Adventitious Shoot Regeneration Research Articles

Rapid Rubber Extraction and NMR Spectroscopy of Rubber, Extracted from the Endemic Species Scorzonera Tau-Saghyz

Scorzonera tau-saghyz Lipsch. et G.G. Bosse is an endemic rubber producing plant, growing in mountain regions in South Kazakhstan. The rubber content in plants and the quality of biopolymer has an important impact on industrial rubber production. The results of this study showed that the amount of rubber in S. tau-saghyz roots fluctuates between 7.74% and 38.75%. The amount of synthesized and deposited rubber biopolymer particles depends on various factors such as physiological age of plant, origin, temperature, moisture and environmental conditions. We optimized the extraction method of natural rubber by using n-hexane as a solvent for direct extraction. This method allows extracting the maximum amount of rubber from 3‒4-year-old plants. NMR results show structural links of natural isoprene rubber in the root extract sample. There is a clear relationship between methyl, methine and methylene protons which corresponds to isoprene rubber structure. The samples having strongly marked singlets that are inherent for rubber functional groups confirms the stereospecific structure of rubber. Good solubility of the root extract in deuterated chloroform can characterize the low molecular weight of the polymer. NMR characterization of rubber, extracted from S. tau-saghyz roots, is reported for the first time. Regeneration in vitro provides an important opportunity for endemic preservation by rapidly increasing the number of plants. The best regeneration of adventitious shoots was obtained on MS medium containing 5.5 μM kinetin and 0.5 μM NAA. The plants were successfully acclimatized in a glasshouse with 75% of S. tau-saghyz plantlets, respectively surviving after transfer to ex vitro conditions.

Hypericum adenotrichum SPACH‘UN KALLUS İNDÜKSİYONU VE ADVENTİF SÜRGÜN REJENERASYONU

In this study, A standart protocol to callus formation and adventitious shoot regeneration of Hypericum adenotrichum Spach via direct or indirect organogenesis has been described.Callus induction was carried out by using leaves which were collected from their native environment. The maximum callus induction frequency has been observed on MS media containing 4 mgL-1 BA and 0.2 mgL-1 NAA. These calli were not induced to shoot regeneration. Shoot formation was obtained by transferring the calluses from BA-containing media to MS medium containing 0.5 mgL-1 KIN. Maximum shoot number was observedin transferred calluses from callus induction medium containing 3 mgL-1 BA to media containing 0.5 mgL-1 KIN.Direct shoot formation developed on leaf explants of H. adenotrichum on MS media containing KIN alone. The highest direct shoot development was observed on MS medium with 1 mgL-1 KIN.

High frequency in vitro regeneration of adventitious shoots in daylilies (Hemerocallis sp) stem tissue using thidiazuron

BackgroundDaylilies are a lucrative crop used for its floral beauty, medicinal proprieties, landscaping, fire prevention, nutritional value, and research. Despite the importance, daylilies remain extremely challenging for multiplying in vitro. The response difficulty is exacerbated because a few good protocols for daylilies micropropagation are generally difficult to reproduce across genotypes. An efficient strategy, currently applied at Langston University, is to systematically explore individual tissues or organs for their potential to micropropagation. This article is a partial report of the investigation carried out under room environmental conditions and focuses on developing an efficient daylilies in vitro propagation protocol that uses the stem tissue as the principal explant.ResultsIn less than three months, using thidiazuron, the use of the stem tissue as the in vitro experimental explant was successful in inducing multiple shoots several folds greater than current daylilies shoot organogenesis protocols. The study showed that tissue culture can be conducted successfully under unrestricted room environmental conditions as well as under the controlled environment of a growth chamber. It also showed that splitting lengthwise stem explants formed multiple shoots several folds greater than cross-sectioned and inverted explants. Shoot conversion rate was mostly independent of the number of shoots formed per explants. The overall response was explant and genotype-dependent. Efficient responses were observed in all thidiazuron treatments.ConclusionAn efficient protocol, which can be applied for mass multiple shoots formation using the daylilies stem tissue as the main explant, was successfully developed. This could lead to a broad and rapid propagation of the crop under an array of environmental conditions to meet the market demand and hasten exogenous gene transfer and breeding selection processes.

