Phenocritical times in the process of in vitro shoot organogenesis

Shoot organogenesis occurs when leaf explants of Convolvulus arvensis are cultured on Murashige and Skoog salts, sucrose, vitamins, and 0.05 mg/liter IAA with 7.0 mg/liter 2-isopentenyl adenine. Under the influence of this shoot inducing medium (SIM), the explants become competent for the organogenic effects of SIM and eventually become determined for shoot formation. The induction process includes five separate transient sensitivities to inhibitors. Such stage-specific inhibitions reflect phenocritical times in development rather than general metabolic toxicities. The phenocopying agents are tri-iodobenzoic acid (TIBA), sorbitol, ribose, ammonium ion, and acetylsalicylic acid. The process of in vitro shoot organogenesis from leaf explants is now seen to include a series of discrete steps which precede morphological differentiation. An initial dedifferentiation process results in the formation of competent callus tissue along the cut edges of the explant. Under the influence of the phytohormone balance in SIM, shoot organogenic induction proceeds. This process involves a time which is sensitive to inhibition by salicylates followed by a time sensitive to TIBA which is followed in turn by a time sensitive to sorbitol and culminates in cells or groups of cells determined for shoot formation. This process also includes a time sensitive to inhibition by ribose, although its place in the order of events is not yet firmly assigned. There is also a sensitivity to ammonium ion (or lack of nitrate) at or near the time the explant becomes determined for shoot production.     

CUC2 as an early marker for regeneration competence in Arabidopsis root explants

CUP SHAPED COTELYDON 2 (CUC2) was tested as a marker for shoot induction to monitor and facilitate the optimization of in vitro regeneration of Arabidopsis thaliana. The expression of a pCUC2::3XVENUS-N7 fluorescent marker allowed the observation of early steps in the initiation and development of shoots on root explants. The explants were first incubated on an auxin-rich callus induction medium (CIM) and then transferred to a cytokinin-rich shoot induction medium (SIM). CUC2-expression occurred prior to visible shoot formation during the incubation of the root explant on CIM. Shoot formation was invariably preceded by the accumulation of CUC2 expression at dispersed sites along the root explant. These patches of CUC2-expression also marked the site of lateral root primordium formation in root explants that were transferred to hormone free medium. Thus, CUC2 is a predictive marker for the acquisition of root explant competence for root and shoot organogenesis.     

Developmental steps in acquiring competence for shoot development in <Emphasis Type="Italic">Arabidopsis</Emphasis> tissue culture

Arabidopsis shoots regenerate from root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium (CIM) followed by incubation on a cytokinin-rich shoot induction medium (SIM). During CIM preincubation, root explants acquire competence to respond to shoot induction signals. During CIM preincubation, pericycle cells in root explants undergo cell divisions and dedifferentiate, losing the expression of a pericycle cell-specific marker. These cells acquire competence to form green callus only after one day CIM preincubation and to form shoots after 2–3 days CIM preincubation. Reversible DNA synthesis inhibitors interfered with the acquisition of competence to form shoots. Genes requiring CIM preincubation for upregulation on SIM were identified by microarray analysis and included RESPONSE REGULATOR 15 (ARR15), POLYGALACTURONASE INHIBITING PROTEIN 2 (PGIP2) and WUSCHEL (WUS). These genes served as developmental markers for the acquisition of competence because the CIM preincubation requirements for ARR15 and PGIP2 upregulation correlated well with the acquisition of competence to form green callus, and the CIM preincubation requirements for WUS upregulation matched those for shoot formation. Unlike ARR15, another cytokinin inducible, A-type ARR gene, ARR5, was upregulated on SIM, but the induction did not require CIM preincubation. These findings indicate that competencies for various events associated with shoot regeneration are acquired progressively during CIM preincubation, and that a set of genes, normally upregulated on SIM, are repressed by a process that can be relieved by CIM preincubation.     

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

Influence of 5′-azacitidine on promoting recovery of cell competence for shoot organogenesis in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, adventitious shoots are formed at a high frequency when the calli are induced from roots or hypocotyls cultured on callus induction medium (CIM) and then transferred to shoot induction medium (SIM). The prolonged duration of culture on CIM decreased the frequency of shoot regeneration. However, when 5′-azacitidine (AzaC), an inhibitor of DNA methylation, was added to CIM, the excess culturing on CIM did not decrease the frequency of shoot regeneration. The level of methyl cytosine was up-regulated when hypocotyl explants were cultured on CIM for 2 weeks. We examined the expression patterns of genes that are involved in the formation or regeneration of shoots. Prolonged duration of culture on CIM up-regulated the CUC1, CLV1, CLV3, ESR1, and WUS mRNA levels, and the addition of AzaC to CIM reduced their expression levels. Our results suggest that an increase in DNA methylation decreased the shoot-forming ability and that AzaC can partially recover this ability.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: Effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledonnodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

Production of glycyrrhizin in callus cultures of licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

A protocol toward multiplication of the medicinal tree,Eucommia ulmoides oliver

Summary A protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.     

A two-step protocol for shoot regeneration from hypocotyl explants of oilseed rape and its application for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations of 6-benzylaminopurine (BA; 2–5 mg dm⁻³) and naphthaleneacetic acid (NAA; 0.05–0.15 mg dm⁻³). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm⁻³ BA and 0.1 mg dm⁻³ NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7 d on a CIM medium containing 1.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved to be transgenic.     

