杉木再生系统的比较研究

通过对杉木(Cunninghamia lanceolata Hook)再生系统的比较研究,建立了子叶、下胚轴、茎段和针叶等四个再生体系。子叶在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率为93.7%±0.45%,平均芽数为3.76±0.25;下胚轴在附含2mg/L BA和0.2mg/L NAA的DCR培养基中再生频率为96.2%±0.35%,平均芽数为17.4±0.18;茎段在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达100%,平均芽数为3.28±0.11;针叶在附含1.5mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达84.5%±0.45%,平均芽数仅为1.42±0.08。不定芽在附含0.2mg/L BA和0.02mg/L NAA的DCR培养基中有效伸长。芽苗经预处理后在附含0.3mg/L IBA的1/2 DCR培养基中生根效果最佳。     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Direct organogenesis and plantlet regeneration from mature zygotic embryos of masson pine (Pinus massoniana L.)

Mature zygotic embryos of masson pine were cultured as initial explants to investigate the process of direct organogenesis. Adventitious buds were initiated on DCR medium (Douglas-fir cotyledon revised medium) supplemented with 0.5 mg l⁻¹ N⁶-benzyladenine (BA) and 0.05 mg l⁻¹ indolebutyric acid (IBA) or α-naphthaleneacetic acid (NAA). The highest induction frequency of adventitious buds was 99.3%. Subsequent transfer of buds to medium with lower concentrations of plant growth regulators in time was necessary for differentation of high quality adventitious buds. After culturing on elongating medium, in which the proportion of cytokinins to auxins was reduced, shoots higher than 2 cm were transferred for root induction to GD medium with half of the concentration of macro-salts (½ GD) and with 2 mg l⁻¹ IBA and 0.05 mg l⁻¹ BA. The average root frequency was over 70%. After adventitious roots had appeared, the shoots were transferred to ½ GD medium with a lower concentration of IBA (0.2 mg l⁻¹) for further root development.     

Plantlet regeneration from mature zygotic embryos and embryonic explants of masson pine （Pinus massoniana Lamb.）

Excised zygotic embryos,cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones.BA(1.0mg/L) in combination with NAA(0.05mg/L) in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos,but most of them were formed at the tips of embryonic cotyledons.Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L.Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal(0.5%).Root initiation was achieved with full or half strength DCR medium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L.Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated(3-20h) with BA 50-100 mg/L,followed by transfer to hormone-free DCR medium.The maximum number of shoots obtained per explant within six months was 33.     

Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg?l^?1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg?l^?1 TDZ. The WPM medium supplemented with 2.5 mg?l^?1 BA?+?0.4 mg?l^?1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg?l^?1 BA and 3.0 mg?1^?1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg?l^?1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Organogenesis of vegetative shoots from in vitro cultured flower buds of Mammillaria albicoma (Cactaceae)

We tested the organogenetic capacity of floral buds of Mammillaria albicoma Böed. (Cactaceae). Buds were incubated on solid MS medium supplemented with 0.1 mg l^?1 α-naphthaleneacetic acid (NAA) and 5.0 mg l^?1 6-benzylaminopurine (BA). Callus growth was observed from the cut explant base and from within the perianth. These calli during subsequent subcultures to the same medium gave rise to adventitious shoots. Shoots formed also directly from the perianth, as confirmed by observations in the light microscope and scanning electron microscope (SEM). On transfer to a fresh medium, the shoots produced proliferating cultures. This is the first report of regeneration of cactus shoots from floral explants.     

In Vitro Regeneration of Achillea millefolium L from Shoot-tips and Root Segments of Seedlings

This report describes, for the first time, an efficient plant regeneration system for Achillea millefolium L (yarrow), a medicinal plant, via shoot multiplication from shoot-tips and adventitious shoot regeneration from root segments. Higher numbers of shoots were obtained when shoot-tips were cultured on MSMO medium supplemented with 3.0 mg l^?1 BA and 0.5 mg l^?1 IAA, or 5.0 mg l^?1 KIN and 1.0 mg l^?1 IBA, producing 17.3 and 17.0 shoots per explant at 100% frequency, respectively. For adventitous shoot regeneration, only root segments developed shoots when cultured on medium containing a combination of 1 mg l^?1 TDZ, 0.5 mg l^?1 IAA and 0.5 mg l^?1 GA3 (18.9 shoots per explant at 100% frequency), while other types of explants (i.e., cotyledons, leaf lamina and petiole segments) or hormonal combinations tested were found ineffective. Regenerated shoots rooted readily on MSMO medium containing different concentrations of IAA, IBA, NAA or 2,4-D, however, NAA at 0.5 mg l^?1, or IBA at 0.5 or 1.0 mg l^?1 were found to be the most productive. Nearly all of the regenerated plants (98%) survived through the hardening process when the rooted plantlets were kept at 55–65% relative humidity for 2 weeks, which were then planted in pots containing potting soil and kept at 25–35% humidity.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

High efficiency in vitro organogenesis from mature tissue explants of Citrus macrophylla and C. aurantium

