Frequency Of Embryogenesis Research Articles

In vitro somatic embryo productions from Curculigo orchioides derived gold nanoparticles: Synthesis, characterization, its biomedical applications, and their eco-friendly approaches to degradation of methylene blue under solar light irradiations

Curculigo orchioides rhizome explants were employed to develop a rapid and effective strategy for increased plant regeneration using somatic embryogenesis. Direct somatic embryo development was shown on rhizome explants cultivated on Murashige and Skoog (MS) making with 2, 4-D (1.0–3.0 mg/L). Rhizome explants cultivated on MS media supplemented with 2.0 mg/L 2, 4-D yielded the highest frequency of embryogenesis (87.5%) and the maximum number of somatic embryos (1596.7/explant). Somatic embryo germination was accomplished using MS media with 2.0 mg/L 6-benzylaminopurine (BAP). With an 80% survival rate, the germination plantlets were acclimated in the greenhouse. The current study is the first evidence of the efficacy of in vitro-produced plants and C. orchioides somatic embryo callus cultures of stable gold nanoparticles. The UV–Vis spectrophotometric absorbance, at 510 nm, revealed the absorption spectra of the AuNPs. The FT-IR revealed functional groups and reaction processes in green AuNP formation. High-resolution transmission electron microscopy (HR-TEM) was used to assess the surface morphology and structure of the AuNPs after their elemental composition was determined using a dispersive energy X-ray (EDAX) spectrum. The average size of AuNPs was around 35 nm in diameter. The crystalline nature of the AuNPs was investigated by X-ray diffraction (XRD). The highest growth inhibition was found for C. orcthioides against Klebsiella pneumoniae (17.5 mm) and Serratia marcescens (16.5 mm). The AuNPs exhibited antioxidant activity against free radicals such as DPPH and ABTS. Furthermore, the cytotoxicity of AuNPs was assessed, and inhibitory concentration (IC50) was 20 μg/mL and 80 μg/mL for breast carcinoma (MDA-MB-231) and Vero cell lines. The degradation of methylene blue measures the photocatalytic activity of the manufactured AuNPs when subjected to visible sunlight (MB). Thus, the result showed a maximum degradation efficiency of MB (84%).

Successful application of microspore culture in pakchoi (Brassica campestris L. ssp. chinensis Makino var. communis Tsen et Lee) for hybrid breeding.

Microspore embryogenesis is an effective method of obtaining double haploid (DH) lines in only 1year. However, the microspore embryogenesis protocol was not efficient in pakchoi. This study aimed to establish an effective microspore culture protocol in pakchoi for hybrid breeding. The embryos were obtained from three genotypes (18SY01, 18SY02, 18SY03), but the frequency of microspore embryogenesis was significantly different. Globular embryos from three genotypes were placed into a rotary shaker (50 r/min, 25 ℃) for further culture to improve microspore embryogenesis and plantlet regeneration without callus development. Shake culture not only increased the frequency of cotyledonary embryos but also accelerated microspore embryogenesis in the NLN-13 liquid medium. Moreover, the doubled haploid rates of regenerated plants for the three genotypes were above 50%. The morphological characters and plot yield of DH lines were identified, and there were significant differences between them. According to the measurement of the self-compatibility index, all the DH lines were self-incompatible. Furthermore, the hybrid combination was prepared with the selected DH lines and the pakchoi genic male sterile line GMS010 to develop excellent hybrids. This work contributes to accelerating the application of microspore embryogenesis and supplying the DH lines in pakchoi hybrid breeding.

Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee).

