Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree.

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White's, Gamborg's B5 or woody plant medium (WPM) supplemented with 2.5 µM benzyl adenine (BA) and 0.1 µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5 µM) and NAA (0.1 µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83 cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13 cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2 mM indole-3-butyric acid (IBA) for 5 min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.

The effect of silver nitrate on micropropagation of Moringa oleifera Lam. an important vegetable crop of tropics with substantial nutritional value.

An improved micropropagation protocol facilitating continuous multiplication of elite germplasm of Moringa oleifera has been developed. Initial culture of nodal explant in MS medium supplemented with 2.5 µM BA resulted in the formation of 12.5 shoots per explant with high frequency of leaf fall (84.3%). To confirm whether the leaf fall is due to accumulation of ethylene in the culture vessel, effect of ethylene releasing agent CEPA in the medium was tested. In order to reduce leaf fall and improve multiplication, varying concentration of anti-ethylene agent, AgNO3 was incorporated in the medium. Addition of 2.5 µM AgNO3 in combination with 2.5 µM BA produced maximum number of shoots (17.6) including shoots originated from the base of the explant and shoots from the axillary buds of the primary shoots, where significant reduction in leaf fall (20.6%) was noticed. This enabled sustained multiplication of M. oleifera through continuous subculture without adversely affecting shoot number or shoot quality in terms of shoot length. Microshoots obtained from fourth subculture onwards were used for ex vitro rooting and found that by treating 50 µM NAA for 30 s, maximum numbers of microshoots (83.3%) were rooted. Rooted plants were acclimatized, survived and were successfully transferred to field. Genetic fidelity analysis using 10 ISSR primers revealed more than 95% monomorphic bands among plants raised in MS medium containing low concentration (2.5 µM) of AgNO3 and BA (2.5 µM). The addition of AgNO3 in the medium sustained in vitro growth and effectively prevented leaf fall compared to control, thus demonstrating efficient micropropagation of M. oleifera.

An improved micropropagation and ex vitro rooting of a commercially important crop Henna (Lawsonia inermis L.).

An improved micropropagation protocol has been developed for a cosmetically important, dye yielding crop, henna (Lawsonia inermis). Quality of henna product is governed by naphthoquinone based pigment lawsone, thus in vitro multiplication of superior healthy plant to achieve enhanced productivity in terms of dye content and biomass deserve due attention. In the present study, nodal explants collected from an elite plant screened on the basis of superiority in lawsone content was cultured on MS medium supplemented with 0.5 µM benzyl adenine (BA) gave significantly (p < 0.05) high number of shoots (24.33). The explants placed on MS medium augmented with 0.5 µM BA and 2-isopentenyladenine (2-iP) resulted in the formation of maximum number of shoots (43.67) and was elongated (12.57 cm) within 4 weeks of culture period. Enhanced axillary bud proliferation and production of mass number of micro shoots was achieved by the continuous subculture in MS medium containing 0.5 µM BA and 2-iP. In vitro raised micro shoots were dipped in 0.44 mM NAA for 5 min followed by planting in polyethylene pots containing a soil: vermiculite (1:1 v/v) mixture produced rooted plantlets (100%). Different auxin types and its concentrations had significant role rooting of L. inermis. Rooting response of various size shoots of L. inermis treated with 0.44 mM NAA showed 100% rooting in 4.1-5 cm size class shoots. After two months of potting, survived (95%) plants were successfully transferred to medicinal plant garden of the Department. The lawsone content of one-year-old micropropagated plants (23.04 mg/g dw) growing in normal environmental conditions and elite mother plant (22.84 mg/g dw) was almost similar. Through the present study, efficient cloning of superior germplasm of L. inermis was established.

An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop.

