Adventitious shoot regeneration fromFagus sylvatica leaf explantsin vitro

Summary Adventitious shoots were induced on transversally divided expanding leaves fromFagus sylvatica shoot cultures of juvenile origin. Adventitious shoot buds formed mainly on callus that developed on the petiole stump or on the cut across the midrib of distal leaf halves. However, sometimes they arose directly from leaf tissue. An anatomical study confirmed both the direct and indirect origin of the adventitious buds. The best results were obtained by culturing proximal leaf sections on woody plant medium supplemented with 2.9 μM indole-3 acetic acid in combination with 8.9 μM benzyladenine or 2.3 μM thidiazuron (TDZ). Proximal explants were more responsive than distal explants in terms of both callus formation and bud regeneration, regardless of the induction medium or clone tested. Bud formation capacity was influenced by the genotype of the stock shoot culture and was enhanced by an initial 10 d darkness, but was inhibited by longer periods of darkness. Caulogenic competence was significantly affected by the duration of exposure to TDZ; in particular, adventitious shoot length was depressed by increasing the exposure period. Three weeks culture with TDZ was the most efficient treatment for shoot production and elongation. Further shoot development was promoted by subculturing the explants to the same medium used for the maintenance of the stock shoot cultures. Shoots so obtained were multiplied and rooted producing plantlets of adventitious origin.     

In vitro adventitious shoot production inPaulownia

In vitro adventitious shoot formation was examined in petioles and laminae ofPaulownia elongata, P. fortunei, andP. Henan 1. Fully expanded, dark green, thick, older leaves were healthier and exhibited greater callus and shoot production than did young leaves. The ten clones used varied greatly in adventitious shoot formation. When single explants from the center of each lamina were cultured,P. Henan 1 andP. elongata exhibited similar shoot production, and both were more prolific thanP. fortunei. Shoot production per leaf fromP. elongata andP. Henan 1 could be greatly increased if petioles were cut in half and leaf laminae were cut into three strips, rather than leaving petiole and laminae explants intact. The growth regulator concentrations required for maximal shoot production differed among clones, but all required 0.2 or 0.5 mg/l naphthaleneacetic acid and 5.0 or 7.0 mg/l benzyladenine. The average adventitious shoot production after 4 weeks in culture for the two most prolific genotypes was 63 shoots per leaf fromP. Henan 1 and 48 shoots per leaf fromP. elongata.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962)     

Axillary shoot proliferation and growth of Populus deltoides shoot cultures

Shoot cultures of four genotypes of Populus deltoides Bartr. ex Marsh. were established from adventitious shoots regenerated from internodal stem explants. Stable shoot cultures for all four genotypes were maintained in a continuous culture regime for over one year. The stable shoot cultures were used as explants to investigate the effects of zeatin concentration and genotype on axillary shoot production and growth. The concentration of zeatin significantly affected the production of axillary shoots, with 1.0 mgL^–1 zeatin producing the greatest number of shoots (31.0 shoots per culture vessel) while 0.25 mgL^–1 zeatin produced the greatest growth (5.9 mg per axillary shoot) when measured by dry weight accumulation per shoot. Genotypic differences were significant in the production and growth of axillary shoots.Abbreviations DKW Driver and Kuniyuki Walnut medium - PAR Photosynthetically Active Radiation Journal Series No. 9111, Agricultural Research Division, University of Nebraska     

Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures

Experiments were performed to determine the effects ofcarbon source and concentration on shootmultiplication in shoot cultures of Fagussylvatica (one clone) and F. orientalis (twoclones) and on the induction of adventitious shootbuds from leaf and internode explants of F.orientalis. In general, glucose was the best carbonsource for both axillary branching and adventitiousshoot regeneration. Shoot-tip explants grown on 3–4%glucose medium produced more shoots than those onsucrose or fructose. For maximum shoot length, glucosemedium was best for two of the three clones, and 4%sucrose for the other. The number of shoots was theparameter most influenced by glucose concentration inthe adventitious shoot regeneration experiments, thenumber increasing with sugar concentration. The lowesthyperhydricity rate occurred in the presence ofsucrose in both species. Shoot growth and quality wasnegatively affected by fructose supplied media. Theuse of filter-sterilized rather than autoclavedfructose neither stimulated shoot growth nor reducedthe incidence of hyperhydricity in all three clones.The response of shoot cultures to differentcarbohydrate treatments appears to some extent to begenotype dependent.     

