplasm.

Induction, Culture and Differentiation of Callus from Immature Rachises, Seeds and Embryos of Triticum

Summary Callus was induced from immature rachises, embryos and seeds of Triticum monococcum, T. longissimum, T. speltoides, T. tauschii, T. turgidum, T. timopheevii, T. aestivum and several aneuploid lines of the latter species. No difference could be detected between the various Triticum species in respect to their potency to form callus. On the other hand, the developmental stage of the plants appears to strongly affect the success of callus induction. The cultures were maintained in the dark on various basal media containing 1 to 2 mg/l of 2,4-D for callus induction and proliferation. The frequency of callus production from rachis explants (R-callus) was about 70 0/0. Higher and more consistent results were obtained when embryos were used. In these explants, regardless of plant source, callus (E-callus) induction reached 95 0/0. However, only 15 Ofo of explanted seeds initiated callus (S-callus). During the early subcultures, the R-calli showed high root differentiation in media supplemented with 1 to 2 mg/l of 2,4-D, while E- and S-calli exhibited only occasionaly root regeneration under these conditions. The high frequency of root differentiation in R-calli could be induced consistently, even after one year of culture - when the calli were subcultured in medium devoid of auxin. The ability to differentiate shoots was examined in R-calli, S-calli and E-calli during the first to the fourth subculture periods. When 2,4-D was replaced by IAA and zeatin and the calli were transferred to light, shoot differentiation was induced during all these subculture periods, but the frequency of induction tended to decrease with time. The frequency of shoot differentiation in E- and S-calli was higher than in R-calli. Moreover, in many of the former calli a massive and abrupt regeneration of several shoots was observed. When calli containing regenerated shoots were transferred to media devoid of hormones, roots could be easily induced. Up to now, 32 out of 62 so regenerated plantlets attained maturity. The high potency of shoot and root differentiation of the E-calli combined with the high frequency of primary callus induction from immature embryos implicates the E-callus as a powerful tool for future use of Triticum cell culture in crop improvement and breeding of wheat.