High Frequency Plant Regeneration System Research Articles

Establishment of a high-frequency plant regeneration system from rhizome-derived embryogenic cell-suspension cultures of Curcuma longa L.

We developed an efficient plant regeneration technique for turmeric (Curcuma longa L.) via somatic embryogenesis derived from embryogenic cell suspensions cultured from petioles. We initiated in vitro callus cultures from the plantlets of turmeric cultured on Murashige and Skoog (MS) medium. Then leaf, petiole, stem, and root explants of in vitro-grown plantlets were cultured on Schenk and Hildebrandt (SH). The stem and root explants formed white nodular calluses at a frequency of 86.7% and 85.8%, respectively, when cultured on SH medium; however, we used the calluses derived from the root explants for initiating a cell-suspension culture. In this study, the actively growing cell suspension culture comprised small, highly cytoplasmic cell aggregates and large vacuolated cells. On being transferred to a basal SH medium (without growth regulators), a few of these calluses differentiated into somatic embryos, which indicated that the initial calluses were embryogenic cells. We stimulated additional shoot differentiation by adding 0.5 mg L−1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) and various concentrations of N6-benzyladenine (BA) to the embryogenic cell suspension. The highest frequency at which shoot primordia developed from embryogenic cell clusters was 70% (when cultured in the dark on the supplemented MS medium), with more than 83.3% of regenerated shoot primordia producing roots. We successfully transplanted rooted plantlets into a soil mixture of sterile vermiculite and potting soil (1:1) and grew them to maturity in a growth chamber, achieving a survival rate of > 95%. To our knowledge, this is the first report of complete plant regeneration of turmeric from embryogenic cell-suspension cultures. The embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and gene manipulation for improving turmeric quality.

High-frequency Plant Regeneration from Cultured Flower Bud Receptacles of Allium hookeri L.

Allium hookeri L. (Alliaceae family) is an important ethnomedicinal plant native to the Himalayan region of Asia. The aim of this research was to establish a high-frequency plant regeneration system for in vitro propagation of A. hookeri. Among the tissue types examined, receptacle explants derived from immature flower buds showed the highest regeneration rate of shoots (<TEX>$93.33{\pm}4.63%$</TEX>), roots (<TEX>$76.67{\pm}7.85%$</TEX>), and calli (<TEX>$80.00{\pm}7.43%$</TEX>) when cultured on Gamborg B5 (B5) medium containing <TEX>$10{\mu}M$</TEX> 6-benzylaminopurine (BA) + <TEX>$1{\mu}M$</TEX> naphthalene acetic acid (NAA), <TEX>$0.5{\mu}M$</TEX> BA + <TEX>$5{\mu}M$</TEX> NAA, and <TEX>$1{\mu}M$</TEX> BA + <TEX>$10{\mu}M$</TEX> NAA, respectively. Shoot multiplication was superior when cultured in liquid rather than on solid medium and relatively high concentrations of BA, ranging from 5 to <TEX>$10{\mu}M$</TEX>. Efficient bulblet formation following root induction from shoot clumps was achieved with culture in liquid B5 medium containing 7% (w/v) sucrose. Regenerated bulblets were successfully acclimatized to ex vitro conditions with a greater than 95% survival rate. By this method, a maximum of 62 plantlets per receptacle could be propagated within 9 weeks of initial culture. The in vitro propagation system established in this study will promote A. hookeri biotechnology, including large-scale production of healthy and aseptic clones, preserving parental genotypes with desirable traits, and genetic manipulation to enhance medicinal value.

테프그라스 조직배양을 통한 캘러스 형성 및 식물체 재분화 효율

Teff grass is a warm season C4 annual grass that is used for dry hay, silage and haylage. We have developed a high-frequency plant regeneration system for teff grass via callus culture using mature seeds. It was revealed that mature seeds cultured on MS medium supplemented with 2 mg/l 2,4-D, 0.5 g/L proline, 0.5 g/L casamino acid and 3 g/L Gelrite under light condition produced the highest percentage of callus formation (91.9%). Addition of cytokinins (BA) at 0.0~0.5 mg/L to media containing 2 mg/l 2,4-D enhanced callus growth. The most suitable medium for plant regeneration from dehydrated calli was MS agar medium supplemented with 0.1 mg/l NAA, 1 mg/l BA, 0.5 g/L proline, 0.5 g/L casamino acid 3 g/L Gelrite which induced the highest percentage of calli forming shoots (47.0%). The shoots were rooted at the highest rate (100%) when transferred onto 1/2 MS medium and acclimated in greenhouse conditions.

Efficient plant regeneration from cotyledonary node explants of Cucumis melo L.

