Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.

Plants have capability to optimize its architecture by using CDK pathways. It involves diverse types of cyclin dependent kinase enzymes (CDKs). CDKs are classified in to eight classes (CDKA to CDKG and CKL) based on the recognized cyclin-binding domains. These enzymes require specific cyclin proteins to get activated. They form complex with cyclin subunits and phosphorylate key target proteins. Phosphorylation of these target proteins is essential to drive cell cycle further from one phase to another phase. During cell division, the activity of cyclin dependent kinase is controlled by CDK interactor/inhibitor of CDKs (ICK) and Kip-related proteins (KRPs). They bind with specific CDK/cyclin complex and help in controlling CDKs activity. Since cell cycle can be progressed further only by synthesis and destruction of cyclins, they are quickly degraded using ubiquitination-proteasome pathway. Ubiquitylation reaction is followed by DNA duplication and cell division process. These two processes are regulated by two complexes known as Skp1/cullin/F-box (SCF)-related complex and the anaphase-promoting complex/cyclosome (APC/C). SCF allows cell to enter from G1 to S phase and APC/C allows cell to enter from G2 to M phase. When all these above processes of cell division are going on, genes of cyclin dependent kinases gets activated one by one simultaneously and help in regulation of CDK pathways. How cell cycle is regulated by CDKs is discussed.

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm^-3 α-naphthalene acetic acid (NAA) + 0.5 mg dm^-3 6-benzyladenine (BA) for embryos production and 0.03 mg dm^-3 NAA + 0.5 mg dm^-3 BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5-42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.

Effect of plant growth regulators, explant size, season of explant collection, temperature (20, 25 and 30 °C) and photoperiod on in vitro lotus (Nelumbo nucifera Gaertn.) shoot formation and growth were examined. Shoots formation was greatly influenced by growth regulators, explant size and season of explant collection. The maximum number of shoots were induced from bud explants on Murashige and Skoog (MS) medium containing 4.44 µM benzyladenine (BA) + 0.54 µM α-naphthalene acetic acid (NAA). Explants formed by bud of one expanded and one unexpanded leaf, which was collected in spring gave encouraging results of shoot production. Higher temperature favoured shoot induction and subsequent growth was much better at 25 °C compared to that at 20 and 30 °C.

Effects of plant hormones indole-3-yl-acetic acid (IAA), gibberellic acid (GA), benzylaminopurine (BAP), abscisic acid (ABA) and ethrel (ETH) in 5 μM concentration on gas exchange, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39) activity, pigment content and yield in cotton (Gossypium hirsutum L. cv. H-777) under drought were studied. At reproductive stage (55 - 60 d after sowing) these hormones were sprayed on shoots one day prior to stress imposition by withholding irrigation. The soil moisture of control plants was kept at field capacity. Net photosynthetic rate (P_N), stomatal conductance (g_s), transpiration rate (E), carboxylation efficiency (CE), water use efficiency (WUE), RuBPCO activity, boll number per plant, seed number per plant and lint mass per plant significantly decreased at drought while chlorophyll (Chl) b content and flower number per plant increased. ABA and ETH significantly reduced gas exchange parameters, Chl a and Chl b content. Detrimental drought effect on P_N, g_s, E, CE, RuBPCO and lint mass per plant was significantly alleviated by BAP and also its effect on seed number and lint mass per plant was significantly alleviated with the ABA treatment.

Sugarcane is cultivated in tropical and subtropical regions where cold stress is not very common, but lower yields and reduced industrial quality of the plants are observed when it occurs. In our efforts to enhance cold tolerance in sugarcane, the gene encoding the enzyme isopentenyltransferase (ipt) under control of the cold inducible gene promoter AtCOR15a was transferred via biolistic transformation into sugarcane (Saccharum spp.) cv. RB855536. Semiquantitative RT-PCR using GAPDH encoding glyceraldehyde-3-phosphate dehydrogenase as the normalizer gene showed the increased expression of the ipt gene under cold stress. The detached leaves of genetically modified plants subjected to low temperatures showed visible reduction of leaf senescence in comparison to non-transgenic control plants. Induced overexpression of ipt gene also enhanced cold tolerance of non-acclimated whole plants. After being subjected to freezing temperature, leaf total chlorophyll contents of transgenic plants were up to 31 % higher than in wild type plants. Also, lower malondialdehyde content and electrolyte leakage indicated less damage induced by cold in transgenic plants. Thus, the expression of ipt driven by the stress inducible COR15a promoter did not affect plant growth while providing a greater tolerance to cold stress.

