Adventitious Bud Regeneration Research Articles

The Impacts of Plant Growth Regulators on the Rapid Propagation of Gardenia jasminoides Ellis. in Tissue Culture

The optimization of explant selection and adjustment of plant growth regulators (PGRs) ratio may enhance the efficiency of micro-propagation of Gardenia jasminoides Ellis. The findings of the study suggest that the shoot tip proved to be the optimal explant for regenerating adventitious buds, with an impressive regeneration rate of 77.78% and the average number of adventitious buds being 2.86. The ideal medium consisted of Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (6-BA) at a 2 mg L−1, indoleacetic acid (IAA) at a 0.2 mg L−1, kinetin (KT) at 0.15 mg L−1, resulting in an outstanding regeneration rate of adventitious buds reaching up to 91.11%. For rooting purposes, the best medium was found be half-strength MS supplemented with indoleacetic acid (IAA) 0.5 mg L−1, achieving an rate for adventitious roots amounting to as high as 97.78%. The culture plantlets ultimately thrived, achieving an impressive transplanting survival rate of 93.33%. The application of PGRs was also found to enhance the regeneration of adventitious buds by increasing the ratios of endogenous hormones ZR/IAA and GA3/IAA. Additionally, it facilitated the differentiation of adventitious roots by elevating the ratios of endogenous hormones IAA/ZR, IAA/GA3, and ABA/GA3. Our study would provide a theoretical reference for the establishment of an efficient gardenia tissue culture system and the industrial production of gardenia.

Improvement of Culture Conditions and Plant Growth Regulators for In Vitro Callus Induction and Plant Regeneration in Paeonia lactiflora Pall.

Owing to its high ornamental, medicinal and horticultural values, herbaceous peony (Paeonia lactiflora Pall.) has been widely used as a landscaping and economical plant around the world. However, the lack of an efficient and stable regeneration system in P. lactiflora restricts its rapid propagation and large-scale production. By testing the key factors affecting callus formation, proliferation, adventitious bud induction and rooting, here, we developed an in vitro system for callus induction and regeneration in P. lactiflora. Our results show that callus formation was affected by explant types, culture environment, basal medium and plant growth regulators. Using cotyledons as explants, we established good conditions for P. lactiflora callus induction and callus proliferation. We effectively obtained adventitious buds differentiated from callus in Murashige and Skoog (MS) medium containing kinetin (KT) and thidiazuron (TDZ). Adventitious bud growth can be further promoted by adding gibberellin 3 (GA3), 1-naphthaleneacetic acid (NAA) and 6-benzyleaminopurine (6-BA) into the MS medium. A high percentage of rooting can be achieved by adding indolebutyric acid (IBA) and activated carbon (AC) to ½ MS medium. Overall, our system promotes callus induction and adventitious bud regeneration for P. lactiflora through improved culture conditions and plant growth regulators in the culture media, and lays a foundation for subsequent genetic engineering research.

Molecular Regulation of Bud Regeneration from Callus of Hybrid Sweetgum (Liquidambar styraciflua × Liquidambar formosana)

Adventitious bud regeneration is an effective means of rapid propagation of plants, which can be used in the study of plant development and genetic transformation. It can be divided into direct and indirect adventitious bud regeneration. Of the two kinds of adventitious bud regeneration mentioned, indirect adventitious bud regeneration includes callus formation in vitro and organ regeneration. In the process of callus formation, some cells acquire the pluripotency of tissue regeneration, which is the key to regeneration of adventient buds. It is not clear which molecular processes and genetic factors are involved in establishing cellular pluripotency. The object of the study is hybrid sweetgum (Liquidambar styraciflflua × L. formosana). At present, the reproductive efficiency of hybrid sweetgum is low and the reproductive cycle is long. Improving its reproductive efficiency by improving the differentiation speed of callus may be a decent approach. In order to explore the mechanism of pluripotency acquisition during forming hybrid sweetgum callus, we used RNA-seq to perform transcriptomic analysis of the regenerable calli (RC) and non-regenerable (NRC) calli of hybrid sweetgum. A dataset of differentially expressed genes (DEG) was obtained and several genes probably involved in bud formation were analyzed to explain the molecular processes of acquiring and maintaining pluripotency. In this study, a total of 665 significantly expressed DEGs were identified in the RC and NRC of hybrid sweetgum, among which, 585 differentially expressed genes were up-regulated and 80 differentially expressed genes were down-regulated. GO, KEGG analysis and qRT-PCR results showed phenylpropanoid is a key factor regulating the bud regeneration of hybrid sweetgum; WOX1, WOX11, BGLU12 and BGLU13 were also important regulatory factors. These results provide a pivotal reference point for future sweetgum propagation research.

