Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Improvement of caper (Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper (Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%.     

秋福红树莓叶片离体再生植株研究

以红树莓品种秋福(Rubus L.cv.Autumn Bliss)离体叶片为外植体,研究适合其再生植株的叶片部位、放置方式、植物生长调节剂以及暗培养时间等条件。结果表明,叶片外植体接种于MS+TDZ2.00mg/L+IAA0.10mg/L的培养基,暗培养2～3周转移至正常光照下培养效果最好,愈伤组织形成率、不定芽再生率和外植体不定芽数分别为100.00%、95.83%和(5.57±0.27)个。将再生芽接种于1/2MS+IBA0.10mg/L的培养基中,35d后生根率达100.00%。再生植株炼苗后移栽,30d时成活率达97.30%,成功地建立了红树莓叶片的离体再生体系。     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (Malus × domestica Borkh.)

Summary

The conventional selection system for apple transformation is based on the selectable marker gene, nptII, encoding antibiotic resistance against kanamycin. We tested an alternative selection system based on the use of D-amino acids using the gene, D-amino acid oxidase 1 (dao1) as the selectable marker, in order to avoid the presence of antibiotic resistance genes in the resulting transgenic apple plants. In addition, dao1 allowed the selection as well as the elimination of dao1-transgenic plants, based on differences in the toxicity of different D-amino acids. Regeneration experiments using apple leaf explants revealed that 2 mM D-serine or D-alanine inhibited shoot regeneration. We performed transformation experiments using the apple cultivars ‘Gala’, ‘Holsteiner Cox’, and a progeny of the apple cultivar ‘Pinova’, and the vector p35S::dao1-intron, containing the dao1 and nptII selectable marker genes. Several shoots regenerated successfully on selection media containing various concentrations of D-serine or D-alanine, but transgenic shoots were not obtained. However, three dao1/nptII transgenic apple lines were obtained after selection with kanamycin, indicating that the vector was functional. Furthermore, we showed that 20 mM D-serine could be used to select dao1-transgenic shoots from non-transgenic in vitro shoots, whereas 13 mM D-isoleucine had the opposite effect.     

Adventitious shoot regeneration in a bioreactor system and EST-PCR based clonal fidelity in lowbush blueberry (Vaccinium angustifolium Ait.)

Adventitious shoots were regenerated from leaf explants of three lowbush blueberry (Vaccinium angustifolium Ait.) clones: ‘QB1’, ‘QB2’ and ‘PB1’ by culturing on a gelled basal medium (BM) with 2.3–4.5 μM thidiazuron (TDZ) for four weeks followed by in a bioreactor system containing the same liquid medium but with 1.2–2.3 μM TDZ for another four weeks. Young expanding basal leaf segments with the adaxial side touching the culture medium and maintained for two weeks in darkness, produced the best results. Callus development and shoot regeneration were genotype dependent. Adventitious shoots were elongated in the liquid BM with 1 μM zeatin and rooted on a three peat: two perlite (v/v) medium. Acclimatized plantlets were grown actively in the greenhouse with an apparent normal leaf and shoot morphology. Ten random ‘QB1’ regenerated plants were screened using 14 expressed sequence tag-polymerase chain reaction (EST-PCR) markers and showed similar monomorphic amplification profiles confirming clonal fidelity of in vitro-derived ‘QB1’ plants. Results obtained suggested the possibility of adventitious shoot regeneration and true-to-type lowbush blueberry micropropagation using a bioreactor system combined with gelled medium.     

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.     

Micropropagation of Cryptanthus with leaf explants with attached intercalary meristems excised from greenhouse stock plants

By using the leaves with attached intercalary meristems from greenhouse grown stock plants, five cultivars of Cryptanthus were cultured on modified MS media with 4.5 μM NAA and IBA and 3 μM BA to induce adventitious shoot formation from callus tissue. Contamination was 17–21% for explants taken from stock plants which were sprayed weekly with Agribrom and 27–75% for those taken from stock plants which were not treated. More than 99% true to type plantlets were obtained from non-chimeric plants. Green and albino plantlets were obtained from chimeric plants. The chimeric C. ‘Coster's Favorite’ DeCoster also produced a few chimeric plantlets with intermarginal pink stripes in addition to the green and albino plantlets. Most of the non-chimeric plants took a shorter time to produce plantlets of transplantable size (8–12 mm) than the chimeric ones. Except for albino plantlets, survival rate of plantlets exceeded 95%. A minimum average of 500 rooted plantlets can be obtained in a year from a single well-callused leaf explant. The protocol in this report should speed up the mass production and introduction of desirable new cultivars and hybrids of non-chimeric Cryptanthus.     

