Adventitious Shoots Research Articles

High-frequency adventitious shoot organogenesis from in vitro stem explants of Scutellaria araxensis Grossh.

The present study introduced an in vitro shoot organogenesis protocol for the medicinal plant Scutellaria araxensis (Lamiaceae). Stem, leaf, and petiole explants were cultured in half-strength Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP) alone or in combination with thidiazuron (TDZ), indole-3-butyric acid (IBA), or α-naphthalene acetic acid. Callus formation occurred from stem and petiole explants in most cultures; however, in leaf explants, it was observed only in cultures containing 0.5 mg/l BAP supplemented with TDZ at all concentrations. The highest frequency of indirect shoot induction (100 and 90%) with an average of 20.33 and 12 shoots per explant was observed in stem-derived calli cultured on half-strength MS medium containing 2.0 mg/l BAP plus 0.5 and 1.5 mg/l TDZ, respectively. The best direct shoot organogenesis (40%) was observed in stem explants cultured on half-strength MS medium containing 0.5 mg/l BAP and 0.5 mg/l IBA with a mean of 18 shoots per stem explant. The regenerated micro-shoots were elongated on a medium fortified with 0.5 mg/l gibberellic acid and then successfully rooted in half-strength MS medium supplemented with 0.5 mg/l IBA. The obtained plantlets were acclimatized in a growth chamber with a survival rate of 100%. This study is the first report of a simple and efficient in vitro shoot organogenesis and regeneration protocol for S. araxensis by using stem explants, which could be useful for the conservation, genetic manipulation, and exploitation of biological molecules of this valuable genetic source.

Synergistic effect of benzylaminopurine and meta-Topolin combination for micropropagation of gerbera ‘Pink Melody’

ABSTRACT Gerbera (Gerbera jamesonii Bolus ex Hook. F.; Asteraceae), is one of the most economically important ornamental plants due to its aesthetic value. In the present study, we established a micropropagation method for the large-scale production of quality planting material of gerbera ‘Pink Melody’. Eighty-six percent of the capitulum explants produced adventitious shoots (15.44 ± 0.34 shoots per capitulum) on Murashige and Skoogs (MS) medium containing 2 mg L−1 6-benzylaminopurine (BAP) after six weeks of incubation. The highest shoot multiplication rate (17 shoots per explant) was obtained on MS medium supplemented with BAP and meta-Topolin (each at 2 mg L−1) after eight weeks. The micro-shoots were successfully rooted (91.35%) on half-strength MS medium containing 2 mg L−1 indole-3-butyric acid (IBA) within four weeks. The micropropagated plantlets were acclimatized with a 97.5% survival rate and produced flowers with no visible morphological aberrations.

Mutagénesis in vitro en auturio inducida mediante colchicina

Developing new varieties of anthurium by hybridization can take 8-10 years; therefore, induced mutagenesis can be an alternative strategy to hybridization. The objective of this work was to induce mutations in A. andreanum by exposing explants obtained from vitroplants to colchicine. Explants of leaves, nodes and roots obtained from vitroplants were exposed to 0.1 % colchicine for 0, 2, 3 and 4 h. The mean lethal dose (LD50), survival, number of explants that generated callus, number of explants that formed shoots and the number of shoots per explant were evaluated. The karyotype of the presumed mutated regenerated plants was determined by the root apex squash technique. The leaves showed the highest sensitivity to cochicine. The survival of the root explants treated with colchicine was 100 %; 4 % of roots exposed for 2 and 3 h formed adventitious shoots (120 shoots). For nodes, the LD50 was found at 3.98 h; 76 and 56 % of the nodes cultivated for 2 and 3 h with colchicine formed adventitious shoots (4.4 and 3.6 shoots). The plants regenerated from the explants exposed to colchicine showed morphological changes. The chromosomal number of the regenerated vitroplants from the explants exposed for 2 and 3 h to colchicine was 2n = 29, while that of those obtained from the explants that remained on the colchicine for 4 h was 2n = 31. The sensitivity to colchicine was a function of the type of explant and the dose used. Colchicine caused the loss (monosomy) or gain of chromosomes (trisomy).

