Optimization of in vitro conditions for stigma-like-structure production from half-ovary explants of Crocus sativus L

Summary Studies were conducted to optimize the in vitro production of stigma-like-structures (SLS) that yielded the important biochemical constituents responsible for the color, taste, and aroma naturally found in the stigmas of autumn crocus. Immature half-ovary explants were evaluated for the frequency of proliferation of SLS by culture on five basal media supplemented with different combinations of plant growth regulators and vitamins. The optimum proliferation of SLS was observed on B5 basal medium containing α-naphthalene acetic acid (NAA) (5.4 μM), N⁶-benzyladenine (BA) (44.4 μM), MS organics, casein hydrolysate (0.05%), and l-alanine (11.2 μM) 50–60 d after inoculation. Some explants formed other structures (roots, corms, petals, leaves), the growth and development of which substantially reduced the development of SLS. Removal of brown tissues and other tissues during subculture (3-wk intervals) allowed continuous culture of halfovary explants for 9–10 mo. SLS that had deep red pigment and reached more than 0.5 cm length were removed from the explants and with concomitant reculturing of the ovaries, enabled SLS to be harvested three times. Activated charcoal (1%) added to B5 basal containing NAA (5.4 μM), BA (44.4 μM), and sucrose (3%) was found to be a helpful addendum to prevent browning of explants and accelerate the initiation, growth, and development of SLS. The amounts of crocin, crocetin glucosyl esters, picrocrocin, and safranal in SLS, as determined by high performance liquid chromatography analysis, were found similar to those in natural saffron.     

Micropropagation of Rollinia mucosa (JACQ.) baill

Summary A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l⁻¹ polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l⁻¹ sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.     

3种抗褐化剂影响水曲柳体胚发生的生理分析

水曲柳体胚发生过程中往往伴随褐化现象的产生,并且大多体胚生长在褐化的外植体上,为解析外植体褐化和体胚发生之间的联系,本研究通过在培养基中添加PVP（聚乙烯毗咯烷酮）、L-Glu（L-谷氨酸）和AgNO₃（硝酸银）,探究其对外植体褐化、体胚发生和生理生化的影响。研究结果表明：（1）低浓度（0.1、0.5 g·L^-1）PVP和100 mg·L^-1 L-Glu处理加剧了外植体褐化,但显著促进了体胚发生,并且体胚发生率高达60%以上（分别比对照组提高了6.59%、24.08%和22.88%）;（2）200 mg·L^-1 L-Glu处理有效降低了外植体褐化,褐化率为68.11%（相比对照组降低了5.83%）,但是体胚发生率降低,其发生率为46.32%（相较于对照组降低了22.8%）;（3）3种抗褐化剂处理后外植体细胞内的多酚氧化酶活性（PPO）和超氧化物歧化酶（SOD）活性均低于对照组,过氧化物酶（POD）活性和丙二醛（MDA）含量均高于对照组。因此,研究表明低浓度的抗褐化剂可以促进体胚发生,在这个过程中涉及到POD活性升高和MDA含量增加。本研究为解析水曲柳体胚发生伴随外植体褐化的生物学机理以及水曲柳体胚高频发生提供理论依据。     

Influence of form of activated charcoal on embryogenic callus formation in coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis>)

Development of micropropagation protocols for Cocos nucifera has progressed slowly. Activated charcoal is included in the culture medium of each protocol, mainly to prevent tissue browning. Charcoal production procedures can affect the properties of different brands. In this study, eight types of activated charcoal were evaluated for their effects on free 2,4-dichlorophenoxyacetic acid level, pH, conductivity, and osmolarity of the culture medium and on the frequency of embryogenic callus induction. Moreover, the effect of particle size of the optimum charcoal type on embryogenic callus development was also studied. Charcoal type had a significant effect on (Y3) culture medium properties. Free 2,4-D was highest in Reactivos y Productos Químicos Finos-containing medium and pH was lowest in MERCK-containing medium. Charcoal type also influenced embryogenic callus induction, with acid washed for plant cell and tissue culture-, DARCO- and United States Pharmacopeia-containing media promoting ~60% embryogenic callus, but with different optimal 2,4-D concentrations. Particle size profiles varied among all charcoal types, although small particle fraction (<38 μm) was abundant in all. Use of small particle fractions produced higher frequencies of embryogenic callus (70%) than either large particle or whole charcoal fractions.     