Influences of Various Factors on Inducing Adventitious Shoot Regeneration From Stem Explants of Cassia Mimosoides Linn.

In the present study, an efficient plant regeneration method was established in Cassia mimosoides by using stem explants. Low regeneration frequency with poor quality advantageous shoot buds were obtained when the explants were grown only Kinetin (KN) or thidiazeuron (TDZ) enriched media. For improving the regeneration efficiency and production of good quality adventitious buds, Cu2+, Mg2+, Fe2+ and Ag+ were supplemented in the regeneration medium at different concentrations. The highest (63.96%) adventitious bud regeneration rate, the maximum number of buds (5.39) per explant and the longest regenerated buds (3.49 cm) were obtained by adding 26 mg/l Ag+ in the regeneration medium. In order to improve the rooting in shoot buds, diethyl aminoethyl hexanoate (DA-6) at different concentrations was added into rooting medium. The optimal rooting was obtained by supplementing 0.6 mg/l DA-6. The highest rooting rate (90.23%), the highest number of roots per bud (6.08) and the longest roots (4.26 cm) were developed within 20 days of culture in this medium. By applying this method culture cycle for obtaining complete regenerated plantlets was significantly shortened (in 60 days), Additional culture for proliferation and elongation of regenerated adventitious buds was not needed. Therefore, the method established in the present study was helpful to improve the efficiency of plant regeneration from stem explants in C. mimosoides. Besides the establishment of this culture method could lay a foundation for the future large-scale cultivation and genetic transformation of C. mimosoides. Bangladesh J. Bot. 51(4): 873-882, 2022 (December) Special

Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × E. grandis DH32-29 in Southern China.

Eucalyptus, as an economically important species for wood and paper industries, remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones. Eucalyptus urophylla × E. grandis clone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system of Eucalyptus was established using E. urophylla × E. grandis DH32-29 as material. The in vitro leaves from microshoots that were subcultured for 20-25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L-1 α-naphthaleneacetic acid (NAA) and 0.24 mg·L-1 forchlorfenuron [callus-inducing medium (CIM)]. After 15 days, explants were transferred to a medium containing 0.10 mg·L-1 NAA and 0.50 mg·L-1 6-benzyladenine (shoot-inducing medium, SIM) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Moreover, an Agrobacterium tumefaciens-mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min with A. tumefaciens strain EHA105 grown to a bacterial density of 0.3 (OD600). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L-1 cefotaxime (Cef), 100 mg·L-1 timentin, and 15 mg·L-1 kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After inducing roots in root-inducing medium supplemented with 200 mg·L-1 Cef and 75 mg·L-1 Kan, completely transgenic plants were obtained. Using the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement of E. urophylla × E. grandis DH32-29 and gene function analysis in Eucalyptus.

Morphological and transcriptome analyses reveal mechanism for efficient regeneration of adventitious buds from in vitro leaves of Rhododendron delavayi regulated by exogenous TDZ

Morphological and transcriptome analyses reveal mechanism for efficient regeneration of adventitious buds from in vitro leaves of Rhododendron delavayi regulated by exogenous TDZ

Establishment and optimization of agrobacterium-mediated transformation in blueberry (Vaccinium species)