Use of Thin Cell Layer Technique for Induction of Somatic Embryogenesis of Agave fourcroydes.

Agave fourcroydes (henequén) is a plant used for the extraction of hard fiber from its leaves. Due to its long-life cycle, it is very difficult to genetically improve. Somatic embryogenesis (SE) is a very useful micropropagation technique, that can be used for genetic improvement programs and increase the micropropagation of this species. SE is a morphogenic process by which somatic embryos are generated from somatic cells reprogramming. To initiate the regeneration program, the loss of cell-cell communication is suggested to be important. The Thin Cell Layer (TCL) technique allows for the isolation of specific cell or tissue layers, and in conjunction with strictly controlled growth conditions, may lead to the in vitro induction of specific morphogenic programs. Here, we describe a new protocol for the induction of somatic embryogenesis through TCL culture technique, from stem of elite clonal A. fourcroydes vitroplants previously generated through micropropagation of adventitious shoots.

ФАК ТОРЫ ПРЯМОГО ПРОРАСТАНИЯ МИКРОСПОРОГЕННЫХ ЭМБРИОИДОВ BRASSICA NAPUS L.

Isolated microspore culture is the main method of producing doubled rapeseed haploids and is widely used in research institutions and commercial companies. The protocol of rapeseed embryo production is well developed and efficient for many genotypes, but some issues remain due to the low regeneration frequency of plantlets from embryos. When the standard protocol is applied, regeneration of plantlets from microspore-derived embryos usually involves a callus-forming stage followed by regeneration of adventitious shoots or secondary embryos, which prolong the period of plantlet regeneration and makes production of doubled haploids complicated. Identifying factors, which affect the frequency of direct embryo germination will increase the frequency of plantlet formation and reduce the period of DH plant production. In this work, we studied the effect of medium pH on the duration of plantlet regeneration from rapeseed microspore-derived embryos and effect of their low temperature treatment of +1 and +5℃ for 3, 6, 8, 9, 12 days in complete darkness on the embryo maturation and germination. Rising the pH of the nutrient medium from 5.8 to 6.1 increased the frequency of direct embryos germination up to 18% and the overall frequency of plantlet regeneration up to 76%. Culturing embryos at low temperatures effected the frequency of direct germination of embryos into plantlets. The maximum frequency of 44–53% direct embryo germination was observed when cultured at +1℃ for 6 and 9 days, when embryos were cultured at +5℃ the frequency of direct germination was 0–10%. In the control variant without cold treatment it was 16%.

Effects of different BA and IBA concentrations on proliferation and rooting of ‘GARNEM’ rootstock in vitro propagation

In this study, the regeneration shoot tip and nodal explants grown in vitro conditions of ‘Garnem’ hybrid rootstock were investigated, the effect of different phytohormone and concentrations on obtaining adventitious shoots from different explants was investigated, so an efficient protocol was developed for in vitro regeneration of ‘Garnem’ hybrid rootstock These outputs of the study can be a reference resource for future in vitro and biotechnological studies on the rootstock in question. The differences of PGR in MS medium culture containing node explants infinite (0.5-2.0 mg / l) BA (benzyladenine) were investigated. Upon the research, it was observed that the number of shoots andproliferationwere achieved in explants of nodal cuttings that were taken to culture in MS medium containing 2.0 mg / l BA. It has been determined that the most effective culture medium for the elongation of shoot length is MS medium containing 0.5 mg / l BA, 30 g / l sucrose and 5.5 g / l agar. Shoots growing in length were transferred to a new culture with ½ MS medium 0.5-2.0 mg / l IBA (indole-3-butyric acid) to be rooted. While rooting at a rate of 42.8% was achieved in ½ MS medium containing 2 mg / l IBA, 47.2% of rooted plantlets were acclimatized to ex- vitro conditions. Rooted plantlets obtained under in vitro conditions were transferred to plastic containers with 3 different environments in order to get accustomed to external conditions. At the end of the 8th week, the vitality rates of the plantlets were determined. While the viability rate of the plantlets transferred to the medium containing peat: perlite at the ratio of 1: 1 was found to be 47.2%, the viability rate of the plantlets in the environment containing only perlite was found to be 32.8%, and the viability rate of the plantlets in the environment containing only peat was found as 23.6%

