High-frequency conversion of abnormal peanut somatic embryos

Peanuts (Arachis hypogaea L.) are widely cultivated as a rich source of protein and oil. Although protocols for the regeneration of peanut via somatic embryogenesis and organogenesis have been developed, most of them have resulted in low frequencies of plant recovery. In this report, we describe a protocol for plantlet formation at high frequency from somatic embryos. Morphologically abnormal somatic embryos germinated and produced roots only in medium devoid of growth regulators. Shoots emerged from the undeveloped plumule of these rooted embryos in medium containing both 6-benzyladenine (BA) and kinetin (KN), or in medium with thidiazuron (TDZ) alone. In Murashige and Skoog basal medium supplemented with 8.9 μm BA and 14 μm KN, 86% of the embryos developed shoots. Substitution of BA and KN with 22.7 μm TDZ increased plant recovery from 86% to 92%. Plants grown on TDZ had multiple shoots. Eighty-four percent of these plants survived in sandy soil and were grown to maturity. Received: 12 February 1996 / Revision received: 11 July 1996 / Accepted 30 April 1997     

Inhibition of shoot induction by 5-azacytidine and 5-aza-2′-deoxycytidine in Petunia involves DNA hypomethylation

Shoot bud regeneration from Petunia leaf disks was inhibited when they were cultured with the demethylating agents, 5-azacytidine (AzaC) and 5-aza-2′-deoxycytidine (AzadC), in shoot induction (SI) medium. Explants induced shoot primordia if they were transferred after 1 week from the medium containing the drugs to medium without drugs. The fresh weight of leaf disks cultured on SI medium for 2 weeks in the presence of the drugs was 60–80% lower when compared to control shoot-forming cultures. Internode length was reduced when shoots were transferred to phytohormone-free Murashige and Skoog medium containing the drugs. However, no other morphological abnormalities were seen in these shoots, even at 20 μm AzaC or 5 μm AzadC. Coupled restriction enzyme digestion (with HpaII and MspI) and random amplification of genomic DNA was performed to detect the level of methylation of CCGG sites in the DNA of the explants exposed to AzaC and AzadC. Over 15 amplified bands were detectable in the control. Five of these bands were absent in the amplified products when digested DNA from the drug-treated explants was used as the template, showing that hypomethylation of DNA had occurred. This suggests that inhibition of shoot bud formation in the presence of the drugs AzaC and AzadC may be due to the altered methylation status. Received: 7 January 1997 / Revision received: 17 February 1997 / Accepted: 1 March 1997     

Micropropagation of gum karaya (Sterculia urens) by adventitious shoot formation and somatic embryogenesis

Nodal explants from selected trees of gum karaya (Sterculia urens Roxb.) in the adult growth phase cultured on Murashige and Skoog (MS) medium supplemented with 6.62 μm N⁶-benzylaminopurine (BAP) produced an average of six adventitious shoots in 30 days. Shoots were rooted in vitro on 1/4-strength MS medium containing 9.82 μm indole-3-butyric acid. Nodulated callus was produced from hypocotyl explants cultured on MS medium supplemented with 4.52 μm 2,4-dichlorophenoxyacetic acid and 8.90 μm BAP. Somatic embryos developed when the nodulated callus was transferred to MS medium containing 0.45 μm thidiazuron (TDZ). TDZ treatment for 2 days gave the optimum response. Over 30% of the somatic embryos developed into plantlets when transferred to 1/4-strength MS basal medium without any growth regulators. Plantlets produced from adventitious shoots and somatic embryos were acclimatized to ex vitro conditions and established in the field. Received: 26 November 1997 / Revision received: 14 April 1998 / Accepted: 11 May 1998     

Somatic embryogenesis from mature leaves of rose (Rosa sp.)

Several plant growth regulators (0.3–53.3 μm 6-benzyladenine, 2,4-dichlorophenoxyacetic acid, gibberellic acid, 3-indoleacetic acid, p-chlorophenoxyacetic acid, kinetin and α-naphthylacetic acid), alone or in combination, and culture conditions were tested for their capacity to induce somatic embryogenesis from mature leaf and stem explants of rose (Rosa sp.) of four commercial rose cultivars (Baccara, Mercedes, Ronto and Soraya). Somatic embryos were only induced from mature leaf explants derived from Soraya on Murashige and Skoog (MS) medium supplemented with 53.5 μm p-chlorophenoxyacetic acid and 4.6 μm kinetin, although satisfactory callus induction rates were obtained from all cultivars. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto a MS medium supplemented with 5.2 μm 6-benzyladenine and 5.7 μm 3-indoleacetic acid. Germination of mature embryos took place after subculturing them onto medium of the same composition. Plantlets regenerated from embryos and bearing three to four leaves were transferred to a greenhouse. Received: 4 February 1997 / Revision received: 28 August 1997 / Accepted: 1 October 1997     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

In vitro plant regeneration in Melia azedarach L.