Effect of genotype on rooting of hypocotyls and in vitro-produced shoots of Pinus radiata

Significant genotypic variation at both the family and clone-within-family levels was seen for hypocotyl rooting and the rooting of adventitious shoots produced in vitro for Pinus radiata D. Don. High rooting frequencies for hypocotyls were obtained in the absence of exogenous auxin; auxin greatly stimulated the rooting of adventitious shoots. No correlation was seen between the rooting of hypocotyls and shoots; families whose hypocotyls rooted at high frequencies did not necessarily produce shoots that rooted at high frequency. No correlation was seen between adventitious shoot production and subsequent rooting at either the family or clone level. The lack of a negative correlation indicated that selecting families or clones for high levels of shoot production will not automatically select for low rooting ability, obviating a possible bottleneck for commercial propagation of Pinus radiata. Significant family by replication interaction suggests that rooting protocols could be optimized through manipulations of the rooting environment for each family.     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

In vitro adventitious shoot formation on mature-phase leaves and petioles of Liquidambar styraciflua L.

Adventitious shoots were induced to form on leaves and petiole segments of mature-phase Liquidambar styraciflua L. Shoot organogenesis occurred directly, without the formation of a distinct callus stage, and well-defined shoots were visible in 6–9 weeks. Prolific shoot production was supported by Woody Plant Medium supplemented with relatively high levels of benzyladenine (2.5 mg/l). Changes in benzyladenine concentration and the addition of 0.1 mg/l naphthaleneacetic acid to the medium altered the relative abundance of shoots on various parts of a leaf. Shoot formation occurred most frequently at or near breaks in major vasculature. Wounding of the leaves by slashing across the lamina and vasculature significantly increased the total number of shoots formed per explant and also altered the pattern of organogenesis. Differences in organogenic response were seen between the cultivars ‘Moraine’ and ‘Variegata’. Shoots derived from leaves were easily rooted and acclimated to greenhouse conditions. Three new variegation types arose in vitro as a result of adventitious shoot formation on ‘Variegata’ leaves.     

Adventitious shoot formation from embryonic explants of red pine (Pinus resinosa)

Adventitious shoots were induced from excised embryos of Pinus resinosa Ait, on half-strength Le-Poivre (LP) medium containing 1–70 μ M N⁶-benzyladenine (BA). At lower concentrations of BA, only 2–3 shoot primordia (from as many as 22 formed per embryo) developed into shoots when subcultured onto medium containing 0.5% activated charcoal. Concentrations of 10 to 70 μ M of BA produced significantly higher numbers of shoot primordia and most of them developed into shoots. Ten to 17 day culture on medium containing 10–25 μ M BA proved optimal for maximum adventitious shoot production. Less than three days of incubation on the cytokinin medium did not stimulate the formation of adventitious shoots. Twenty-four day culture on the same medium produced several shoots, but most of them failed to develop normally and formed callus. Coconut milk (0.1–5% v/v) inhibited adventitious shoot formation. Using optimal conditions, seeds from 11 open-pollinated selected trees were compared to test for genetic differences in the potential to produce adventitious shoots from embryos. No significant differences were observed with regard to the shoots produced per embryo among the different seed collections. More than 200 plants produced through this technique were tested for variation in several isozymes by electrophoresis. No variations were observed.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Cytokinin production by Asparagus shoot apex cultured in vitro

The cytokinin producing capacity of asparagus (Asparagus officinalis L.) shoot apex was examined by means of shoot apex culture in vitro, where adventitious roots were never formed. The cultured shoot apices continued to diffuse a small but constant amount of cytokinin into the medium throughout five passages of subculture. The cytokinin content in the apices at the end of the subculture was not different from that at the beginning of the subculture. These results indicate a production of cytokinin by the apices. However, the finding is not in conflict with the hypothesis that the root tip is the major source of cytokinin supply, because the root tip of asparagus produced more cytokinin than the shoot apex and the decline of shoot growth observed during the subculture was partially restored by an application of zeatin into the medium.     