Use of Biotechnological Methods to Support the Production of New Peach Hybrids

Peach [Prunus persica (L.) Batsch] is among the most demanded fruit crops in the world. Biotechnological methods help to originate new hybrid forms in order to increase the cultivar diversity and create new valuable genotypes. Cross combinations between the cultivars Clyde Wilson, Jerseyglo, Loadel, Summerglo and the promising cultivar ‘Nikitskiy Podarok’ have been done. The embryos of these hybrids germinated and formed plantlets after stratification at 4 °C for 45–60 days. The best regeneration rates in the hybrids ‘Loadel’ × ‘Nikitskiy Podarok’ and ‘Summerglo’ × ‘Nikitskiy Podarok’ (96.30% and 92.59%, respectively) were noted on hormone-free Monnier culture medium supplemented with 400.0 mg L−1 casein hydrolyzate. When the newly formed plantlets had necrosis of the shoot apex or immature roots, nodal shoot segments were used. At the same time, a high regeneration capacity was noted in the hybrids ‘Summerglo’ × ‘Nikitskiy Podarok’ and ‘Loadel’ × ‘Nikitskiy Podarok’ on B5 culture medium with 0.75 mg L−1 6–benzyl–aminopurine (BAP) + 0.1 mg L−1 indole–3–butyric acid (IBA). After the second subculture, the number of new adventitious shoots was 5.18 ± 0.18 and 4.95 ± 0.18 shoots per explant, respectively. The plants obtained from the hybrid embryos in a soil mixture soil: peat: sand (3:1:1) were adapted. The main morphological and anatomical features of the leaf blades in newly originated peach hybrids have been studied: the thickness of their tissues and the distribution of stomatal apparatus, as well as the physiological parameters of the photosystem II activity in regenerants cultured in vitro and during their in vivo acclimatization. The high capacity to post aseptic adaptation in the obtained hybrids has been shown.

An Effective Protocol for Carnation (Dianthus caryophyllus L.) cv. 'Geolei' Explants Sterilization for Successful Callusing and Shoot Regeneration

Carnation is a popular floricultural crop grown widely for its attractive cut flowers. Micro-propagation can be used to create large-scale carnation output. For growth and development, plants require some necessary nutrients as well as growth regulators. Due to the importance of carnation, the present work is carried out using leaf and nodal segments to examine the potential of several plant growth regulators for in vitro callus formation and adventitious shoot regeneration. Explants were sterilized properly with bavistin, sodium hypochlorite and mercuric chloride. The minor contaminated cultures were created by consecutively treating the explants with 0.25% bavistin, 0.50% sodium hypochlorite, and 0.1% mercuric chloride for ten, fifteen, and two minutes.
 MS media with 2.5 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in combination with 0.75 mg/l naphthalene acetic acid (NAA) resulted in the maximum callus induction (90.47%) from leaf explants. Maximum shoots (76.47%) were produced in MS media supplemented with 2.0 mg/l Thidiazuron (TDZ) + 0.25 mg/l NAA. NAA at 1.25 mg/l was most efficient for maximum root induction (83.32%). In the present study, an effective protocol of carnation explants sterilization was optimized for successful callusing and shoot regeneration.