In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 μM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per explant can be obtained within 36 weeks from culture initiation.

The leaf apoplast is a dynamic compartment in contact with plant pathogenic bacteria after infection. Among the very first interaction events is the receptor-mediated perception of bacterial surface molecules such as flagellin or other conserved microbe-associated molecular patterns (MAMPs). Apoplast proteins likely play a role in basal resistance (BR) or pattern-triggered immunity (PTI). Here, a proteomic approach was carried out on water soluble - potentially the most mobile - apoplast proteins from flagellin-treated tobacco (Nicotiana tabacum) leaves. As the quickness of BR/PTI seems crucial for its efficacy, samples were taken as early as 2.5 and 7 h post inoculation. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Forty-nine different proteins from 28 protein spots changed in their density compared to the water-inoculated control. Eleven protein spots appeared de novo in response to EBR induction. There are glycohydrolases and redox-active proteins besides pathogenesis-related proteins among them, predicting plant cell wall structural modifications and more direct antimicrobial effectors as earliest changes related to BR/PTI.

Branching is an important step in higher plant development, which not only determines the configuration of the plant directly, but also affects its adaptability to the environment. The interactions between hormones, genes, environmental and other factors subtly regulate the process of branching. Strigolactone is a newly recognized phytohormone and its content and distribution might be a key factor affecting branching. This review is focused on the genes related to synthesis and transduction pathway of strigolactone, and summarizes the inhibitory role of strigolactone in plant branching. Discussions about the issues to be clarified and prospects of the future research were also proposed.

Small heat shock proteins (sHSPs) are crucial components of the plant response to heat shock. We identified and analyzed eight sHSP genes of Capsella bursa-pastoris to better understand the ability of this species to adapt. Eight genes were initially cloned and sequenced from the mature embryo cDNA pool. They belong to the cytosolic I (CI), cytosolic II (CII), and cytosolic III (CIII) subfamilies. One CI sHSP gene was homologous to that of C. rubella. Sequence analysis using 3' RACE revealed that there are two or more variable 3'-untranslated regions (UTRs) in these sHSP transcripts. The transcriptional levels of the eight sHSP genes were analyzed in different organs and developmental stages via qRT-PCR. Eight genes were significantly up-regulated in young leaves exposed to heat stress at 42 °C, and also showed differential responses to ABA treatment. We also compared expression of these genes with corresponding Arabidopsis sHSP genes and found some differences between the two species.

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm^-3 each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm^-3 kinetin.

Brassinosteroids (BRs) have been proposed to increase the resistance of plants to drought stress. The effect of foliar application of 0.1 μM 24-epibrassinolide (EBR) on chlorophyll (Chl) content, photosystem 2 (PS 2) photochemistry, membrane permeability, lipid peroxidation, relative water content (RWC), proline content, and the antioxidant system in drought-stressed Chorispora bungeana plants was investigated. The results showed that polyethylene glycol (PEG) induced water stress decreased RWC, Chl content and variable to maximum Chl fluorescence ratio (F_v/F_m) less in plants pretreated with EBR than in non-pretreated plants. In addition, lipid peroxidation, measured in terms of malondialdehyde content, membrane permeability and proline content in drought-stressed plants were less increased in EBR pretreated plants, while antioxidative enzyme activities and reduced ascorbate and glutathione contents were more increased in EBR pretreated than in non-pretreated plants. These results suggested that EBR could improve plant growth under drought stress

Leaf hydraulic conductance (K_leaf) plays a significant part in plant-water regulation. In walnut leaves, K_leaf is stimulated by irradiance and closely relates to the accumulation of JrPIP2s aquaporin transcripts, but it is independent of stomatal aperture. To provide an insight into the early molecular events occurred during light-induced K_leaf, a large-scale transcriptomic analysis consisting of the cDNA-amplified fragment length polymorphism (AFLP) was carried out on walnut leaves maintained under irradiance or in darkness. Of the total 12 000 transcript-derived fragments (TDFs) obtained using cDNA-AFLP with 128 primer pairs, 187 TDFs were selected after sequencing, and only 93 (49 %) that had been ascribed known functions through BLAST searching of the GenBank databases. Most of these TDFs correspond to genes whose protein products are involved in cellular regulation (57.9 %) and global metabolism (39.8 %). To validate cDNA-AFLP expression patterns, 30 TDFs were further analyzed using real-time quantitative polymerase chain reaction. Moreover, exposure of leaves to irradiance was accompanied by the modification of the Ca²⁺-signaling pathway, ubiquitin-proteasome pathway, vesicle trafficking process and expression of multiple transcription factors.