Momordica dioica Roxb. ex Willd., is a perennial and dioecious (2n = 28) plant of family Cucurbitaceae. Conventional methods of propagation through seeds, stem cuttings and rhizomatous/tuberous roots are inadequate for its mass cultivation as a vegetable crop. This paper reports an improved and efficient micropropagation method for wild female M. dioica using nodal explants. Shoot amplification was achieved using subculturing of in vitro raised shoots on MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid (IAA). The maximum number of shoots (45.30 ± 3.83) with an average length 6.52 ± 0.89 cm were differentiated on MS medium containing 0.5 mg L-1 BAP, 0.1 mg L-1 IAA and additives (50 mg L-1 ascorbic acid, 25 mg L-1 each of adenine sulphate, citric acid and l-arginine). The cloned shoots were rooted ex vitro. Each shoot treated with 250 mg L-1 IBA for 5 min produced 12.3 ± 1.33 with a mean length 5.4 ± 0.73 cm. More than 85% (46 plants) of ex vitro rooted plantlets were successfully hardened in a greenhouse with normal growth characteristics. In order to evaluate the genetic stability of micropropagated plants, the two PCR-based techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used. The amplification patterns of the micropropagated and mother plant were monomorphic thus depicting genetic stability of the micropropagation system. This protocol could be effectively employed for the mass multiplication of wild female M. dioica, a popular summer vegetable crop.

Optimization of in vitro and ex vitro regeneration and micromorphological studies in Basella alba L.

The optimum concentrations of the plant hormones for in vitro regeneration and subsequent effect of auxins on rooting (in vitro and ex vitro) of shoots of Basella alba L. have been investigated in present study. Nodal shoot segments were used as explants to initiate the cultures. The bud breaking from explants was observed within 1 week of incubation on agar gelled Murashige and Skoog's (MS) medium. Multiple axillary shoots (7.30 ± 0.56 shoots per explant) were induced on MS medium supplemented with 2.0 mg/L 6-benzylaminopurine (BAP). The shoots were multiplied (maximum 17.10 ± 0.44 shoots per explant) on the same medium fortified with 0.5 mg/L each of BAP and Kin (Kinetin) +0.1 mg/L IAA. These shoots were excised and rooted in vitro (10.73 ± 0.92 roots per shoot) on half-strength MS medium augmented with 2.0 mg/L indole-3 butyric acid (IBA). Hundred percentage success rates have been achieved by ex vitro rooting of the in vitro regenerated shoots with IBA at 300 mg/L. The in vitro and ex vitro rooted shoots were acclimatized in greenhouse and subsequently transferred to the natural field conditions where 100 % survival rate was reported. The ex vitro rooting method was found more advantageous than in vitro rooting in terms of time, energy and survival percentage of B. alba. A comparative foliar micromorphological study of B. alba was conducted to understand the micromorphological changes in plants while shifting from in vitro to the in vivo conditions in terms of variations in stomatal index, venation pattern and vein density, and the arrangement of crystals. The study could help in understanding the response of in vitro raised plants towards in vivo environment.

In vitro propagation, micromorphological studies and ex vitro rooting of cannon ball tree (Couroupita guianensis aubl.): a multipurpose threatened species.

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l(-1) indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l(-1) 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l(-1) each of BAP and Kinetin (Kin) + 0.5 mg l(-1) α-naphthalene acetic acid (NAA) with additives (50 mg l(-1) of ascorbic acid and 25 mg l(-1) each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l(-1) IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l(-1) of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7-8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0 mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25 mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1 mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500 mg l(-1) of IBA (indole-3 butyric acid) for 3 min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85 % survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting.

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20-25-year-old lopped tree, on MS medium containing 8.88 µM benzylaminopurine (BAP). Multiple shoots differentiated (20-21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 µM of BAP and 0.002 µM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO3)2, K2SO4, KCl, and NH4(SO4)2. The maximum shoot multiplication (29-30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 µM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2-3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.-an important multipurpose plant of the Pacific traditional Medicine.

A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0 µM was most effective in inducing multiple shoots where 90 % explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1 cm) after 6 weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1 cm were obtained on completion of 12 weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500 µM indole-3-butyric acid for 15 min which was the most effective in inducing roots, as 95 % of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92 % survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert.

Ephedra foliata Boiss. & Kotschy ex Boiss., (family - Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l(-1) of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR's) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l(-1) ammonium sulphate {(NH4) 2SO4} (MMS3) + BA (0.25 mg l(-1)), Kinetin (Kin; 0.25 mg l(-1)), Indole-3-acetic acid (IAA; 0.1 mg l(-1)) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l(-1) of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.

In Vitro Propagation and Conservation of Lavandula stoechas subsp. luisieri and Pterospartum tridentatum, Two Important Medicinal and Aromatic Species from Portugal.

