Frequency Of Embryogenic Callus Formation Research Articles

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Embryogenic Callus Induction of Aquilaira Malaccensis Lam . a nd Aquilaria Subintegra Ding Hou

Aquilaria malaccensis Lam. and Aquilaria subintegra Ding Hou belong to the family of Thymelaeaceae which is commonly known as gaharu or agarwood. It is a commercially important tree and identified as a potential aromatic plant. The overwhelming responses in the lodging sector reduce gaharu species in the forest. Mass propagation through plant tissue culture technology will substitute this problem. The present study was conducted to investigate the embryogenic callus induction between these two species. The most optimum sterilization method for both species was sodium hypochlorite 5.0% which gave the highest percentage of aseptic culture (95%) with the absence of tissue browning. The leaves of both species were cultured on Murashige and Skoog, (1962) (MS) media supplemented with combination of various concentrations of 6-benzylaminopurine (BAP) (0.5, 1.0, 2.0 and 2.5 mg/L) and 2,4-dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5 and 2.0 mg/L) and kept under dark condition. The explants produced embryogenic, white and compact callus at the end cut of the explants after two weeks of culture in all treatments. The highest frequency of embryogenic callus formation was observed in explants cultured on 2.0 mg/L BAP and 0.5 mg/L 2,4-D for both species. From the present study, the optimum sterilization technique and embryogenic callus induction for A. malaccensis Lam. and A. subintegra were established.

An efficient plant regeneration through embryogenic callus formation and direct somatic embryogenesis via immature embryo culture in Ipomoea purpurea and I. tricolor

We established an efficient plant regeneration system to form embryogenic calli in common morning glory (Ipomoea purpurea) and blue morning glory (I. tricolor). Immature embryos of both morning glories cultured on media containing 1 mg l−1 4-fluorophenoxyacetic acid (4FA) and 6% sucrose formed many embryogenic calli. The frequency of embryogenic callus formation was highest in I. purpurea strain Q74 (42.5%) and in I. tricolor cultivar ‘Flying Saucers’ (36.7%). Embryogenic callus formation differed with the genotypes in both morning glories. Numerous somatic embryos were formed from the embryogenic calli when the calli were transferred onto plant growth regulator-free medium.

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).

Somatic embryogenesis and plant regeneration from callus cultures of Cleome rosea Vahl

This paper describes a protocol for the efficient vegetative propagation of Cleome rosea by somatic embryogenesis. Leaf and stem explants from nursery-grown seedlings of C. rosea were cultivated on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA), a -naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) or 2,4-dichlorophenoxyacetic acid (2,4-D). Nodular calli were produced from both explant types in the presence of 4.5 and 9.0 µM 2,4-D. Embryo development and maturation were achieved when calli from stem explants were transferred to media containing a ten-fold reduction of 2,4-D concentration initially used (0.45 and 0.90 µM). Leaf-derived calli did not form embryos with the same treatments. The highest frequency of embryogenic callus formation (85%) and number of embryo per callus (13.45 ± 2.8) were achieved during the first subculture on medium supplemented with 0.90 µM 2,4-D. Embryo conversion into plantlets was achieved following transfer to growth regulator-free MS medium solidified with 2 g.L-1 phytagel. An acclimatization rate of 53% was found three months after transfer to ex vitro conditions and the recovered plants presented a normal phenotypic aspect.

어리연꽃 (Nymphoides indica (L.) O. Kuntze) 화경 배양으로부터 체세포배발생을 통한 식물체 재생

Plant regeneration system from floral stem of Mymphoides indica via somatic embryogenesis was established. After four weeks of culture onto 1/2MS medium containing 2,4-D, pale-yellow globular structures and calluses were formed on the cut surface of floral stem explants. Upon transfer to 1/2MS basal medium, pale-yellow globular structures were developed into somatic embryos and normal plantlets. These results indicated that pale-yellow globular structures and calluses from floral stem were globular embryos and embryogenic calluses, respectively. The frequency of embryogenic callus formation from floral stem was reached to nearly 100% when floral stem was cultured onto 1/2Ms medium supplemented with low concentration of 2,4-D (0.1 to 0.3 mg/L). However, the higher concentration of 2,4-D resulted in decrease of the frequency of embryogenic callus formation. In this study, low concentration of 2,4-D had a stimulative role in embryogenic callus formation, whereas BA showed inhibitory role in callus formation. In comparison to floral stem, leaf explants showed low frequency of embryogenic callus formation. The highest frequency of embryogenic callus formation from leaf explants was 9.5% when leaf explants were cultured onto 1/2MS medium supplemented with 0.3 mg/L of 2,4-D. The plant regeneration system of Nymphoides indica established in this study, might be applied to mass proliferation, conservation of genetic resources and genetic transformation for molecular breeding.

Seasonal effect on tissue culture response and plant regeneration frequency from non-bombarded and bombarded immature scutella of barley (Hordeum vulgare) harvested from controlled environment

Scutella of immature embryos from two barley cultivars were used for cell culture and transformation. Explants were supplied by continuous growth of donor plants in a 2-week schedule under defined conditions at first plants were grown for 6–7 weeks in a growth chamber, followed by 10–13 weeks in a greenhouse with stringent control of temperature and light round the year. Strong seasonal variation in plant regeneration frequency was observed for both genotypes in non-bombarded (control) as well as bombarded and subsequent selected explants. Scutella from immature embryos of cv. Salome showed increased frequencies of plant regeneration from January to March, reaching highest values in March/April and followed by a continuous and strong decrease from May to December. This tendency was observed in all 3 years studied, although absolute numbers of plant regeneration varied between the years. The same seasonal effect was evident for plant regeneration from immature scutella of cv. Golden Promise. Frequency of embryogenic callus formation was also found to be influenced by season but this effect was not so pronounced as for plant regeneration.

