Validating field regeneration capacity for selected accessions of Gossypium hirsutum using callus induction and regeneration capacity

Background Gossypium hirsutum undergoes rapid clonal propagation to regenerate a mature plant through tissue culture.However,the correlation between cotton leaf regeneration,callus induction,and regeneration ability was still obscure.In this research,cotton leaf regeneration level for 21 accessions in the field(new leaves)was observed after the first harvest,and a comparison between field regeneration level and callus induction with its regeneration capacity(new shoots and roots)for the same 21 accessions was carried out.Agronomic traits,including plant height,leaf area,fresh leaf weight,dry leaf weight,the number of flowers and bolls,and biochemical(proline content)and physiological(chlorophyll and carotenoid content)traits during the flowering stage of 21 upland cotton accessions,were investigated.Result A significant correlation between physiological parameters and callus induction was discovered.Callus induction and regeneration capacity of roots and shoots for hypocotyl,cotyledons,and shoot tip tissues were used to validate field leaf regeneration level after the first harvest.CCRI 24 showed significant leaf regeneration in the field and callus induction capacity through callus induction and regeneration.Conclusion We found a substantial relationship between field regeneration capability and callus induction with its regeneration capacity for the hypocotyl,cotyledons,and shoot tip.The results showed that ZS061,Lumian 378,Jimian 863,and ZS065 have the highest moisture retention capacity,while CCRI 24,Liaoyang Duomaomian,and Beizhe Gongshemian have the lowest moisture retention capacity.CCRI 24 has the highest leaf regeneration capacity in the field,while Beizhe Gongshemian has the lowest leaf regeneration capacity.All our result provides a clue for checking the regeneration capacity through leaf regeneration level in the field.

Optimization of Callus Induction Medium for Schisandga chinensis Baill via Uniform Design

[Objective]This study was to optimize callus induction medium for Schisandga chinensis Baill.[Method]Using callus induction rate as an indicator,uniform design was employed to optimize hormone combination at poly-factors and poly-levels for callus induction from Schisandga chinensis Baill.[Results]Optimal hormone combinations depended on different explants：optimum medium for both tender leaf and petiole was MS ＋3 mg/L 6-BA,for stem segment was MS ＋3 mg/L 6-BA ＋0.6 mg/L NAA.[Conclusion]Uniform design is a time-saving and convenient method for the optimum medium for callus and yields a higher callus induction rate.

Callus Induction from Mature Embryos of Maize

In this study we studied the factors influencing the callus induction from mature embryos of maize inbred lines Qi 319, Zhen 58, Chang 7 -2, Lx 9801 and 81162, such as genotype, combination of plant growth regulators, and low-temperature pretreatment. The results showed that the induction rate of Qi 319 was the highest among the four genotypes tested; combination of 4.0 mg/L 2,4-D ＋ 0.5 mg/L 6-BA was suitable for inducing callus from mature embryos; three days of 4℃ pretreatment can promote the callus induction significantly. The indices optimized in the present study are helpful for establishing genetic transformation system in maize without considering seasonal variation.

Callus induction from leaves of different paulownia species and its plantlet regeneration

The experiment was carried out on five different species of Paulownia for callus induction from leaves. MS medium was adopted as basic medium, and from different combinations of NAA and BA the suitable media were determined for callus induction, bud differentiation, and root differentiation of five different species. MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5NAA+4BA, MS+0.3NAA+6BA, and MS+0.3NAA+8BA were suitable media of callus inductions of leaves, respectively, for Paulownia tomentosa, Paulownia australis, Paulownia fortunei, Paulownia elongata and P. tmentosa x P. fortunei, and MS+0.3NAA+12BA, MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA, and MS+0.7NAA+12BA were suitable media for bud differentiation from leaf callus respectively for above five species. The rooting media was determined as 2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, 1/2MS+0.3NAA, and 1/2MS+0.5NAA. These results provide reference data for breeding new fine va-rieties with different kinds of Paulownia protoplasts fusions.

A Preliminary Study on Callus Induction and Proliferation of Cylocarya paliurus

[Objective] To carry out preliminary study on callus induction and prolifer- ation of Cylocarya pafiurus. [Method] The leaf and stem segments of Cylocarya paliurus as explants were cultured on MS, WPM and improved DKW3 mediums, added 6-BA and IBA with different concentration ratios to explore the optimum medium for callus induction and proliferation. [Result] It was found that MS ＋ 4.0 mg/L 6-BA ＋ 2.0 mg/L IBA was the optimum medium for inducing stem callus of Cylocarya pali- urus, MS ＋ 2.0 mg/L 6-BA ＋ 0.5 mg/L IBA was the optimum medium for inducing leaf explants callus of Cylocarya paliurus, and MS ＋ 1.0 mg/L 6-BA ＋ 1.0 mg/L IBA was the optimum medium for callus proliferation. 200 mg/L Vitamin C was best to inhibit Cylocarya paliurus callus from browning with the browning rate of only 5.71%. [Conclusion] This study provided some theoretical basis for the establishment of tis- sue culture and rapid propagation system and the development of molecular breed- ing study of Cylocarya paliurus.