Lavandula stoechas subsp. luisieri and Pterospartum tridentatum are two valuable aromatic and medicinal plants. Their biometric and morphological parameters, such as the number of new shoots, length of the longest shoot, multiplication rate, and fresh weight, were evaluated using the multiplication MS medium protocol. The rooting protocols involved immersing the explants in IBA (1 g L-1) and a commercial IBA (3.3 g L-1) preparation (Clonex®). Slow-growth conservation assays were carried out using two different sucrose concentrations (15 g L-1 and 30 g L-1), and a control, with the cultures kept at 4 °C for 12 months. The multiplication rate for L. stoechas subsp. luisieri was 6.8, and that of P. tridentatum was 13.3, achieved using the MS medium supplemented with 0.2 mg L-1 BAP, 1 mg L-1 BAP, and 0.5 mg L-1 IBA. The application of Clonex® showed the best ex vitro rooting results in L. stoechas subsp. luisieri (77%) and P. tridentatum (90%). In the slow-growth conservation assays, at 4 °C, in darkness for 12 months, an excellent survival rate was achieved in L. stoechas subsp. luisieri (>80%) and P. tridentatum (>90%), even at the reduced sucrose concentration. This study demonstrates the effectiveness of in vitro multiplication and ex vitro rooting protocols for two valuable aromatic and medicinal plants. These findings are significant for the ex situ conservation of these species, as they provide effective long-term preservation and utilization strategies.

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 µM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 µM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

Micropropagation as a Tool for the Conservation of Autochthonous Sorbus Species of Czechia.

Members of the genus Sorbus are the only endemic tree species that occur in Czechia. They are important components of endangered plant communities. Their natural regeneration is usually problematic because of their mode of reproduction and because they can survive in rare populations with small numbers of individuals. The aim of this study was to develop a successful micropropagation protocol for selected Sorbus species, of which two are endemic (S. gemella and S. omissa) and two are hybrid (S. × abscondita and S. × kitaibeliana). We found significant differences in shoot induction and rooting ability between the Sorbus species under study. With the exception of S. × abscondita, N6-benzyladenine had a significantly greater effect on shoot regeneration, both in terms of shoot number and total shoot length, than meta-topolin. Root induction was key to the successful micropropagation of the Sorbus species studied. Our results show that four Sorbus species can be successfully rooted under ex vitro conditions, without a rooting powder treatment in a steamed peat-perlite substrate. Auxin-untreated microcuttings of S. gemella, S. × kitaibeliana and S. omissa, but not S. × abscondita, rooted better than ones treated with indole-3-butyric acid. This is the first time a micropropagation protocol for S. omissa, S. × abscondita and S. × kitaibeliana has been published.

Micropropagation of commercially cultivated Henna (Lawsonia inermis) using nodal explants.

Lawsonia inermis Linn. (Mehandi) is cultivated as cash crop in India particularly in Sojat area of Pali district, Rajasthan. Present investigation describes an efficient regeneration system for elite genotype of L. inermis using nodal segments. Optimum response in terms of percent cultures responding, days to bud break and average shoot length was observed on MS medium supplemented with 6-benzylaminopurine (BA; 2.0 mg l(-1)). Shoot multiplication was influenced by plant growth regulators, repeated transfer of explants and addition of ammonium sulphate. Maximum shoots were regenerated on MS medium supplemented with BA (0.25 mg l(-1)), kinetin (Kn; 0.25 mg l(-1)), indole-3-acetic acid (IAA; 0.1 mg l(-1)) and ammonium sulphate (150 mg l(-1)). To reduce resources, time and labours costs, we have also attempted ex vitro rooting of shoots. About 95 % shoots were rooted ex vitro on soilrite after treatment with indole-3-butyric acid (IBA; 300 mg l(-1)) and 2-naphthoxy acetic acid (NOA; 100 mg l(-1)) and establishment in soil successfully.

Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.