Nodal explants of 3- 6-week-old seedlings cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) (17.75 μm) produced multiple shoots. Shoots were isolated and induced to root on 1/2-strength MS medium supplemented with indole-3-butyric acid (4.92 μm). In-vitro-rooted shoots resumed growth after a short period of acclimatization and resulted in plantlets which were successfully established in soil. In vitro flowering was observed in some of the nodal explants in the above medium, and also in cotyledonary leaves and internodal explants on MS medium supplemented with a combination of indole-3-acetic acid (IAA) (0.06 μm)+BA (4.44 μm) and IAA (0.06 μm)+kinetin (4.65 μm). Received: 25 October 1997 / Revision received: 1 May 1998 / Accepted: 15 May 1998     

In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds

In vitro shoot regeneration from sterile leaf and petiole explants and from in-situ-collected inflorescence buds of Petasites hybridus was achieved by a simple two-step protocol. Murashige and Skoog (MS) nutrient medium was supplemented with 17.6 μm benzyladenine (BA)+0.54 μm naphthaleneacetic acid (NAA) to induce shoots. After 5 weeks of culture, 40% of the petiole and 27% of the leaf explants produced shoots compared to 76% of the inflorescence buds. Single shoots were excised and subcultured on MS medium supplemented with various cytokinins (N⁶-(Δ²-isopentenyl)adenine, BA, kinetin and thidiazuron). A concentration of 8.8 μm kinetin+0.54 μm NAA performed best in terms of shoot multiplication rate, average shoot length and spontaneous root induction. Received: 20 August 1997 / Revision received: 29 December 1997 / Accepted: 5 February 1998     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

Micropropagation of Spathoglottis plicata

A rapid and reliable micropropagation method was established for Spathoglottis plicata. Nodal and leaf explants dissected from 8-month-old pot-grown seedlings were cultured on charcoal-amended Murashige and Skoog medium supplemented with 16 combinations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) at concentrations of 0.54–10.74 μm. Regeneration of protocorm-like bodies (PLBs) and subsequent plantlet development were observed from 98.5% of the nodal explants. Only 6.5% of leaf explants and occasionally some root segments (dissected from regenerated plantlets) were able to produce PLBs and then plantlets. The optimum plant growth regulator (PGR) combination for maximal PLB regeneration was 5.37 μm NAA and 0.44 μm BA. The best combination of PGR for plantlet development was 2.69–10.74 μm NAA and 8.88 μm BA. The NAA to BA ratios for maximal PLB induction and plantlet development were 12.2 and 0.3–1.2, respectively. Regenerated PLBs and plantlets, when cut into pieces of less than 1 mm and subcultured onto the above media, regenerated new PLBs and plantlets in another 3 months. Received: 20 February 1997 / Revision received: 27 May 1997 / Accepted: 16 June 1997     

Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture

A protocol for micropropagation of Lilium longiflorum using pseudo-bulblets from in vitro shoot-tip-derived stem nodes was developed. Shoot tips from stems of dormant bulbs were cultured on one-half Murashige and Skoog (MS) medium. Stems from plantlets derived from the shoot tips were cut into nodal segments, which were then cultured on one-half MS medium supplemented with 1 μm 6-benzyladenine (BA). Pseudo-bulblets formed on each node after 1 month. An average of 32 pseudo-bulblets were formed on all nodes of the plantlet. Pseudo-bulblets gave rise to multiple shoots when cultured on one-half MS medium supplemented with 2.3 μm BA. Shoots were rooted on one-half MS medium containing 1.1 μm α-naphthaleneacetic acid. A continuous system of propagation by multiplication of pseudo-bulblets with no dormancy period was developed. The 80 flowering plants produced from shoot tip culture were acclimatized in the greenhouse for 3 months and then grown in the experimental garden for 8 months. Received: 29 October 1997 / Revision received: 28 April 1998 / Accepted: 3 May 1998     

Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis

A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media fortified with 22.2 μm N⁶-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS) basal medium supplemented with 2.22 μm N⁶-benzylaminopurine and 0.54 μm α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may be useful for improving the crop through genetic manipulations. Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998     

Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.)