Differences in Primary Callus Induction among Different Anthurium andraeanum Varieties

[Objective] This research aimed to optimize continuously the highly efficient regeneration system of Anthurium andraeanum. [Method] The leaves and petioles of four A. andraeanum varieties were used as explants to investigate the differences in primary callus induction among different A. andraeanum varieties. [Result]The callus formation capacity of SAM and SST was stronger than that of SDM and SHG. Among the four varieties, the leaf regeneration capacity of SAM, SDM and SHG was stronger than the corresponding petiole regeneration capacity. However,the petiole regeneration capacity of SST was stronger. The optimum medium for petiole callus induction of SST was 1/2 MS ＋ TDZ 4.0 mg/L ＋ 2, 4-D 0.2 mg/L with induction rate of 87.5%; the optimum medium for leaf callus induction of SAM was 1/2 MS ＋ TDZ 2.0 mg/L ＋ 2, 4-D 0.2 mg/L with induction rate more than90%; the optimum medium for leaf callus induction of SDM and SHG was all 1/2MS ＋ ZT 2.0 mg/L ＋ 2, 4-D 0.2 mg/L with induction rates of 59.34% and 79.63%,respectively. [Conclusion] In addition to variety differences, the differences in differentiation ability among different types of calluses should be also taken into account in the establishment and optimization of tissue culture and rapid propagation technology system of A. andraeanum.

A Genetic Transformation System for Rosa multiflora Thunb. var. cathayensis Rehd. et Wils through Callus Induction

An efficient genetic transformation system is a preparation for Rosa multi-flora Thunb. var. cathayensis Rehd. et Wils to diversify its flower color through ge-netic engineering. We firstly optimized the explants and culture conditions on callus induction, hormone concentrations and dark period of culture time on bud differentia-tions in particular, with sterilized seedlings to establish the regeneration system of R. multiflora. It showed that callus induction frequency reached 100% after the ex-plants being cultured in dark for 21 d when MS was chosen to be the initial culture medium. The bud differentiation rate was 48% after cal i being cultured under dark for 8 d on MS medium supplemented with TDZ （1.5 mg/L） and NAA （0.05 mg/L）. The cal i was used as the explants that were infected with Agrobacterium tumefa-ciens harboring a DFR-RNAi construct. The transformation rate reached as high as 50%. The establishment of a highly efficient rose gene transformation system out-lined in this report is prerequisite for genetic improvement in rose flower colors.

In Vitro Propagation and Artificial Seed Production of Fritillaria cirrhosa D. Don, an Endangered Medicinal Plant

Fritillaria cirrhosa D.Don(Liliaceae)is an endangered perennial bulbous plant and its dry bulb is a valuable med-icinal material with antitussive and expectorant effects.Nevertheless,lack of resources and expensive prices make it difficult to meet clinical needs.This study presents a regeneration system aimed at overcoming the challenge of inadequate supply in F.cirrhosa,focusing on:(1)callus induction,(2)bulblets and adventitious bud induction,and(3)artificial seed production.Callus development was achieved in 84.93%on Murashige and Skoog(MS)medium fortified with 1.0 mg·L^(-1) picloram.The optimal medium for callus differentiation into regenerated bulb-lets was MS medium supplemented with 1.0 mg·L^(-1)6-benzyladenine(6-BA)and 0.2 mg·L^(-1)α-naphthaleneacetic acid(NAA).Subsequently,bulblets and adventitious buds were induced from regenerated bulblet sections cul-tured on MS medium fortified with 0.3 mg·L^(-1)6-BA+1.0 mg·L^(-1)2,4-dichlorophenoxyacetic acid(2,4-D),2.0 mg·L^(-1)6-BA+0.5 mg·L^(-1),and the induction rates were 88.17%and 84.24%,respectively.The regenerated bulblets were transplanted into a substrate of humus soil,river sand,and pearlite(1:1:1)after low-temperature treatment.The germination rate was 42.80%after culture for 30 days.Regenerated bulblets were used for encap-sulations in liquid MS medium containing 3%sucrose(w/v)+0.5 mg·L^(-1) NAA+2.0 mg·L^(-1)6-BA+3%sodium alginate(w/v)with a 10 min exposure to 2%CaCl_(2).Under non-aseptic conditions,the germination rate reached 81.67%,while the rooting rate was 20.56%after 45 days.The capsule added 1.0 g·L^(-1) carbendazim and 1.0 g·L^(-1) activated carbon was the best component of artificial seeds.This study successfully established an efficient regen-eration system for the rapid propagation of F.cirrhosa,involving in vitro bulblet regeneration and artificial seed production.This method introduces a novel approach for efficient breeding and germplasm preservation,making it suitable for large-scale industrial resource production.

