Plant regeneration from shoot tips and callus of papaya

Summary Two methods of in vitro culture were employed to regenerate papaya plants. One involved regeneration of plants from callus and the other, production of multiple plants from single shoot-tip explants. Callus was induced from stem sections of papaya seedlings in a medium containing 1 mg per 1 NAA and 0.1 mg per 1 kinetin. The callus regenerated shoots and/or embryoids when transferred to a medium of lower auxin, 0 to 0.05 mg per 1 IAA, and higher cytokinin, 1 to 2 mg per 1 kinetin Multiple shoots were produced when the excised shoot-tip explants were cultured in a medium supplemented with 0.05 mg per 1 IAA and either 5 mg per 1 kinetin or 0.5 to 1.0 mg per 1 benzyladenine. Root formation of the shoots or embryoids that derived from callus or shoot tips occurred in a medium containing 5 mg per 1 IAA and in a light intensity of 3000 to 4000 Ix. The rooted plants could be established in soil and under standard greenhouse conditions after they had been acclimated by initially growing them in moist vermiculite contained in polyethylene-covered pots. This research was supported by the National Science Council, Republic of China.     

Plant regeneration from callus culture of a Paphiopedilum hybrid

Totipotent calli of a Paphiopedilum hybrid (Paphiopedilum callosum ‘Oakhi’ × Paph. lawrenceanum ‘Tradition’) were induced from seed-derived protocorms on a 1/2 strength Murashige–Skoog medium plus 1–10 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–1 mg l⁻¹ 1-phenyl-3-(1.2.3-thiadiazol-5-yl)urea (TDZ). These calli grew well when subcultured on the same medium, but proliferated more on 1/2 MS medium plus 5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ TDZ. Calli developed further along a route of production of protocorm-like bodies and eventually formed plantlets that could be transplanted to pots and grew well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from seedling cotyledons, leaves and petioles of Glycine clandestina

A B5-based culture medium containing 4.4 μ M N⁶-benzyladenine (BA) and 0.025 μ M indole-3-butyric acid (IBA) induced callus from seedling cotyledons, leaves and petioles of Glycine clandestina Wendl. Only hard, green, nodular callus tissues were capable of producing shoot buds and of five accessions examined, only two (G1231 and G1145) were morphogenetically competent. Callus that did not regenerate could often be induced to produce shoot buds after subculture to fresh regeneration medium. Buds developed into shoots following transfer of callus to a medium containing 0.9 μ M BA and 0.025 μ M IBA. Shoots were rooted in hormone-free, half-strength B5 medium supplemented with 0.2% activated charcoal. The application of these results is discussed in relation to somatic hybridisation between the cultivated soybean and wild Glycine species.     

Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava

Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×10⁶ protoplasts/g fresh weight). Protoplasts plated at a density of 10⁵–10⁶/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N⁶-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP. Received: 30 August 1996 / Revision received: 9 June 1997 / Accepted: 1 October 1997     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis

Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 M -naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0 M thiamine HCl increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30 M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3 M 6-benzylaminopurine, 0.2 M and -naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner. The cultures retained the ability to regenerate complete plants for at least 12 months and 12–15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetio acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Shoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Plant regeneration through callus initiation from anthers and ovules of Scabiosa columbaria

Anthers and ovules of Scabiosa columbaria L. were cultured in vitro to determine whether gametophytic cells would proliferate and/or a protocol for plant regeneration could be developed. Several factors were tested, including explant type, donor plant, cold pre-treatment, and medium composition. Callus induction frequency varied among treatments, indicated by significant effects of explant type, medium composition, and their interactions. Histological analysis revealed numerous sites of callus induction, however, gametophytic cells did not proliferate. Stepwise removal of growth regulators and simultaneous lowering of sucrose from the nutrient medium, resulted in initiation of embryogenesis or shoot organogenesis, and allowed plant regeneration. Under the conditions tested, regeneration capacity was donor related, because only material of one donor responded. Regenerants were diploid (except one mixoploid individual), but showed various types of flower heads. They were probably of sporophytic origin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Type II callus production and plant regeneration in tropical maize genotypes

A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally, four media combinations of 15 or 30 μm dicamba with or without 88 μm AgNO₃ were used to study the effect of dicamba and AgNO₃ on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 μm and when AgNO₃ was added to the medium. A synergistic effect between 88 μm AgNO₃ and 30 μm dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best tissue culture performance and plant regeneration capacity. Received: 5 October 1996 / Revision received: 21 April 1997 / Accepted: 9 May 1997     

In vitro plant regeneration from callus derived from root explants of Lathyrus sativus

Protocols have been developed for the in vitro production of plants from callus derived from root explants of Lathyrus sativus cv. P-24. Callus and shoot regeneration were achieved only in MS medium supplemented with 10.7 M naphthaleneacetic acid and an increased concentration of kinetin (0.9 M for 14 days to 1.4 M for 18 days) during callusing. The shoots obtained rooted in 1/2 MS supplemented with 0.5 M indolebutyric acid. During the year plants have been regenerated several times. The requirement for growth regulators is very specific and narrow.Abbreviations IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - BA benzyladenine     

Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus

Summary Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 μM kinetin and 22.3 μM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 μMTDZ, A11 TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 μM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 μM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.     