In Vitro Adventitious Regeneration of Artemisia annua L. Influencing Artemisinin Metabolism

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using ‘Artemis’ progeny was established and the potential of tissue culture for inducing new variability in terms of AN metabolism of in vitro regenerated plants was investigated. Among the plant growth regulators tested, the cytokinin 6-benzyladenine (BA) at 4.4 μM in combination with the auxin indole-butyric acid (IBA) at 0.35 μM yielded the greatest frequency of shoot induction. The optimal multiplication medium contained BA at 0.9 μM and naphthaleneacetic acid (NAA) at 0.05 μM. Regenerated plants (RPs), after transferring to the greenhouse and subsequently to the field, were analyzed during the growth cycle at different sampling times, showing a peak of AN content 20 days before blossom. Variability among different RPs and sampling times, in terms of AN and its precursors dihydroartemisinic acid (DHAA) and artemisinic acid (AA) was observed. This suggests that adventitious shoot induction could provide a useful strategy to induce variability influencing artemisinin metabolism as a consequence of in vitro manipulation.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots.

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2–13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.

Micropropagation of Rare Scutellaria havanensis Jacq. and Preliminary Studies on Antioxidant Capacity and Anti-Cancer Potential.

We report the development of in vitro propagation protocols through an adventitious shoot induction pathway for a rare and medicinal Scutellaria havanensis. In vitro propagation studies using nodal explants showed MS medium supplemented with 10 µM 6-Benzylaminopurine induced the highest number of adventitious shoots in a time-dependent manner. A ten-day incubation was optimum for shoot bud induction as longer exposures resulted in hyperhydricity of the explants and shoots induced. We also report preliminary evidence of Agrobacterium tumefaciens EHA105-mediated gene transfer transiently expressing the green fluorescent protein in this species. Transformation studies exhibited amenability of various explant tissues, internode being the most receptive. As the plant has medicinal value, research was carried out to evaluate its potential antioxidant capacity and the efficacy of methanolic leaf extracts in curbing the viability of human colorectal cancer cell line HCT116. Comparative total polyphenol and flavonoid content measurement of fresh and air-dried leaf extract revealed that the fresh leaf extracts contain higher total polyphenol and flavonoid content. The HCT 116 cell viability was assessed by colorimetric assay using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, showed a steady growth inhibition after 24 h of incubation. Scanning electron microscopy of leaf surface revealed a high density of glandular and non-glandular trichomes. This research provides a basis for the conservation of this rare plant and future phytochemical screening and clinical research.

A Novel and Efficient In Vitro Organogenesis Approach for Ajuga lupulina Maxim.

This work was aimed at establishing an effective approach for in vitro propagation of Ajuga lupulina Maxim, a medicinal and ornamental plant mainly found in eastern Xizang, in the western Sichuan region of China. We report an optimum response in the proliferation of axillary shoots from nodal segment explants (10.2 shoots/explant) on MS medium containing 3.0 mg L−1 of 6-benzyladenine (BA). When BA and TDZ individually or in combination with NAA were employed for adventitious shoot regeneration, shoots and embryo-like structures (ELSs) were noted in the callus from leaf explants. The maximum response of 26.4 shoots /explant (81.6%) and 12.0 ELSs/explant were ascertained on MS medium with 4.0 mg L−1 TDZ and 0.1 mg L−1 NAA. The leaf despite browning still demonstrated a high regeneration capacity. TDZ (2.0 mg L−1) and BA (2.0 mg L−1) along with NAA (0.01 mg L−1) were found to perform well for shoot regeneration via callus from shoot tip explants. The best for rooting was MS medium (half-strength) containing indole-3-butyric acid (IBA: 1.5 mg L−1) and (NAA: 0.5 mg L−1) with the maximum number of roots (25.8 per shoot) and the highest rooting frequency (81.71%). The survival of the plantlets in the greenhouse was 78.2% indicative of successful acclimatization. This work is the first report of a consistent, definitive, and unique protocol for A. lupulina regeneration, paving the way for the in vitro preservation of such significant genetic resources and also further allied systems based on such callus-based or embryo-based approaches.