A reproducible protocol for adventitious shoot regeneration from leaves of apple rootstock Merton793 and assessment of genetic stability

The present studies were aimed to develop an efficient and reproducible method for shoot regenerationfrom leaf explants of apple rootstock Merton793. The influence of two cytokinins BA and TDZ with three auxinsIAA, IBA and NAA in different combinations were evaluated for shoot induction using light and dark incubation.Direct and healthy shoots were obtained from leaves on MS medium with BA-NAA and BA-IAA combinations inlight incubation and 100% regeneration was achieved on medium with 4 mg/l BA and 1 mg/l NAA. Both directand indirect shoot regeneration was obtained on TDZ-NAA and TDZ-IBA. Maximum shoot regeneration waspromoted on 0.8 & 0.6 mg/l TDZ with 1 mg/l NAA, however, it showed abnormal, vitrified, small and rosettetype of shoots. Dark treated leaf explants showed low rates of shoot induction with TDZ combinations. Healthyadventitious shoots regenerated directly from leaves were multiplied, rooted and hardened successfully. RAPD analysis of regenerants revealed that all the banding profiles from directly originated shoots were monomorphicand similar to those of the mother plant, while shoots originated through intervening callus revealed 61.2%polymorphism. The results show that genetic fidelity of directly produced adventitious shoots was maintained,and thus, our regeneration protocol can be an useful method in transformation research in Merton793 withoutthe induction of variation.

Sodium Nitroprusside Stimulates Micropropagation and TDZ Induces Adventitious Shoots Regeneration in Red Flesh Apple Malus niedzwetzkyana Koehne Dieck ex

Abstract The aim of the present investigation was to optimize protocols for micropropagation and adventitious shoot regeneration from leaf explants of two wild ecotypes of red flesh apple Malus niedzwetzkyana for future breeding programs. At the proliferation stage, different concentrations of sodium nitroprusside (SNP) and triacontanol (TRIA) were compared. To optimize shoot regeneration from leaf explants, interactive effects of 1-phenyl-3-(1,2,3-thidiazol-5-yl)-urea – thidiazuron (TDZ), indole-3-butyric acid (IBA) and two explant types were investigated. At rooting stage, the effect of exposure time of microshoots to darkness and exposure time to different concentrations of IBA and α-naphthalene acetic acid (NAA) were compared. The results showed that SNP affected the growth rate significantly and the maximum multiplication rates per explant (9.6 in the first ecotype and 8.8 in the second) were produced in the Quoirin and Lepoivre medium containing 17 SNP µM, in addition to 4 µm 6-benzylaminopurine (BAP) and 3 µm gibberellic acid (GA3). IBA and TDZ affected the adventitious shoot regeneration from leaf explants significantly, the highest number of regenerated shoots (18.3 per explant) was obtained from the basal section of the leaves cultured on the medium containing 2 μM IBA and 15 μM TDZ. At rooting stage, the maximum rooting (88.6%) was obtained in the result of one weak exposure to darkness on medium containing 3 μM IBA.

Chromosome doubling ofToreniahybrids by using efficient adventitious shoot regeneration in vitro

Chromosome doubling of<i>Torenia</i>hybrids by using efficient adventitious shoot regeneration in vitro

Rejuvenation of chicory and lettuce plants following phase change in tissue culture

BackgroundA frequent problem associated with the tissue culture of Compositae species such as chicory (Cichorium intybus L.) and lettuce (Lactuca sativa L.) is the premature bolting to in vitro flowering of regenerated plants. Plants exhibiting such phase changes have poor survival and poor seed set upon transfer from tissue culture to greenhouse conditions. This can result in the loss of valuable plant lines following applications of cell and tissue culture for genetic manipulation.ResultsThis study demonstrates that chicory and lettuce plants exhibiting stable in vitro flowering can be rejuvenated by a further cycle of adventitious shoot regeneration from cauline leaves. The resulting rejuvenated plants exhibit substantially improved performance following transfer to greenhouse conditions, with increased frequency of plant survival, a doubling of the frequency of plants that flowered, and substantially increased seed production.ConclusionAs soon as in vitro flowering is observed in unique highly-valued chicory and lettuce lines, a further cycle of adventitious shoot regeneration from cauline leaves should be implemented to induce rejuvenation. This re-establishes a juvenile phase accompanied by in vitro rosette formation, resulting in substantially improved survival, flowering and seed set in a greenhouse, thereby ensuring the recovery of future generations from lines genetically manipulated in cell and tissue culture.