The Effect of Activated Charcoal Supplemented Media to Browning of In Vitro Cultures of Piper species

With the aim to effectively minimise the browning of tissue cultures of different Piper species (P. longum, P. attenuatum, P. betle, P. nigrum) explants from stem (nodal and internodal segments), petiole, and leaf were planted on Murashige and Skoog's basal medium supplemented with activated charcoal (AC). AC in the concentration of 200 mg dm^-3 proved to be the best. Among the taxa studied, P. longum showed the least browning whereas, wild P. nigrum showed maximum browning.     

Improved culture ability of potato protoplasts by use of activated charcoal

Protoplasts were obtained from in vitro grown plants of Solanum tuberosum L. The protoplasts were cultured in X-plate petri dishes with the culture medium joined to a reservoirmedium. When activated charcoal was added to the reservoirmedium the culture ability of the protoplasts was significantly increased. The effect of activated charcoal was mainly due to a less pronounced browning of the developing protoplasts and this technique might be of help in protoplast cultures where browning is a problem.     

Cultural manipulations affecting callus formation from seedling explants of the pearl lupin (Lupinus mutabilis Sweet)

The influences of several cultural factors were investigated in relation to callus formation from seedling explants of L. mutabilis Sweet. The interaction of low culture temperature (18°C), light differences, rapid callus transfer, and media additions led to the successful abolition of media browning which usually accompanied callus senescence. The supplement of activated charcoal slowed down the growth rate of the callus produced, and made it appear friable. Low levels of picloram and dicamba were found to produce large quantities of friable callus especially when combined with kinetin. The availability of nitrogen, as well as type of explant, markedly influenced callus induction and growth, whilst the addition of glutamine enhanced callus formation.     

Influence of some adjuvants on <Emphasis Type="Italic">in vitro</Emphasis> clonal propagation of male and female jojoba plants

Summary The influence of different adjuvants, activated charcoal (AC), casein hydrolyzate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA), has been assessed on the shoot production potential of the nodal explants derived from in vitro-raised male and female jojoba (Simmondsia chinensis) shoots. Nodal explants of each sex were cultured separately on Murashige and Skoog medium supplemented with different levels of AC, CW, CH, PVP, and TIBA either alone or along with optimum levels of N ⁶-benzyladenine (BA; 10 μM for male, 20 μM for female). Some differences in response of the explants of both the sexes have been observed in terms of (1) percentage of explants developing shoots, (2) average shoot number, and (3) average shoot length. AC alone proved beneficial for elevating morphogenic response in male as well as female explants in comparison to basal medium or media containing AC and the optimum level of BA. When used alone, CH proved inhibitory for shoot differentiation in both sexes, especially in male explants. Addition of PVP to MS enhanced shoot proliferation in female explants only, but along with BA it increased the response of male explants. BA in combination with different levels of TIBA promoted shoot multiplication in female explants. Thus, explants of both male and female shoots exhibited differential morphogenic behavior under the influence of various adjuvants. However, BA alone proved to be the best for differentiation of shoots in both male (10 μM) as well as female (20 μM) explants.     

四数九里香组织培养(简报)

以四数九里香新生带腋芽茎段为外植体,研究取材时期、6-BA浓度及生长素种类对外植体生长的影响,探讨不同浓度活性炭对外植体褐化的影响。结果表明,四数九里香组织培养适宜的取材时期为4月;外植体腋芽诱导率在6-BA浓度为2.5 mg·L^-1时表现较高;IBA是较适宜外植体生长的生长素;活性炭浓度为1.5 g·L^-1可有效减轻外植体褐化;组织培养适宜的启动培养基为MS + 6-BA 2.5 mg·L^-1 + IBA 0.1 mg·L^-1 + GA3 0.3 mg·L^-1 + 活性炭1.5 g·L^-1。     

Studies on the Browning of Kori-tofu

Kori-tofu was stored under RH 95% at 60°C and the changes of its nitrogenous compounds in the course of browning were examined. The water-soluble and 10% TCA-soluble fractions prepared from the ether extraction residue of Kori-tofu were analyzed. The nitrogen content of water-soluble fraction temporarily decreased and then increased gradually in the course of browning, while that of 10% TCA-soluble fraction increased gradually during the browning process. Amino acid analyses of 10% TCA-soluble fraction and its hydrolyzate revealed that no free amino acid was detected in unbrowned Kori-tofu, whereas in browned Kori-tofu occurred peptides, ammonia and free amino acids such as aspartic acid, serine, glutamic acid, glycine and alanine.