Vaccinium species such as blueberry, cranberry and bilberry are widely planted in the world and have high economic values. Current varieties are mostly generated by conventional breeding methods, and application of biotechnology especially genetic modification in the breeding process is seldom. Moreover, mechanistic studies of orphan genes in Vaccinium species are more and more important to facilitate the improvement of yield, quality, resistance to abiotic and biotic stresses in breeding, which requires efficient genetic transformation systems. In this study, we select 19 Vaccinium varieties to investigate the effects of variety specificity, explant types and culture schemes on callus development, adventitious bud regeneration and transformation efficiency. The wood plant medium (WPM) contains sufficient nutrients for the effective callus induction from mature leaves of plants grown in the field. Both zeatin and indole-3-butyric acid with the concentration of 2.0 mg L−1 and 1.0 mg L−1, respectively, are critical for adventitious bud regeneration from a variety of Vaccinium leaves. Different Vaccinium varieties have distinct regeneration efficiencies from leaf discs, and the diploid wild Vaccinium reticulatum ‘Red Button’ has the highest efficiency of callus formation and adventitious bud regeneration. Furthermore, we establish an efficient Agrobacterium-mediated genetic transformation system using ‘Red Button’, in which the transformation efficiency of green fluorescent protein (GFP) encoding gene is about 12.82%. Taken together, our study establishes an efficient tissue culture and regeneration system from leaves of Vaccinium varieties, and identify the diploid material ‘Red Button’ as an ideal variety for genetic transformation, which would greatly accelerate the mechanistic studies and breeding program of Vaccinium genus using emerging plant biotechnologies in the future.

Integrated transcriptome and endogenous hormone analysis provides new insights into callus proliferation in Osmanthus fragrans

Osmanthus fragrans is an important evergreen species with both medicinal and ornamental value in China. Given the low efficiency of callus proliferation and the difficulty of adventitious bud differentiation, tissue culture and regeneration systems have not been successfully established for this species. To understand the mechanism of callus proliferation, transcriptome sequencing and endogenous hormone content determination were performed from the initial growth stages to the early stages of senescence on O. fragrans calli. In total, 47,340 genes were identified by transcriptome sequencing, including 1798 previously unidentified genes specifically involved in callus development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) was significantly enriched in plant hormone signal transduction pathways. Furthermore, our results from the orthogonal projections to latent structures discrimination analysis (OPLS-DA) of six typical hormones in five development stages of O. fragrans calli showed jasmonic acid (JA) could play important role in the initial stages of calli growth, whereas JA and auxin (IAA) were dominant in the early stages of calli senescence. Based on the weighted gene co-expression network analysis, OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b were selected as hub genes from the modules with the significant relevance to JA and IAA respectively. The gene regulation network and quantitative real-time PCR implied that during the initial stages of callus growth, the transcription factors (TFs) OfERF4 and OfMYC2a could down-regulate the expression of hub genes OfSRC2 and OfPP2CD5, resulting in decreased JA content and rapid callus growth; during the late stage of callus growth, the TFs OfERF4, OfMYC2a and OfTGA21c, OfHSFA1 could positively regulate the expression of hub genes OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b, respectively, leading to increased JA and IAA contents and inducing the senescence of O. fragrans calli. Hopefully, our results could provide new insights into the molecular mechanism of the proliferation of O. fragrans calli.

Genome-wide identification and characterization of AINTEGUMENTA-LIKE (AIL) family genes in apple (Malus domestica Borkh.).

AINTEGUMENTA-LIKE (AIL) genes play a key role in various growth and developmental processes in plants. Thus far, the genome-wide identification of AIL genes has been reported for some plant species. However, genome-wide identification of AIL genes has not been conducted in apple (Malus domestica Borkh.). The current study focused on a comprehensive analysis of the AIL genes in the apple genome (i.e., MdAIL genes). In total, 27 MdAIL genes in the apple genome were identified and then divided into four groups according to phylogenetic analysis. The chromosomal locations, gene and protein structures, and physicochemical characteristics of MdAIL genes were analyzed. Synteny analysis revealed that segmental duplication events played a major role in the expansion of the AIL gene family in apple. The analysis of cis-regulatory elements in MdAIL promoter regions indicated that most of the MdAIL genes are involved in embryo development and seed germination. Moreover, the analysis of tissue-specific expression patterns and transcript levels in adventitious bud regeneration indicated that MdAIL genes play an extensive regulatory role in apple growth and development, especially in the regulation of germination and adventitious bud regeneration from in vitro leaves of apple. In conclusion, this is the first genome-wide analysis of the AIL genes in apple. The current results may help in better understanding the evolution and function of MdAIL genes and thus facilitate further research on plant growth and development.

Genome-Wide Identification, Classification, and Expression Analysis of the HD-Zip Transcription Factor Family in Apple (Malus domestica Borkh.)