Optimization of the Callus Induction System of Chenopodium quinoa Willd

Callus induction effects of nine varieties of Chenopodium quinoa Willd. were compared by taking stem segments and cotyledons of C. quinoa as the ex- plants. At the same time, callus JnductJon of stem segments was optimized, as well as the callus proliferation system. Research results showed that the optimal explant for callus induction was stem segment. The average callus induction rate of nine varieties reached 90% in culture medium MS ＋ 0.5 mg/L 2, 4-D. In the callus opti- mization test, treatment VI （MS ＋ 0.5 mg/L 2, 4-D ＋ 0.5 mg/L KT ＋ 0.5 mg/L NAA） and treatment II （MS ＋ 0.5 mg/L 2, 4-D） had close induction rate, but the callus morphology was greatly different. The latter had loose, glossy and yellowish white calluses. Therefore, culture medium MS ＋ 0.5 mg/L 2, 4-D was the optimal for callus induction. And using 2, 4-D together with KT and NAA could significantly increase the proliferation rate of calluses.

Plant Regeneration by Callus-Mediated Protocorm-Like Body Induction of Anthurium andraeanum Hort.

This research aims at developing a plant regeneration system from leaf and petiole explants of Anthurium andraeanum Hort., thereby establish a foundation for mass production and transformation. Using tissue culture technique, the conditions for callus induction, protocorm-like body （PLB） formation and plant regeneration from leaf explants and petiole of A. andraeanum, such as basal medium and plant growth regulator, were investigated. Totipotent callus was induced on a 1/2-strength MS medium containing 0.90 μmol L^-1 2,4-dichlorophenoxyacetic acid （2,4-D） and 8.88μmol L^-1 N6-benzyladenine （BA）. The callus exhibited complete hormone autonomy for growth and differentiation of PLBs. This callus proliferated well and was maintained by subculturing on 1/2 MS medium containing 0.90 μmol L^-1 2,4-D and 4.44 μmol L^-1 BA. On average, 8 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the 1/2 MS medium with 4.44 μmol L^-1 BA after 8 wk of culture. The regenerated PLBs formed shoots and roots on 1/2 MS medium. After 24 wk of culture on these medium, well-developed plantlets for potting were produced. An efficient micropropagation method was established for indirect PLB formation and plant regeneration from leaf and petiole ofA. andraeanum.

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.

Proline and Glutamine Improve in vitro Callus Induction and Subsequent Shooting in Rice

This study was conducted to evaluate the effects of proline and glutamine on in vitro callus induction and subsequent regeneration and to develop a reproducible and highly efficient plant regeneration protocol in four rice genotypes, viz. Pawana, Jaya, Indrayani and Ambemohar. Considerable variation in response to plant growth regulators and amino acid supplements used was observed in all the four genotypes. Medium supplemented with proline and glutamine was shown to be superior to medium without proline and glutamine. The best callusing from mature embryo was observed on Murashige and Skoog （MS） medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid （2,4-D）, 500 mg/L proline and 500 mg/L glutamine. Shoot induction was higher in the callus obtained from medium supplemented with 500 mg/L proline and 500 mg/L glutamine. The highest shoot regeneration frequency （83.2%） was observed on MS medium with 2.0 mg/L benzylaminopurine, 0.5 mg/L 1-naphthaleneacetic acid, 500 mg/L proline, and 500 mg/L glutamine in the callus obtained from MS medium supplemented with 2.0 mg/L 2,4-D, 500 mg/L proline and 500 mg/L glutamine. Among the four genotypes, Pawana has the highest regeneration efficiency （83.2%）, whereas the regeneration efficiency of the rest three rice genotypes was in the range of 32.0% to 72.3%. This optimized regeneration protocol can be efficiently used for Agrobacterium mediated genetic transformation in rice.