Free amino acids similar to those detected in browned Kori-tofu occurred in the model system consisting of lysozyme and methyl linoleate, crotonaldehyde, glyoxal, methyl-glyoxal and also the brown substance prepared from glycine and methyl linoleate during storage. The formation mechanism of free amino acids from proteins was discussed.     

Unfertilized ovary: a novel explant for coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) somatic embryogenesis

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 μM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 μM abscisic acid, followed by plant regeneration medium (with 5 μM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.     

High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants

An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal.     

Effects of sodium nitroprusside on callus induction and shoot regeneration in micropropagated Dioscorea opposita

The effects of sodium nitroprusside (SNP) on callus induction and shoot regeneration of Dioscorea opposite Thunb. have been studied. Application of 40 μM of SNP depresses accumulation of H₂O₂ in tuber explants of Dioscorea opposita markedly. Supplementation of 40 μM of SNP to the Murashige and Skoog medium with combinations of benzylaminopurine (3 mg dm⁻³) and naphthaleneacetic acid (0.5 mg dm⁻³) reduces the browning of explants and increases the frequency of callus induction from tuber explants significantly. The regeneration frequency of adventitious shoot shows a significant increase in the presence of SNP. Further analysis indicates that treatment with 40 μM of SNP results in significant decreases in catalase and peroxidase activity, while increasing the activity of superoxide dismutase. Supplementation with 40 μM of SNP also promotes the accumulation of non-enzymic antioxidants, including proline and glutathione. The effects on callus induction and shoot regeneration promoted by SNP were reversed by the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)- 4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide. These results indicate that the exogenously applied NO-donor SNP alleviates browning of tuber explants by reducing H₂O₂ accumulation, thereby promoting a higher in vitro proliferation frequency of D. opposita.     

In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal

The ability for adventitious rooting of micropropagated shoots from juvenile and mature Pinus pinaster Ait. explants was assessed in vitro on a rooting expression medium. The different rooting traits observed, namely the rooting rate, the number and the length of the adventitious roots, and the root score, were greatly influenced by the age of the donor plant: 98% of juvenile explants rooted, while only 49% of mature explants did. Addition of activated charcoal in the rooting expression medium improved the overall rooting capacity of the mature explants to an average of 78%. Whatever the plant material, the number and the length of the adventitious roots, as well as the root score, fluctuated according to the sampling date.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid - REM rooting expression medium - RIM rooting induction medium     

Factors influencing somatic embryogenesis induction in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill.: basal medium and anti-browning agents

The low induction rates of somatic embryogenesis (SE) in Eucalyptus globulus hamper scaling up the process for commercialization. We analyzed the effectiveness of several media (MS, 1/2MS, B5, WPM, DKW and JADS) during SE induction and expression. MS and B5 were the best media for SE induction and embling regeneration. In general, MS was the best medium for expression, independently of the medium previously used during induction. Several anti-browning compounds (ascorbic acid, charcoal, DTE, DTT, PVP, PVPP and silver nitrate) were added to the expression medium (MS), but all decreased SE potential and only DTE, charcoal and silver nitrate reduced explant browning. When added only during the induction period, anti-browning agents reduced accumulation of phenolics but also severely reduced SE potential. Continuous exposure completely inhibited the SE response. The negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.     

An efficient method for regeneration of lavandin (Lavandula x intermedia cv. 'Grosso')

An efficient protocol for the regeneration of lavandin (Lavandula x intermedia cv. 'Grosso') is reported. Thiadazuron (9 μM), a plant growth-modulating phenylurea, was used to induce callus formation and shoot initiation from cultured leaf explants. Newly emerged shoots were maintained on media containing 0.05 μM naphthaleneacetic acid to allow maturation, and then transferred to media containing 2.9 μM indole-3-acetic acid to allow root formation. The phenolic control agents polyvinylpyrrolidone (PVP), ascorbic acid, 2-aminoindane-2-phosphonic acid, and activated charcoal were tested for their ability to prevent shoot browning and death in culture. All agents except PVP were found to be effective, with ascorbic acid being most consistent in promoting development of healthy mature shoots. The effect of light type (red light vs. white light) and culture medium composition (full- and half-strength Murashige and Skoog or Llyod and McCown’s woody plant medium (WPM)) on rooting efficiency was also evaluated. Cultures on half-strength WPM in white light were found to have the highest rooting efficiency. Additionally, application of the polyamines putrescine, spermine, and spermidine were tested for their effect on rooting. While rooting efficiency was not improved with any of the treatments, spermine and spermidine were found to have an inhibitory effect at concentrations greater than 10 μM.     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