Reinvigoration of diploid strawberry (Fragaria vesca) during adventitious shoot regeneration

Diploid strawberry (Fragaria vesca ‘Baiguo’) is a model plant for studying functional genomics in Rosaceae. Adventitious shoot regeneration is essential for functional genomics by Agrobacterium tumefaciens-mediated transformation. An efficient shoot regeneration method using diploid strawberry leaf explants was conducted on 1/2MS + 1/2B5 medium that contained 2.0 mg L−1 TDZ over 14 days of dark culture; this induced the maximum percentage of shoot regeneration (96.44 ± 1.60%) and the highest number of shoots per explant (23.46 ± 2.14) after 11 weeks of culture. The explants considerably enlarged after 12 days; then, turned greenish brown after 30 days, yellowish brown after 36 days, and completely brown and necrotic after 48 days. Large numbers of adventitious shoots were produced from 48 to 66 days, and the shoots elongated from 66 to 78 days; this represents a critical period of reinvigoration, which included 30 days for leaf explant chlorosis, 36 days for adventitious shoot appearance, and 48 days for generation of numerous shoots. During the reinvigoration process, higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days. Our results indicate that these genes may regulate reinvigoration of shoot regeneration.

Silver and gold nanoparticles impact on in vitro adventitious organogenesis in chrysanthemum, gerbera and Cape Primrose

Despite an increasing interest in nanoparticles influence on plant metabolism and growth is recently observed, still it is an under-explored area of research, especially in the context of nanoparticles impact on the in vitro adventitious organogenesis in ornamental plants. The work presents the results of three independent experiments, in which silver and gold nanoparticles, 20 nm-in-size, were poured on the culture medium immediately after explants inoculation. The aim of the Experiment 1 was to evaluate the effect of 10 and 30 ppm silver and gold nanoparticles on the adventitious roots regeneration in Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Bydgoszczanka’ and Gerbera × jamesonii H. Bol ‘Suri’. Chrysanthemum shoot fragments and gerbera single rosettes were polarly inoculated on the modified MS medium with 2 mg∙L−1 indole-3-acetic acid (IAA) for rooting. Chrysanthemum explants treated with 10 and 30 ppm silver nanoparticles regenerated less adventitious roots than control explants. These roots were also of the smallest area and diameter, but the length of the longest root was the highest in the experiment. Roots parameters after gold nanoparticles treatment were similar to those reported for the control object, expect for one parameter – the biggest values of root diameter (for 10 and 30 ppm). As for gerbera, the efficiency of adventitious roots regeneration was also reduced by 10 and 30 ppm silver nanoparticles, but the addition of 10 ppm gold nanoparticles stimulated rhizogenesis. Similarly to chrysanthemum, the greatest length of the longest root, the smallest root system area and root diameter were also observed under the influence of silver nanoparticles. In the Experiment 2 the effect of 10 and 30 ppm silver and gold nanoparticles on the adventitious shoots regeneration in Streptocarpus × hybridus Voss. was tested. Leaf explants were horizontally inoculated on the modified MS medium with 0.1 mg∙L−1 1-naphthaleneacetic acid (NAA). The best effects in the stimulation of the rosulate and phyllomorphs regeneration were reported for both silver and gold nanoparticles at the concentration of 10 ppm. The aim of the Experiment 3 was to evaluate the effect of 50 and 100 ppm silver nanoparticles on the adventitious shoots regeneration in Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’. Internodes used as explants were horizontally put onto the modified MS medium with 0.6 mg∙L−1 BAP (benzylaminopurine) and 2 mg∙L−1 IAA. Formation of adventitious shoots was inhibited as a result of silver nanoparticles application at high concentrations.

Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus.

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 μM 6-Benzyladenine (BA), 0.1 μM 1-Naphthaleneacetic acid (NAA) and 6.0 g L−1 plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 μM). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach.

Adventitious Shoot Regeneration and Agrobacterium tumefaciens-mediated Transient Transformation of Almond × Peach Hybrid Rootstock ‘Hansen 536’

‘Hansen 536’ (Prunus dulcis × Prunus persica) is an important commercial rootstock for peach and almond. However, susceptibility to wet soil and bacterial canker has limited its use primarily to areas with less annual rainfall. Genetic engineering techniques offer an attractive approach to improve effectively the current problems with this cultivar. To develop an efficient shoot regeneration system from leaf explants, 10 culture media containing Murashige and Skoog (MS) or woody plant medium (WPM) supplemented with different plant growth regulators were evaluated, and adventitious shoot regeneration occurred at frequencies ranging from 0% to 36.1%. Optimal regeneration with a frequency of 32.3% to 36.1% occurred with WPM medium containing 8.88 µm 6-benzylamino-purine (BAP) and 0.98 to 3.94 µm indole-3-butyric acid (IBA). The regenerated shoots had a high rooting ability, and 80% of the in vitro shoots tested rooted and survived after being transplanted to substrate directly. Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using all three Agrobacterium tumefaciens strains tested with a concentration of OD600 0.5 to 1.0 for 4 days of cocultivation. The protocols described provide a foundation for further studies to improve shoot regeneration and stable transformation of the important peach and almond rootstock ‘Hansen 536’.

Adventitious shoot regeneration from leaf, petiole and root explants in triploid (Populus alba × P. glandulosa)× P. tomentosa

Leaf, petiole and root of triploid (Populus alba × P. glandulosa) × P. tomentosa were cultured on a medium containing benzyladenine (BA) and naphthaleneacetic acid (NAA) either alone or in combination with thidiazuron (TDZ) for the study of adventitious shoot regeneration. The study demonstrated that TDZ played a primary and distinctive role in shoot regeneration: the frequency of shoot organogenesis of explants cultured with TDZ (71.16%) far surpassed those cultured without TDZ (30.42%). Among explant types, high variation was observed in the regeneration capacity: petiole and leaf had better regeneration capacity than that of root. To understand this difference, an analysis of endogenous hormones (zeatin (ZT), abscisic acid (ABA) and indole-3-acetic acid (IAA)) was conducted on leaf, petiole and root explants. The results showed that the regeneration ability of explants varied dependently on their IAA and ABA contents. The organogenic capacity varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (98.33%) and shoot number (up to 9.4 shoots/explant) were obtained in leaf explants harvested from the intermediate node (node 3). Regenerated shoots transferred to the medium containing 0.5 mg l−1 indole-3-butyric acid (IBA) achieved the highest frequency of rooting (100%), with means of 5.33 roots per shoot and 6.32 cm length per root. The shoots with an abundant root system were later transferred to the greenhouse for further growth and development. The effect of plant growth regulators (PGRs), explant type, endogenous hormone contents and maturity of explant was evaluated on adventitious shoot regeneration of triploid (Populus alba × P. glandulosa) × P. tomentosa in this study.

Establishment of an Efficient Leaf Regeneration System for ‘Jinyan’ Kiwifruit

In this experiment, ‘Jinyan’ kiwifruit field leaves were used as explants for tissue culture to study the effects of different plant growth regulators on the regeneration of leaves of ‘Jinyan’ kiwifruit, including the induction of adventitious shoots, the proliferation of adventitious shoots and the rooting of adventitious buds. Establish a rapid propagation system for ‘Jinyan’ kiwifruit. The results showed that the optimal medium for the regeneration of adventitious shoots of ‘Jinyan’ kiwifruit leaves was MS + 6-BA 4.0 mg·L−1+NAA 0.1 mg·L−1, the budding rate was 76.67%; and the best medium for adventitious shoots proliferation was MS+6-BA 2.0 mg·L−1+NAA 0.4 mg·L−1+GA3 0.1 mg·L−1, the average proliferation coefficient was 3.33; the optimal rooting medium was 1/2MS +IBA 0.6 mg·L−1, the rooting rate was 86.67%, and the average root length was 2.30 cm.