In vitro plant regeneration of spinach from mature seed-derived callus

Summary A system for the regeneration of spinach (Spinacia oleracea L.) from mature dry seed explants has been established. The response of two commercial spinach cultivars, ‘Grandstand’ and ‘Baker’, was examined. Callus proliferation was most prominent on MS medium supplemented with 9.3 μM of 6-furfurylaminopurine (kinetin) and 3.39 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoot formation was observed within 8 wk after callus was transferred onto regeneration medium. Shoot regeneration was best from callus induced on 9.3 μM kinetin and 4.56 μM 2,4-D. The regeneration medium contained 9.3 μM kinetin, 0.045 μM 2,4-D, and 2.89 μM gibberellic acid (GA₃). Shoots were rooted on hormone-free medium, and plants grown in a greenhouse showed normal phenotype. This system is beneficial in rapid propagation of spinach plants, particularly when only a limited number of seeds are available.     

In vitro regeneration of plantlets from shoot callus of mature trees ofDalbergia latifolia

A successful procedure was established for in vitro mass multiplication of Indian rosewood (Dalbergia latifolia Roxb.). In vitro regeneration of plantlets was achieved from callus of shoot tips and shoot segments of over 50-year-old elite trees on Murashige & Skoog's medium containing naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). For rooting, regenerated shoots from the calli were excised and first treated with White's liquid medium or half-strength Murashige & Skoog's medium, supplemented with indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid for 48 h to 72 h. Following this treatment, plantlets were transferred to hormone-free half-strength MS medium. Rooted plantlets were then transferred to pots and grown in the greenhouse.Abbreviations BAP 6-benzylamino pruine - CH casein hydrolysate - CM coconut milk - 2, 4-D dichlorophenoxyacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PVP-10 polyvinyl pyrrolidone - YE yeast extract     

Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae)

Summary Embryogenic calluses were induced from 73% of Phalaenopsis shoot-tip explants excised from flower stalk buds by culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 μM α-naphthaleneacetic acid (NAA), 4.4 μM 6-benzylaminopurine and 29.2 mM sucrose. The sucrose concentration was increased to 58.4 mM 4 mo. after initiation of the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented with 5.4μM NAA and 58.4 mM sucrose. By simply reducing the sucrose concentration to 29.2 mM, the cells grew into plantlets through a developmental process similar to that of Phalaenopsis seedlings. The occurrence of somaclonal variants was less than 10% in six out of eight genotypes examined. These results suggest that the embryogenic callus and cell suspension culture could be utilized as the materials for micropropagation and breeding of Phalaenopsis orchids.     

Plant regeneration from callus and protoplasts in Medicago polymorpha

Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l^-1 2iP and 0.1 mg l^-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l^-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.Abbreviations NAA -naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - 2iP N⁶-²-isopentenyl-adenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - GFMS growth regulator free MS medium - Prol proline - Malt maltose     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

籼稻珍汕97B成熟种子胚愈伤组织诱导及其再生体系的建立

以珍汕97B成熟种子为材料,研究了影响其盾片诱导、愈伤组织诱导的主要因素,建立了籼稻珍汕97B愈伤组织的再生体系。结果表明,消毒剂NaClO的浓度及灭菌时间对盾片的诱导率影响极大,2,4—D的浓度不仅影响愈伤组织的数量也影响愈伤组织的质量。继代培养中添加1mg/L的ABA能显著改善愈伤组织的质量。     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

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).     

陆地棉中棉所24胚性愈伤组织的诱导及植株再生

以陆地棉“中棉所24”为材料进行了全固体体细胞培养,获得了愈伤组织和再生植株。愈伤组织诱导阶段采用0．01IAA 0．01KT 0．012,4-D的培养基效果好,继代时间多为30～50d;激素由高到低的继代可明显提高胚性愈伤分化率,IAA和KT含量均较低,IAA／KT比例为1：1～1：6,胚性愈伤最高分化率为50．22％。