Browning and necrosis of transformed cells/tissues, and difficulty to regenerate transgenic plants from the transformed cells/tissues (recalcitrance) are common in Agrobacterium-mediated transformation process in many plant species. In addition, most crop transformation methods that use NPTII selection produce a significant number of nontransgenic shoots, called “shoot escapes” even under stringent selection conditions. These common problems of plant transformation, (browning and necrosis of transformed cells/tissues, recalcitrance, and the occurrence of shoot escapes) severely reduces transformation efficiency. Recent research indicates that reactive oxygen species (ROS) such as superoxide radical (O₂⁻), the hydrogen peroxide (H₂O₂), the hydroxyl radical (OH'), and the peroxyl radical (RO₂) may be playing an important role in tissue browning and necrosis during transformation. This review examines the role of ROS in in vitro recalcitrance and genetic transformation and the opportunities to improve transformation efficiency using antioxidants.

The stable insertion and expression of multiple transgenes in crops is highly desirable, as the manipulation of complex agronomic traits and the introduction of novel biosynthetic pathways are dependent upon it. This study was performed to explore the frequency and efficiency of introducing multiple genes in soybean by using somatic embryogenesis and microprojectile bombardment transformation. The co-transformation frequency of six selectable marker or reporter genes (GusA, bleomycin resistance, glufosinate resistance, hygromycin resistance, green fluorescent protein, and kanamycin resistance) were followed throughout the T0, T1, and T2 generations. Three bombardment strategies were compared to determine the best method to generate transgenic plants that express the introduced transgenes and have a simple insertion pattern that would facilitate any downstream breeding. The plasmid bombardment treatments were (1) a six-gene-containing plasmid, (2) an equimolar treatment of five individual plasmids that collectively contained the six transgenes of interest (genes of glufosinate and hygromycin resistance were on the same plasmid), and (3) a 1:9 ratio mixture of the five plasmids, in which the plasmid containing the selectable marker used in the regeneration process, hygromycin resistance, was used in ninefold excess to all the other plasmids. Of the six bombardments performed per plasmid treatment, the results of seven independent events for the six-gene plasmid, four events for the 1:9 treatment, and a single regenerated event for the equimolar treatment indicate that containing all the transgenes on one plasmid just had an advantage in terms of frequency of a successful transformation events. Based on Southern analysis, the only events that contained all six transgenes was the one obtained by the equimolar treatment. No event was obtained that expressed all six transgenes, and certain transgenes seem to be non-randomly lost, namely gusA, bleomycin resistance, and glufosinate resistance, regardless of treatment. The addition of elements to optimize the expression of each gene cassette when multiple genes are in close proximity needs to be further investigated.

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA) 1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.

An efficient system was established for a higher frequency of protocorm-like body (PLB) formation from the callus of Dendrobium candidum Wall ex Lindl. The calluses were induced from longitudinally bisected segments of protocorms and subcultured two times every 40d on Murashige and Skoog medium with macronutrients at half strength, micronutrients at full strength, 2% sucrose, and with 8.8μM 6-Benzylaminopurine. PLB formation was achieved when calluses were transferred onto the same basal medium without any plant growth regulators. PLBs developed into intact plantlets about 2cm in height and with four roots when on basal medium with 2.7μM 1-naphthaleneacetic acid. Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Histological observations proved that globular somatic embryos could be produced from the inside and surface of the embryogenic callus during PLB formation.

Helicteres isora is a medicinal plant effective against asthma, diabetes, hypolipidemia, HIV, polio besides a good source of diosgenin. Seed dormancy and low natural fruit production rate make this plant a perfect candidate for developing an in vitro regeneration method. However, to date, no such work has been procured in this plant. An efficient method for plant regeneration via shoot organogenesis from callus cultures has been developed using nodal explants in H. isora. Murashige and Skoog (MS) media counting 2,4-Dichlorophenoxyacetic acid (2,4-D, 2.26 to 13.57 μM), Indole-3-acetic acid (IAA, 2.85 to 17.13 μM), Indole-3-butyric acid (IBA, 2.46 to 14.70 μM), 6-Benzylaminopurine (BA, 2.22 to 13.32 μM) and Kinetin (Kin, 2.32 to 13.92 μM) either singly or in the following combinations (IAA BA; IAA Kin, and BA Kin) produced granular callus except BA Kin which resulted in compact, hard, greenish-white (CHGW) callus. The optimum CHGW callus (2.62 g fresh weight/explant) was produced on MS media with 13.32 μM BA 2.32 μM Kin with over 93% callus induction frequency. Optimum shoot organogenesis (67% frequency) was achieved in CHGW callus with lower level of BA (2.22 μM) and Kin (2.32 μM) and produced 3.2 shoots/0.5 g callus within 35 d of culture. Microshoots were rooted successfully (62% frequency) after 35 d of culture on 1/2MS containing 4.90 μM IBA and hardened off. Antioxidant enzymes such as catalase, peroxidase, polyphenol oxidase, and biochemical parameters viz. hydrogen peroxide, reducing and nonreducing sugars, starch, proteins, phenols, and proline contents were studied in regenerating and nonregenerating CHGW calluses to establish a correlation between these parameters and shoot morphogenesis. All the enzyme activities and biochemical parameters were found more in regenerating callus than in nonregenerating except phenols.