In vitro propagation via organogenesis and formation of globular bodies of Salvia plebeia: a valuable medicinal plant

As a valuable medicinal plant, Salvia plebeia R. Brown (S. plebeia) belongs to the Lamiaceae family that has been subjected to over-exploitation in its natural habitat for phytochemical and pharmacological studies. The present study focuses on the development of a micropropagation protocol for sustainable propagation and conservation of S. plebeia. Direct organogenesis (from shoot tips and cotyledonary nodes explants) and globular bodies (GBs) induction (from hypocotyl explants) systems have been established in S. plebeia. Alternative collection methods need to be developed for the large-scale propagation of S. plebeia. In addition, roots as explants can also induce adventitious shoots via callus. The highest and number of regenerated shoots (7.0 ± 0.8) per shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 0.1 mg L−1 indole-3-acetic acid (IAA) and 1.0 mg L−1 6-benzyladenine (6-BA), the proliferation of shoots and shoots rooted were carried out on the same medium treatments almost synchronously. Similarly, MS medium supplemented with 0.1 mg L−1 IAA and 1.0 mg L−1 thidiazuron (TDZ) yielded the maximum number of shoots (37.5 ± 1.3) with 100% shoot sprouting frequency. Simultaneously, a protocol was developed for GB induction from hypocotyl explants, and it produced 17.4 GBs per explant with 82.7% response on MS medium supplemented with TDZ (1.0 mg L−1) and IAA (0.1 mg L−1), and produced GBs were characterized by globular, heart-shaped, and cotyledonary stages and successfully germinated on hormone-free MS medium, the developmental process of which was similar to that embryo. The acclimatized plantlets with well-developed root systems were successfully shifted to the natural soils with a 100% survival rate. Taken together, it is the foremost report on in vitro regeneration of S. plebeia; this work will facilitate in germplasm preservation and genetic transformation and provides necessary plant materials for various biotechnological and pharmaceutical applications.

The use of biotechnology in hop (Humulus lupulus L.) improvement

Hop (Humulus lupulus L.) is a clonally propagated, dioecious, perennial, climbing plant used commercially for their secondary metabolites. The resins containing α- and β-acids, and essential oils produced by the lupulin glands, present on the female flowers are used to add bitterness, aroma and flavour to beer. Recently, flavonoids, including chalcones and flavanones, of hops have been shown to exert a variety of biological activities, including oestrogenic and anticancerogenic characteristics. In this review, we provide a overview of the techniques and opportunities presented by the integration of plant biotechnology into hop improvement. The use of tissue culture techniques such as micropropagation, meristem culture, in vitro storage, adventitious shoot induction, callus culture and cell suspension culture in hops are briefly reviewed. The usefullness of genetic transformation technology to introduce novel traits into hop is also discussed.

Effect of medium composition, genotype and age of explant on the regeneration of hexaploid plants from endosperm culture of tetraploid kiwiberry (Actinidia arguta)

Endosperm, an ephemeral and storage tissue, serves as a source of nutrition and protection during embryo development and germination. It can be used for the cultivation of polyploid plants in vitro. Here, results of plant regeneration and acclimatization from the endosperm-derived calli of four cultivars of Actinidia arguta has been presented. Seeds excised from fresh fruit and dry seeds stored for one year served as the sources of endosperm explants of selected tetraploid cultivars of A. arguta. Callus Induction Medium (CIM; containing 0.25, 0.5, or 1 mg/l of TDZ) and Actinidia Endosperm Medium (AEM; containing 2 mg/l of 2,4-D and 5 mg/l of kinetin) were used to study the organogenic responses of the calli. On AEM, the source of explant did not significantly affect the rate of callus induction for any of the tested cultivars; no organogenic events were observed. In contrast, on CIM both the source of explants and the cultivar origin caused significant differences in callus formation and subsequent organogenic events. Histological and ultrastructural analyses revealed the adventitious nature of shoot bud formation on these media. The most efficient elongation of shoot buds was achieved after transferring organogenic calli with adventitious shoot buds to a medium supplemented with zeatin or meta-topolin. Robust root induction with minimal basal callus formation occurred on the medium with indole-3-acetic acid. Flow cytometric analysis revealed that the nuclear DNA content in the leaves of some regenerants was approximately 50 % higher (4.5 pg/2C) than that in leaves from the tetraploid seedlings (3.1 pg/2C),which confirmed that those regenerants originated from the endosperm. The regeneration of such hexaploid plants was more efficient when endosperm from fresh seeds served as an explant; therefore, fresh rather than dry seeds are recommended for endosperm-derived plant production. The hexaploid plants of A. arguta can serve as an important source of breeding material.

