<Emphasis Type="Italic">Brassica</Emphasis> biotechnology: Progress in cellular and molecular biology

Summary Considerable progress has been accomplished in the cellular and molecular biology of Brassica species in the past few years. Plant regeneration has been increasingly optimized via organogenesis and somatic embryogenesis using various explants; with tissue culture improvements focusing on factors such as age of the explant, genotype, and media additives. The production of haploids and doubled haploids using microspores has accelerated the production of homozygous lines in the Brassica species. Somatic cell fusion has facilitated the development of interspecific and intergeneric hybrids in the sexually incompatible species of Brassica. Crop improvement using somaclonal variation has also been achieved. The use of molecular markers in marker-assisted selection and breeding, transformation technology for the introduction of desirable traits, and a comparative analysis of these as well as their future prospects are important parts of the current research that is reviewed.     

Shoot organogenesis and somatic embryogenesis from leaf and shoot explants of <Emphasis Type="Italic">Ochna integerrima</Emphasis> (Lour)

Ochna integerrima is a medicinal and ornamental plant in Southeastern Asia. It has been listed as a rare and endangered species in China. Here we studied the effects of plant growth regulators and their concentrations on the induction of somatic embryogenesis and shoot organogenesis from leaf and shoot explants of O. integerrima for the first time. Cytokinins played a crucial role in somatic embryogenesis and shoot organogenesis. Among them, a higher concentration of thidiazuron (10.0–15.0 μM TDZ) could induce both somatic embryogenesis and adventitious shoot formation whereas low concentrations of TDZ (5.0 μM) could only induce adventitious shoots. However, 6-benzyladenine (BA at 5–15 μM) could only induce adventitious shoots. Shoot explants induced more adventitious shoots and somatic embryos than leaf explants when cultured on medium with the same concentration (5–15 μM) of TDZ or 15 μM BA. Medium containing 0.5 μM α-naphthaleneacetic acid and 8 μM indole-3-butyric acid and 0.1% activated charcoal could induce adventitious roots within 1 month. An efficient mass propagation and regeneration system has been established.     

Antioxidant Enzyme Activities during in vitro Morphogenesis of Gladiolus and the Effect of Application of Antioxidants on Plant Regeneration

Activity of antioxidant enzymes was evaluated during somatic embryogenesis and shoot organogenesis from cultured leaf segments of Gladiolus hybridus Hort. The effect of exogenous antioxidants on somatic embryogenesis and shoot organogenesis has also been monitored. Activity of superoxide dismutase (SOD) gradually increased during somatic embryogenesis. while activities of catalase (CAT) and peroxidase (POX) decreased. In contrast, increase in CAT and POX activity and a concomitant decrease in SOD activity were noted during shoot organogenesis. Exogenous application of antioxidants such as glutathione (GSH), α-tocopherol and ascorbate (AA) inhibited somatic embryogenesis but stimulated shoot organogenesis. The frequency of somatic embryogenesis increased with the addition of H₂O₂. However, H₂O₂ inhibited shoot organogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Leafy cotyledon genes are essential for induction of somatic embryogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.     

<Emphasis Type="Italic">Crambe tataria</Emphasis>: actions for ex situ conservation

In vitro micropropagation by direct organogenesis and somatic embryogenesis via callus was developed for Crambe tataria (Brassicaceae). C. tataria is an endemic species of the Pontic-Pannonic region, but it is also present in Italy, where it is localized in Friuli on a characteristic grassland formation, called “magredi”. C. tataria is regarded as an endangered species. Leaf and root explants were subjected to plant regulator treatments, which invoked different morphogenic responses. Leaf explants produced more callus than root explants and a higher amount of callus was obtained with 1 mg l⁻¹ 2,4-D in combination with 2 mg l⁻¹ Kin. Somatic embryogenesis was obtained in calli maintained in a delayed subculture regime on media containing BAP in combination with NAA. Root explants cultured with BAP combined with NAA developed adventitious rosette shoots. Shoots rooted on half-strength MS media, and the number of roots per plantlet and their length were heavily dependent on sucrose content. The in vitro regenerated plantlets were acclimatized ex vitro and a mean of 50% of the plantlets survived and showed a true-to-type growth habit. This study describes the development of two in vitro micropropagation protocols, via direct organogenesis and via embryogenesis from callus, that are the basis for the application of in vitro tools for the establishment of basal collections with representative genetic diversity and for the long-term storage of plant genetic material.     

