High-frequency somatic embryo production from unfertilized ovules of grapes

Somatic embryos were produced in large numbers from cultured, unfertilized ovules of Vitis vinifera, cultivars ‘Cabernet Sauvignon’ and ‘Grenache’, and of the hybrid grape ‘Gloryvine’ (V. vinifera × V. rupestris). Callus of nucellar origin was produced by culturing the ovule explants initially in Nitsch medium containing 5 μM 2,4-dichlorophenoxy acetic acid (2,4-D) or 5 μM β-naphthoxyacetic acid (NOA) plus 1 μM benzyladenine (BA), and then in a medium supplemented with NOA (10 μM) plus BA (1 μM). Embryos were produced when callus was transferred to basal medium containing no auxin or cytokinin. Secondary and tertiary embryos were formed by budding of primary somatic embryos. Prolonged culture of ovular callus in NOA (10 μM) plus BA (1 μM) gave rise to cell aggregates which developed into free-floating roots when transferred to basal medium.     

Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)

Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 μM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 μM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 μM 2,4-D and 1 μM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 μM 2,4-D and 1 μM BA and subsequently transferred to media containing 2–4 μM 2,4-D and 0 or 0.5 μM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 μM 2,4-D and 1 μM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 μM 2,4-D and 0.5 μM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks.     

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

Summary

Secondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.     

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ Kn, which contained only half concentration of NH₄NO₃. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm²) was transferred to solid media (MS + 0.2 mg l⁻¹ 2,4-D + 5.0 mg l⁻¹ BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.     

Plantlet regeneration and stability from callus cultures of Freesia × hybrida Bailey cultivar ‘Royal’

True-to-type plantlets of Freesia × hybrida Bailey cultivar ‘Royal’ were generated from callus after 27 months of sub-culture in liquid medium. Callus was initiated from young flower pedicels cultured on semi-solid Linsmaier—Skoog (LS) medium supplemented with 5 mg/l of 2,4-D and 0.5 mg/l kinetin, and transferred to the same medium in liquid form without hormones and thereafter sub-cultured every 7–10 days. Liquid cultures with 2.4–4.3 g of callus per 25 ml medium produced largest increases in callus fresh weight. Callus generated the most shoots when cultured on LS medium supplemented with 5 mg/l of kinetin and incubated in the light, while fewer plantlets were produced when no growth regulator or GA₃ or PBA were used. Callus cultures incubated in continuous darkness did not form shoots.     

Callus induction and culture of Rosa

Leaf and stem explants of Rosa manetti Hort. and R. hybrida L. ‘Tropicana’ were placed on Murashige and Skoog (MS), Schenk and Hildebrandt (SH) and Gamborg and Wetter (1-B5C) media, containing growth regulators, casein hydrolysate (CH) or coconut milk (CM), to determine the optimum conditions for callus initiation and maintenance. The explants were cultured either in dark or in light (2 Klux 16 h/day) at 26±2°C. Friable, fast growing callus was evident after 3 weeks for both rose species on MS + 2.0 mg/l 2,4-D, 0.25 mg/l kinetin (K) and 2.0 g/l CH as well as on SH + 0.5 mg/l 2,4-D, 2.0 mg/l p-CPA and 0.1 mg/l K. Callus initiation occurred sooner on SH than on MS. However, during sub-culture of callus arising on SH, rapid oxidation often occurred, resulting in severe browning of the callus, which made it unsatisfactory for further experimental use. Callus initiation occurred faster in dark than in light, but deteriorated when continuously sub-cultured in the dark regardless of media. Although ‘Tropicana’ initiated callus sooner than R. manetti, very fast growing callus of the latter was obtained when leaf callus was transferred to MS + 2.0 mg/l NAA and 0.5 mg/l BA. Unsuccessful attempts to regenerate callus and induce adventitious shoots on the 2 explants are discussed.     