An efficient regeneration system was developed using cotyledonary node of two inbred line of melon, Cucumis melo 'CM-15' and 'CM-23'. The induction of shoots was achieved on Murashige and Skoog (MS) solid medium supplemented with different combinations of 6-benzylaminopurine (BA) and indole- 3-acetic acid (IAA). The best shoot induction was observed when the explants were cultured on MS medium supplemented with BA (1.5 mg/L) and IAA (0.1 mg/L), and on MS medium supplemented with BA (1.5 mg/L) and IAA (0.5 mg/L) for 'CM-15' and 'CM-23', and inbred line CM-15 had the best regeneration rate. The shoot clumps were transferred to MS with BA (0.05 mg/L), resulting to the differentiation of most of the shoots initially into well developed shoots. Regenerated shoots were rooted on half-strength MS medium without plant growth regulators (PGRs). The rooted plants were established in soil with 100% success rate. This high frequency plant regeneration system provides improved technology to assist in genetic transformation of melon. Key words : Cucumis Melo L., cotyledonary node, shoots regeneration, plant regeneration.

High frequency plant regeneration from the mature seeds of Garcinia indica

A high frequency plant regeneration system was developed for the production of high yielding elite clones of Garcinia indica via direct organogenesis. A maximum number of 86.2 shoot buds per explant were induced from the mature seed segments cultured on Woody plant medium (WPM) supplemented with 2.0 mg dm-3 N6-benzyladenine and 1 mg dm-3 indole-3-acetic acid. Rooting was achieved on half-strength WPM medium supplemented with 3 mg dm-3 indole-3-butyric acid. Rooted plantlets were acclimatized and transferred to greenhouse for further growth. The highest survival rate of 95 % was recorded using a mixture of garden soil and sand. Histological studies clearly revealed multiple shoot formation from mature seed segments.

High frequency plant regeneration system for Nymphoides coreana via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 mg l−1 of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 mg l−1 with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 mg l−1 of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.

Establishment of high frequency plant regeneration system from leaf explants of Pinellia koreana via bulblets formation

Pinellia koreana K-H Tae & J-H Kim is a recently discovered Korea endemic medicinal plant species whose natural habitat is rapidly destroyed by industrial development. Described in this paper are culture conditions for high frequency plant regeneration via bulblet formation from leaf explant cultures of P. koreana. Leaf explants formed white nodular structures and off-white calluses at a frequency of 91.2% when cultured on MS medium supplemented with 2 mg/L BA and 0.5 mg/L NAA. However, the frequency of white nodular structures and off-white calluses formation was slightly decreased with an increasing concentration of NAA up to 4 mg/L, where the frequency reached 31.7%. Most petiole explants did not form white nodular structures and off-white calluses except the combination treatment of 2 mg/L BA and 2 mg/L NAA. Upon transfer onto MS basal medium, over 90% of nodular structures gave rise to numerous bulblets and developed into plantlets. Plantlets regenerated from bulblets were transplanted to potting soil and grown to maturity at a survival rate of over 95% in a growth chamber. Therefore, the in vitro plant regeneration system of P. koreana obtained in this study will be useful for mass propagation and long-term preservation of genetic resources of P. koreana. *Corresponding author Tel 042-860-4646 Fax 042-860-4677 E-mail: kimsw@kribb.re.kr Introduction Pinellia koreana K-H Tae & J-H Kim is a new plant species discovered at Chrisan in Korea (Tae and Kim, 2005). This plant differs from other plants belonging to the same genus Pinellia by having 3-foliolate leaves with bilobed lateral leaflets at maturity. The genus Pinellia is herbaceous medicinal plants belonging to the Araceae. The chemical composition of Pinellia is not well established as there are only a few reports on the occurrence of small amounts of alkaloids ephedrine in the tuber. Also a glycoprotein fraction was reported to have notable antiemetic effects (Kurata et al. 1998). And root contains toxins (presence of calcium oxylate), that are destroyed by drying or cooking. Pinellia is mostly used for indigestion, nausea, vomiting, gastritis, and ulcer (Lee and Cho 1987; Bown 1995). The genus of Pinellia consists of a few species in eastern Asia (Kitamura et al. 1980). Pinellia is propagated by tubers and bulbils. However, the yield and quality of tubers are gradually reduced over cultivation because of viral diseases. Therefore, introduction of a mass propagation system using in vitro culture technology such as development of virus-free strains and genetic manipulation of tubers is necessary to enhance the productivity of tubers. Despite its medicinal importance, few scientific studies of P. koreana have been reported. Only several in vitro culture studies have reported on the related species P. ternata including protoplast culture and plant regeneration (He et al. 1996), formation of micro-tubers and plantlet regeneration by thidiazuron (Yoo and Lim 1997), and clonal propagation by bulbils (Chen et al. 1989; Tsay et al. 1989). Also natural habitats of P. koreana decreased gradually because of industrialization. Tissue culture techniques may provide an alternative mean for its mass multiplication and ex-situ conservation of genetic resources. This study describes culture conditions for high frequency plant regeneration via bulblet formation from leaf explants cultures of P. koreana. 식물생명공학회지 제36권 제2호 J Plant Biotechnol Vol. 36, No. 2, 193-196 (2009) 194 ‧ Journal of Plant Biotechnology Figure 1. Plant regeneration from leaf explants of P. koreana via bulblets formation. A: Leaf explants; B: Off-white callus formation; C: White nodular structure formation; D: Bulblets formation from leaf-derived callus; E: Leaf and root development from bulblets; F: Regenerated plantlets transferred to soil; G: Morphological variation in leaf shape of regenerated plantlets; H: Morphological variation of plants after 2 year cultivation; I: Flowering of mature plants. Scale bars represents 1 mm (A, B, C, D and E), 2 cm (F, H) and 5 cm (G, I), respectively Materials and methods