<Emphasis Type="Italic">Arabidopsis tanmei/emb2757</Emphasis> embryo mutant is defective for in vitro plant morphogenesis

A mutation in the Arabidopsis TANMEI/EMB2757 (TAN) gene with an embryo defective phenotype was analysed for its effect on the morphogenic potential of somatic tissue cultured under in vitro conditions. The capacity for in vitro morphogenesis was evaluated using cultures of immature zygotic embryos, and seedling explants of the tan mutant and the parental Col-0 genotype. The explants were cultured on media supplemented with different plant growth regulators, and the capacity for two alternative pathways of morphogenesis, somatic embryogenesis (SE) and shoot organogenesis, was evaluated. Reporter genes (GUS, GFP) were used to monitor auxin and LEC2 and FUS3 gene activity in the tan explants. Moreover, the expression pattern of the TAN gene was analyzed during SE and in callus tissue of Col-0. It was indicated that the tan mutation resulted in a total lost of embryogenic and organogenic capacity of cultured tissues, suggesting the involvement of the TAN gene in basic cellular processes related to cell growth and differentiation. However, differential expression of the TAN gene during SE, and its increased activity at advanced stages of embryogenesis, implicate a specific role for the gene in the development of somatic embryos.     

Simple hormonal regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of Pogonatherum paniceum (Poaceae)

Pogonatherum paniceum (Poaceae) is a perennial plant with good potential for eco-recovery and ornamental function. This study presents in vitro culture systems of simple hormonal regulation of somatic embryogenesis and shoot organogenesis from mature caryopses. Mature caryopses of P. paniceum were grown on Murashige and Skoog medium with 3% sucrose (w/v) and various concentrations or combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Morphological development was analyzed by light microscope after histological sectioning. Four types of callus were induced by different concentrations of 2,4-D. Type I callus was regenerated via somatic embryogenesis; type II callus failed to produce any regeneration; type III callus had both somatic embryogenesis and shoot organogenesis capacities; and type IV callus only displayed shoot organogenesis capacity. Regarding hormone combinations used in this study, NAA only induced type IV callus and BAP only induced direct multiple shoot formation. The combinations of 2,4-D and NAA induced type III callus. Several of the regeneration pathways were simply controlled by one or two kinds of plant hormones. The established systems will be helpful for further research on the developmental mechanism of switch between somatic embryogenesis and shoot organogenesis.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Exogenous Spermidine Promotes Somatic Embryogenesis of <i>Cunninghamia lanceolata</i> by Altering the Endogenous Phytohormone Content

In order to study how exogenous hormones in C. lanceolata (gymnosperm) regulate somatic embryogenesis, we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic embryogenesis. By applying exogenous spermidine, we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone content. Based on these results, further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic embryogenesis. Our research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.     

Early expression of WUSCHEL is a marker for in vitro shoot morphogenesis in tobacco and <Emphasis Type="Italic">Beta palonga</Emphasis>

We have studied the role of growth regulators behind in vitro shoot organogenesis and somatic embryogenesis in two plant systems, viz. tobacco (Nicotiana tabacum L. var. Jayasri) and Beta palonga R.K. Basu & K.K. Mukh. We have also correlated the phenomena of de differentiation with the relative expression of WUS (WUSCHEL) gene in a time-dependent manner. The results indicated that early WUS gene expression is a definite marker for in vitro shoot organogenesis in tobacco and Beta both in direct and indirect modes of regeneration. Additionally, we have performed a comparative homology modeling and in silico structural analysis of WUSCHEL proteins of B. palonga, B. vulgaris, and Arabidopsis to find out the commonality of the ligand binding site. The amino acids of the binding sites were identical (Arginine, Tryptophan, Proline, Asparagine, and Tyrosine) in the three materials under study; except two additional amino acids (Isoleucine and Alanine) in B. vulgaris.     