Rapid germination and development of <Emphasis Type="Italic">Taxus baccata</Emphasis> L. by <Emphasis Type="Italic">in vitro</Emphasis> embryo culture and hydroponic growth of seedlings

A highly promising procedure to obtain seedlings of Taxus baccata L. has been developed, which involves a combination of in vitro embryo culture and growth under hydroponic conditions. Embryos isolated from freshly collected seeds were 100% sterile, even though the seeds were not treated with acid or soaked in water prior to culturing. The embryo germination level of non-leached seeds was slightly lower (85%) than those leached in running water for 7 d (100%). The leached embryos germinated with extended roots while the non-leached embryos had abnormal shapes. The embryos cultured on media supplemented with an absorbent (PVP or activated charcoal) had extended roots and shoots and were a larger size without any browning, as compared to those grown without the supplement; activated charcoal gave better results. There were no significant differences in germination rates of T. baccata embryos between the media with differing strengths of macronutrients; however, for further development of the shoot, it was necessary to sub-culture the seedlings in MS in the light. To obtain seedlings with longer roots, they had to be maintained in one-half strength MS in darkness. Approximately 90% of the plants survived when grown hydroponically for 2 mo. The surviving plants showed well-extended roots and were a good starting material for genomic, proteomic, and conservational studies as well as Taxol permeabilization investigations.     

景宁木兰组织培养外植体选择与抗褐化研究

该研究以景宁木兰(Magnolia sinostellata)为材料,从外植体类型的选择、消毒时间、预处理方法、离体培养条件和抗褐化剂类型选择等方面进行综合研究,以期筛选出景宁木兰组织培养的最佳方案。结果表明:利用0.1%氯化汞(HgCl_2)浸泡处理14和8 min分别是景宁木兰叶片和带芽茎段、根部的最佳消毒时间;景宁木兰叶片及茎段的褐化率在暗培养7 d时,分别为60.00%和56.67%,14 d时显著升高,而根部则在暗培养14 d时褐化率最低(45%);利用1 g·L~(-1)聚乙烯吡咯烷酮(PVP)浸泡预处理景宁木兰外植体6 h可显著降低(P0.05)其褐化程度,处理后叶片、带芽茎段和根部的褐化率分别降至45.00%、28.33%和63.33%。不同的抗褐化剂均可减轻外植体的褐化程度,但针对不同外植体其效果不同。景宁木兰叶片最佳抗褐化剂为0.02 g·L~(-1)硝酸银(AgNO_3),可使其褐化率降低至16.67%,成活率为11.67%;带芽茎段和根部的最优抗褐化剂为2 g·L~(-1)柠檬酸(CA),褐化率分别降至30.00%和5.00%,成活率依次为50.00%和76.67%。综上所述,带芽茎段和根部抗褐化处理后褐化率较低且成活率较高,为景宁木兰组织培养最佳外植体。     

The Effect of Continued Training with Crocin on Apoptosis Markers in Liver Tissue of High Fat Diet Induced Diabetic Rats

Diabetes mellitus (DM) disease can affect process of apoptosis by increasing oxidative stress, nevertheless exercise and crocin can improve apoptosis; therefore present study aimed to investigate the effect of continued training with crocin on apoptosis markers in liver tissue of diabetic rats. In this experimental study 32 diabetic rats based on fasting glucose divided into four groups of eight rats including: 1) sham, 2) training, 3) crocin, and 4) training with crocin also for investigate the effect of DM induction on apoptosis markers, eight healthy rats assigned in healthy control group. During eight weeks groups 2 and 4 ran 60 minutes on treadmill with intensity of 50–55% maximum speed for three sessions per week and groups 3 and 4 received 25 mg/kg/day crocin peritoneally. Shapiro–Wilk, one-way ANOVA with Tukey’s post-hot tests were used for statistical analysis of data (P ≤ 0.05). DM induction significantly increased Bcl-2 as well as decreased Bax and P52 (P ≤ 0.05) nevertheless training and training with crocin significantly decreased Bcl-2 and increased Bax and P53 (P ≤ 0.05); crocin significantly decreased Bcl-2 and increased P53 (P ≤ 0.05) and training with crocin had higher effect on increase of Bax and P53 compare to training (P ≤ 0.05) also increase of Bax compare to crocin. Although training and crocin alone can improve apoptotic markers in diabetic rats, nevertheless training simultaneously with crocin have better effects than training alone.