Influence of growth regulators and light on in vitro shoot regeneration in M.26 apple roostock

Summary

The effects of different BA and IBA concentrations and dark/light combinations, applied during both the last proliferating subculture (LPS) and the regeneration phase (RP), on shoot regeneration from leaves were evaluated in M.26 apple rootstock. A positive influence on caulogenesis was found with a low cytokinin/auxin ratio in the medium during the LPS. The increase in IBA concentration from 0.49 μM to 4.92 μM in the LPS, along with the absence or the use of a low cytokinin concentration (0.89 μM) in the medium, enhanced the subsequent shoot regeneration from leaves. During the RP, a concentration of 22.2 μM BA gave the best caulogenesis results. During both the LPS and the RP, high IBA concentrations were able to replace the combined effect of a low IBA concentration and dark treatment; this could indicate that dark treatments interact with the auxin metabolism in the leaf caulogenesis response. Auxin application could not reproduce all the effects of dark treatments, suggesting that dark also affects other biochemical and/or physiological aspects, such as gibberellin metabolism. Moreover, cytokinins applied during both the LPS and the RP influenced the size of regenerated shoots.     

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。     

In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers

This study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

悬铃木下胚轴及叶片再生不定芽的影响因素分析

 以悬铃木的下胚轴和离体叶片为材料, 研究了基因型、植物生长调节剂、光照条件、外植体部位等因素对其不定芽分化的影响。结果表明: 1) 6-BA与IBA的浓度比对下胚轴不定芽的分化影响较大,以6-BA ∶IBA小于20 ∶1较为合适; 而叶片的不定芽再生受基因型的影响显著, 在4种供试材料中, PH2的再生能力最强, 但0.1～1.0 mg·L^-1 TDZ不利于其不定芽再生。2) 黑暗培养对下胚轴的不定芽分化有一定的促进作用, 但不利于叶片的不定芽再生, 50～100 lx的弱光有利于叶片的不定芽分化。3) 不同部位的下胚轴切段及叶片的再生能力差异显著, 其中靠近子叶的下胚轴切段和试管苗顶端的2枚叶片再生能力较强。此外, 刻伤叶片的再生效果明显优于叶片切块, 说明悬铃木的叶片与下胚轴的离体培养存在明显的生物全息现象。     

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.     

Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

Summary

An efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.     

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.     

Adventitious shoot regeneration from leaf thin cell layers in apple

The present investigation was conducted to study organogenesis of a conventionally easy-to-regenerate (‘Royal Gala’) and a difficult-to-regenerate (‘Freedom’) apple cultivar using leaf transverse thin cell layers (tTCLs). The position of the leaf source, explant type and light regime applied to tTCLs were investigated as was their influence on the organogenic outcome. Light during the onset of regeneration delayed morphogenesis or, in ‘Freedom’, completely inhibited organogenesis on leaf tTCLs. Genotype and initial position of the leaf also influenced percentage regeneration and number of regenerated shoots per explant. This study demonstrates the successful application of leaf tTCLs for adventitious shoot regeneration in apple, even though further studies are needed to increase the number of shoots more than control explants. Shoot regeneration from apple leaf tTCLs serves as an alternative protocol for studying fundamental and applied aspects of regeneration.     

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。     

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.     

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.     

朱顶红离体培养快速繁殖体系及胚状体发生

 以朱顶红鳞茎基部鳞片为外植体, 在MS + NAA 1 mg/L + BA 2 mg/L 培养基上愈伤组织的诱导频率最高, 为93. 3 % , 愈伤组织上可以直接生芽, 生芽频率为90. 0 %。NAA 的诱导效果优于2 ,42D , BA 优于KT。愈靠近鳞茎基部愈容易诱导产生愈伤组织和不定芽。愈伤组织继代培养4 次后有胚状体产生。诱导产生的不定芽和胚状体在MS0 培养基上可以100 %生根。生根后的再生植株移栽到蛭石中, 浇MS 无机盐营养液, 成活率达98 %以上。不生根直接将不定芽移栽到消毒蛭石中, 成活率为85 %以上。     

Adventitious shoot regeneration from leaf segments of in vitro cultured shoots of the apple rootstock Jork 9

SUMMARY

Adventitious shoot formation was investigated using leaf segments of in vitro cultured shoots of the apple rootstock Jork 9. Regeneration capacity was influenced by the pretreatment of the mother shoots, macroelements, hormone concentrations, the gelling agent and the carbohydrate source. The highest regeneration rate and most shoots per leaf explant resulted from young leaves on a medium based on MS macroelements supplemented with 22 µM BAP and 0.1 µM_NAA together with sorbitol, at concentrations of 165 mM or 220 mM. Sorbitol was more effective than sucrose, glucose, fructose or a combination of these sugars. A cold and dark pretreatment of the shoots enhanced the formation of adventitious shoots.     

Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants,and protocorm-like bodies

In vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly priced commercial cut flower cultivars through direct organogenesis from in vitro derived foliar explants was established. Rapid clonal propagation was achieved by subsequent induction of protocorm-like bodies (PLBs) and its conversion to shoots. No significant differences were observed in the induction of direct shoots, shoot multiplication, PLBs formation and subsequent shoot development and rooting of shoots between the two cultivars. Leaf explants from flower stalk node derived shoots cultured on half-strength Murashige and Skoog (MS) medium supplemented with 44.4 μM N⁶-benzyladenine (BA) developed more than seven shoots per explant. The isolated shoots transferred onto the same medium induced more than eight PLBs from the base within 60 days, which upon transferral to fresh medium having the same level of BA facilitated rapid proliferation. More than 200 PLBs were yielded from fifth subculture. Half-strength MS medium containing 6.97 μM kinetin (Kn) facilitated conversion of more than 90% PLBs to shoots. PLBs exhibited proliferation without decline up to the 15th subculture. Half-strength MS medium with 2 g l⁻¹ activated charcoal was the best for in vitro rooting. Plantlets of the hybrids exhibited more than 80% ex vitro establishment.     

In vitro propagation of GF677 hybrid rootstock (Prunus persica × Prunus amygdalus) from mature cotyledons

Adventitious shoot regeneration from mature cotyledons of GF677 rootstock (Prunus persica × Prunus amygdalus) was achieved in vitro. Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl-urea; TDZ) at 32 µM gave the highest percentage of cotyledons forming adventitious shoots (68.8%) and the highest number of shoots per cotyledon (4.8) on Quoirin and Lepoivre (QL) medium. On QL medium containing 32 µM TDZ, exposure of the proximal segments of cotyledons to darkness at the start of culture increased the percentage of cotyledons forming adventitious shoots (62.5%) when compared with those kept under light conditions (15%). A combination of 0.72 µM gibberellic acid and dark treatment resulted in at least 2.7-fold more elongated shoots than non-treated shoots. The highest rooting percentage (100%) occurred on 0.5× Murashige and Skoog medium supplemented with Gamborg (B5) vitamins and 2 mg l^?1 indole-3-butyric acid. Rooted plantlets were acclimatised under greenhouse conditions with a 70% survival rate.     

Rejuvenation influences indirect organogenesis from leaf explants of Pomegranate (Punica granatum L.) ‘Kandhari Kabuli’

Sequential subculturing leads to a gradual physiological change in cells that may be termed ‘rejuvenation’. The effect of repetitive subculturing on callus induction and shoot regeneration from leaf explants of Punica granatum L. ‘Kandhari Kabuli’ were investigated. Surface-sterilised leaves were cultured on 1.0× Murashige and Skoog (MS) medium supplemented with 4.0 mg l^–1 α-naphthaleneacetic acid (NAA) and 2.0 mg l^–1 6-benzyladenine (BA) for callus induction. Shoots were regenerated from callus on 1.0× MS medium supplemented with 1.5 mg l^–1 BA, 0.5 mg l^–1 kinetin, and 0.25 mg l^–1 NAA. Subculturing of callus onto fresh medium maintained the rate of shoot formation and substantially increased the production of shoot buds up to the second subculture. Following further subculture passages, a lower shoot regeneration potential from callus was observed. A maximum shoot bud induction from callus of 63.9% was observed at the second subculture passage. The rate of multiplication of in vitro shoots increased until the fourth subculture, then became constant. Similarly, in vitro rooting of micro-shoots increased up to the third subculture, followed by a decline during further subculturing.     

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.     

不同培养条件对‘丰香’草莓离体叶片再生的影响

 以草莓品种‘丰香’离体叶片为外植体, 探讨了基本培养基、不同细胞分裂素、暗培养、硝酸银浓度以及不同植物生长调节剂组合对不定芽再生的影响。结果表明, 基本培养基中以MS 最为适合,WPM、QL 、AS 培养基均不利于不定芽的再生, 而TDZ 的诱导效果好于BA。以MS 基本培养基附加TDZ2.0 mg·L - 1和IBA 0.8 mg·L ^-1可以使‘丰香’叶片不定芽的再生率高达72.33 % , 平均每叶再生芽5.59个。暗培养14 d 可以将‘丰香’叶片的不定芽再生率提高到90.09 %。硝酸银对于提高‘丰香’叶片的不定芽再生没有明显效果, 但在一定程度上改变了细胞分化的方向。     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.