Media and gelling agent effect on cotton callus initiation from excised seed hypocotyls

A factorial experiment was performed to develop a medium which would support initiation and proliferation of callus in a diverse group of exotic lines of Gossypium hirsutum. Seed hypocotyls of T1, T25 and T133 were cultured on Linsmaier and Skoog (LS) basal medium (1965) with NAA or 2,4-D tested in combination with BA or kinetin. The best medium from this study was then compared to five published media for support of callus initiation and growth of the varieties Acala 1517-75, Coker 500, Dunn 120, Paymaster 303 and TM1. Furthermore, the effects of two gelling agents, Difco-Bacto agar and Kelco Gelrite, were investigated with each of the six media. Significantly more callus was initiated on media solidified with Gelrite than with agar. The best callus production occurred on LS medium supplemented with 30gl^-1 glucose, 0.1 mgl^-1 BA and 0.1 mgl^-1 2,4-D.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Plant regeneration from callus of Puccinellia distans (L.) Parl

Callus was induced from seeds of Puccinellia distans (L.) Parl. on MS medium supplemented with 2 mgl^-1 2,4-dichlorophenoxyacetic acid and 0.5 mgl^-1 kinetin. Morphogenesis initiation was achieved during subculture on medium containing 0.1 mgl^-1 2,4-D. From the point of morphogenetic capacity, 3 types of callus were selected. High frequency of plant regeneration was obtained by selection of embryogenic type of callus, and culture on N₆ medium and N₆ medium supplemented with kinetin (5–10 mgl^-1), or kinetin (2 mgl^-1) and IAA (0.5 mgl^-1). A high ratio of albinos among regenerants was observed.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

Somatic embryogenesis from immature leaves of in vitro grown tea shoots

Immature leaves of in vitro grown shoots of tea were cultured on various levels of 2,4-D. Somatic embryos were induced directly on leaves or via embryogenic callus produced at the basal regions of the leaves. Induction of embryogenesis appeared to be correlated with the maturity of the leaf explants, with younger leaves responding better. The embryogenic response of leaf explants also was correlated with the period of culture in 2,4-D containing liquid medium. Embryogenic calli or repetitive somatic embryos maintained their regeneration capacity for more than 3 years. Histological observation revealed somatic embryos were formed on various regions of the leaf midrib. Somatic embryos germinated and developed into plantlets on agar medium containing BA and IBA.Abbreviations BA 6-benzylaminopurine - IBA indole-3-butyricacid - 2,4-D 2,4-dichloro phenoxyacetic acid     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum

Tissue culture methods were developed for the induction, maintenance, and regeneration of embryogenic callus in sweet sorghum (Sorghum bicolor) cultivars Keller, Rio, and Wray. No significant differences were observed in production of embryogenic callus in cultures established from developmentally immature or mature embryo explants cultured on LS medium with 2 mg/1 2,4-D plus 0.5 mg/1 kinetin. Prolific callus production did not occur until the third four-week culture period. Long-term maintenance of embryogenic callus was dependent upon the selective transfer of embryogenic callus, with other callus types discarded. High-frequency plant regeneration was achieved and quantified on a fresh weight basis of embryogenic callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - LS Linsmaier and Skoog basal medium (Linsmaier and Skoog, 1965)     

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     

Clonal propagation of papaya in vitro

A procedure for the rapid tissue culture propagation of papaya is being developed. Tissue culture methods using apices of nursery and orchard trees of Carica papaya cv. Sunrise Solo were evaluated. The explants were established in a modified Murashige and Tucker (1969) basal medium with half-strength inorganic salts, 0.5mgl^-1 6-benzylaminopurine (BA) and 0.2mgl^-1 naphthaleneacetic acid (NAA). Established explants were transferred to a proliferation medium consisting of Murashige and Tucker (1969) basal medium, 0.5mgl^-1 BA and 0.1mgl^-1 NAA, which caused extensive multiplication of shoots. Rooting was induced at a higher frequency by subculturing plantlets onto media with indole-3-butyric acid (IBA) than with NAA.     

Tissue culture and plant regeneration from immature embryo explants of Calotropis gigantea (Linn.) R. Br.

