Plant regeneration of an endangered medicinal plant Hydrastis canadensis L.

Goldenseal (Hydrastis canadensis L.) is an endangered medicinal plant used to treat sore eyes and mouths, cold and flu and also as a dye. The objective of this study was to develop an efficient in vitro propagation protocol for goldenseal. Significantly more shoots (26 shoots per leaf explants) were induced on a medium containing 2.5 μM thidiazuron (TDZ) and 5.0 μM 1-naphthaleneacetic acid (NAA) than any other treatment. Sub-culturing regenerated shoots on a medium with 5.0 μM 6-benzylaminopurine (BA) induced the maximum rate of shoot multiplication. Growth of the regenerated shoots in a temporary immersion bioreactor resulted in significant increases in biomass, shoot height and shoot multiplication. The regenerated shoots from the temporary immersion bioreactor formed roots when transferred onto a medium with 1.0–2.0 μM indole-3-butyric acid (IBA). Regenerated whole plantlets were acclimatized and maintained in standard greenhouse conditions for further growth. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of this rare, medicinally important species.     

Two-way germination system of encapsulated clonal propagules of Vitex trifolia L.: an important medicinal plant

In the present study we have developed an efficient and effective method of synthetic seed production and its two-way germination system of Vitex trifolia, for easy transport of the propagules and efficient utilization of its in vitro regeneration system. Nodal segments harvested from 8-week-old in vitro cultures were encapsulated in calcium alginate beads. Three percent (w/v) Na-alginate polymerized in 100 mmol/L CaCl₂.2H₂O for 30 min produced clear and uniform beads. Germination of encapsulated beads with shoot and roots was achieved on Murashige and Skoog (MS) medium augmented with 6-furfurylaminopurine (KN, 2.5 µmol/L) + α-naphthalene acetic acid (NAA, 1.0 µmol/L). For multiple shoot production, synseeds were incubated on 6-benzyladenine (BA, 5.0 µmol/L) + NAA (0.5 µmol/L) augmented MS medium followed by in vitro rooting on MS + indole-3-butyric acid (IBA, 1.0 µmol/L). The synseeds produced retained about 90% regeneration potential even after 4 weeks of storage at 4°C. Genetic stability of the regenerated plants was evaluated using 13 inter simple sequence repeats (ISSR) primers. The study thus provides an efficient system for production of synthetic seeds, their storage and subsequent conversion into genetically identical plants.     

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

Summary

Secondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

In vitro grafting of cashew (Anacardium occidentale L.)

A successful micrografting technique in cashew was developed using in vitro germinated seedlings as rootstocks and axenic shoot cultures (shoot-tip and nodal cultures) established from mature tree source as microscions. In vitro germinated seedlings, which emerged 20–25 days after inoculation on absorbent cotton, were decapitated and used as rootstock. Mature tree explants initiated on hormone-free Murashige and Skoog [Physiol. Plant. 15 (1962) 473] (MS) modified medium were made into scion of 3–15 mm length for grafting. Micrografts could be easily cultured on hormone-free liquid half-MS medium and were potted out after 10–12 weeks of culture growth. Grafting success was dependent on the method of grafting and size of the scion. Shoot-tip grafting and side grafting were equally successful (79.5–100%). Length of scion shoot had significant effect on micrografting success. Graft success was high (79.5%) when the scion length was >5 mm and it was less (0.5%) when size of scion was small (3–5 mm). Scion presoaked in either water or 0.01% ascorbic acid and 0.015% citric acid (1:1) reduced phenolic browning and drying of scion. Micrografting techniques standardized could be used for rejuvenation of shoot explants of mature tree.     

Effects of culture conditions on in vitro bulblet induction in the medicinal plant Amana edulis (Miq.) Honda

Amana edulis (Miq.) Honda is an important medicinal plant with a variety of anti-cancer properties, and it is of great importance to improve its reproductive rate through micropropagation technology to meet increasing demand. In the present study, in order to establish a rapid in vitro bulblet propagation protocol for A. edulis, an L₁₆ (4⁵) orthogonal design was used to investigate the effects of sucrose, 6-benzyladenine (6-BA), α-naphthaleneacetic acid (NAA), and macro-elements on bulblet induction. The results show that the sucrose concentration was the crucial factor for A. edulis bulblet initiation, followed by 6-BA and macro-elements, while NAA had the weakest effect. The optimal medium for in vitro bulblet induction was Murashige and Skoog medium supplemented with 0.1 mg·L^?1 6-BA, 0.01 mg·L^?1 NAA, and 100 g·L^?1 or 80 g·L^?1 sucrose (pH 5.8), in which A. edulis shoot clusters (without roots) were cultured at 5(±2)°C for 35 d or 60 d and then incubated at 23(±2)°C for 90 d. The entire cultivation process occurred in the dark. The present study demonstrates that this protocol can be used for the propagation of A. edulis.     