Homeodomain-leucine zipper (HD-Zip) family genes are considered to play an important role in plant growth and stress tolerance. However, a genome-wide analysis of HD-Zip genes in apples (Malus domestica Borkh.) has not been performed. We detected 48 MdHDZ genes in the apple genome, and categorized them into three subfamilies on the basis of phylogenetic analysis. The chromosomal locations, gene/protein structures, and physiological and biochemical properties of these genes were analyzed. Synteny analysis revealed that segmental duplications were key in the expansion of the apple HD-Zip family. According to an analysis of cis-regulatory elements and tissue-specific expression patterns, MdHDZ genes may be widely involved in the regulation of apple growth and tolerance to environmental stresses. Furthermore, the transcript levels of apple HD-Zip I and II genes were up-regulated in response to fungal treatments. Expression of apple HD-Zip Ⅲ genes was enhanced during adventitious bud regeneration. This suggested possible roles of these genes in regulating the apple response to fungal infection, as well as adventitious bud regeneration. The current results may help us to better understand the evolution and function of apple HD-ZIP genes, and thus facilitate further research on plant resistance to fungal infection and in vitro regeneration.

In vitro amphidiploid induction of a distant hybrid Populus simonii × P. euphratica cv. ‘Xiaohuyang-2’ and its effect on plant morphology and anatomy

Abstract Highly gametic sterility of a distant hybrid Populus simonii × P. euphratica cv. ‘Xiaohuyang-2’ restricts its utilization in breeding programs of Populus. Amphidiploid induction by somatic chromosome doubling is expected to restore its gametic fertility. In this study, nodal-segment and leaf explants of ‘Xiaohuyang-2’ were used to induce chromosome doubling with colchicine in vitro. Although chromosome doubling of the nodal-segment explants only produced mixoploids, the treatments of leaf explants on adventitious bud regeneration medium successfully produced 4 amphidiploids, which might be attributed to the direct organogenesis of the adventitious buds on the leaf explants. This is the first report of amphidiploid induction in a distant hybrid between Populus section Tacamahaca and sect. Turanga. The highest amphidiploid induction frequency was 16.7 %. Both the explant survival rate and polyploidization frequency were significantly affected by colchicine concentration and exposure time. The amphidiploid plants significantly differed from the diploid and mixoploid plants in morphological and anatomical characteristics. They had larger, thicker, and greener leaves than the diploids and mixoploids. The increase in ploidy level also resulted in changes in stomatal features. The induced amphidiploid plants of the distant hybrid ‘Xiaohuyang-2’ are expected to play important roles in breeding programs of Populus in the future, which can be used as a bridge parent with the ability of unreduced gamete formation to cross with fast-growth germplasms to produce triploids pyramiding desirable traits of fast growth, easy cutting propagation, and salt and drought tolerances.

IN VITRO CLEANING UP AND MICROPROPAGATION OF PERS PECTIVE CHRYSANTHEMUM CULTIVARS AND NEW HYBRID FORMS OF THE NIKITA BOTANICAL GARDENS' BREEDING

An in vitro micropropagation protocol has been developed for garden chrysanthemum cultivars Mokroe Serebro, Predrassvetnyiy Ayu-Dag, Eldorado, and Ermitage of the NВG's breeding. 10-20 mg/1 of ribavirin promoted the primary explants cleaning up. А high frequency of regeneration of adventitious buds and microshoots was observed in cultivars Eldorado and Ermitage (21.0 and 23.5 microshoots/explant) cultured on MS medium with 0.75 mg/1 kinetin and Mokroe Serebro - on MS medium with 0.75 mg/1 ВАР (24.5 microshoots/explant). Spontaneous root formation facilitated 100% rooting of microshoots.

Effects of different factors on adventitious bud induction from stem explants of Ludisia discolor

In this study, the stem explants of Ludisia discolor were used as experimental materials to investigate the effects of 6-BA, NAA, Cu2+ and Ag+ on the induction of adventitious bud regeneration, and to analyze the most suitable culture conditions for stem explants regeneration. The results showed that when the medium was supplemented with 1.0 mg/L 6-BA, 0.75 mg/L NAA, 0.25 mg/L CuSO4, or 6.4 mg/L AgCl, the best regeneration effects would be gained, respectively. And the adventitious bud regeneration rate reached the maximum, which were 61.67%, 83.67%, 80.95% and 87.63%, respectively. The results of this study provided a theoretical basis for tissue culture and rapid propagation of L. discolor.