In vitro multiple shoot regeneration and plant production in Alysicarpus rugosus DC. var. heyneanus Baker

A protocol for in vitro multiple shoot regeneration and plant production through seedling (shoot tip) culture was established for Alysicarpus rugosus DC. var. heyneanus Baker. Maximum number of adventitious shoots (14.4) per shoot tip explant were initiated after two subcultures on MS solid medium supplemented with IAA (2.85 microM) plus BAP (2.22 microM) after 4 weeks. Shoot elongation (3.0-3.5 cm) was achieved on MS medium without any hormones. Stunted shoots elongated on half MS medium without growth hormones. Rooting occurred in MS medium containing IAA (1.14 - 2.85 microM) alone or in combination with IBA (0.89 - 2.46 microM) and or NAA (1.07 - 2.69 microM). Maximum rooting was established in MS medium supplemented with IAA (2.85 microM). The plants were acclimatized successfully with 55% survival in pot containing cocoa peat and sand (1:1). After a month, hardened plants were transferred to pots with manure, garden soil and sand (1:2:1) for further growth and finally planted in field.     

Shoot doubling time: A quantitative parameter for characterizing shoot cultures in vitro

Shoot doubling time is proposed as a suitable parameter for characterizing in vitro propagation rates in shoot cultures. Doubling times were estimated for cultures of black currant and apple; for black currant, doubling times ranging from 14 to 115 days were obtained, depending on the concentration of benzyladenine in the culture medium. These doubling times, which were constant for each culture, were maintained for periods of up to 76 days and could apparently be sustained indefinitely. For one subculture period only, a shoot doubling time as short as 5.6 days was obtained for black currant at high cytokinin concentration (3 × 10⁻⁵ M), but this rate could not be sustained. Shoot doubling time is a convenient parameter for use in optimizing proliferation rates in shoot cultures; its use may also facilitate investigations into the mechanisms of processes underlying shoot proliferation in vitro.     

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.     

A Comparative Anatomical Study of Organogenesis in Cultured Tissues of Maize, Wheat and Oats

Five cereals and two related grasses were tested for adventitious shoot production from tissue cultures using methods concordant with those reported to be successful for cereals. The five cereals I wheat (Triticum aestivum L.), oats (Avena sativa L.), and maize (Zea mays L.) Pioneer hybrid 3369A, the Bolivian race Pororo and the Equadorian race Chococenõl were all found to proliferate in culture through an aberrant root-like mechanism of growth which had the external appearance of callus. Two related species, teosinte (Zea mexicana Reeves and Mangelsdorf) and tripsacum (Tripsacum dactyloides L.), were less successful in culture, but grew in the same way. Oats, and probably Pororo and Chococeño, initiated presumptive shoot meristems directly from root vascular tissues within this root-like growth. Hybrid maize and wheat initiated no shoot meristems and produced only roots. The occasional shoot production observed in wheat was discounted as simple carryover of existing shoot apices from the primary embryo cultures. This study suggests that the incidence of shoot regeneration in cultures of these cereals may be related more directly to adventitious bud formation on roots than to any controlled de novo organogenesis from undifferentiated callus.     

In vitro adventitious shoot regeneration from leaves of Prunus spp.

A high level of adventitious shoot regeneration was obtained from proliferating shoots in vitro for a range of Prunus spp. There was a significant variability in clone response to a range of adventitious shoot regeneration treatments. Treatment of apricot clone H.152 with Quoirin macroelements (C.R. Rech., Stu. Cult. Fruit. Maraîchères Gemblaux (1977) 93–117), and both apricot clone H.146 and hybrid plum clone P.1869 with half-strength Murashige and Skoog medium, consistently induced regeneration. Thidiazuron (TDZ) alone, or in combination with naptthaleneacetic acid (NAA), was most effective in stimulating adventitious shoot production, the optimum concentration being clone-dependent. Addition of silver nitrate (AgNO₃) to regeneration media enhanced regeneration by 10–40% and reduced the variability between experiments. Regeneration with AgNO₃ was obtained also for three other plum clones belonging to the P. marianna, P. domestica and P. insititia species.     