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2mg·L-1) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1mg· L-1 sodium nitrophenol and 0.01 ~ 0.2mg· L-1 of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1mg· L-1 sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01mg·L-1 significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

Effects of genotype and sodium p-nitrophenolate on microspore embryogenesis and plant regeneration in broccoli (Brassica oleracea L. var. italica)

Isolated microspore culture has been applied in breeding to obtain doubled haploids (DH) and therefore shorten the time of breeding process; however, the production of pure broccoli lines using DH produced in isolated microspore culture has not been sufficiently developed. This study aimed to establish an effective microspore culture protocol for broccoli for hybrid breeding purposes. Microspores of thirteen broccoli genotypes were successfully induced to form embryos in NLN-13 liquid medium. Three genotypes, BB1, BB4, and BB6, were cultured in NLN-13 medium containing sodium p-nitrophenolate (SPN) ranging from 0 to 0. 2 mg•L-1, to improve microspore embryogenesis and plantlet regeneration without callus development. The addition of 0.01–0.2 mg•L-1 SPN enhanced the frequency of microspore embryogenesis. Adding 0.1 mg•L-1 SPN yielded the maximum number of embryos per bud in BB1, BB4, and BB6, which were 1.92-, 2-, and 2.1-folds higher than the control, respectively. The suitable concentration (0.1 mg•L-1) of SPN application could enhance the frequency of direct plantlet regeneration for broccoli. Further, average spontaneous diploidization rate for BB1, BB4, and BB6 was 51.28%. This is the first study to utilize SPN in improving the frequency of microspore embryogenesis and plantlet regeneration, and offer an efficient method for the large production of DH lines that needed in hybrid seeds and genetic study in broccoli.

Effect of sowing dates of donor plants and concentration of 2,4-D on the purity of productive anther formation of common barley (Hordeum vulgare L.) in anther culture in vitro.

Creating dihaploid lines of agricultural plants is a labour-intensive but essential step in variety production in modern plant breeding. This stage allows significantly accelerate the process of creating new varieties of common barley and other crops. Barley digaploids are produced mainly by anther culture and microspore culture. The authors preferred anther culture in vitro. In the present study, the influence of climatic factors in the cultivation of donor plants on the yield of productive anthers at different sowing dates was established. The authors also identified the more stable culti- vars with a high anther production regardless of sowing date (Signal, Laureate and Eifel). Varieties showed the highest number of embryo-like structures formation at the first and third sowing dates (Zu Suren, Zu Zaza); and sorts with a high rate of productive anther formation at the second sowing date (Acha, Exploer) were identified. Different concentrations of 2,4-D in N6 medium on the frequency of embryogenesis and yield of productive anthers were studied. As a result of this study, the authors found that different concentrations of 2,4-D (1 mg/l and two mg/l) had no significant effect on the for- mation frequency of productive anthers in all the varieties studied. When the embryogenesis capacity of the cultivars was reviewed, all the samples were found to be positive in anther culture. However, the array Zu Suren had a significantly lower effective anthers yield than the samples Signal and Acha. As a result of correlation analysis, the authors found a close relationship between the length of the ear tube of donor plants and the frequency of formation of productive anthers (r = -0.69). A close relationship with the development of optimal microspore phase for the induction of androgenesis in anthers extracted from the ear tube with an average length of 6 cm was determined. This information can significantly speed up the selection of donor plants, but it is recommended to confirm the stage of microspore development microscopically for each new cultivar used.

Methodological approaches for producing doubled haploids in sugar beet and red beet (Beta vulgaris L.).

The in vitro production of doubled haploids is a biotechnological path of an accelerated development of parental lines in F1-hybrid breeding programs. Unlike the traditional inbreeding method requiring 5 to 6 generations to reach a suf­f icient homozygosity of lines, the number of generations to produce pure lines of beet by haploid technologies is reduced to 2. The production of doubled haploids by gynogenesis is the most common biotechnological approach in sugar and red beets. Protocols for the production of doubled haploids for B. vulgaris species are few and have been developed mainly for sugar beets. There are no protocols for the production of doubled haploids for red beet (B. vulgaris convar. esculenta Salisb.), and the protocols developed for sugar beet (B. vulgaris convar. saccharifera Alef.) are ineffective for red beet, even though these two crops belong to the same species. The greatest success has been achieved in the production of doubled haploids by gynogenesis through isolated ovule culture, especially in sugar beet. Studies on the production of doubled haploids by androgenesis were actively carried out in the 1970s and 1980s and did not lead to the production of regenerated plants. However, at present, there is renewed interest among researchers in this approach, and scientists in different countries are conducting studies of Beta vulgaris androgenesis through isolated microspore culture. This article provides an overview of studies devoted to the production of doubled haploids, addressing the main problems of doubled haploid technologies, and methods to increase the frequency of embryogenesis and doubled haploid plant formation in B. vulgaris crops