阔叶猕猴桃叶片离体器官发生和植株再生(英文)

以阔叶猕猴桃(A ctinidia latifolia M err.)叶片为外植体,通过器官发生途径诱导形成不定芽,探讨了不同激素组合、暗培养时间以及不同浓度的蔗糖对叶片器官发生和植株再生的影响。结果表明,BW +0.1μm ol/L N AA +5μm ol/L Zeatin 附加20g/L 蔗糖,先进行21d 暗培养然后转到光下培养效果最好,6周后不定芽再生频率达91.67%,平均每个外植体再生芽数6.87个。再生芽生根良好,生根率可达100%。首次报道了阔叶猕猴桃的器官发生和植株再生,建立了叶片的高效再生体系,为今后的遗传转化研究奠定了基础。     

余甘子下胚轴离体培养中的器官形成与植株再生(英文)

以余甘子(phyllanthusemblicaL.)下胚轴为外植体,通过器官发生途径诱导形成不定芽,探讨不同激素组合对下胚轴器官发生和植株再生的影响,以期建立有效的再生体系,为以后的遗传转化研究奠定基础。结果表明,在附加0.1μmol/LNAA、5μmol/LBA和30g/L蔗糖MS培养基上培养,5周后不定芽再生频率达87.5%,平均每个外植体再生芽数为9.7个,为最佳组合。将分化的不定芽转移至含有10μmol/LIBA的MS培养基上,5周后生根,发育成健康的植株,生根率在29.2%。     

香榧茎段离体培养再生植株的研究

对香榧茎段不定芽诱导研究的结果表明,不定芽诱导的最佳培养条件为:培养基为B5+KT0.1mg/L+IBA0.5mg/L+0.08%活性炭+2%葡萄糖,培养温度为20℃,光强不高于400lx,该条件下的不定芽诱导率可达55%。将不定芽培养在1/2B5+IBA0.1mg/L+0.08%活性炭+2%葡萄糖的生根培养基上,生根率最高达到40%。通过器官发生途径国内外首次建立了香榧离体再生体系和快繁体系,并获得了完整的再生植株,为今后开展香榧的遗传转化和品种改良奠定了基础。     

In vitro propagation of Pistacia vera L. from seedling tissues

Proliferating shoot cultures were established from shoot tips and nodal bud segments excised from seedlings germinated aseptically and cultured on Murashige and Skoog medium supplemented with BAP plus NAA. Shoot tip necrosis occurred in some cultures. Cultured shoots were rooted in vitro using MS medium (half strength macronutrients) containing IBA for root initiation, followed by subculture onto hormone-free medium for root development. Rooted shoots were readily established in peat-based compost.     

蓬溪县药用植物资源调查及开发利用

采用实地考察、走访和查阅文献等方法,对蓬溪县药用植物资源状况进行了调查。结果表明蓬溪县共有药用植物241种,其中菌类3种,地衣1种,苔藓2种,蕨类10种,裸子植物5种,被子植物220种。其中较有特色的有川白芷,半夏、黄姜、栝楼和白芨等。针对药用植物资源变化情况,应采取合理采挖,加强药用植物升值研究,扩大人工种植,积极开展异地保护等措施合理开发蓬溪县的药用植物资源。     

The complete chloroplast genome sequence of the folk medicinal and vegetable plant purslane (Portulaca oleracea L.)

Portulaca oleracea is an important and widely distributed medicinal and edible plant that has great economic value in the medical and food industries. We sequenced the complete chloroplast (cp) genome of P. oleracea,whichis156,533 bp in length, including a pair of inverted repeats (IRs) of 25,501 bp separated by a large single copy (LSC) region of 87,436 bp and a small single copy (SSC) region of 18,095 bp. The genome contains 85 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. There are 40 repeat units and 111 simple sequence repeats (SSRs) in the genome. We also carried out complete cp genome comparison with other Caryophyllales species. The size, gene content, and structural organisation of P. oleracea are relatively conserved, but the genes ycf1 and ycf2 have larger introns. The simple sequence repeats (SSR) in the introns of ycf3, ycf1, atpB, rrn23 and ndhH have significant polymorphism. Comparisons of IR boundaries among 8 Caryophyllales species showed contraction and expansion. The complete cp genome sequence will contribute to a better understanding of the evolutionary mode of the cp genome and to the development of new markers for Caryophyllales classification.     

In vitro shoot multiplication and plant regeneration in four Capsicum species using thidiazuron

A procedure of in vitro plant propagation using shoot meristem explants (∼0.5 cm) has been developed for Capsicum annuum cv CA960, C. baccatum, C. frutescens and C. praetermissum on Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497] medium containing various cytokinins. Among various concentrations of cytokinins tested; adenine (Ad), N⁶-benzyladenine (BA), kinetin (Kn), zeatin and thidiazuron (TDZ) individually. TDZ regenerated maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM l⁻¹) + IAA (0.48 μM l⁻¹). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM l⁻¹ indole-acetic acid (IAA). Rooting was observed in 72–94% of shoots obtained from TDZ-containing regeneration medium followed by elongation treatment in contrast to 8–22% of shoots without elongation treatment. Plantlets obtained from TDZ-containing media were normal diploid (2n = 24) and could readily be established in the soil under green house conditions with a survival frequency of 68–84%. Regenerated plants were developed into morphologically normal, fertile plants and able to set viable seeds.     