A simple and efficient protocol for obtaining organogenesis from mature nodal explants of Citrus macrophylla (alemow) and Citrus aurantium (sour orange) has been developed by optimizing the concentrations of the plant growth regulators, the incubation conditions, the basal medium and by the choice of explant. In order to optimize the plant growth regulator balance, explants were cultured in the regeneration medium supplemented with several N ⁶-benzyladenine (BA) concentrations or with 2 mg?l^?1 BA in combination with kinetin (KIN) or 1-naphthaleneacetic acid (NAA). The presence of BA was found to be essential for the development of adventitious buds; the best results were obtained using BA at 3 and 2 mg?l^?1 for alemow and sour orange, respectively. The combination of BA with KIN or NAA in the culture medium decreased the regeneration frequency, with respect to the use of BA alone. The effect of three different basal media was rootstock-dependent. For C. macrophylla the best results were obtained with Woody Plant Medium or Driver and Kuniyuki Walnut Medium (DKW). However, for C. aurantium, although high percentages of regenerating explants were obtained independently of the basal medium used, the highest number of buds per regenerating explants was obtained with DKW medium. Attempts were made to identify the type of explants which had a higher regeneration ability using particular regions along the mature shoots of C. macrophylla. When nodal segments, where the buds were completely removed, and internode segments were compared, the highest percentage of responsive explants was obtained with nodal segments. The existence of a morphogenetic gradient along the shoot was observed and the organogenic efficiency was highest when explants from the apical zone were used. Incubation in darkness for 3 or 4 wk was essential for regeneration process in both rootstocks.     

Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmano?lu’ and ‘Sar?a?lama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmano?lu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L)?+?kinetin (KIN; 2 mg/L)?+?indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L)?+?1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L)?+?IBA (0.01 mg/L), and (3) KIN (2 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sar?a?lama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L)?+?KIN (2 mg/L)?+?IBA (0.01 mg/L), (2) BA (1 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmano?lu’ and between 0 and 0.49 per ‘Sar?a?lama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA₃), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA?+?0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA₃ (3 mg/L).     

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l^?1 6-benzyladenine (BA) and 0.1 mg l^?1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l^?1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.     

Micropropagation of <Emphasis Type="Italic">Asparagus macrorrhizus</Emphasis>, a Spanish endemic species in extreme extinction risk

Asparagus macrorrhizus: is a new species, which has been recently described. It is limited to the area surrounding the “Mar Menor” lagoon, in Murcia (Spain), and is the only “Critically Endangered” species of the genus Asparagus. Despite being protected, the number of plants has decreased in the last years due to the urbanization of its natural habitat. This species is a valuable genetic resource for asparagus breeding because of its special characteristics. So, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The micropropagation protocol from asparagus rhizome buds previously developed by our research group has been adapted for A. macrorrhizus. Rhizome buds of A. macrorrhizus were extracted, disinfected, and then cultured on Asparagus Rhizome Bud Medium (ARBM) consisting of MS medium supplemented with 0.3 mg l^??1 NAA, 0.1 mg l^??1 KIN, 2 mg l^??1 ancymidol and 6% sucrose. A percentage of 69.7?±?8.0% of the rhizome buds developed shoots, but only 17.4?±?7.9% of them rooted. To increase this low rooting rate, the shoots were cultured on Macrorrhizus Rooting Media (MRM) supplemented with three different concentrations of IBA. The highest rooting rate (55.0?±?7.9%) was reached when shoots were incubated in MRM-2 consisting of MS medium supplemented with 2 mg l^??1 IBA and 4% sucrose. The acclimatization rate of the micropropagated plantlets was 90%. The method developed in this study allows the micropropagation of A. macrorrhizus, offering a new option to preserve this almost extinct species.     

High-frequency shoot regeneration through transverse thin cell layer culture in <Emphasis Type="Italic">Dendrobium Candidum</Emphasis> Wall Ex Lindl.

An efficient in vitro propagation protocol for Dendrobium candidum Wall ex Lindl. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration and the number of adventitious buds produced from the regenerated shoots significantly relied on the concentration of plant growth regulators, and the position and orientation of the explant. Murashige and Skoog (MS) medium with half-strength macronutrients and 2% sucrose, supplemented with 1.2 mg l⁻¹ naphthaleneacetic acid (NAA) and 1.2 mg l⁻¹ 6-benzyladenine (6-BA), was optimal for shoot regeneration. Upon this medium, the youngest explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (92%), and the highest number of adventitious buds (an average of 24.5) per explant. Rooting of shoots and adventitious buds was achieved on MS medium with half-strength macronutrients and 2% sucrose with 1.0 mg l⁻¹ NAA and 1.0 mg l⁻¹ indole-3-acetic acid (IAA). Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Ontogenetic studies revealed that the shoots originated from the stem vascular bundles.     

Meristematic nodule culture: a new pathway for in vitro propagation of<Emphasis Type="Italic"> Eucalyptus globulus</Emphasis>

Morphogenesis was induced in Eucalyptus globulus seeds, cotyledons, hypocotyls and leaves from in vitro clonal plantlets. Globular structures were observed after 2 weeks induction on B5 culture medium supplemented with 10% coconut water, 0.05–0.5 mg l^?1 6-benzylaminopurine (BAP) and 0.5 mg l^?1 indole-3-butyric acid (IBA). These continued to proliferate under dark conditions until the 2nd to 3rd subculture. Following transfer to a photoperiod of 16 h light, shoots evolved from these globular structures and developed further to plantlets. The influence of several factors, including culture medium composition, sucrose concentration, the type, concentration and combination of growth regulators and the presence of coconut water was studied. The percentage of explants showing globular structure formation and the number of globular structures per explant were evaluated. Macroscopic, histological and scanning electron microscopic studies revealed that the morphogenic process involved mainly organogenic nodules with fewer globular somatic embryos. The nodules gave rise to shoots and subsequently complete plants following incubation on B5 Gamborg medium containing 0.5 mg l^?1 IBA and 30 g l^?1 sucrose, which promoted root formation.