In this study, we aimed to maximize the rates of somatic embryogenesis achievable in anther cultures of Chinese pink (Dianthus chinensis L.) (2n=2x=30). The genotype of the donor plant was found to be a major factor in determining the success rate. Conditions imposed during anther culture (notably medium composition and light conditions) and pretreatments (namely, cold, heat, and mannitol incubations) were also found to influence somatic embryo induction. For example, the highest levels of embryogenic callus induction were achieved when the donor buds had been cold pretreated and the subsequent anther culture was maintained in darkness. Furthermore, there appeared to be an interaction of genotype with culture conditions. Thus, in cultures of the cultivar (cv.) ‘Carpet’, the highest rates of embryogenesis were obtained when the anthers had received a 5-d heat-shock, but such a thermal treatment did not generally produce a significant effect. Likewise, a 3-d mannitol pretreatment was optimal only for the cross-hybrid line ‘HC’ Assessment of the ploidy of the plants regenerated from the anther cultures revealed both diploid and tetraploid plants. Histological and cytological observations showed that all of these (both from n-pollen and 2n-pollen lines) derived from anther wall cells. Spontaneous chromosome doubling was inferred to have occurred during the embryogenic callus culture period.

An efficient in vitro plant regeneration system was established from callus culture of Scopolia parviflora. Callus was induced from adventitious roots on B5 medium with 0.45–9.04 μM 2,4-dichlorophenoxyacetic acid (2, 4-D). In vitro plantlet regeneration was achieved on B5 medium supplemented with 44.38 μM benzyladenine (BA), 3% sucrose, and 0.38% gelrite. Plantlets were transplanted to artificial soil and grown to maturity successfully in a greenhouse. The tropane alkaloid contents in regenerated plants were analyzed using high-performance liquid chromatography (HPLC), and were found to be higher than those of adventitious roots, native growing plants, and acclimated plants. Regenerated plants from organogenic callus cultures produced a greater amount of tropane alkaloids.

Valeriana glechomifolia, a southern Brazilian endemic species commonly known as Valerian, accumulates the bioactive terpene derivatives valepotriates in all of its organs. In vitro growth of V. glechomifolia on solid Murashige and Skoog (MS) without phytohormones at full, 75% (MS 75), or on a modified formulation (M μ) was compared in stock cultures kept for up to 9 mo. without subculture. Changes in biomass accumulation, development of roots and shoots, and the production of the valepotriates acevaltrate, didrovaltrate, and valtrate were monthly evaluated. The highest biomass accumulation and root development was observed in plants grown on M μ, whereas better leaf development was detected in M-μ- and MS-medium-grown plants after 8 and 9 mo. of culture, respectively. Maximal didrovaltrate and valtrate yields were observed in M-μ-grown plants harvested after 5 and 6 mo. of culture, respectively, whereas acevaltrate concentration was highest on M-μ- and MS-75-grown plants after 7 mo. of culture. Plants grown for 6 mo. without subculture in M Δ were successfully propagated, showing stable growth and valepotriate yields three- to sixfold higher that those observed in field-grown plants. The results showed a positive effect of combined moderate reduction in salt concentration and increases in selected micronutrients and myoinositol amounts on both growth and valepotriate yields of extended period stock cultures of V. glechomifolia.

The internodes of Cephaelis ipecacuanha elongated when cultured on Gamborg B5 solid medium supplemented with 0.5 or 1 mg/L gibberellic acid (GA₃). The size of the elongated internode doubled in length compared to the untreated shoots, and the adventitious shoots formed on the elongated internodes. The shoots grew easily into plantlets without the use of auxin for rooting. The ex vitro regenerates cultivated in the greenhouse showed normal characteristics. Emetic alkaloids were detected in the leaves of in vitro shoots and the roots of regenerates cultivated in the greenhouse. This method using GA₃ propagated numerous plants at a rate of more than 100 times compared to the method without GA₃.