Abscisic acid promotes shoot regeneration in Arabidopsis zygotic embryo explants

An efficient shoot organogenesis protocol for Arabidopsis zygotic embryo explants of Landsberg erecta ecotype was established. This de novo shoot organogenesis protocol has three different steps, i.e., induction of callus in an auxin-rich callus induction medium, the formation of green-organogenic callus in the shoot induction medium (SIM), and the final morphological differentiation of shoot in the hormone-free shoot development medium (SDM). Abscisic acid (ABA), auxin, and cytokinin (CK) were used in the SIM. Individual plant growth regulators as well as their combination were studied to understand their importance in the shoot induction treatment. We found that a combination of ABA + CK and ABA + CK + auxin induced higher shoot organogenic ability in the callus than ABA, CK, and auxin alone. Optimum ABA concentration on shoot organogenesis was determined to be 10^?5 M. Morphological characterization of callus induction and shoot organogenesis events indicated that calli were derived from the cotyledons of zygotic embryo explants and the formation of green organogenic calli was specific to the exogenous inclusion of ABA + CK in the SIM. During the time of shoot development, the green organogenic callus became darker green due to the formation of anthocyanins. Shoot organogenic calli in the SIM and the SDM were easily identified by the green-colored calli and anthocyanin pigments, respectively. Furthermore, we demonstrated the significance of exogenous and endogenous ABA in shoot organogenesis by fluridone treatments. The inclusion of ABA in SIM has a significant effect on shoot formation.     

A rapid and efficient method for regeneration of plantlets from embryo explants of cumin (Cuminum cyminum)

A new, simple and efficient method was developed for multiple shoot regeneration of cumin from imbibed embryo cultures. This method yielded a large number of shoots within short period of time (30–50 days) without any subculturing. The effects of different media, different embryo explants and various combinations of plant growth regulators (PGRs) on callus formation and shoot regeneration in cumin were investigated. Simultaneous callus formation and shoot regeneration was obtained. The best response for multiple shoot regeneration was observed on B5 medium containing 1.0 mg l^–1 BAP, 0.2 mg l^–1 NAA and 0.4 mg l^–1 IAA, with an average of 140 shoots per explant.     

Effect of plant growth regulators on evolution of ethylene and methane by different explants of chickpea

Shoot tips, cotyledonary nodes and hypocotyls of chickpea (Cicer arietinum L.) were grown on 3 media: plant induction medium (PIM), callus induction medium (CIM), and shoot induction medium (SIM). Maximum growth and differentiation was seen in PIM, whereas minimum was observed in CIM. Shoot tips which differentiated to multiple shoots evolved negligible amounts of ethylene. Maximum ethylene evolution was recorded by hypocotyls in PIM. Ethylene appears to have stimulatory effect on shoot bud differentiation in cotyledonary nodes. But in hypocotyls, increased ethylene inhibited growth and differentiation. Calli on media containing only auxin (PIM) evolved significantly more ethylene, whereas those on media with cytokinin (SIM) evolved more methane. Callus forming explants like cotyledonary nodes and hypocotyls evolve more ethylene than shoot tips. This revised version was published online in August 2006 with corrections to the Cover Date.     

Efficient plant regeneration of native spearmint (Mentha spicata L.)

Summary Protocols and media constituents for efficient in vitro plant regeneration of Native Spearmint (Mentha spicata L. cultivar ‘Native Spearmint’) have been defined. Adventitious shoots were initiated either directly from morphogenetically competent cells of explants or primary callus. Leaf explants from at least 2-mo.-old in vitro-maintained shoots exhibited the greatest morphogenetic capacity. Explants derived from basal portions of leaves at the bottom of the shoot were most responsive, with up to a 100% regeneration frequency and greater than nine shoots per explant. Highest frequency of meristemoids and morphogenetic callus were initiated from explants cultured onto a basal medium containing Murashige and Skoog (MS) salts, supplemented with 4 mg thidiazuron (TDZ) per L and 25% (vol/vol) coconut water (CW) for 10 to 14 d in darkness. Bud and shoot development required removal of both TDZ and CW from the medium. Shoot propagules were transferred to basal medium supplemented with 0.01 mg α-naphthaleneacetic acid (NAA) per L and grown under low light for about 2 wk to facilitate shoot elongation. Individual shoots about 1 cm tall were dissected and retransferred onto the same medium. Root initiation began within 4 to 6 d and a functional root system developed within 2 to 3 wk. These plantlets were transferred to soil and acclimated successfully for growth and development in a greenhouse. This is the first report of an efficient regeneration system for Native Spearmint based on adventitious organogenesis.     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains

Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM Agrobacterium infection medium - CIM callus-inducing medium - CTAB cetyltrimethylammonium bromide - 2,4-D 2,4-dichlorophenoxy-acetic acid - GUS ß-glucuronidase - hph hygromycin phosphotransferase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip N -(2-isopentenyl) adenine - NPTII neomycin phosphotransferase II - RIM root-inducing medium - 35S cauliflower mosaic virus 35S promoter - SIM shoot-inducing medium