Callus cultures were established from immature embryos of Calotropis gigantea (Linn.) R. Br. on a modified basal medium of Murashige & Skoog supplemented with 1 mgl^-1 2,4-D. In addition to 0.1 mgl^-1 of NAA the optimal BAP concentration for promoting shoot bud formation and growth was 2 mgl^-1. Rooting was induced when shoots were transferred to auxin-supplemented Bonner's solution or half-strength MS basal salt solutions.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-butyric acid - BAP 6-benzylaminopurine - Kin kinetin     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

High frequency plant regeneration from stem explants of Brassica alboglabra Bailey in vitro

Culture conditions for shoot regeneration and proliferation, and rooting of Brassica alboglabra Bailey were optimized by a judicious selection of explants and manipulation of hormonal combinations in the culture medium. Both half and whole stem explants were more regenerative than cotyledons and hypocotyls. The highest shoot regeneration frequency (100%) accompanied by high number of shoots was obtained using half stem explants grown on Murashige & Skoog [14] medium supplemented with 2 mgl^-1 BA in combination with 1 mgl^-1 NAA, or 4 mgl^-1 2iP with 0.5 mgl^-1 NAA. For shoot proliferation, 4 mgl^-1 kinetin was most effective. The presence of auxin reduced shoot proliferation significantly. Maximum rooting (100%) of shoot cuttings was obtained either in the absence or in the presence of 0.5 mgl^-1 NAA, or IBA or IAA ranging from 0.1 to 8 mgl^-1.     

Comparison of 2,4-D and picloram for selection of long-term totipotent green callus cultures of sugarcane

Long-term regeneration of sugarcane (Saccharum spp. hybrid and Saccharum spontaneum L.) callus cultures was achieved by selection of green callus on MS agar medium containing 0.5 mgl^-1 picloram or 2,4-D. Newly initiated sugarcane callus cultures were a complex mixture of different tissue types including white, nonregenerative and green, regenerative tissues. The proportion of the tissue types changed as a function of time in culture, genotype, and amount and kind of auxin. Green callus on picloram media always regenerated green plants. Nine hybrids and ten wild relatives of sugarcane produced green calli on picloram media whereas only three hybrids were grown as green calli on 2,4-D media in long-term culture. Green calli were inoculated into liquid MS medium with 0.5 mgl^-1 picloram for suspension culture. These cultures were totipotent after 19 months. For routine culture, we initiated callus cultures on modified MS medium with 3 mgl^-1 2,4-D, then in two to three weeks we subcultured callus on MS medium with 0.5 mgl^-1 picloram and selected for green callus. Green calli regenerated large numbers of green plants after more than four years.     

Isolation and culture of protoplasts from mesophyll tissue of the legume Cyamopsis tetragonoloba L.

Protoplasts of Cyamopsis tetragonoloba were isolated from leaves of in vitro grown plants. The yield of the protoplasts, their viability and subsequent divisions were greatly influenced by the pH of the media used for isolation and culture of protoplasts. Sustained divisions of the cultured protoplasts were best supported by a modified Kao and Michayluk (1975) nutrient medium containing glucose (0.4 M), NAA (4 mgl^–1), 2,4-D (1 mgl^–1) and KIN (2 mgl^–1 ). The protoplast derived cells developed calli on transfer to Murashige and Skoog (1962) medium supplemented with 1 mgl^–1 each of 2,4-D, NAA and KIN.     

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 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)     

杜仲叶片愈伤组织诱导的激素优化研究

以杜仲优树L33的幼叶为材料,在B5培养基上添加不同浓度的生长素与细胞分裂素进行愈伤组织诱导研究,结果表明：无激素的培养基上不能诱导出愈伤组织,单独加入2,4-D、NAA和IBA均可诱导出愈伤组织,以0.5 mg·L^-1 2,4-D、0.5～1.0 mg·L^-1 NAA、1.0 mg·L^-1 IBA出愈率最高,达100%,且愈伤组织生长较好;在适宜浓度的生长素的培养基上加入细胞分裂素时,KT的加入对愈伤组织的诱导及生长起抑制作用;1.0 mg·L^-1 NAA+0.3～0.5 mg·L^-1 BA与1.0 mg·L^-1 IBA+0.3～0.5 mg·L^-1 BA 的组合可明显促进愈伤组织的诱导和生长,适合进一步继代培养。