韭菜组织培养高频植株再生体系的研究

 通过对韭菜组织培养过程中激素配比、外植体、基因型、苗龄及生根条件的研究, 建立了一次性诱导成芽的高频植株再生体系。适于愈伤组织和不定芽分化的最佳培养基为MS + NAA 1 mg/ L+ BA mg / L, 在此分化培养基上91-1、91-8、保定红根、寿光马蔺韭和兰州小韭根尖培养的芽分化频率分别为78. 7%、83. 7%、81. 9%、76. 7 %和73. 3 %, 平均出芽数分别为40. 1 、46. 7 、36. 3 、35. 4 和44. 5 个,苗龄为7~ 10 d 的根尖最适于组织培养。随苗龄增加, 芽的分化频率呈下降趋势。无任何激素的MS0 培养基最适于不定芽的生根, 生根率达100%, 平均根数14. 5 条。卡那霉素( Km) 对植株再生有很强的抑制作用, 在Km 为20 mg/ L 时就完全抑制愈伤组织和不定芽的发生; 羧苄青霉素( Carb) 和噻孢霉素( Cef) 也抑制韭菜根尖培养的植株再生, Cef 的抑制作用更加显著, Carb 500 mg/ L 或Cef 300 mg/ L 完全抑制不定芽的再生; Timentin 对愈伤组织和芽的分化影响不大, 抑制性主要表现在出芽数随浓度升高而降低。当Timentin 浓度为500 mg/ L 时, 平均出芽数仍可达到23. 8 个, 为对照的49. 4% 。     

In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L.

Shoot tip explants obtained from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100 mM calcium chloride for the production of synthetic seed in Solanum nigrum L., a medicinally important plant. Morphogenic responses of encapsulated shoot tips to various sowing media (full or half-strength 0.8% agar-solidified or liquid MS medium or full-strength MS medium containing BAP) were evaluated in vitro. Of the six media evaluated, maximum conversion was obtained on 0.8% agar-solidified growth regulator free full-strength MS medium. The addition of MS nutrients in alginate matrix had a pronounced effect on the length of shoots that emerged from alginate beads. Encapsulated shoot tips also converted when directly sown in sterile soil moistened with liquid MS medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

Morphogenetic responses of Salpiglossis sinuata L. leaf explants and callus

Leaf explants of Salpiglossis sinuata L. were cultured on MS medium containing the auxins 2,4-D, IAA, NAA, or the cytokinins 6-BAP, 2ip, or K singly or NAA + 6-BAP combinations (0.01 to 10.0 mg/l) to determine their morphogenetic responses. IAA and NAA induced callus or roots at all levels except IAA at 0.01 mg/l. Callus varying in friability was obtained on MS + 2,4-D at various concentrations. Friable, uniform dividing callus was obtained on UM medium. Optimum adventitious shoot formation occurred on leaf sections and from callus cultures placed on MS + 2ip. Rooting-difficulties were encountered, but 75% rooting efficiency was obtained on shoots cultured in MS + 0.001 mg/l 2,4-D. Rooted plants were readily transferred to the greenhouse, and flowered.     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

魔芋茎尖组织培养及快速繁殖技术研究

以魔芋茎尖为外植体,进行愈伤组织诱导、芽分化及植株再生的研究。研究结果表明,最佳茎尖剥取材料是无菌试管苗。刚剥离的茎尖需要预培养30d,然后将膨大的茎尖四周给予伤口后再转接到MS 6-BA 0.6 mg/L NAA 0.1 mg/L上进行愈伤组织诱导;芽分化最适培养基是MS 6-BA 2.0 mg/L NAA 0.5 mg/L;切割后的芽在生根培养基1/2 MS NAA 0.1 mg/L上能100%形成完整植株。     

The effects of explant and cytokinin type on regeneration of Prunus microcarpa

We investigated in vitro regeneration ability of Prunus microcarpa subsp. tortusa using various explants (root, cotyledon and hypocotyl pieces) and cytokinins [benzyladenine (BA), meta-Topolin (mT) and thidiazuron (TDZ)]. Sectioned cotyledon, root and hypocotyl pieces of in vitro grown seedlings were cultured on Nas and Read Medium (NRM) containing BA (7.5, 10, 12.5, 15 or 17.5 μM), mT (2.5, 5.0, 7.5, 10 or 12.5 μM) or TDZ (2.5, 5.0, 7.5, 10 or 12.5 μM). As a measurement of morphogenetic reaction, the ratios of regenerating explants and the numbers of primary adventitious shoots per regenerating explant were analyzed. Cotyledon explants exhibited higher regeneration ratios than hypocotyl explants, and the root explants were inappropriate for regeneration. Both BA and mT were effective on shoot regeneration but higher regenerating explant ratios were obtained when BA was used. In comparison with BA and mT, the effect of TDZ on enhancing explant regeneration ability was insignificant. Mean number of adventitious shoot per regenerating explant was between 1 and 4, and regenerating explant ratios were between 0% and 77%. The practical appliacations of the results are discussed.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.     

樱桃砧木CAB-6p离体叶片再生体系的优化

以欧洲甜樱桃优良矮化砧木CAB-6p试管苗幼嫩叶片为试材,从基本培养基、激素配比、叶片生理状态和培养基中琼脂用量等方面对影响离体叶片再生的关键因素进行了研究。结果表明,CAB-6p试管苗幼嫩叶片以WPM为基本培养基再生效果最好,明显优于QL和DKW培养基,1/2MS培养基再生效果最差;最佳激素配比是BA2mg/L+IAA2mg/L,用IBA或NAA替代IAA出现愈伤组织生长量大但再生率低;CAB-6p试管苗顶部新发出合拢的幼嫩叶片再生能力最高,半展开的幼嫩叶片和完全展开的幼嫩叶片未能再生植株;用4.5g/L琼脂配制偏软的再生培养基明显有利于提高离体叶片的再生效率。通过以上几个方面的优化,建立CAB-6p试管苗幼嫩叶片高效离体再生技术体系,再生率可稳定地保持在90%左右,平均每叶再生4～5芽。