Improvement of in vitro donor plant competence to increase de novo shoot organogenesis in rose genotypes

A procedure was developed for in vitro propagation of Rosa genotypes along with an efficient de novo shoot organogenesis (DNSO) method. We tested, on one genotype (hybrid of Rosa wichurana), the effects of MS basal medium complemented with two growth regulators to achieve either shoot elongation or shoot multiplication of plants. These media were complemented with carbohydrate concentrations from different sources. Then, the impacts of various carbohydrates (fructose, glucose, maltose, sorbitol, sucrose) on the growth and development of several rose genotypes during donor plant subculturing were studied on SMM. The results showed high variability in growth and development between genotypes. Contrary to other members of the Rosaceae family, no correlation was found between the shoot size and number when the amount of sorbitol was increased.Murashige and Skoog medium supplemented with 3.0 mg L−1 BAP and containing fructose or glucose at 30 g L−1 was chosen to induce leaf explants for the DNSO experiments. MS basal medium complemented with TDZ/IBA at three ratios and the same range of carbohydrate sources were tested for DNSO. Significant genotypic variations with regard to the percentage of regeneration was demonstrated with six genotypes. For two genotypes, a hybrid of Rosa wichurana and Rosa ‘White Pet’, we defined the conditions required to obtain 100% DNSO. For Rosa chinensis ‘Old blush’ and the rootstock genotype Rosa ‘Natal Briar’, we obtained 74 and 87.5% DNSO and only 56.67% and 37.5% for Rosa GUY SAVOY® (‘Delstrimen’) and Rosa ‘Félicité et Perpétue’ respectively. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies in Rosa.

Histological Investigations During &lt;i&gt;In-Vitro&lt;/i&gt; Adventitious Shoot Regeneration in Leaflet Explants from Nodal Segments of a Mature Tree of &lt;i&gt;Albizia procera&lt;/i&gt; Benth.

This is a 60 day study of transformation from leaf to callus and adventitious shoot regeneration. For initial 30 days leaf explants were kept on MS medium supplemented with 3% sucrose, 2.5 μM 2,4-D and followed by a subsequent adventitious shoot differentiation phase for another 30 days on half MS medium supplemented with 0.25 μM each of BA and IBA. The histological investigation revealed sequential array of events starting from initiation of callus on upper and lower epidermis of inoculated leaflet to initiation of green shoots from the mass of callus in the culture medium.

Development of markers for shoot organogenesis in roses

Shoot organogenesis is an essential step for genetic engineering and the study of developmental processes in roses. Several adventitious shoot regeneration protocols have been established, but the number of responding cultivars is very limited. Furthermore, large differences in regeneration capacity between genotypes were reported rendering many cultivars as recalcitrant for shoot regeneration. Therefore, knowledge about the genetic complexity of the capacity for adventitious shoot regeneration and genes influencing this trait would be helpful for optimising regeneration protocols. Previously we analysed an association panel of 96 genotypes for in vitro shoot regeneration from petioles, and we detected a number of markers associated with the phenotypic traits. Here we present results of experiments to verify significantly associated markers in independent populations of garden roses using the KASP (Kompetitive Allele Specific PCR) technology.

In vitro adventitious shoot regeneration from leaf explants of some apricot cultivars