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5–7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 μm 2,4-dichlorophenoxyacetic acid and 0.93 μm kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology. Received: 29 December 1997 / Revision received: 9 April 1998 / Accepted: 24 April 1998     

Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb

Friable callus was obtained from styles and flower pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige and Skoog (MS) media containing either 2 μm dicamba or 2 μm picloram. Cell suspension cultures were established by suspending the callus of L. longiflorum Snow Queen in liquid medium containing 2 μm dicamba. Through a purification process, a fine fast-growing cell suspension was obtained. This suspension was composed of a homogenous population of small dense cells, which tended to organise into embryo like structures (ELS). In liquid culture with the auxin dicamba, the ELS underwent continuous callus formation. When transferred to solidified hormone-free MS medium, the ELS germinated, forming complete plantlets. Histological investigation showed that in the ELS both shoot and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact somatic embryos. Received: 4 April 1997 / Revision received: 13 May 1997 / Accepted: 15 June 1997     

Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp)

Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 μm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 August 1997     

Somatic embryogenesis and plant regeneration from stem explants of Moricandia arvensis

Efficient and reproducible plant regeneration has been established from stem internode explants of Moricandia arvensis, a crucifer of special interest due to its C₃-C₄ intermediate photosynthetic activity. Somatic embryogenesis was induced in one-third of explants cultured on Murashige and Skoog based medium containing 9 mm 2,4-dichlorophenoxyacetic acid. High frequencies of plant regeneration (>90%) resulted when somatic embryos were germinated on medium lacking growth regulators. Regenerated plants were diploid, fertile and morphologically similar to seed-derived plants of M. arvensis. This is the first report of somatic embryogenesis in M. arvensis. This plant regeneration system should facilitate gene identification and localisation studies of C₃-C₄ physiology by insertional mutagenesis, a prerequisite for the isolation and transfer of genes involved in C₃-C₄ metabolism from Moricandia to cultivated brassicas. Received: 28 November 1996 / Revision received: 30 January 1997 / Accepted: 15 February 1997     

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     

Micropropagation of nodal explants from adult trees of Cleistanthus collinus

The micropropagation of adult Cleistanthus collinus was accomplished. The nodal segments from terminal twigs of a 15-year-old tree and basal sprouts of a comparable chronological age were used for initiating shoot bud cultures. Washing explants with sterile mixtures of citric acid (520.5 μM) and PVP 40 (3.75 μM) three to four times controlled the leaching of brown inhibitory substances into the establishment medium. Axillary shoots proliferated best on MS medium containing citric acid (104.1 μM), and PVP 40 (12.5 or 25 μM) supplemented with 0.44 μM BA. The number of new shoots from nodal segments of explants placed on MS medium supplemented with 0.44 μM BA increased when the remaining lengths of nodal segments were transferred to fresh medium after the longer microshoots were harvested. The microshoots derived from basal sprouts rooted best (50%) when treated with 11.4 mM IAA for 2 min, whereas only 40% of the microshoots derived from terminal twigs produced roots after a 2-min exposure to 28.5 mM IAA. The placement of BA-soaked agar cubes on the apex-decapitated shoots controlled shoot-tip necrosis considerably. In general, explants from basal sprouts were more suitable than terminal twig explants for the micropropagation of adult trees of C. collinus. Received: 26 February 1997 / Revision received: 13 September 1997 / Accepted: 29 September 1997     

Induction of lincomycin and streptomycin resistance by nitrosomethylurea and ethyl methanesulphonate in Capsicum annuum L.

A high frequency of plastid-encoded antibiotic-resistant variants of Capsicum annuum were isolated on selective media following treatment by ethyl methanesulphonate (EMS) and nitrosomethylurea (NMU). Seeds and explants were mutagenized with 0.1% EMS and 5 mm NMU separately. Non-mutagenized cotyledons (controls), mutagenized cotyledons from EMS-treated seedlings and NMU-treated cotyledons were placed on regeneration medium supplemented with the antibiotics streptomycin or lincomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. The stability of streptomycin and lincomycin resistance was confirmed by leaf assay. NMU-treated cotyledons gave a higher frequency of variants than cotyledons from EMS-treated seedlings. The mutagenic effect of EMS was more pronounced using whole seeds rather than cotyledons; in contrast, NMU was more effective in inducing variations in cotyledons than in seeds. Received: 1 October 1996 / Revision received: 12 May 1997 / Accepted: 3 June 1997     

Factors influencing regeneration from protoplasts of Asparagus densiflorus cv. Sprengeri

This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7 mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt solution with 7 mm CaCl₂ ^.2H₂O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks. Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997