Effect of Different Treatment on Anther Callus Formation and Browning in Balsam Pear （Mornordica charantia L.）

Brown callus derived from anther limited the application of anther culture in balsam pear. In order to establish a more perfect regeneration system from anther cultuer, the effects of low temperature pretreatment, 2,4-dichlorophenoxyacetic acid （2,4-D）, vitamin C （Vc） and silver nitrate （AgNO3） on callus induction and browning in anther culture of balsam pear （Momordiea charantia L.） were investigated. The results showed that after pretreatment at 4 ℃ for 1 day, callus induction rate was the highest and browning rate was the lowest. Anthers on MS medium supplemented with 2,4-D 0.5 mg/L formed more and better callus. The medium supplemented with Vc or AgNO3 was advantageous to the induction of callus and reduction of browning. When cultured on medium supplemented with 50 mg/L Vc or 5 mg/L AgNO3, callus induction rate was the highest and browning rate was rather low.

Effects of Cultivar and Explant Sources on Callus Induction and Plant Regeneration in Rice （Oryza sativa L.）

In vitro callus induction and plant regeneration potentiality were studied from mature embryo of three Indian rice （Oryza sativa L.） groups at Field Crops Department, Agricultural Faculty, Ondokuz May？s University, Samsun, Turkey. The study was done by using callus induction MS medium having different concentration of four. The present research was conducted according to the design of randomized blocks trial. A total of 696 calluses, 193 plants and 917 seeds were obtained from Indica group; 2,110 calluses, 103 plants and 235 seeds were obtained from Japonica group; 1,243 calluses and 13 plants were obtained from Javanica group. With regard to number of calluses obtained from each explant source, 52 calluses were obtained from whole-plant explants, 1,668 calluses from root explants, 629 calluses from shoot explants, 649 calluses from the 1st node explants, 240 calluses from the 2nd node explants, 269 calluses from the 1st internode explants and 12 calluses from the 2nd internode explants. With regard to number of plants obtained from each explant source, 27 plants were obtained from whole-plant explants, 195 plants from shoot explants, 43 plants from the 1st node explants, 40 plants from the 2nd node explants and four plants from the 1st internode explants. With regard to number of seeds, 823 seeds were obtained from shoot explants and 329 seeds were obtained from the 2nd node explants. Germination rate of harvested seeds was over 90%. The establishment of this regeneration system is essential for the development of a genetic transformation system for commercial rice cultivars.

Study on the Callus Induction and Petiole Proliferation of the Wild European Plum

[Objective]To establish the callus induction and proliferation system of the petiole of the wild European plum.[Methods]The petiole of the wild European plum was used as an explant.The effects of disinfection time,type and concentration of carbon source,dark treatment time,basic medium,growth regulator and sampling time on callus induction and petiole proliferation were studied.[Results]The best sampling time was from mid-May to mid-June.The lowest pollution rate was 3.33%when they were disinfected with 75%alcohol for 45 s and 0.1%mercuric chloride for 7 min.21 d of dark treatment was the best dark treatment time.The best formula for callus induction and proliferation was:B5+0.2 mg/L 6-BA+2 mg/L NAA+30 g/L sucrose+7 g/L agar.[Conclusion]A theoretical foundation was laid for the tissue culture and regeneration system of the wild European plum.

Callus Induction of Young Leaf Coconut cv. MATAG with Combination of 2,4-Dichlorophenoxyacetic Acid (2,4-D), α-Naphthalene Acetic Acid (NAA) and Benzyl Amino Purin (BAP)

This research was to study in vitro callus induction in Coconut cv MATAG from young leaf explants. Young leaf segments from mature coconut were cultured on Y3 medium supplemented with different concentrations of 2,4-D and a combination of NAA and BAP. Each of these plant growth regulators (PGR) gives different responses toward callus formation, the percentage of explants producing callus, the percentage of callus proliferation, and the morphology of callus. A series of different concentrations were used for 2,4-D (1, 5, 10, 20, 40, 60, 100 mg/L), NAA (1, 3, 5 mg/L) and BAP (1, 3, 5 mg/L) respectively. The range of days of callus formation using 2,4-D treatments is 7 - 12 months, while the 2,4-D combined with NAA is recorded at 2 - 5 months. Despite the variety of different months between these plant growth regulators for callus formation, the percentages of explants producing callus and callus proliferation are different. The highest percentage of explants producing callus (2.9%) was observed at 2,4-D (40 mg/mL), followed by 2.7% at 2,4-D (10.0 mg/mL) with NAA (1 mg/mL). At a concentration of 100 mg/mL of 2,4-D, the highest percentage of callus proliferation was found, as well.