ABSTRACT Apricot is one of the most valuable commercial fruits. In vitro propagation of apricot is very important for rapid multiplication of cultivars with desirable traits and production of cleaning up and virus-free plants. Low frequency of multiplication is the main limiting factor for traditional propagation methods. In this regard, the objective of our investigation was to study the morphogenetic capacity of apricot leaf explants of the promising cultivars ‘Iskorka Tavridy’, ‘Magister’ and ‘Bergeron’ for regeneration system development and solving some breeding questions. The source of explants was in vitro plants regenerated and cultured on QL medium. Leaves were maintained in the dark at 24±1 °C in thermostat for three-four weeks. Morphogenic callus and structures were mainly formed at the central and proximal parts of leaves on MS, QL and WPM media with 1.5 or 2.0 mg L-1 BAP and 1.5 or 2.0 mg L-1 IAA in different combinations, or TDZ (0.6 and 1.3 mg L-1). Callus with adventive buds was transferred to regeneration medium and placed into a growth chamber at 24±1 °C and 16-hour photoperiod with a light intensity of 37.5 μmol m-2 s-1. The best results were obtained when adaxial leaf surface was in contact with the culture medium. Frequency of leaf callus formation on MS medium with 1.5 mg L-1 BAP and 1.5 mg L-1 IAA was higher in the explants of ‘Iskorka Tavridy’ (80.0%) than in - ‘Bergeron’ (50.0%) and ‘Magister’ (36.7%). The best results of callogenesis for ‘Magister’ was obtained on MS medium with 1.3 mg L-1 TDZ (53.3%). Active microshoot regeneration in ‘Iskorka Tavridy’ cultivar was shown on MS medium with BAP and IAA and in ‘Magister’ cultivar - on MS medium with TDZ. Rhizogenesis was obtained on half strength MS medium with 2.0 mg L-1 IBA.

Optimisation of Adventitious Shoot Regeneration and Agrobacterium-mediated Transformation in Canna × generalis (Canna Lily)

Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine (6-BA), thidiazuron (TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata’ was the best responding cultivar. And 2 mg · L−1 6-BA or 1.5 mg · L−1 TDZ along with 0.1 mg · L−1 IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata’ (36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L−1 ascorbic acid (AsA), while, 100 mg · L−1 of l-cysteine or 100 mg · L−1 dithiothreitol (DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L−1 indole-3-butyric acid (IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100% survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in Agrobacterium suspension. The explants were washed with 500 mg · L−1 cefotaxime solution, subjected to 100 mg · L−1 kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis.

Assessment of genetic stability and analysis of alkaloids potential in micropropagated plants of Croomia japonica Miquel, an endangered, medicinal plant in China and Japan

Croomia japonica (Stemonaceae) is an endangered species both in China and Japan. We have developed an efficient regeneration system through adventitious buds organogenesis in C. japonica using rhizome buds as explants. Multiple buds regenerated directly on the explants without calli within 2 months when explants were cultured on Murashige and Skoog’s (MS) medium with 8.88 µmol 6-benzylaminopurine (BAP) and 1.07 µmol α-naphthaleneacetic acid (NAA). The adventitious buds of newly forming were proliferated by subsequent subcultures on MS medium with 2.66 µmol BAP and 2.69 µmol NAA. We evaluated the kinds and concentrations of plant growth regulators on adventitious shoot regeneration and root induction and also inspected the adsorbent (polyvinyl pyrrolidone and activated charcoal) and antioxidant (ascorbic acid, AS) on the inhibition of tissue browning. The results showed that soaking the explants with 1.14 mmol AS was the best approach for controlling browning. The maximum number of stout shoots per explant was achieved on MS medium containing 8.88 µmol BAP. In vitro regenerated shoots were rooted on MS medium supplemented with three different concentrations of auxin. The highest rooting rate (84.0 ± 3.6%) was reached on MS medium with 5.71 µmol Indole-3-acetic acid (IAA). One-step culture was developed when the adventitious buds cultured on the medium were supplemented with 2.66 µmol BAP and 0.54–2.69 µmol NAA. Rooted plantlets were acclimatized to the green house and development with a 87% survival rate. Genetic stability assessment of in vitro plants compared to the wild plants was revealed by simple sequence repeat markers. Similarly, flow cytometric analysis confirmed that the ploidy level of in vitro plantlets was stable. Total alkaloid content in wild and in vitro plants was tested by acid dye colorimetric analysis with tuberostemonine as the reference. Our results showed that alkaloids content in 11-week cultures reached 40.7–88.4% of the content of wild plants. The protocol described here could be employed for effective mass propagation of C. japonica for commercial and conservational purposes.

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (Saccharum officinarum L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant−1) and multiple shoots (657 secondary shoots explant−1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.