High frequency plant regeneration from zygotic-embryo-derived embryogenic cell suspension cultures of watershield (Brasenia schreberi)

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on half-strength MS medium supplemented with 0.3 mg l−1 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to 3 mg l−1, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus were established using half-strength MS medium supplemented with 0.3 mg l−1 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.

오차드그래스 성숙종자로부터 캘러스 유도 및 고효율 식물체 재분화

In an effort to optimize tissue culture conditions for genetic transformation of orchardgrass (Dactylis glomerata L.), an efficient and high-frequency plant regeneration system from seed-derived calli was established. Embryogenic calli induced on MS medium containing 3mg/L 2,4-D and 0.1mg/L BA had significantly improved regeneration ability. Plant regeneration rate was 92％ when embryogenic calli were cultured on N6 medium supplemented with 1mg/L 2,4-D and 3mg/L BA. Among three kinds of medium, MS and N6 medium were optimal for embryogenic callus induction and plant regeneration, respectively. Ho difference in callus induction frequency was observed among four cultivars of orchardgrass, however, "Roughrider" cultivar showed higher regenerability with the frequency of 61％. Addition of maltose to the regeneration medium as a carbon source dramatically increased regeneration frequency up to 69％. A short tissue culture period and high-frequency regeneration system would be beneficial for molecular breeding of orchardgrass through genetic transformation.

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l-1 kinetin.

Plant regeneration from seed-derived embryogenic callus and cell suspension cultures of bahiagrass ( Paspalum notatum)

We have established a high-frequency plant regeneration system via somatic embryogenesis from seed-derived callus and cell suspension cultures in 6 genotypes of bahiagrass ( Paspalum notatum). Embryogenic callus was initiated from mature seeds on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3.0% sucrose and 0.3% Gellan Gum in the dark. Culture response was found to be correlated with genotype. ‘Pensacola’ had the best response in embryogenic callus formation, and 74% of the calli regenerated plants. Cell suspension cultures were established from embryogenic callus of ‘Pensacola’ in modified N 6 medium containing 1.0 mg/l 2,4-D. The suspension was composed of compact cell clusters. When smaller clusters (approx. 2.0–3.3 mm in diameter) were transferred to a solid MS medium with 3.0% sucrose but without hormones, plant regeneration was initiated at high frequency (28.6%). Morphological evidence is provided that regeneration of suspension cells occurred via embryogenesis. Regenerated plants were established to soil after culture in water at room temperature for 14 days for acclimatization.

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 μM dicamba, addition of 2.25 to 9 μM BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 μM dicamba and 2.25 μM BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.

Hormonal effects on &lt;i&gt;in vitro&lt;/i&gt; regeneration of &lt;i&gt;ricinus communis&lt;/i&gt; L. Cultivar shabje

Context: Castor is the best candidate plant to the venture of green energy sector. In Bangladesh castor is widely grown and we can reap the benefit from castor by the application of different biotechnological approaches. Genetic engineering appears as one of the necessary tools for the improvement of cultivars of castor. Therefore, an efficient regeneration protocol for castor needs to be established, which is rapid, reproducible and applicable to a broad range of genotypes. Objectives: To establish a high frequency plant regeneration system from the seedling explants of Ricinus communis L. cultivar shabje using tissue culture technique. Materials and Methods: The proliferating axillary shoots were established on modified MS medium supplemented with different concentrations of BA, Kn and 2ip using explants from the in vitro grown seedlings of R. communis. Results: Explants from the seedlings raised on cytokinin enriched medium showed early sprouting and development of axillary shoots than those taken from the seedlings grown on cytokinin omitted medium. Shoot proliferation efficiency of cotyledonary node explant was noted two times greater than that of shoot tip explant. MMS1 medium fortified with 1.5-2.5 mg/l BA showed the best performance and yield 5-10 axillary shoots within 6 weeks of culture. Microcuttings prepared from in vitro proliferated shoots were rooted on MMS2 medium. The best rooting (92.7%) obtained on ½MS fortified with 0.2 mg/l of IBA accompanied by 0.6 mg/l of AgNO3. Conclusion: Best media formulation for shoot proliferation in Ricinus cultivar was MS medium fortified with 2.0 mg/l BA and its recalcitrant property could be minimized in some extent by using of AgNO3 and charcoal in case of root induction. Keywords: In vitro regeneration; seedling explants; Ricinus communis DOI: http://dx.doi.org/10.3329/jbs.v18i0.8787 JBS 2010; 18(0): 121-127