Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Calli and suspension cultures for biomass production ofEuphorbia characias L. subsp.characias

Summary Friable calli and cell suspension cultures were obtained from leaf segments ofEuphorbia characias L. subsp.characias, in Murashige and Skoog (MS) basal medium supplemented with 1g.l^–1 casein hydrolyzate (CH), 5mg.l^–1 ascorbic acid, 1.0mg.l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.75mg.l^–1 benzyl adenine (BA). The highest callus specific growth rate (=0.085.day^–1), calculated for 1 year old calli cultures, was obtained with 0.25 mg.l^–1 2,4-D and 0.50mg.l^–1 BA. Suspension cultures started with an inoculum of 8.0×10⁴ cells.ml^–1 in supplemented liquid MS medium, gave a specific growth rate =0.256.day^–1.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

In vitro plant regeneration from leaf callus inPiper colubrinum Link

Callus-mediated shoot regeneration from leaf explants ofPhytophthora resistant pepper (Piper colubrinum Link.) is described. The effect of basal media composition and growth regulators onin vitro response of explants was evaluated. Shoot buds were induced and elongated on half-strength MS medium containing 2.0 mg l^–1 BA and 0.5 mg l^–1 NAA , as well as 1.0 mg l^–1 BA and 0.5 mg l^–1 2,4-D. The shoots were rooted in half-strength MS medium with or without IAA or IBA, and then were transferred to soil with 100% survival.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.)

Morphogenic calli were obtained efficiently from ab initio cultures of isolated microspores in eggplant. Initial culture of freshly isolated microspores in sucrose-free medium at high temperature (35°C) for 3 d was a prerequisite for callus induction. The microspores were re-cultured in modified NLN medium containing 2% sucrose and phytohormones (NAA 0.5 mg l^–1, BA 0.5 mg l^–1) in the dark. After 4 weeks of re-culture, small calli derived from microspores were transferred to MS medium containing 4 mg l^–1 zeatin and 0.2 mg l^–1 IAA for shoot regeneration. The ploidy of 12 randomly selected regenerants was assessed by chromosome counts in root tips. Only one of the regenerants was haploid, 7 were diploid, 3 were triploid and one was tetraploid. The diploids set seeds after self-pollination and showed no segregation for morphological traits in the progeny, suggesting that they were spontaneously doubled haploids.Abbreviations BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - DAPI 4,6-diamidino-2-phenylindole dihydrochloride     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted. The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6). Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction. Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 3.0 mg l^–1 of 2,4-D and 30 g l^–1 of sucrose. Agar concentration in the regeneration medium was also critical for the shoot induction. Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (2.0 mg l^–1) of NAA. The optimal concentration of kinetin for the highest shoot regeneration frequency (6777%) was different among the cultivars tested. The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds.     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

Micropropagation of Morus laevigata Wall. from mature trees

Multiple shoots were obtained from nodal explants of 10-year-old tree of Morus laevigata on Murashige and Skoog's medium supplemented with different concentrations (0.5–5.0 mg.l^–1) of benzyladenine (BA). Nodal segments taken from in vitro proliferated shoots gave further multiple shoots when cultured on the same basal medium containing 2.5 mg.l^–1 BA. Repeated subculture resulted in rapid shoot multiplication at the average rate of 6-fold per subculture. In vitro raised shoots rooted on MS medium containing 0.1 mg. l^–1 each of 3-indolebutyric acid (ISA) and -naphthaleneacetic acid (NAA). The regenerated plantlets were successfully established in soil under field conditions after a few days of indoor acclimatization.     

Regeneration of `juvenile' plants of black plum,Syzygium cuminii Skeels,from nodal exp of mature trees

Nodal explants, excised from young shoots from mature trees of Syzygium cuminii, were cultured on MS medium supplemented with different concentrations of BA or kinetin. Among these, BA (0.5 or 1.0 mg l^–1) induced greening and opening of the incipient shoot buds, which however did not elongate. Elongation of the shoot buds was facilitated on MS medium with 1.0 mg l^–1 BA supplemented with casein hydrolysate (1.5 g l^–1) or glutamine (200 mg l^–1). Nodal explants (microcuttings), taken from shoots developed in vitro, also developed multiple shoots when cultured on MS with1 mg l^–1 BA. These explants did not require an additional supply of reduced nitrogen, for further normal development. Shoots developed from explants from mature trees and microcuttings were rooted by sub-culturing them on Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The plants that developed in vitro were planted in soil and were transferred to the field after an acclimatization period of 7–8 months. These plants have been thriving well for more than three years and have no apparent phenotypic aberrations.     

Micropropagation of Capparis decidua (Forsk.) Edgew. — a tree of arid horticulture

A method for micropropagation of mature trees of Capparis decidua was developed. Multiple shoots were obtained from nodal explants on Murashige and Skoog's (1962) medium+0.1mgl^–1 NAA+5.0mgl^–1BAP+additives (50mgl^–1 ascorbic acid and25 mgl^–1 each of adenine sulphate, L-arginine and citric acid) at 28 ± 2°C, 12 h/dphotoperiod and 35–40 mol m^-2s^–1 photon flux density. The shoots were multiplied by (i) subculture of nodal shoot segments onto MS +0.1 mgl^-–1 IAA+1.0mgl^–1 BAPH+additives, and (ii) repeated transfer of original explant onto MS+ 0.1mgl^–1 IAA+mg l^–1 BAP+additives, at intervals of 3 weeks. Sixty to 70% of the shoots rooted when pulse treated with 100 mg l^–1 IBA in half strength MS liquid medium for 4h, and then transferred onto hormone-free half-strength agar-gelled MS basal saltmedium. Incubation in dark at 33 ± 2°C for 6d favoured root induction. In vitro hardened plants were transferred to pots.Abbreviations IAA Indole-3-aceticacid - IBA Indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - 2-ip Isopentenyl adenine - B₅ Gamborg et al. (1968) medium - MS Murashige and Skoog's (1962) medium - WP Woody plant medium (Lloyd and McCown 1981)     

In vitro propagation of green-foliaged Dracaena fragrans Ker.

Callus tissue was induced in young stem segments cultured on MS based media supplemented with 0.25–0.5 mg l^-1 2, 4-D. Shoots were differentiated on media containing 0.5–1.0 mg l^-1 BA and 0.5–2.0 mg l^-1 IBA or 0.1–0.2 mg l^-1 NAA. The same media were suitable for shoot multiplication. Shoot elongation and rooting were strongly inhibited by BA and stimulated by auxins IBA and NAA. Medium containing 0.5 mg l^-1 IBA was optimal for rooting. Root elongation was stimulated by light and inhibited in darkness. Transfer of rooted plantlets to outdoor conditions was feasible and special hardening procedures were not required. Among more than 5000 plants produced by this procedure only 9 off-type plants with variegated leaves were found.