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l⁻¹ KNO₃, 0.29 g l⁻¹ NH₄NO₃, 3 mg l⁻¹ thiamine HCl, 0.5 mg l⁻¹ pyridoxine HCl, and 5 mg l⁻¹ nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

De novo differentiation of shoot buds from leaf-callus of Dianthus caryophyllus L. and control of hyperhydricity

A method is described for producing de novo shoots from leaf derived callus of carnation (Dianthus caryophyllus L.). Plants were regenerated in four steps, viz. callus induction, shoot regeneration, removal of hyperhydricity from regenerated shoots and root development. Callus induction medium contained 2,4-D and BAP. Shoot buds were formed when the callus was further subcultured on 2,4-D- and BAP-containing medium, or MS medium without any growth regulators. The shoots so formed were hyperhydric, bushy in appearance with reduced stem length and watery leaves. The normal conformation of shoots was restored by culturing the hyperhydric shoots onto medium supplemented with GA₃ and bactopeptone. The recovered shoots were rooted on MS medium added with NAA (1 mg/l) or IBA (2 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions after initial acclimation.     

In vitro culture of cotyledon tissue of Castanea sativa Mill

Establishment of callus tissue culture from cotyledon fragments of Castanea sativa Mill. is described. The explants were grown on various combinations of auxins (IAA, IBA, NAA, 2,4-D) and cytokinins (K, BA) to induce callus and regeneration of organs. The best growth occurred on basal medium with 2,4-D (1 and 10 mg/l) plus both K or BA (0.5 mg/l). Root regeneration was achieved with both IBA and NAA (10 mg/l) supplemented with K or BA.     

番茄愈伤组织诱导的初步研究

以4个番茄品种为试材,从中筛选出发芽率最高的品种为"法国粉玫瑰",并以此品种为材料,针对不同外植体种类、激素组合等因素对愈伤组织生成的影响进行了初步的研究。结果表明:子叶愈伤生成的最佳培养基为MS+2.0mg/L BA+1.0mg/L 2,4-D;茎段愈伤生成的最佳培养基为MS+2.0mg/L BA+0.5mg/L 2,4-D。     

芹菜胚性愈伤的诱导及高频植株再生体系的建立

通过对芹菜离体培养过程中激素配比、基因型、不定芽产生等条件的研究,建立了芹菜高频植株再生体系。研究表明,芹菜苗期的下胚轴最适于诱导愈伤组织,其愈伤组织诱导和继代培养的最佳培养基是MS+2,4-D1.0mg·L-1+KT1.0mg·L-1和MS+2,4-D0.5mg·L-1+BA0.2mg·L-1+CH500mg·L-1+4%甘露醇;基因型对愈伤组织和胚状体的诱导影响很大;60%以上的愈伤组织在继代培养基上可以从非胚性状态向半胚性和胚性状态转化。愈伤组织在1/2MS+1.5%蔗糖+CH500mg·L-1+KT0.25mg·L-1上获得不定芽或小试管苗;在无激素的MS或1/2MS固体培养基上继续培养不定芽或小试管苗,即可生根或长大,获得芹菜的完整再生植株。     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

Morphogenetic responses of Salpiglossis sinuata L. leaf explants and callus

Leaf explants of Salpiglossis sinuata L. were cultured on MS medium containing the auxins 2,4-D, IAA, NAA, or the cytokinins 6-BAP, 2ip, or K singly or NAA + 6-BAP combinations (0.01 to 10.0 mg/l) to determine their morphogenetic responses. IAA and NAA induced callus or roots at all levels except IAA at 0.01 mg/l. Callus varying in friability was obtained on MS + 2,4-D at various concentrations. Friable, uniform dividing callus was obtained on UM medium. Optimum adventitious shoot formation occurred on leaf sections and from callus cultures placed on MS + 2ip. Rooting-difficulties were encountered, but 75% rooting efficiency was obtained on shoots cultured in MS + 0.001 mg/l 2,4-D. Rooted plants were readily transferred to the greenhouse, and flowered.     

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou (Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l⁻¹ 6-benzylaminopurine (BA) and 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l⁻¹ 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.     