We investigated whether changes in abscisic acid (ABA) content in leaves of Mikania micrantha infected by the holoparasite Cuscuta campestris at five growth stages, influenced the host stomatal conductance (g_s), transpiration rate (E) and net photosynthetic rate (P_N). C. campestris infection caused a negative effect on g_s, E and P_N of the host plants. ABA content in host leaves infected by C. campestris was significantly lower at 6 d after parasitization (DAP) and significantly higher at 13 and 33 DAP, relative to uninfected controls. In the parasite, ABA content was lowest at 13 DAP and then sharply increased to the maximum at 26 DAP. Moreover, the ABA content in the parasite was always lower than in the infected host leaves. The results suggest that an increase in host ABA concentration contributes to reduced host g_s, E and P_N in the holoparasitic C. campestris - M. micrantha association.

Phytohormones are indispensable factors regulating plant cell dedifferentiation. In this paper, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) were incorporated in the culture medium and the anatomy of dedifferentiated cells prior to callus formation from Arabidopsis thaliana petiole explants was examined. The results indicated that the cytoplasm of parenchyma cells in the vascular bundle gradually became denser with time of culture only if 2,4-D was included in the medium. The WUSCHEL (WUS) gene was expressed in derivative cells of the vascular bundle after culture for 24 h in the presence of 2,4-D and there was no obvious signal in these cells of cultured petioles with KIN alone. These results suggest that 2,4-D plays an important role in the process of dedifferentiation of vascular bundle cells in Arabidopsis petioles and KIN has no obvious effect on it.

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.

An efficient in vitro regeneration protocol was developed for medicinally important aromatic plant Anethum graveolens. Nodal segments were cultured onto Murashige and Skoog (MS) basal medium supplemented with different auxins and cytokinins singly as well as in combinations. The optimum callus induction (93.33 %) was obtained on medium fortified with 2.2 μM N⁶-benzyladenine (BA) and 0.21 μM α-naphthaleneacetic acid. The best shoot regeneration (85.7 %) with 12.86 shoots per explant was achieved in two weeks when callus was subcultured on MS medium amended with 2.2 μM BA and 1.85 μM kinetin. The average length of regenerated shoots varied from 3.15 to 4.8 cm. The rooting of regenerated shoots was nearly 100 % on 1/4 MS augmented with 4.9 μM indolebutyric acid with a maximum root length of 5.1 cm. Plantlets were successfully acclimatized with 60 % survival rate. During organogenesis, catalase and ascorbate peroxidase activity increased while superoxid dismutase activity decreased. Clonal fidelity of in vitro raised plants has been checked by random amplified polymorphic DNA using 10 selected decamer primers. It has been found that regenerated plants are true to type plants.

We report here for the first time that fully differentiated stomatal guard cells have the ability to form directly secondary somatic embryos. Histological and scanning electron microscopy studies reveal that the stomata are formed on primary embryos of both Oncidium cultivars Gower Ramsey and Sweet Sugar. Secondary embryogenesis from these guard cells could be induced by several plant growth regulators (PGRs), including N⁶-benzyladenine, kinetin, thidiazuron, 1-aminocyclopropane-1carboxylic acid (ACC), ancymidol and 2,3,5-triiodobenzoic acid. When compared with other PGRs, ACC at concentration 1 mg dm^-3 resulted in highest number of secondary embryos that were derived from guard cells. The present communication provides a model system for studying factors and mechanism affecting totipotency or embryogenetic capacity of guard cells.

Cucumber (Cucumis sativus L.) is considered a model plant for the investigation of Fe deficiency responses, since it strongly exhibits typical strategy I, i.e. increased activities of Fe(III)-chelate reductase, H⁺-ATPase and iron regulated transporters. In this study, cDNA amplified fragment length polymorphism analysis was employed to identify genes differentially expressed in the root apex following Fe deficiency. The expression patterns of the most interesting transcript derived fragments were validated by semiquantitative reverse trascriptase - polymerase chain reaction. A set of new genes overexpressed under Fe deficiency, such as those coding for calmodulin, SNAP, TIM23 and V-PPase were identified. Furthermore, we also observed that calmodulin protein accumulated in Fe-deficient root apexes.