Development of highly tissue culture responsive lines of Lolium temulentum by anther culture

Most of the important forage and turf grasses are outbreeders, require vernalization to flower, and in some cases are polyploid. With the development of gene sequencing information in grasses, there is an urgent need for a model system to perform large-scale functional analysis of candidate genes. We propose to use Lolium temulentum L. (Darnel ryegrass) as a model system for genetic manipulation studies in forage and turf grasses because L. temulentum has the following advantages: self-fertile, short life cycle (11 weeks), diploid, easy to grow, and closely related with other major grass species. In order to improve tissue culture response of L. temulentum, two relatively responsive lines were crossed and putative hybrid seeds obtained. Analysis of the F 1 plants by SSR markers confirmed that the F 1 plants were true hybrids. Anthers were dissected from F 2 plants of the cross, 3.0% of the cultured anthers responded with the formation of calluses. Green haploid plants were recovered from 48.9% of the anther-derived calluses. Seeds were harvested from doubled haploid plants and mature embryos were used as explants for comparing tissue culture responses with other lines. Besides anther culture, F 2 seeds of the cross were subjected to a cycle of selection for callus formation and plant regeneration, and F 4 seeds were obtained from the regenerated plants. Comparison of tissue culture response of different lines revealed that the doubled haploid plants had a much higher frequency of embryogenic callus formation than that of the parental lines and the F 4 seeds. This is the first report on the generation of green haploid and doubled haploid plants in L. temulentum. The technique was successfully used for the rapid production of homozygous diploid plants that are highly tissue culture responsive. The anther culture-derived new L. temulentum lines could be valuable material for functional test of genes in grasses.

Somatic Embryogenesis and Morphogenetic Potential of Spring Barley (Hordeum vulgare L.) in the System of Technological Genetic Transformation

We studied somatic embryogenesis and morphogenetic potential in young embryos of 17 spring barley cultivars. A considerable effect of the genotype as well as of certain biologically active compounds in the growth medium on the frequency of embryogenic callus formation and regenerative capacity was observed. The Murashige and Skoog medium complemented with myoinositol and casein hydrolysate is suitable for embryogenic callus induction. The regeneration medium should be complemented by hormones, 2.5 mg/l auxin 2,4-D and 0.1–0.5 mg/l cytokinin 6-BAP, to increase the frequency of somatic embryo maturation. The presence of cefotaxime and high level of copper are desirable to increase the efficiency of callogenesis, regeneration, and callus quality. Three cultivars with economically valuable characters and high morphogenetic potential were recommended for development of efficient technology for barley transformation.

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng.

Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA3, the embryos were transferred to 1/2-MS medium without GA3. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield.

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens

Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba x Abies cephalonica, and Abies alba x Abies numidica)

Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.

Somatic Embryogenesis and Plant Regeneration in Cucumber

The embryogenic capacity of seven cucumber (Cucumis sativus L.) cultivars was examined by tissue culture of cotyledon, young first-leaf, and internode explants. Somatic embryogenesis frequencies differed significantly among the tested cultivars, and `Fushinarimidori' produced the highest number of embryos from either cotyledons or young first leaves. Cotyledon- and first-leaf-derived calluses produced more embryos than calluses from internodes. Somatic embryos were induced from `Aonaga F1' internodes. With relatively high sucrose levels (6% and 9%) in the initiation medium, the frequency of embryogenic callus formation from `Fushinarimidori' cotyledon explants was >90%. The highest yield of somatic embryos occurred in cultures initiated with high sucrose levels (9% or 12%), although 12% sucrose inhibited callus formation and growth. Somatic embryos germinated in a basal liquid medium supplemented with 0.5% activated charcoal, and they developed into well-shaped, healthy plantlets on semisolid medium with 1% sucrose.

Callus Induction and Plant Regeneration in Elite U.S. Rice Lines

Four California and one Texas rice (Oryza sativa L.) cultivars were examined for potential use in tissue culture studies. Under varying 2,4‐dichlorophenoxyacetic acid (2,4‐D) and kinetin levels embryogenic calli derived from mature seed ranged in weight from 0 to 502 mg after 4 wk. Addition of kinetin allowed a 2‐ to 10‐fold increase in embryogenic callus size for ‘M‐202’, ‘Calmochi‐101’, and ‘Lemont’. The frequency of embryogenic callus formation across different 2,4‐D and kinetin levels was highest with ‘S‐201’ (0.93 to 1.00) and lowest with Lemont (0.00 to 0.44). Mean green shoot number varied from 0 to 14.2 (100 mg)−1 embryogenic callus 8 wk after initiation of regeneration. Similar numbers of shoots were derived from compact, friable, and leafy callus types. S‐201 and Calmochi‐101 developed green shoots from virtually all regions of the embryogenic callus while the others formed shoots from the underside of regenerating tissue. Frequency of albino shoots ranged from 0.00 to 0.045 for the five cultivars. Albino shoots from California cultivars were detected only in regeneration medium containing 0.1 mg L−1 6‐benzylaminopurine (BAP). L‐202 and S‐201 were identified as the best cultivars for cell culture studies. Data from these cultivars suggest that the California rice germplasm should respond equally as well to in vitro manipulation as other non‐U.S, strains.

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.