Influence of Light Conditions and Medium Composition on Morphophysiological Characteristics of Stevia rebaudiana Bertoni In Vitro and In Vivo

We investigated the influence of different conditions (light composition and plant growth regulators (PGRs) in culture media) on the morphophysiological parameters of Stevia rebaudiana Bertoni in vitro and in vivo. Both PGRs and the light spectra applied were found to significantly affect plant morphogenesis. During the micropropagation stage of S. rebaudiana, optimal growth, with a multiplication coefficient of 15, was obtained in an MS culture medium containing 2,4-epibrassinolide (Epin) and indole-3-acetic acid (IAA) at concentrations of 0.1 and 0.5 mg L−1, respectively. During the rooting stage, we found that the addition of 0.5 mg L−1 hydroxycinnamic acid (Zircon) to the MS medium led to an optimal root formation frequency of 85% and resulted in the formation of strong plants with well-developed leaf blades. Cultivation on media containing 0.1 mg L−1 Epin and 0.5 mg L−1 IAA and receiving coherent light irradiation on a weekly basis resulted in a 100% increase in the multiplication coefficient, better adventitious shoot growth, and a 33% increase in the number of leaves. S. rebaudiana microshoots, cultured on MS media containing 1.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 IAA with red monochrome light treatments, increased the multiplication coefficient by 30% compared with controls (white light, media without PGRs).

Establishment of an efficient micropropagation system in enhancing rooting efficiency via stem cuttings of apple rootstock M9T337

Rootstocks play a vital role in regulating the environmental adaptability and controlling the growth and development of apple trees. M9T337, an excellent apple rootstock widely used in commercial orchards, could confer dwarf tree architectures, early fruiting and suitability for high-density planting. However, the rooting ability of M9T3337 is low when it is vegetatively propagated, and researchers have not yet established an efficient micropropagation system. The present study systematically evaluated the multiplication in adventitious shoots and the in vitro formation of adventitious roots to determine the effects of the culture media and plant growth regulators of M9T337 and a rapid micropropagation system was developed. For the shoot multiplication, the highest multiplication index of 3.93 was obtained on Murashige and Skoog (MS) media supplemented with 2.0 mg/L 6-BA, 0.1 mg/L NAA and 0.3 mg/L GA3 from 12 combinations of 6-BA and NAA. Stronger and taller adventitious shoots were grown on MS supplemented with 1.8 mg/L 6-BA and 0.5 mg/L NAA. The optimal media with 100% rooting was obtained using 1/2 MS supplemented with 0.3 mg/L IBA or MS supplemented with 0.6 mg/L IBA for the rooting induction, resulting in mean rooting numbers of 13.00 and 11.33, respectively. Additionally, the effect on rooting of adding 0.3 mg/L IBA or not on the 1/2 MS and MS media was compared; the results suggested that an appropriate IBA concentration was the key to successful rooting. The rooted plantlets were acclimatised in a shaded greenhouse with an 84% survival rate. The established micropropagation system could be used for the rapid propagation of M9T337 for commercial production.