Increasing the somatic embryogenesis frequency of Panax vietnamensis Ha et Grushv. by disinfection of leaf explant using nano silver and the addition of nano silver in culture medium

Somatic embryo is a developmental method for mass multiplication of valuable medicinal plants. In this study, leaf explants of Ngoc Linh ginseng were disinfected with nano silver at different concentrations and exposure times to eliminate infectious agents and induce embryogenic callus for the production of somatic embryos. The results show that the lowest contamination rate (20.00%) was observed when leaf explants were treated with 0.5 g/L nano silver for 15 minutes while the highest embryogenic callus induction rate (72.22%) and fresh weight (0.77 g) was determined at 0.2 g/L nano silver for 20 minutes. High frequency of somatic embryogenesis formation and germination were occurred on MS medium supplemented 1.0 mg/L 2,4-D; 0.5 mg/L NAA; 0.2 mg/L Kin and 1.6 mg/L nanosilver. After 8 weeks of culture, the number somatic embryos derived from nano silver treated-leaves was increased 2 times than non-treated explants. Addition of 1.0 mg/L NAA and 1.2 mg/L nano silver was showed the highest shoot and root length, root number, fresh and dry weight of plantlets. This research showed that pre-treatment and supplement of nano silver in culture medium is potentially useful for improving embryogenesis frequency, and plantlet formation of Ngoc Linh ginseng cultured in vitro.

Non-ionic surfactants improved microspore embryogenesis and plant regeneration of recalcitrant purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Bailey)

As Brassicaceae species are mostly cross-pollinated, breeding homozygous parental lines by traditional approaches is time-consuming and costly. Alternatively, microspore culture has been widely applied to produce double haploid lines in a short time. This study aimed to establish a highly efficient microspore culture protocol for purple flowering stalk. Among the five genotypes studied, the highest and lowest embryo induction rates were observed in J18 and J17 (13.5 and 7.67 embryos per bud, respectively). Microspores of genotypes J17 and J18 were successfully induced to produce embryos in NLN-13 medium, but the frequency of microspore embryogenesis was low. Three non-ionic surfactants (Pluronic F-68, Triton X-100, Tween-20) were evaluated independently for their effect on microspore embryogenesis of purple flowering stalk. Microspores of the two genotypes were cultivated in NLN-13 medium supplemented with different concentrations (0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, and 1% (w/v)) of the three non-ionic surfactants to enhance microspore embryogenesis and plant regeneration. In both genotypes, supplementation with any of the three non-ionic surfactants at 0.0001% significantly increased the frequency of microspore embryogenesis; furthermore, at that concentration, Tween-20 significantly increased the number of plants regenerated from induced embryoids by 29.9% and 30% in J17 and J18, respectively. Moreover, the rate of double haploid formation among regenerated plants of the five genotypes was above 60%, which allowed the creation of 93 double haploid lines.