Spontaneous spectinomycin resistance mutations of the chloroplast <Emphasis Type="Italic">rrn16</Emphasis> gene in <Emphasis Type="Italic">Daucus carota</Emphasis> callus lines

Bioballistic transformation of carrot (Daucus carota L.) callus cultures with a plasmid containing the aadA (aminoglycoside 3′-adenyltransferase) gene and subsequent selection of transformants on a selective medium containing spectinomycin (100–500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3′ end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.     

Efficient plant regeneration via somatic embryogenesis and organogenesis from the explants of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

High-frequency plant regeneration of C. roseus cv. ‘little bright eye’ via somatic embryogenesis and organogenesis from five out of six explants was standardized. Two factors were found to be important for regeneration: (1) the type of explants, and (2) the combination and concentrations of plant growth regulators. The highest regeneration percentage through somatic embryogenesis was obtained from mature zygotic embryo in MS medium supplemented with 7.5 μM of thidiazuron (TDZ). The mature embryo also regenerated efficiently via organogenesis in MS medium supplemented with either 2.5 μM TDZ or 5.3 μM α-naphthalene acetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA). Hypocotyl and cotyledon did not induce somatic embryogenesis and organogenesis in TDZ-containing medium but gave a maximum percentage of shoots in MS medium supplemented with 5.3 μM NAA and 2.2 μM BA. Stem nodes and meristem tips showed better regeneration via organogenesis in the medium supplemented with NAA and BA and in lower concentrations of TDZ.     

Ectopic overexpression of <Emphasis Type="Italic">Arabidopsis AtmiR393a</Emphasis> gene changes auxin sensitivity and enhances salt resistance in tobacco

To characterize the biological function of microRNA miR393 in tobacco, AtmiR393a gene was isolated from Arabidopsis using PCR and fused downstream to CaMV 35S promoter to make a plant expression construct 35S::AtmiR393a. The resultant construct was then introduced into tobacco with Agrobacterium-mediated transformation. Transgenic tobacco lines ectopically overexpressing AtmiR393a were successfully obtained. Transgenic lines L1 (a weak line), L2 (a middle line), and L3 (a strong line) were confirmed using stem-loop RT-PCRs and used to characterize the function of miR393 in tobacco. The results showed that L1, L2, and L3 exhibited reduced plant size and root length related to the WT control. In addition, seedling growth was less sensitive to IAA treatment and NaCl stress in three transgenic lines than the non-transgenic WT control. Furthermore, L1, L2, and L3 showed reduced phototropism relative to WT. Therefore, the biological function of miR393 is conserved in tobacco, just like in Arabidopsis. It regulates plant growth and development as well as the responses to environmental cues by influencing auxin sensitivity.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

Pattern analysis of stem cell differentiation during <Emphasis Type="Italic">in vitro Arabidopsis</Emphasis> organogenesis

Plant somatic cells have the capability to switch their cell fates from differentiated to undifferentiated status under proper culture conditions, which is designated as totipotency. As a result, plant cells can easily regenerate new tissues or organs from a wide variety of explants. However, the mechanism by which plant cells have such remarkable regeneration ability is still largely unknown. In this study, we used a set of meristem-specific marker genes to analyze the patterns of stem cell differentiation in the processes of somatic embryogenesis as well as shoot or root organogenesis in vitro. Our studies furnish preliminary and important information on the patterns of the de novo stem cell differentiation during various types of in vitro organogenesis.     

Tiba inhibition of <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in excised tobacco leaf explants

Summary Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.