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     

Callus initiation,growth and plant regeneration in Plantago ovata Forsk. cv. GI-2

The technique for callus initiation, growth and plant regeneration from cultured hypocotyl explants of Plantago ovata cv. GI-2 is described. Best initiation and growth of callus was achieved on Murashige & Skoog's medium containing 2,4-dichlorophenoxyacetic acid (1.0 mgl^-1) and kinetin (1.0 mgl^-1). The callus showed maximum shoot differentiation on medium containing kinetin (4.0 mgl^-1) and -naphthaleneacetic acid (0.01 mgl^-1). Root formation of shoots was best on half-strength medium supplemented with 3-indolebutyric acid. The regenerated plants were successfully transferred into pots.     

Impact of cefotaxime on somatic embryogenesis and shoot regeneration in sugarcane

A cephalosporin antibiotic, cefotaxime (Omnatax™) promoted somatic embryogenesis and subsequent shoot regeneration in vitro from spindle in sugarcane irrespective of the genotypes as (CoJ 83, CoJ 88 and CoJ 64) culturered on MS medium with 2,4-D (2.5 mgl⁻¹) and kinetin (0.5 mgl⁻¹). Seven different concentrations of cefotaxime (100, 200, 300, 400, 500, 600 and 700 mgl⁻¹) were tested to find the optimal concentration of cefotaxime for somatic embryogenesis from callus cultures. Among the three varieties, calli of variety CoJ 83 incubated on MS medium with 2,4-D (2.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) + cefotaxime (500 mgl⁻¹) exhibited maximum somatic embryogenesis. To improve shoot regeneration, the callus was transferred to MS medium with BAP (0.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) in combination with different levels of cefotaxime. Highest frequency of shoot regeneration was observed in callus of CoJ 83 in the presence of 500 mgl⁻¹ cefotaxime. The plantlets could be successfully hardened in polybags and transferred to soil, where they exhibited normal growth. Our results convincingly demonstrated that cefotaxime improves somatic embryogenesis from spindle and regeneration from embryogenic calli of sugarcane and hence can be strongly recommended for rapid and large scale multiplication of sugarcane.Key words: Saccharum officinarum L., leaf segments, callus, plant regeneration, antibiotic     

Induction of mitosis in polytene nuclei and hormonal effect on nuclear changes during callus initiation in diploid Urginea indica Kunth. (liliaceae)

Bulb scale and inflorescence explants of Urginea indica Kunth. (2n=20) were cultured in vitro on modified Murashige & Skoog's medium with different hormonal composition. Media containing 2,4-dichlorophenoxy-acetic acid (2,4-D) (2 and 4 mgl^–1) and -naphthalene-acetic acid (NAA) (2 mgl^–1) could induce callus in inflorescence explants. Combination of 2,4-D (4 mgl^–1) + NAA (2 mgl^–1) + Kinetin (2 mgl^–1) only could induce callus formation in scale explants. The bulb scale explants contained mostly diploid cells while the inflorescence explants contained cells with nuclear DNA content ranging from 2C to 64C. The lowest karyological heterogeneity was recorded in callus derived from bulb scale and in callus derived from inflorescence induced with NAA. The highest variability was recorded on media with 2,4-D alone. Induction of division, probably of the pre-existing polytenic nuclei in the inflorescence explant, has been suggested to be the cause of origin of polyploid cells in such cases.     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Cultural behavior of protoplasts from different organs of eggplant (solanum melongena L.), and plant regeneration

Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 Em^-2s^-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl^-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl^-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×10³ protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×10³ and 1,220.4×10³ protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl^-1) + zeatin (0.5 mgl^-1) + NAA (1 mgl^-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl^-1) + IAA (0.2 mgl^-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl^-1) + NAA (0.5 mgl^-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.Abbreviations (IAA) Indol-3-acetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (NAA) naphthale-neacetic - (BAP) 6-benzylaminopurine - (MS) Murashige and Skoog basal medium - (CPW) Cell and Protoplast Washing solution     

Regeneration of pigeonpea (Cajanus cajan) from cotyledonary node via multiple shoot formation

Summary Plant regeneration, which is the major limiting factor for transformation of Cajanus cajan, has been obtained via multiple shoot formation from the cotyledonary node region of seedlings germinated on MS medium containing 2 mgl^–1 6-benzylaminopurine. A mass of multiple shoot-initials formed at the axillary bud region of the cotyledonary node of the seedlings within two weeks. The cotyledonary node along with the mass of shoot-initials excised from the seedling, continued to form new shoot-initials on MS medium containing 6-benzylaminopurine (2 mgl^–1) and supplemented topically with indole-3-acetic acid. The formation of new shoot-initials was also observed from the cotyledonary nodal explant, after cutting off its surface layers to completely remove the pre-existing shoot-initials and culturing it on 6-benzylaminopurine (2 mgl^–1) containing medium. The shoots elongated rapidly on basal MS medium and rooted efficiently in MS medium supplemented with indole-3-butyric acid (0.5 mgl^–1). The procedure described is efficient, and highly reproducible and a common response was observed for all the six varieties tested.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - MS medium, Murashige and Skoog (1962) medium - CN cotyledonary node     