Establishment of plant regeneration system for young leaf explants of Cassia mimosoides

In the present study, a plant regeneration system was established by using young leaf explants from aseptic seedlings of Cassia mimosoides. The results showed that when the explants were inoculated on the 1/2MS medium contained 2.0 mg/L 6-BA and 0.5 mg/L NAA, high rate of callus induction and good growth status of adventitious bud, and the highest number of buds were obtained. Further studies conclusively suggested that IBA could not promote root formation effectively, but the root regeneration could be induced by NAA. However, although the rooting of regeneration buds could be induced when adventitious buds were placed into 1/2MS medium supplemented with 0.5 mg/L NAA, the rate of rooting was reduced, and the roots became shorter. Therefore, the best medium for adventitious bud regeneration using young leaf as explants in C. mimosoides was 1/2MS medium contained 2.0 mg/L 6-BA and 0.5 mg/L NAA, and the best rooting medium was 1/2MS.

Effect of different factors on plant regeneration from leaf explants of Jatropha curcas L.

AbstractThe in vitro regeneration frequency was commonly low, and the quality of adventitious buds was poor, when the traditional methods were used to induce adventitious buds from leaf explants of Jatropha curcas L. through the medium containing low concentration of thidiazuron (TDZ). When treated with high concentrations (6.5–104 mg/L) of TDZ for a certain time (7–120 min), the regeneration frequency and quality of adventitious buds were significantly increased. The best induction rate of adventitious buds (78.58%) and the average number of buds per explant (7.26) were obtained by soaking leaf explants in 26 mg/L TDZ solution for 30 min and then transferring them onto MS medium without hormone for 30 days. Furthermore, the adventitious bud regeneration was affected obviously by source of explants, sterilization time, illumination time and genotype. The subsequent elongation and rooting experiments showed that adventitious bud elongation could be improved by adding diethyl aminoethyl hexanoate (DA‐6) in the medium, and the best elongation effect could be gained when the concentration of DA‐6 was 1.2 mg/L. The elongated shoots induced the roots and developed into small intact plants on the MS medium with 1.5 mg/L sodium nitroprusside (SNP). After domestication, these small plants could be transplanted into the soil and growing normally. Therefore, the method established in this study was helpful to improve the plant regeneration efficiency of leaf explants in J. curcas.

Effects of Hormones and Epigenetic Regulation on the Callus and Adventitious Bud Induction of Fraxinus mandshurica Rupr.

Fraxinus mandshurica Rupr. (hereafter “F. mandshurica”) is known as one of northeast China′s important, valuable hardwood timber species. However, tissue culture and micropropagation of the species are difficult and have low efficiency, limiting asexual propagation. In this manuscript, stem explants were utilized to establish an effective regeneration system through adventitious bud organogenesis. The factors influencing callus regeneration in vitro were determined, and callus regeneration technology was established. The mechanism of adventitious bud formation was analyzed. Thidiazuron (TDZ) played a crucial role in the formation of adventitious buds. Elevated concentrations of TDZ were beneficial to callus induction and low concentrations of 6-benzyladenine (BA) led to loose state callus formation. The order of callus induction rates for different explants was stem cotyledon (100%) > segment (98.54%) > hypocotyl (92.56%) > root (50.71%). The effects of exogenous addition of 6-BA and TDZ on the endogenous hormone content of plants during the regeneration of adventitious buds were also assessed, as well as the expression characteristics of genes related to the regeneration pathway. The comprehensive analysis results showed that the suitable medium for callus induction and adventitious bud differentiation was c12 medium (MSB5 + 30 g/L sucrose + 7 g/L Agar + 5 mg/L 6-BA + 8 mg/L TDZ + 2 mg/L glycine + 0.1 mg/L IBA + 5% coconut water). The induction rates of callus and adventitious buds were 99.15% and 33.33%. The addition of 2.4 mg/L of the DNA demethylation reagent 5-azacytidine (5-aza) and 0.15 mg/L of the histone deacetylase inhibitor trichostatin A (TSA) increased the rates of adventitious bud induction by 17.78% over the control. This further laid the foundation for large-scale cultivation of excellent varieties and genetic transformation techniques.