Influences of Genotype and Explant on Callus Induction and Shoot Regeneration in Sunflower （Helianthus annuus L.）

In vitro regeneration was performed with the aim of developing efficient callus and shoot regeneration from different explants of sunflower （Helianthus annuus L.）. The 6 genotypes （RHAI0, RHA 14, RHA15, PR6404, N Record 109/Sanay 3-5, N Record 109/Sera） were used as plant materials. The roots, hypocotyls and cotyledons were excised from 4 day-old seedlings and cultured on embryo induction medium （EIM） supplemented with Benzylaminopurine （BAP）, Naphthaleneacetic acid （NAA） and Gibberellic acid （GA3）. The experiments were kept in 18/6 hour light/dark photoperiod at 26± 2 ℃for four weeks. The rates of callus and root organ formation on explants were 67%-100% and 7%-31% respectively, depending on the genotype. Root explants produced statistically high callus formation （2.6 score） compared to cotyledon （2.0 score） and hypocotyl explants （2.5 score） at all sunflower genotypes used. The highest shoot regeneration was obtained from RHAI 5 （7%） while PR6404 （100%） produced the highest callus formation.

A single-cell transcriptome atlas reveals the trajectory of early cell fate transition during callus induction in Arabidopsis

The acquisition of pluripotent callus from somatic cells plays an important role in plant development studies and crop genetic improvement.This developmental process incorporates a series of cell fate transitions and reprogramming.However,our understanding of cell heterogeneity and mechanisms of cell fate transition during callus induction remains quite limited.Here,we report a time-series single-cell transcriptome experiment on Arabidopsis root explants that were induced in callus induction medium for 0,1,and 4 days,and the construction of a detailed single-cell transcriptional atlas of the callus induction process.We identify the cell types responsible for initiating the early callus:lateral root primordium-initiating(LRPI)-like cells and quiescent center(QC)-like cells.LRPI-like cells are derived from xylem pole pericycle cells and are similar to lateral root primordia.We delineate the developmental trajectory of the dedifferentiation of LRPI-like cells into QC-like cells.QC-like cells are undifferentiated pluripotent acquired cells that appear in the early stages of callus formation and play a critical role in later callus development and organ regeneration.We also identify the transcription factors that regulate QC-like cells and the gene expression signatures that are related to cell fate decisions.Overall,our cell-lineage transcriptome atlas for callus induction provides a distinct perspective on cell fate transitions during callus formation,significantly improving our understanding of callus formation.

Plant Regeneration through Indirect Organogenesis in Two Cultivars of Pineapple (Ananas comosus L.)

Unavailability of performant planting material of pineapple constitutes a major problem of its cultivation in Africa. For this purpose, indirect organogenesis technique is used to evaluate the in vitro responses of two cultivars of pineapple during the explant’s regeneration. Calli were induced from crown leaf and plantlets leaf of “Smooth Cayenne” and “Sugarloaf cultivars”. Murashige and Skoog medium with vitamins B5 supplemented with different growth regulators combinations were used. BAP and/or 2,4-D have been added to base medium for calli cells’ differentiation while BAP and GA3 have been added for plant elongation. The results indicated that explants from regenerated plantlets leaves cultivated on MS supplemented with copper (II) sulphate 5-hydrate concentrations incorporated had significant (p < 0.0001) influence on callus induction in pineapple cultivars. Likewise, MS medium with NAA (0.5 mg/l) + BAP (1 mg/l) had a highly significant influence with 8.8 differentiated Calli. Also, MS medium supplemented with BAP (3 mg/l) + GA3 (2 mg/l) for the “smooth Cayenne” had significantly influenced (p < 0.0001) Calli regeneration with a high rate of 55.25% plantlets. MS medium containing 0.5 mg/l of NAA + 0 mg/l IBA produced a high number of roots in Sugarloaf whereas the medium containing 1.5 mg/l NAA + 0.5 mg/l (IBA) produced high number of roots in smooth Cayenne. We have established an efficient and reproducible protocol for mass propagation and genetic transformation of pineapple though indirect organogenesis. This protocol may be used in genetics engineering studies for pineapple breeding.

Effects of Genotypes and Basic Medium on Culture of Maize Mature Embryos

[Objective]This study was to screen out suitable genotypes and basic medium for the culture of maize mature embryos.[Method]Using mature embryos of nine maize genotypes as explants,the effects of genotypes and basic medium on callus induction and subculture were investigated.[Result]The genotypes performed better in callus induction and subculture were found in turn 853-35,853-209,Dan 34 and 81162.MS medium is better than N6 medium in the callus induction from maize embryos,while N6 medium is more suitable for callus subculture.[Conclusion]Our study further improved the tissue culture system in maize with mature embryos as explants.