Micropropagation of sour cherry (Prunus cerasus L.) cv. Šumadinka

Sour cherry cv. umadinka (Prunus cerasus L.) is the leading Yugoslav cultivar for production orchards. A method of micropropagation has been developed for the purpose of growing umadinka on its own roots and for rapid multiplication.Aseptic cultures were initiated from shoot explants 1–2 mm long on Murashige & Skoog medium with (in mgl^-1) 6-benzylaminopurine (BAP): 1, indole-3-yl butyric acid (IBA): 1 and gibberellic acid (GA₃): 0–1.The best medium for proliferation was MS with (in mgl^-1): BAP 0.5, IBA 0.1, GA₃ 0.1, but media with (in mgl^-1): BAP 0.5, NAA 0.1, GA₃ 0.1 and BAP 1, NAA 0.1 and GA₃ 0.1 were also shown to be good. A higher degree of proliferation obtained with some media did not necessarily result in a better quality of plantlets produced.For rooting the best combination of culture medium was achieved with pretreatment 10 days in MS 1/2 with 1 mgl^-1 IBA, followed by transfer to a hormone-free medium after 5–10 days, resulting in 88% success.The rooted plants were planted in containers and acclimatized under mist, with over 90% of plants surviving transplantation.     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

Direct shoot regeneration from mature embryo as a rapid and genotype-independent pathway in tissue culture of heterogeneous diverse sets of cumin (Cuminum cyminum L.) genotypes

Summary A rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15–22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mgl^−1 (4.3 μM) NAA in combination with 0.3 mgl^−1 (1.71 μM) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.     

Shoot regeneration of hybrid seed geranium (Pelargonium x hortorum) and regal geranium (Pelargonium x domesticum) from primary callus cultures

Procedures have been developed that increase the rate of shoot regeneration of hybrid seed geranium from month-old primary callus cultures. Hybrid geranium callus tissue covered with green nodular structures was initiated by placing shoot tip explants on solidified Murashige & Skoog medium (MS) supplemented with 2.0 mgl^-1 zeatin and 1.9 mgl^-1 indoleacetic acid. Hybrids Red Orbit, White Orbit and Scarlet Orbit were shown to produce 5–50 shoot primordia per explant when callus was initiated on this medium. Regal geranium callus was initiated by placing leaf explants on MS medium supplemented with 2.0 mgl^-1 6-benzylaminopurine and 2.0 mgl^-1 naphthaleneacetic acid. Regal geranium cultivars Tiny Tot and Lavender Grand Slam were shown to produce between 2–50 shoot primordia per explant when initiated on the same medium.     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid     

Establishment and multiplication of colchi-autotetraploids of Rauvolfia serpentina L. Benth. ex Kurz. through tissue culture

A tissue culture procedure was developed for the establishment and propagation of a colchi-autotetraploid of Rauvolfia serpentina for possible commercial exploitation. Multiplication of autotetraploid shoots was obtained either through axillary bud elongation on Murashige and Skoog [1] medium (MS) containing 2.65 M (0.5 mgl^–1) -naphthaleneacetic acid and 0.33 M (0.05 mgl^–1) kinetin, or via multiple shoot formation on MS medium supplemented with 4.44 M (1.0 mgl^–1) 6-benzylaminopurine and 0.53 M (0.1 mgl^–1) -naphthaleneacetic acid. Rooting could be induced by transferring the shoots to MS medium containing 7.95 M (1.5 mgl^–1) -naphthaleneacetic acid alone. The plantlets, thus formed, were tetraploid in nature by cytological observations of the root tips. They exhibited 80–90% success in establishment under glass house and field conditions.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Plant regeneration from thin cell layers in Spinacia oleracea

Caulogenesis and somatic embryogenesis were induced from transverse thin cell layers (tTCLs) of two European (Spinacia oleracea L.) spinach genotypes. Regeneration occurred mostly when tTCLs had been excised from seedlings grown on a preconditioning medium consisting of White's macroelements, Nitsch's microelements, Murashige and Skoog's (MS) vitamins, 6 g l^–1 agar and 20 g l^–1 glucose. The explants were cultured on MS medium supplemented with sucrose (10, 30, 50 or 80 g l^–1) or fructose (5, 10 or 30 g l^–1) and several combinations of indole-3-acetic acid (IAA), -naphtalene acetic acid (NAA), 6-benzylaminopurine (BAP) and gibberellic acid (GA₃). Most of the regeneration events were obtained from root explants of the cultivar Carpo. The best result was observed on MS medium supplemented with 50 g l^–1 sucrose, 100 M NAA, 1 M BAP and 10 M GA₃. After an 8-week culture, the calluses were transferred onto MS medium where shoots and somatic embryos appeared 1 week later. The best root development was obtained on MS medium supplemented with 4.9 M indole-3-butyric acid (IBA) and 8 g l^–1 Phytagel. The plantlets were, then, transferred to soil and developed into well-conformed, fertile plants.     

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.     

Propagation of Gasteria croucheri Bak. from shoot producing callus

Leaf explants of Gasteria croucheri Bak. cultured on a modified Murashige and Skoog medium supplemented with 8.8 × 10^-6M (2 mgl^-1) benzyladenine and 2.7 × 10^-5M (5 mgl^-1) -naphthalene acetic acid yielded a callus which produced adventitious shoots. This callus could be maintained on a medium with 1.9 × 10^-5M (4 mgl^-1) kinetin only and continued to produce shoots which could be rooted on a hormone free medium. Plantlets could be readily acclimatized.     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl⁻¹ 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl⁻¹ 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl⁻¹ N₆-benzylaminopurine (BA) and 1 mgl⁻¹ NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5–4 mgl⁻¹) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).