拟南芥不定芽发生早期的数字基因表达谱分析

目前,有关不定芽发生的研究主要集中在单基因的调控方面,缺乏转录组方面的系统研究.利用RNA-seq高通量测序技术在全基因组范围内检测了不定芽发生早期的基因表达谱,共检测到2457个差异表达基因.这些基因参与了激素代谢和信号转导、愈伤组织和侧根的形成、茎顶端分生组织的发育和光合作用等过程.进一步的途径富集分析表明,不定芽发生早期苯丙氨酸代谢和苯丙胺素合成等途径相关的基因显著富集.并且苯丙氨酸可以显著抑制不定芽的发生,暗示了苯丙氨酸代谢和苯丙胺素的合成可能在不定芽发生过程起着重要的作用.     

Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree

Rapid clonal multiplication of Aegle marmelos (L.) Corr. (Rutaceae), a medicinal tree, was achieved by enhanced axillary bud proliferation in young single-node segments of a 25-year-old tree cultured in Murashige and Skoog (MS) nutrient medium. Bud break was dependent on cytokinin supply, but the synergistic combination of 2.5 mg l^–1 6-benzylaminopurine (BAP) and 1.0 mg l^–1 indole-3-acetic acid (IAA) induced the formation of 12.1 shoots of up to 5.2 cm length in 48% of the explants after 7 weeks of culture. Explants of in-vitro-grown shoots – node, whole leaf, shoot tip and internode – were subcultured in the presence of 0.05–2.5 mg l^–1 BAP to produce 11.3, 18.4, 5.3 and 3.2 shoots and shoot buds at a 100%, 70%, 95% and 40% rate respectively, in 7 weeks. Different shoot nodes and leaves were equally regenerative and adventitious organogenesis in the latter was confined to cut petiolar ends. Nodal explants responded most favourably at low BAP (0.05–0.1 mg l^–1) and produced uniform (3.8–5.3 cm) shoots facilitating their simultaneous harvest for rooting. Repeated subculturing through five cycles of nodes and leaves of shoot cultures enabled continuous production of healthy callus-free shoots without any sign of decline. Shoot cuttings (3.0–5.2 cm) were best rooted in half-strength MS medium with 0.5 mg l^–1 IAA (70%) or 10.0 mg l^–1 indole-3-butyric acid (90%). Eighty-eight percent of the rooted plants were established in polybags after hardening. Received: 4 April 1996 / Revision received: 8 September 1997 / Accepted: 20 September 1997     

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus

Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about 1cm were transferred to shoot induction media, containing different concentrations (1–50µM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10µM) was the most effective concentration in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants.Abbreviations 2ip N-isopentenyl adenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MES 2-(Nmorpholino)ethane acid - NAA naphthaleneacetic acid - TDZ thidiazuron - BAP 6-benzylaminopurine - PEG polyethylene glycol     

Genetic analysis of in vitro shoot regeneration from cotyledonary petioles of<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

Diallel analysis was used to investigate the genetic control of in vitro shoot regeneration in Brassica oleracea. Twelve doubled haploid (DH) lines, selected to include a range of genotypes with differing shoot regeneration potentials, were crossed reciprocally to produce 132 F₁ and 12 selfed, DH families. Cotyledonary petioles from 4-day-old seedlings, from all families, were excised and maintained on MS medium supplemented with 2 mg/l BAP. Explants were scored after 44 days for both the presence or absence of shoots and the number of regenerating shoots per explant. Diallel analysis showed both shoot regeneration and the production of multiple shoots to be controlled by additive and dominant gene effects, with additive effects being more important. Additive gene effects accounted for 71% and 77% of the genetic variation observed within the diallel for shoot regeneration and multiple shoot regeneration, respectively. By investigating the shoot regeneration potential of subsequent backcross and F₂ populations, the ability to introduce and increase shoot regeneration potential into otherwise recalcitrant lines was demonstrated.Communicated by G. Wenzel