With the objective to develop a practical method of screening potato for drought tolerance, shoot and root growth in plantlets raised in vitro (from nodal cuttings drawn from in vivo as well as in vitro grown plantlets) were studied in three genotypes with known root mass production under field conditions. Different levels of water stress were induced using five concentrations of agar in MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium. Water potential of various media ranged from −0.70 MPa to −0.98 MPa. Water stress in culture adversely affected plantlet growth, and the responses varied with genotype and explant source. Genotype IWA-1 was less affected than Konafubuki and Norin-1. In the experiment with explants from in vivo grown plants, the time to rooting was considerably delayed in Konafubuki and Norin-1 by an increase in agar concentration, but no such effect was observed in IWA-1. In all media, the mean number of roots and root length was greater in IWA-1 than Konafubuki and Norin-1, and the latter two genotypes were at par. At 10 gl⁻¹ agar, IWA-1 had taller plantlets, heavier foliage dry weight, root volume, as well as root dry weight than Konafubuki and Norin-1, whereas the latter two genotypes were at par for all these characteristics. This pattern was similar to the reported pattern of these genotypes for root dry weight under field conditions. However, such similarity in the in vitro and field behavior of the tested genotypes was not observed when nodal cuttings drawn from in vitro plantlets were used as explants. It is concluded that in vitro screening of potato under specific and limited water stress conditions by raising plantlets from nodal cuttings drawn from in vivo grown plants may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions.

Several authors have suggested that plant biotechnologists perform regression or trend analysis to compare means of related quantitative treatments (e.g., doses of inositol). The present paper compares two statistical strategies to determine the effect of inositol (0–400 mg l⁻¹) on proteolytic activity in the culture medium during pineapple growth in temporary immersion bioreactors. Strategy 1 involved one-way analysis of variance (ANOVA) followed by Tukey′s Honestly Significant Difference (HSD). Strategy 2 consisted in the development of different regression analyses to determine the best fit equations to describe the experimental results. Curvefit software (version 2.10-0, May 15, 1987, Thomas S. Cox) was used. Cauchy, Normal, Parabola, and Hoerl equations were the best fitted according to their determination coefficients (R²). The optimal inositol concentrations to increase proteolytic activity were determined from the equations. Quite different results were obtained following strategy 2: 126.76 mg l⁻¹ inositol from Cauchy, 131.29 mg l⁻¹ from Normal, 145.06 mg l⁻¹ from Parabola, and 14.05 mg l⁻¹ from Hoerl equations. In contrast, experimental data identified 200 mg l⁻¹ inositol as the most adequate concentration to increase proteolytic activity in the culture medium. The statistical strategy 1, one-way ANOVA followed by Tukey HSD clearly supported this biological observation. In this paper, regression analysis was not useful to describe our experimental results.

Nodal shoot segments of four grapevine genotypes well known for their Fe-chlorosis characteristic [Vitis vinifera × Vitis berlandieri Fercal, resistant; V. berlandieri × Vitis rupestris 1103 P, mid-resistant; Solonis (Vitis riparia × V. rupestris × Vitis candicans) × Othello (Vitis labrusca × V. riparia × V. vinifera) 1613 C, susceptible; V. vinifera L. cv. Perlette, resistant] were cultured in vitro. The effects of three levels of iron sodium ethylene-diaminotetraacetate (FeNaEDTA; 9, 18, 36 mg l⁻¹) and three mixtures of iron and 840 mg l⁻¹ NaHCO₃ (sodium bicarbonate) in the Murashige and Skoog (MS) medium supplemented 4.9 μM indole-3-butyric acid (IBA) were compared. We assayed the chlorosis rating of leaves, total chlorophyll of leaves, dry shoot weights of the plantlets, and active and total Fe content of the leaves. The most suitable concentration in determining the reaction of genotypes to iron chlorosis was 9 mg l⁻¹ FeNaEDTA. Bicarbonate addition had negative effects on the iron intake and development of plants. While all genotypes were affected by non-ferrous conditions, Fercal and Perlette were found to be the most resistant genotypes and 1613 C rootstock as the most susceptible. The chlorosis rating of the tested genotypes ranked according to their known degree of tolerance and susceptibility to lime-induced chlorosis. The results of this study showed that the in vitro technique could successfully be used in viticulture to get results in shorter times in the studies, aiming at breeding new rootstocks and varieties suitable to calcareous soil conditions and determining the reactions of existing genotypes to Fe chlorosis.