Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed)

Summary Basal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 μM 2,4-dichlorophenoxyacetic acid giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 μM naphthaleneacetic acid, 10 μM gibberellic acid, and 2 μM benzyladenine with either 1 or 20 μM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond’s meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 μM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures.     

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.     

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B₅ medium containing sucrose (1.0 %) with 2,4-D (5.0 μM) and 2-iP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11–13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Plant regeneration through somatic embryogenesis in the forage grass Caucasian bluestem (Bothriochloa caucasica)

Plantlets were regenerated from cultured seed explants of the forage grass Caucasian bluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] via somatic embryogenesis. Embryogenic callus was produced in four weeks when surface sterilized seeds were cultured on a medium containing MS-salts, B-5 vitamins, 12 mM L-proline, 2% sucrose, 0.8% agar and 5M 2,4-D. Plantlets were regenerated in 6–8 weeks after culture initiation. Healthy root and shoot systems were produced within three weeks after the plantlets were transferred to a medium lacking 2,4-D. Approximately 95% of the plantlets survived greenhouse acclimation and produced healthy plants and viable seeds. Caucasian bluestem callus cultures exhibit natural resistance to kanamycin. High levels of kanamycin (up to 800 mg/l) did not completely inhibit callus growth. However, the regeneration of healthy-plantlets was completely inhibited by kanamycin even at low levels (50 mg/l).     

Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of <Emphasis Type="Italic">Landoltia punctata</Emphasis>

The starch-rich duckweed Landoltia punctata is a valuable aquatic plant in wastewater purification, bioenergy production, and many other applications. A highly efficient callus induction and plant regeneration protocol is desirable so that biotechnology can be used to develop new varieties with added value and adaptation. We studied both known and unknown factors that influence callus induction in L. punctata and obtained almost 100 % induction rate in 30 days. The optimum medium for callus induction was MS basal medium supplemented with 1 % sorbitol, 15 mg/L 2,4-D, and 2 mg/L 6-BA. Green fragile callus was induced from the meristematic region in the budding pouches. The optimum photoperiod for callus induction was 16-h day, and the optimum explant orientation was dorsal side down on the medium. The optimum medium for callus subculture was WPM basal medium supplemented with 2 % sorbitol, 4 mg/L 2,4-D, and 0.5 mg/L TDZ. Green callus could be maintained by subculture once every 4 weeks. However, when the subculture cycle was prolonged to 6 weeks or longer, yellow fragile embryogenic callus was obtained. The optimum plant regeneration medium was MS medium supplemented with 0.5 % sucrose, 1 % sorbitol, and 1.0 mg/L 6-BA with frond regeneration rates of approximately 90 %. The regenerated fronds rooted in Hoagland’s liquid medium in 1 week. The callus induction and frond regeneration protocol was tested for its efficiency in geographically distinct strains 5502, 8721, and 9264. Thus, we obtained a rapid and efficient protocol for callus induction and frond regeneration of L. punctata, which takes only 9 weeks.     

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 via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

Callus Induction and Plant Regeneration in Lemna Minor L.

Callus induction was obtained on Murashige and Skogg agar medium with 45 M 2,4-dichlorophenoxyacetic acid under dark at 25°C. Among the four explant types investigated, the best callus induction was obtained from two-week old fronds to which a surgical incision was applied in the basal (meristematic) region. This treatment resulted in 89.11% of fronds producing callus which continued to proliferate for another 24 months. To obtain plant regeneration pieces of calluses were transferred onto Murashige and Skoog agar medium containing 22 M indole-3-acetic acid and 4.6 M kinetin and maintained under 16-h photoperiod (irradiance of 30 mol m^–2 s^–1) at 23°C. Green fronds formed on all callus pieces. The regenerated fronds were later transferred onto Wang medium where they formed roots. The regenerated Lemna minor L. plants obtained through indirect organogenesis did not differ morphologically from individuals forming the stock collection.     

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10¹⁴ plantlets could be produced in a year from one expiant following the proposed protocol.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Somatic embryogenesis in Asparagus officinalis can be an in vitro selection process leading to habituated and 2,4-D dependent embryogenic lines

Long-term embryogenic lines were repeatedly obtained from nine asparagus (Asparagus officinalis L.) genotypes by the selection of rare events, which consisted of the emergence of either a few somatic embryos or an embryogenic callus from a restricted area of a primary callus. In the first case, somatic embryos emerged from 1 % of calli induced with naphtaleneacetic acid and transferred to a medium without auxin. Isolated and subcultured on hormone free medium, these embryos developed habituated embryogenic lines (H lines) growing by adventive embryogenesis. In the second case, 3 % of primary calli developed then subcultured on 2,4-dichlorophenoxyacetic acid (2,4-D) produced a new type of friable and yellowish-white callus, constituted of clusters of globular somatic embryos which can be continuously maintained on 2,4-D (2,4-D lines). Among 2,4-D lines, two types were identified by subculturing them on hormone–free medium. Half of the 2,4-D lines were habituated and half were 2,4-D dependent. Most plants regenerated from H lines exhibited a strong increase in embryogenic capacity compared to control plants, unlike plants regenerated from the 2,4-D dependent lines. This increased embryogenic capacity was transmitted to the progeny as a monogenic dominant trait. H lines would therefore be issued from mutation(s) occurring in vitro, conferring both the embryogenic and habituated phenotypes. On the contrary, in the 2,4-D dependent lines, the embryogenic processes appeared to remain under exogenous auxin control and no evidence of a mutational origin could be inferred from the behaviour of regenerated plants.     