To improve large-scale in vitro production of Oplopanax elatus Nakai, we cultured somatic embryo-derived plantlets under a heterotrophic condition (semi-solid culture with sucrose), photoautotrophic condition (semi-solid culture without sucrose), or modified photoautotrophic condition (liquid culture with forced ventilation). The plantlets grown under the modified photoautotrophic condition had more leaves as well as higher chlorophyll content, and higher net photosynthetic rate than those grown under the conventional conditions. Further, the photoautotrophically grown plantlets acclimatized better and sooner upon ex vitro transplantation than did the conventionally cultured plantlets. Consequently, a photoautotrophic culture method with forced ventilation is effective for enhancing the growth and acclimatization of O. elatus.

Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.

We isolated PaAP3, a homolog of the class B MADS-box transcription factor gene APETALA3 (AP3), from the monoecious plant London plane tree (Platanus acerifolia Willd.). PaAP3 encodes a protein that shares good levels of identity with class B genes from Arabidopsis thaliana (35 and 51 % identity with PISTILLATA (PI) and AP3, respectively), and also with class B genes of other woody species (59 % identity with PTD from Populus trichocarpa and 66 % with TraAP3 from Trochodendron aralioides). Reverse transcription polymerase chain reaction showed that PaAP3 was expressed in both the female and male flowers of P. acerifolia, but almost no signal was detected in the vegetative tissues or mature embryos. The PaAP3 expression in male flowers showed a relationship with developmental stage. There was a small transient increase during differentiation of the flower primordia in June, but maximal levels occurred during December when flower development appeared arrested. Increased PaAP3 expression was also detected in March of the following year, corresponding to meiotic divisions of the microspore mother cells, but this was lost by April when the pollen was mature.

A modified low copy number plant binary vector pUN has been constructed and successfully used to clone unstable DNA sequences. The vector pUN comprises of low copy number, broad host range RK2 replicon from pBin19 and of multiple cloning site (MCS) and T-DNA region, both from a pBINPLUS-derived pLV06 vector. The absence of the ColE1 replicon in the backbone of the binary vector significantly contributed to stability of hardly clonable DNA sequences and enabled their transfer into the tobacco plants through Agrobacterium-mediated transformation.

Embryogenesis and plant regeneration was achieved from callus cultures derived from unpollinated ovaries of Psoralea corylifolia L. Callus was initiated from unpollinated ovaries on Murashige and Skoog (MS) medium supplemented with 2.2 µM N ⁶-benzyladenine (BA) and various concentrations of α-naphthaleneacetic acid (NAA (2.7 to 10.7 µM) or 2,4-dichlorophenoxyacetic acid (2,4-D (2.3 to 9 µM) alone or in combination. Highly organized embryogenic callus induction, embryo development, proliferation and maturation were achieved on transfer of callus clumps to MS medium supplemented with NAA (0.27 µM) or 2,4-D (0.23 µM) alone or in combination with BA (2.2 to 8.8 µM). Addition of abscisic acid (ABA) (0.95 to 5.8 µM) to the medium enhanced average numbers of cotyledonary stage embryos, the maximum number (34.6 ± 0.7) being obtained on MS medium containing 0.27 µM NAA, 2.2 µM BA and 3.8 µM ABA. Embryos germinated on MS medium supplemented with BA (0 to 8.8 µM). MS medium containing gibberellic acid (GA₃ (0.29 to 5.8 µM) enhanced embryo germination frequency, the highest frequency (66.7 %) occurring on MS medium containing 2.2 µM BA and 4.3 µM GA₃. Effect of several concentrations (3.0 to 6.0 %) of sucrose or maltose was also observed on germination of embryos. MS medium enriched with maltose supported high frequency of embryo germination.