An effective method for Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. using cotyledon explants

ABSTRACT Jatropha curcas is one of oilseed crops and has been considered as an energy crop. In the present study, efficient plant regeneration protocol and transformation method were developed for J. curcas. Because the regeneration efficiency of adventitious bud from cotyledon explants of J. curcas induced by traditional methods is low, and it takes a long time to get complete plants. It is necessary to establish a new regeneration system to improve regeneration efficiency. Cotyledon explants were dipped into TDZ solution at different concentrations respectively for various times to obtain higher efficiency of adventitious bud regeneration. This new regeneration method was then applied to genetic transformation of J. curcas. Cotyledon explants were precultured for 1 day after treated with high concentration of Thidiazuron (TDZ) solution (20 mg/L for 40 min), followed by Agrobacterium tumefaciens infection. After co-cultured for 2 days, the explants were placed on the induction hormone-free media for bud regeneration and resistant screening. After 30 days, selected shoot buds were transferred onto elongation medium for 15 days. Young leaf sections of the regenerated shoots were used for PCR (Polymerase chain reaction) detection of the transgenic shoots. The PCR positive shoots were isolated and used for in vitro grafting. The intact plants were obtained within 20 days. GUS (β-Glucosidase) staining and Southern analysis confirmed the transformation events. Briefly, a transformation efficiency of 34.32% was achieved and an intact transgenic plant could be obtained within 65 days.

Isolation of diploid and tetraploid cytotypes from mixoploids based on adventitious bud regeneration in Populus

Somatic chromosome doubling usually results in the production of mixoploids, which are difficult to use in breeding programs. In this study, we developed a method to isolate diploid and tetraploid cytotypes from induced mixoploids of Populus pseudo-simonii × P. nigra var. italica based on adventitious bud regeneration. We produced 75 mixoploids through colchicine-induced seed chromosome doubling in P. pseudo-simonii × P. nigra var. italica. The highest mixoploid induction frequency was 26.67 ± 5.09% following treatment with 0.1% colchicine for 96 h. We then screened a medium for adventitious bud regeneration from leaf explants of multiple genotypes, which was Murashige and Skoog (MS) medium with 0.4 mg L−1 6-benzyladenine (BA) and 0.1 mg L−1 1-naphthaleneacetic acid (NAA). The maximum frequency of leaf differentiation was 96.67 ± 3.33%, and the number of adventitious buds per explant was 6.17 ± 1.02. When the medium was applied for adventitious bud regeneration of 12 mixoploid genotypes, 18 tetraploids and 26 diploids were isolated, of which five mixoploids generated both diploid and tetraploid cytotypes and seven mixoploids generated either diploids or tetraploids. Compared to diploids, tetraploids varied significantly in stomatal characteristics, micropropagation coefficient and rooting ability. Significant differences were also detected among genotypes at the same ploidy level, implying that heterozygosity also affected phenotypic variation during allopolyploidization. This study provides an effective method for isolation of diploid and tetraploid cytotypes from mixoploids and generation of full-sib diploid and tetraploid germplasms for polyploidy Populus breeding programs. This paper reported an efficient method to isolate diploids and tetraploids from Populus mixoploids via adventitious bud regeneration. Paired analysis showed both heterozygosity and ploidy level contributed to morphological variation.

Optimization conditions of adventitious buds regeneration from petiole explants of Echinacea purpurea

Echinacea purpurea as a perennial herb has high ornamental and medicinal value. While the tissue culture technologies of E. purpurea are contradictory. In order to improve the culture conditions of the adventitious buds regeneration of E. purpurea, petioles from the aseptic seedlings were used as the explants. Different concentrations of sodium nitrophenolate (SN), copper sulfate (CuSO4), amine fresh ester (DA-6), sodium nitroprusside (SNP) and ascorbic acid (Vc) were added into MS medium containing cytokinin and auxin, respectively, to investigate their effects on inducing regeneration of adventitious buds. The results showed that the best induction effects of adventitious buds were obtained under 1 mg/L CuSO4, 1 mg/L DA-6, 0.2 mg/L SN, 2 mg/L SNP and 20 mg/L Vc conditions. Our results can provide references for the induction of adventitious buds regeneration and rapid propagation of E. purpurea.

Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven.

Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘White Heaven’, respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino–green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.