Somatic embryogenesis in Cymbopogon pendulus and evaluation of clonal fidelity of regenerants using ISSR marker

An efficient plant propagation system through somatic embryogenesis was established in Cymbopogon pendulus, an aromatic grass followed by analysis of genetic status of regenerants using ISSR markers. Optimum embryogenic callus induction was observed on MS basal medium supplemented with 13.57 μM 2,4-dicholorophenoxyacetic acid (2,4-D) with 8.88 μM N⁶-benzyladenine (BA). Subsequent culturing of embryogenic calli on MS medium containing 4.52 μM 2,4-D and 8.88–13.32 μM BA gave maximum number of somatic embryos. Addition of coconut water (CW) promoted induction, growth and differentiation of callus and somatic embryogenesis. Further development of embryos into plantlets was achieved on MS medium supplemented with lower concentration of biotin and calcium pantothenate (CaP) along with BA (4.44–13.32 μM) and kinetin (2.32–4.65 μM). The root meristems were established on half strength MS medium containing 2% sucrose and 2.46–9.84 μM Indole3-butyric acid (IBA) and successfully established in soil with 77.8% survival rate in field condition. Thirteen randomly selected regenerated clones were screened using six ISSR primers. Nine clones produced similar monomorphic amplification profiles while remaining clones showed minor variation with absence of certain parental bands and appearance of unique band. Majority of the regenerants maintained genetic fidelity with the generation of few variants as evidenced from similarity matrix estimates using Nei Li's coefficient of similarity data.     

Somatic embryogenesis and plantlet regeneration from encapsulated embryos of Selinum tenuifolium

Summary

This paper reports, for the first time, somatic embryogenesis and synthetic seed production in Selinum tenuifolium Wall. Mature leaf explants inoculated in Murishige and Skoog (MS) medium supplemented with 3 µM 2,4-dichlorophenoxyacetic acid (2,4-D), containing 3% (w/v) sucrose and 0.7% (w/v) agar, induced 67% callus. Maximum production of globular structures, their differentiation into embryos and germination, occurred with a combination of 2 µM benzyladenine (BA) and 2 µM indole-3-butyric acid (IBA). To protect somatic embryos and produce synthetic seeds, gel capsules were standardised using a combination of sodium alginate and calcium nitrate concentrations. Gel capsules were most effective when formed with a combination of 3% (w/v) sodium alginate and 100 mM calcium nitrate for 30 min. The addition of MS medium to alginate capsules with 3% (w/v) sodium alginate, 3% (w/v) sucrose, 2 µM BA and 2 µM IBA significantly improved their germination rate to 77.8%, as well as their resulting shoot length (5.6 cm) and root length (7.2 cm), compared to controls (57.8%). Most plantlets (66%) survived under nursery condition. Storage at 4°C for different periods (10 d or 20 d) significantly (P < 0.05) reduced the percentage survival and germination of somatic embryos and artificial seeds compared to controls or 5 d storage.     

苹果砧木M26离体叶片愈伤组织发生及分类研究

对苹果砧木Ｍ２６ 离体叶片愈伤组织的发生研究表明,取继代后３０ ～３５ ｄ 的试管苗顶部１～４ 叶位的叶片,沿与主脉垂直方向横切后,以叶背面朝上方式置于愈伤组织诱导培养基上,经观察在叶片近柄端切口处愈伤组织发生较早,产生量较多。最适的愈伤组织诱导培养基为：ＭＳ＋２ ,４ － Ｄ０ ．２ ～２ ．０ ｍｇ·ｌ－ １ ＋ＩＡＡ０ ．２ ～２．０ ｍｇ·ｌ－ １ ＋ ＢＡ０ ．５ ～１．０ ｍｇ·ｌ－ １ ＋ 蔗糖３ ％ ＋ 琼脂０．６％ 。将诱导产生的愈伤组织按外部形态、细胞特点及生理生化指标等分为三类,其中表面干燥致密、细胞内含物丰富、淀粉、蛋白质及干物质含量均较高的Ⅲ类愈伤组织的芽再生频率最高,可达８３ ．３３ ％ ,是能利用的愈伤组织类型。     

正交设计在黄金鱼花组织培养中的应用

 用正交设计法研究了4 种激素对黄金鱼花叶片诱导愈伤组织的影响。结果显示,诱导黄金鱼花愈伤组织产生的最佳激素配比为: MS + 2 ,4-D 1 mg/L + 6-BA 1 mg/L + NAA 1 mg/L , 有2 ,4-D 的培养基中愈伤组织诱导率明显高于没有2 ,4-D 的培养基, IBA 对愈伤组织的诱导有抑制作用。