The anatomic and functional leaf characteristics related to photosynthetic performance of Castanea sativa growing in vitro and in nursery were compared. The irradiance saturated photosynthesis in in vitro grown plantlets was significantly lower compared to nursery plants (65 vs. 722 μmol m^-2 s^-1). The maximum photosynthetic rate (P_Nmax) was 4.0 and 10.0 μmol(CO₂) m^-2 s^-1 in in vitro microshoots and nursery plant leaves, respectively. Carboxylation efficiency (C_E) and electron transport rate (ETR) were three-folds higher in nursery plants than in microshoots. The nonphotochemical quenching (NPQ) was saturated at 80 μmol m^-2 s^-1 in microshoots suggesting limited photoprotection by thermal dissipation. The microshoots had wide open, spherical stomata and higher stomatal density than nursery plants and they had almost no epicuticular wax. Consequently, the microshoots had high stomatal conductance and high transpiration rate. These anatomic and functional leaf characteristics are likely major causes of the low survival rates of plantlets after ex vitro transfer.

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. Here, we focused on the DNA methylation dynamics during the germination of wheat seeds using the adapted AFLP technique so called methylation-sensitive amplified polymorphism (MSAP). The MSAP profiles of genomic DNA in embryo and endosperm tissues of germinating seeds, as well as dry seeds were characterized and notable changes of cytosine methylation were detected. Comparisons of MSAP profiles in different tissues tested showed that the methylation level in dry seeds is the highest. The alteration analysis of cytosine methylation displayed that the number of demethylation events were three times higher than that of de novo methylation, which indicated that the demethylation was predominant in germinating wheat seeds, though the methylation events occurred as well. Sixteen differentially displayed DNA fragments in MSAP profiles were cloned and the sequencing analysis confirmed that nine of them contained CCGG sites. The further BLAST search showed that four of the cloned sequences were located in coding regions. Interestingly, three of the sixteen candidates were homologous to retrotransposons, which indicated that switches between DNA methylation and demethylation occurred in retrotransposon elements along with the germination of wheat seeds.

Zinc finger proteins are encoded by the genes chiefly involved in stress resistance hormone signal transduction of plants. In this study, a cDNA encoding a zinc finger transcription factor was isolated by the yeast one-hybrid system from Gossypium arboreum using the MYB-box element of the universal stress gene (GUSP1) promoter as bait. The corresponding protein (named GaZnF) can bind specifically to a 13 bp MYB-box region. The GaZnF cDNA is 1093 bp in length, including a 510 bp open reading frame. The predicted GaZnF protein contains ANI-A20 motifs and shares a high sequence similarity with zinc finger proteins from other plants. Spatial expression pattern of GaZnF was studied under drought, heavy metals and salt stresses through real-time PCR. The gene showed enhanced expression under each stress treatment with maximum transcript abundance in root tissues. The results support the hypothesis that G. arboreum zinc finger proteins are involved in plant response to drought, salt and heavy metal stresses.

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05-0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg's B5 medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 µM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 µM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 µM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.

A protocol for induction of direct somatic embryogenesis, secondary embryogenesis and plant regeneration of Dendrobium cv. Chiengmai Pink was developed. Thidiazuron (TDZ) at 0.3, 1 and 3 mg dm^-3 induced 5-25 % of leaf tip segments of in vitro grown plants to directly form embryos after 60 d of culture, and 1 mg dm^-3 TDZ was the best treatment. Somatic embryos mostly formed from leaf surfaces near cut ends, and occasionally found on leaf tips. Higher frequency of embryogenesis was obtained in light than in darkness. During subculture, secondary embryos developed from outer cell layers of primary embryos. All combinations of NAA (0, 0.1, 1 mg dm^-3) and TDZ (0, 0.3, 1, 3 mg dm^-3) increased the multiplication rate of embryos. It takes about 8 months from embryo induction, plantlet formation to eventually acclimatization in greenhouse.

Large-scale gene expression analyses were conducted during embryonic development of Capsella bursa-pastoris using the cDNA-AFLP technique, and 231 differentially expressed genes were identified. Most of the transcript-derived fragments (TDFs) were identified against Arabidopsis sequences and a minority against other plant sequences by searching in NCBI database. A total of 128 TDFs, homologous to genes with known functions, were classified into 13 functional categories, and many of them were involved in transcription, metabolism, disease defense and protein binding. Expression profiles of 90 C. bursa-pastoris genes were compared with those of the corresponding Arabidopsis genes and 64 genes showed similar expression profiles and the expression profiles of the rest 26 TDFs were different from those of the relevant Arabidopsis genes. Besides, some genes related to oxidative stress and ABA response were fiercely up-regulated during embryogenesis of C. bursa-pastoris according to RT-PCR.