In vitro callus-induction in globe artichoke (Cynara cardunculus L. var. scolymus) as a system for the production of caffeoylquinic acids

Summary

Globe artichoke (Cynara cardunculus L. var. scolymus) provides a rich dietary source of bio-active compounds derived from phenylpropanoid metabolism, notably caffeoylquinic acids (CQAs) and flavonoids. Micropropagation techniques have been established for this species, but in vitro cultures have not yet been extended to generate an efficient system for the induction of callus tissue. In this study, we compared more than 100 combinations of media supplements (e.g., phytohormones, absorbers of polyphenols, and inhibitors of polyphenol oxidase), along with various light regimes, and three different genotypes of globe artichoke to define the optimal conditions for callus induction from leaf explants. This led to the elaboration of an in vitro culture protocol which resulted in a high frequency of callus induction after just 1 week in culture. The procedure used leaf explants from virus-free, meristem culturederived plantlets. Quantitative HPLC analysis revealed that, as in globe artichoke leaves, the predominant phenolic esters present in callus were mono- and di-caffeoylquinic acids (diCQA). The concentration of diCQA was three- to five-fold higher in calli than in leaves. The exposure of calli to UV-C light further enhanced the levels of CQAs. In vitro callus culture combined with UV-C irradiation may thus represent a viable production system for diCQA that is suitable for the synthesis of pharmacologically-active compounds.     

In vitro somatic embryogenesis from Mangifera indica L. callus

The nucellus and globular adventitious proembryos were removed from 2-month-old fruits of mango (Mangifera indica L.) cultivars ‘Ono’ and ‘Chino’, and were cultured on sterile, solid Murashige and Skoog (MS) medium that had been modified as follows: half-strength major salts and chelated iron; 20% (v/v) coconut water (CW); 6% sucrose; 100 mg l^?1 ascorbic acid and 400 mg l^?1 glutamine. Embryogenic explants were sub-cultured after 4–6 weeks in liquid modified MS medium containing 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) instead of CW. Rapidly growing cultures were established and were sub-cultured monthly. Somatic embryogenesis was induced following sub-culture from MS medium with 2,4-D to MS without growth regulators and with or without activated charcoal (0.5%). Germination of somatic embryos appeared to be enhanced by 1 mg l^?1 benzyladenine (BA); however, most of the germinating embryos became embryogenic.     

黑莓试管苗叶片植株的再生

以黑莓带芽茎段试管苗叶片为外植体,诱导形成不定芽并进一步形成再生植株。在MS+TDZ2.0mg/L的培养基中叶片不定芽最高分化频率达93.7%(6.52不定芽/外植体),在不定芽伸长培养基BA0.5mg/L上,分化完全的不定芽能够长大伸长,当其高度达到4cm时,切下转接于IBA为0.3mg/L培养基上诱导生根,生根率100%。     

In vitro production of Prunus necrotic ringspot virus-free begonias through chemo- and thermotherapy

Prunus necrotic ringspot virus (PNRSV)-free Begonia spp. plants were raised from petioles of virus-infected plants using in vitro techniques. The petioles were grown on MS medium supplemented with 0.2 mg/l NAA and 0.2 mg/l BAP (pH 5.8). For rooting, half-strength MS medium without any plant growth regulators was used. On rooting medium, shoots were subjected to chemotherapy (virazole, 2-thiouracil or 6-azauracil) and thermotherapy (38 °C for 16 h light period and 22 °C for 8 h dark period) separately or in combination. Regenerated plants (treated with chemo- and thermotherapy) were indexed for PNRSV by DAS-ELISA and RT-PCR. An amplified product of 785 bp was obtained by RT-PCR in PNRSV-infected plants. Virazole at a concentration of 20 mg/l was found to be more effective (30 and 20% of PNRSV-free plants as indexed by ELISA and RT-PCR, respectively) in comparison to the other chemicals. Thermotherapy for 25 days gave 35 and 25% PNRSV-free plants as indexed by DAS-ELISA and RT-PCR, respectively. A combination of both treatments gave a good number of PNRSV-free plants (67.5 and 57.5% as indexed by DAS-ELISA and RT-PCR, respectively). At higher concentrations all three chemicals were found to be toxic. Thermotherapy for more than 25 days caused browning of leaves and shoots died.     

Metabolic stability of plants regenerated from cryopreserved shoot tips of Dioscorea deltoidea – an endangered medicinal plant

Shoot tips of Dioscorea deltoidea, a medicinal yam, were cryopreserved using the vitrification and encapsulation-dehydration technique resulting in high-frequency direct plant regeneration. The biochemical stability of the plants derived from cryopreserved shoot tips was assessed using HPLC analysis. The diosgenin content of plants regenerated from cryopreserved shoot tips was found to be same as that of control plants. Thus plants confirming our cryopreservation experimental conditions ensure the biochemical stability of regenerated D. deltoidea plants.