Stimulatory Effect of Partial Desiccation on Plant Regeneration in Indica Rice (Oryza sativa L)

A simple in vitro protocol was established for high frequency plant regeneration via organogenesis and somatic embryogenesis from the callus cultures derived from immature inflorescence segments of indica rice (Oryza sativa L cvs Safari-17 and Kasturi). Embryogenic and nodular calli were initiated on MSB medium supplemented with 2, 4-D and sucrose (3.0%, w/v). Somatic embryogenesis occurred after transfer of embryogenic calli to MSB medium containing 2.25 μM 2,4-D, 2.2 μM BAP, 2.9 μM thiamine HCl and 244.86 μM L-tryptophan. Plantlet/shoot regeneration occurred after transfer of embryogenic calli to MSB medium containing 17.6 μM BAP and 1.12 μM 2,4-D. Partial desiccation (up to 12, 24, 48, 72 and 96 h) of embryogenic calli prior to transfer to regeneration medium stimulated regeneration frequency. Highly significant (P<0.001) difference was observed for regeneration frequency and average number of plantlets/shoots regenerated per callus in partially desiccated calli in comparison to non-dehydrated calli. Regeneration frequency increased from 33.3% to 80% after 24 h of desiccation treatment to callus cultures of cv. Safari-17, and from 46.7% to 93.3% after 48 h of desiccation treatment to callus tissues of cv. Kasturi. Regenerated shoots were rooted on MSB medium supplemented with 4.9 μM IBA. Plants with well-developed roots were transferred to pots where they grew well and attained maturity.     

Hormone-regulated inflorescence induction and TFL1 expression in Arabidopsis callus in vitro

To study hormone-regulated inflorescence development, we established the in vitro regeneration system of Arabidopsis inflorescences in the presence of cytokinin and auxin. Media containing a combination of thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to induce callus formation. Higher frequencies of calli were obtained by using the inflorescence stems as explants. After transferring the calli to media containing a combination of zeatin and indole-3-acetic acid (IAA), the inflorescences were induced from the calli. The morphology of regenerated inflorescences was similar to that of inflorescences in plants; however, flowers of regenerated inflorescences often lacked a few floral organs. Furthermore, TFL1, a gene involved in floral transition in Arabidopsis, was activated during the inflorescence induction. Our results suggest that the TFL1 gene plays an important role in hormone-regulated inflorescence formation.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses

Objectives of this research were to test the effects of plant genotypes and auxin 2,4-D (2,4-dichlorophenoxyacetic acid) medium concentrations on embryogenic (E) callus production of two grass species. Two Old World bluestem,Bothriochloa ischaemum, accessions (A-8793 and A-8911c) and three bermudagrass,Cynodon dactylon (L.) Pers., accessions (A-10978b, A12164, and Brazos) supplied the explant material. Immature inflorescences 9 mm in length were placed on modified Murashige-Skoog (MS) agar medium containing 0, 1, 3, or 5 mg L^-1 of 2,4-D. Explants of all genotypes produced callus by the end of a 4-week dark incubation period at 25°C. When subcultured onto fresh media and maintained at 25°C with a 16 hr photoperiod, calli became embryogenic within 8 weeks of inoculation. Three mg L^-1 of 2,4-D in the media maximized E callus production in both bluestem genotypes and in A-10978b and A-12164 bermudagrass genotypes. Maximum E callus production from Brazos bermudagrass resulted from the 1 mg L^-1 treatment. Somatic embryos developed after subculture under light. Embryos showed scutellum-like structures and coleoptile-coleorhiza bipolar organization. Plantlets were regenerated from all genotypes except Brazos, whose embryoids failed to germinate. All callus from Brazos eventually senesced. Light and scanning electron microscopy confirmed regeneration through somatic embryogenesis.     

Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum

Crown and leaf slices of in vitro plantlets of a non-flowering Vetiveria zizanioides from Java were used to induce compact calli and to regenerate plantlets. The influence of different growth regulators (2,4-dichlorophenoxy acetic acid, 6-benzylaminopurine), sucrose concentrations (10–100 g l⁻¹), cultivation in light or dark, and cultivation time on callus induction medium (6 or 12 weeks), on the induction of compact callus and the subsequent regeneration of plantlets was studied. Up to 75% of crown slices cultured on modified Murashige and Skoog medium supplemented with 2.26 μM 2,4-dichlorophenoxy acetic acid, 2.22 μM 6-benzylaminopurine and 75 g l⁻¹ sucrose developed compact callus. For subsequent regeneration of plantlets, callus induction in the light for 6 weeks on the callus induction medium containing 10 g l⁻¹sucrose, and subsequent transfer to the regeneration medium, was the best procedure, regenerating plantlets on around 60% of the crown or leaf slices, with up to 100 plantlets per slice. We have compared the efficiency of the above mentioned procedure with several other methods to regenerate plantlets. Our findings indicate that the procedure developed in this study was best in regenerating plantlets for the used vetiver variant. This revised version was published online in June 2006 with corrections to the Cover Date.     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.