The effective reversion of hyperhydricity (HH) in Dianthus chinensis L. facilitated efficient in vitro production of hyperhydricity-free plantlets. Under routine sub-culture practice, the problem of HH arises after third sub-culture in agar (0.85%) gelled Murashige and Skoog (MS) medium containing 2.5 µM 6-benzyladenine (BA). To confirm the role of ethylene on hyperhydricity induction, an ethylene releasing compound ethephon (5 µM) was used in combination with 2.5 µM BA and demonstrated 100% HH with reduced stomatal aperture. Supplementation of 10 µM silver nitrate (AgNO3) to 2.5 µM BA containing medium resulted HH reversion with reduced shoot number (19.0); however, addition of 5 µM cobalt chloride (CoCl2) produced highest microshoots (202.0). The combination effect of AgNO3 (10 µM), CoCl2 (5 µM), and BA (2.5 µM) showed complete HH reversion and upheld normal microshoots (55.0) with reduced relative water content (78.3%). The Ag and Co salts regulate ethylene biosynthesis and thereby 50% reductions in H2O2 content characterized by formation of green healthy shoots with proper stomatal morphology. The gene expression profile of 1-Amminocyclopropane-1-carboxylase synthase (ACS1) and 1-Amminocyclopropane-1-carboxylic acid oxidase (ACO1) showed reduced expression after the retroversion of microshoots in anti-ethylene reversion medium compared to hyperhydric shoot. In vitro raised shoots were rooted (93.3%) ex vitro by 10 mM IBA treatment and 92.2% plants were survived. The genetic stability of micropropagated plants were analyzed and proved that addition of low levels of heavy metal salt in the medium does not cause any variation in banding pattern. The protocol forwards a novel method to revert HH of in vitro cultures by adopting intermittent exposure of anti-ethylene compounds added in the medium and the procedure can be applied to many other plants facing similar HH problems. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02645-7.

An improved micropropagation protocol for the ex situ conservation of Mitragyna parvifolia (Roxb.) Korth. (Rubiaceae): an endangered tree of pharmaceutical importance.

Mitragyna parvifolia (Roxb.) Korth., commonly known as "Kadam," is an endangered and pharmaceutically valued tree of the family Rubiaceae. The numerous medicinal properties are attributed to the various alkaloids of this plant. Poor seedling survival (due to very small size of seeds, approximately 10,000 per gm), overexploitation and habitat destruction are the major constraints in conserving the wild stocks of this species. This paper reports a significant, improved, and repeatable micropropagation protocol of M. parvifolia using nodal explants of a mature tree. Nodal explants harvested during spring season from the lopped tree differentiated the maximum number of axillary shoots (5.3 ± 0.82 per node) on full-strength Murashige and Skoog (MS) medium containing 3.0 mg L-1 6-benzylaminopurine (BAP) and additives (25 mg L-1 each of adenine sulfate, L-arginine, and citric acid and 50 mg L-1 ascorbic acid). Shoots were amplified in vitro through (1) recurrent transfer of mother explants and (2) subculturing on fresh nutrient medium. The greatest number of shoots (13.4 ± 1.26) with an average length of 6.2 ± 1.03 cm was produced after 4 wk on MS medium containing 0.5 mg L-1 BAP, 0.25 mg L-1 kinetin (Kin), 0.1 mg L-1 Indole-3-acetic acid (IAA), additives, 100 mg L-1 activated charcoal (AC), and 0.8% (w/v) agar. This is the first report of concurrent ex vitro rooting and acclimatization (CEVRA) in M. parvifolia. About 90% micropropagated shoots rooted ex vitro on pulse treatment of 500 mg L-1 Indole-3-butyric acid (IBA; for 5 min) and produced 8.5 ± 0.97 roots per shoot with an average length of 9.40 ± 1.06 cm, after 5 wk. Over 80% of CEVRA plantlets were successfully transplanted to the soil in field. The defined protocol can be employed for conservation ex situ and restoration/rehabilitation/reintroduction in situ of M. parvifolia.

Effects of Growth Regulators and Gelling Agents on Ex Vitro Rooting of Raspberry.

Successful acclimatization and ex vitro rooting are among the key factors reducing the cost of micropropagated plants. We compared the survival of seven Russian cultivars of raspberry (Rubus idaeus) after rooting in vitro and ex vitro. Rooted shoots adapted to nonsterile conditions much better than nonrooted ones, with survival rates of 81%⁻98% versus 43%⁻76%, respectively. We studied the effects of different combinations of plant-growth regulators and gelling agents added to a proliferation medium on ex vitro rooting of primocane-fruiting raspberry cultivar "Atlant". Reducing the agar concentration from 8 to 6.5 g/L increased the multiplication rate, but caused shoot hyperhydricity. The highest survival rate (97.2%) was observed for shoots grown in a medium containing 0.2 and 0.1 mg/L IBA, and gelled with 5 g/L agar and 0.2 g/L Phytagel. The microshoot height at the multiplication stage did not correlate with the plant growth during acclimatization. The obtained results can be used in the commercial micropropagation of the raspberry.