Mass Propagation of Nauclea diderrichii (de Willd. & Th. Dur) Merr. Seedlings through Tissue Culture Technique

Nauclea diderrichii is a tree species of economic importance. However, its plantation establishment is limited by inadequate seedling production. Hence, there is ample scope of tissue culture for its mass propagation. Its in vitro plantlets development as affected by media strengths indicated that 100 % seed germination was obtained in full MS basal medium while the least (3.35 %) was from quarter-strength at 8 Weeks after inoculation (WAI). The effects of BAP and NAA assessed on the growth of its sub-cultured plantlets showed that highest number of leaves (17) and adventitious shoots (3) were obtained from MS basal medium supplemented with 0.1 mg/l BAP only. Whereas, highest shoot length (3.61 cm) and average number of roots (5/plantlet) were obtained from the same medium without hormone(s) at 8 WAI. Further sub-culturing into MS with 0.05 mg/l NAA resulted into plantlets having optimum shoot and massive root growth ready for acclimatization in 6 WAI. The plantlets were successfully acclimatized using coconuthusk/ topsoil mixture with 90 % survival.
 Plant Tissue Cult. & Biotech. 31(1): 51-60, 2021 (June)

Protocols for Adventitious Regeneration of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica 'Jugana'.

The aim of this work was to assess the regeneration capacity of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica cv. ‘Jugana’ from different types of explants under various hormonal treatments. The whole leaves, petioles, and internodal segments of in vitro plants were examined as explants. Several plant growth regulators (cytokinins and auxins) were evaluated for their ability to induce adventitious regeneration. Direct and indirect organogenesis was achieved under certain culture conditions in both species. The frequency of shoot regeneration was strongly dependent on concentrations of plant growth regulators in the induction media (L. kamtschatica ‘Jugana’) or concentrations of plant growth regulators in the induction media and type of explant (A. alnifolia var. cusickii). Results showed that leaves were not suitable explants for A. alnifolia var. cusickii. Both species were able to regenerate shoots from internodal segments and petioles. The highest induction of shoots was obtained on Murashige and Skoog (MS) medium enriched with 2 mg/L thidiazuron (TDZ) and 0.5 mg/L indole-3-butyric acid (IBA) for Amelanchier alnifolia and with 1 mg/L TDZ and 0.2 mg/L indole-3-acetic acid (IAA) for L. kamtschatica ‘Jugana’. Obtained adventitious shoots were further proliferated in order to investigate their multiplication capacity. The multiplication of shoots was successful in all cultivars, with the best results reported in A. alnifolia var. cusickii (7.07 shoots/explant on average).

An efficient in vitro propagation protocol for direct organogenesis from root explants of a multi-purpose plant, Broussonetia papyrifera (L.) L’Hér. ex Vent.

Broussonetia papyrifera (L.) L’Hér. ex Vent. is a perennial woody plant used as source material for papermaking, as woody forage, and as traditional Chinese medicine. In this study, an in vitro adventitious shoot induction procedure for B. papyrifera root explants was developed and evaluated the frequency of adventitious shoot regeneration. It showed that the culture medium, treatment with plant growth regulators, explant developmental stage, explant inoculation mode and genotype significantly affected explant regeneration efficiency. The results suggested that the best medium was half-strength Murashige and Skoog (1/2 MS) medium supplemented with 1.0 mg/L 6-benzyladenine (BA), 0.1 mg/L α-naphthalene acetic acid (NAA), 0.05 mg/L indole-3-butyric acid (IBA), explants derived from roots at the third development stage, inoculation by horizontal planting without cutting, and the genotype was GZ2, delivering a regeneration frequency of 84.44% and the maximum proliferation coefficient was 5.02. The optimal condition for inducing adventitious shoot rooting was 1/2 MS supplemented with 0.1 mg/L NAA. Under this condition, the maximum rooting rate of adventitious shoot was 82.22%, and the root grew strongly. Furthermore, the survival rate of transplanted seedlings was > 90%. Although the regeneration system was influenced by the B. papyrifera genotype, it is still effective for all genotypes studied. Using this novel system, B. papyrifera root explants can attain high induction rates and proliferation coefficients.