In vitro regeneration of shoots and ex vitro rooting of an important medicinal plant Passiflora foetida L. through nodal segment cultures.

Methods were developed in the present investigation for cloning and large scale plant production of Passiflora foetida L. germplasm selected from the East-Coast region of South India. Nodal shoot segments were used as explants. The explants were dressed and surface sterilized with 0.1% (w/v) HgCl2. Multiple shoots were induced (6.13 ± 0.22 shoots per explant) by proliferation of nodal shoot meristems on Murashige and Skoog (MS) semi-solid medium + 2.0 mg l-1 6-benzylaminopurine (BAP). The shoots of P. foetida were further multiplied (16.45 ± 0.44 shoots per explant) on MS medium + 0.5 mg l-1 each of BAP and Kinetin (Kin). The in vitro generated shoots were rooted on half-strength MS medium containing 2.5 mg l-1 indole-3 butyric acid (IBA). By this method 67% shoots were rooted. About 97% shoots were rooted ex vitro (8.33 ± 0.29 roots per shoot) when the cut ends of the shoots were treated with 300 mg l-1 IBA for 5 min. The in vitro rooted plants were hardened and acclimatized in the greenhouse and successfully (100%) transplanted to the field.

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron.

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.

Enraizamento de microestacas de Lavandula angustifolia / Rooting of Lavandula angustifolia microcuttings

A eliminação da etapa de enraizamento in vitro na micropropagação de plantas é desejável do ponto de vista econômico, além de proporcionar a melhoria na qualidade do sistema radicial formado. Dois experimentos foram realizados com os objetivos de avaliar diferentes concentrações (0; 2,5; 5,0 e 10mM) de ácido indolbutírico (AIB) no enraizamento ex vitro de lavanda (L. angustifolia), cv. 'Provence Blue' e avaliar a capacidade de enraizamento ex vitro das cultivares 'Vera', 'Provence Blue', 'English' e 'Elegance Ice'. Após 30 dias, foi avaliado o número de microestacas enraizadas, comprimento das raízes principais, porcentagem de enraizamento e porcentagem de sobrevivência. A concentração de 5,0mM de AIB foi mais efetiva para o comprimento de raízes e porcentagem de enraizamento das microestacas de lavanda cv. 'Provence Blue', apesar de reduzir o número de raízes formadas. Entre as cultivares estudadas, a porcentagem de sobrevivência das plantas variou de 82 por cento a 100 por cento. As cultivares apresentaram diferenças no enraizamento ex vitro das microestacas, sendo as maiores médias de porcentagem de enraizamento registradas na 'Provence Blue' e 'Elegance Ice'. Conclui-se que a microestaquia pode ser uma técnica eficiente para a propagação de lavanda, pelo tratamento das microestacas com 5,0mM de AIB, por proporcionar alta porcentagem de enraizamento e sobrevivência das plantas.

Two experiments were carried out aiming to evaluate the ex vitro rooting of L. angustifolia cv. 'Provence Blue' treated with different concentrations (0, 2.5, 5.0 and 10mM) of indolebutyric acid (IBA) with talc as a vehicle to evaluated the ex vitro rooting of 'Vera', 'Provence Blue', 'English' and 'Elegance Ice' lavender cultivars. The experiments were carried out in a greenhouse using three concentrations of AIB plus control. After the 30th day, it was evaluated: surviving microcuttings percentage, percentage of rooted microcuttings, roots number, roots length. It was concluded that the microcutting can be an efficient technique for lavender plantlets production, by using micropropagated plants as explants donor. The concentration of 5.0mM IBA was more effective on root length and rooting rate of microcuttings lavender cv. 'Provence Blue', despite reducing the number of roots. Among the cultivars the percentage of plant survival ranged from 82 percent to 100 percent. The lavender cultivars evaluated showed differences in ex vitro microcuttings rooting. The cultivars 'Provence Blue' and 'Elegance Ice' achieved higher percentage of rooting. The cultivars had no influence on the percentage of microcuttings survival. It was concluded that the microcuttings can be an efficient technique for the propagation of lavender since microcuttings treated with 5.0mM IBA have higher survival and rooting percentage of rooting and survival.