Impact of Osmotica and Plant Growth Regulators on Somatic Embryogenesis of Date Palm

An efficient somatic embryogenesis system is reported for date palm cv. Al-Fayda, a genotype resistant to the bayoud disease. Callus induction was achieved from adventitious bud explants cultured for 6 months on semi-solid Murashige and Skoog (MS) medium containing 4.5 μM 6-(dimethylallylamino) purine (2iP) and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram. The highest somatic embryogenesis frequency (89%) was obtained on MS medium supplemented with 225 μM 2,4-D. Subsequently, embryogenic cultures were transferred to agitated liquid MS medium (maturation medium) containing various concentrations of mannitol, polyethylene glycol (PEG) or sorbitol. The highest rate of somatic embryo maturation (71.4 mature embryos per 100 mg callus) was achieved on the medium supplemented with 40 g l-1 PEG. Mature somatic embryos were then transferred to MS medium supplemented with gibberellic acid (GA3) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) at various concentrations. The highest frequency of germination and conversion (26%) was obtained on the medium containing 5 μM NAA and 5 μM BAP. The developed plants were then transferred to ex vitro conditions, where a survival rate of 77.02% was observed. The regeneration protocol established in the present investigation will be used for mass propagation of date palm cv. Al-Fayda.

Improving the efficiency of wheat microspore culture methodology: evaluation of pretreatments, gradients, and epigenetic chemicals

The objective of the study was to develop efficient widely adaptable microspore culture methodology for spring and winter wheat genotypes. Current wheat microspore culture methods are still inadequate for routine application, especially for winter genotypes. The present study involved increasing the frequency of microspore embryogenesis and improving the conversion of microspore-derived embryos to fertile green plants. A systematic evaluation was conducted testing numerous factors at all phases of the microspore embryogenesis process. Results from pretreatments, maltose gradients, and histone deacetylase inhibitors are presented. A 28 days cold pretreatment of spikes was optimal for winter wheat genotypes whereas a 21 days cold pretreatment was best for spring genotypes. Microspore suspensions containing a high frequency of embryogenic microspores were collected by resuspension in a 23% maltose gradient with subsequent pelleting with a mannitol containing solution. The application of trichostatin A (TSA) enhanced embryogenesis and/or green plant regeneration. Other epigenetic chemicals like scriptaid, BIX-01294, and sodium butyrate were also evaluated. Scriptaid enhanced embryogenesis but regenerated plantlets showed abnormal shoot and root development. The improved methodology was validated using a range of spring and winter wheat genotypes. Enhanced microspore embryogenesis and green plant regeneration in spring and winter wheat was observed with 21 or 28 days cold pretreatments, a 23% maltose gradient, and the application of trichostatin A (TSA).

Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

AbstractTraditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid breeding.

Application of Adaptive Neuro-Fuzzy Inference System-Non-dominated Sorting Genetic Algorithm-II (ANFIS-NSGAII) for Modeling and Optimizing Somatic Embryogenesis of Chrysanthemum

A hybrid artificial intelligence model and optimization algorithm could be a powerful approach for modeling and optimizing plant tissue culture procedures. The aim of this study was introducing an Adaptive Neuro-Fuzzy Inference System- Non-dominated Sorting Genetic Algorithm-II (ANFIS-NSGAII) as a powerful computational methodology for somatic embryogenesis of chrysanthemum, as a case study. ANFIS was used for modeling three outputs including callogenesis frequency (CF), embryogenesis frequency (EF), and the number of somatic embryo (NSE) based on different variables including 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), sucrose, glucose, fructose, and light quality. Subsequently, models were linked to NSGAII for optimizing the process, and the importance of each input was evaluated by sensitivity analysis. Results showed that all of the R2 of training and testing sets were over 92%, indicating the efficiency and accuracy of ANFIS on the modeling of the embryogenesis. Also, according to ANFIS-NSGAII, optimal EF (99.1%), and NSE (13.1) can be obtained from a medium containing 1.53 mg/L 2,4-D, 1.67 mg/L BAP, 13.74 g/L sucrose, 57.20 g/L glucose, and 0.39 g/L fructose under red light. The results of the sensitivity analysis showed that embryogenesis was more sensitive to 2,4-D, and less sensitive to fructose. Generally, the hybrid ANFIS-NSGAII can be recognized as a powerful computational tool for modeling and optimizing in plant tissue culture.

Effects of brassinolide on microspore embryogenesis and plantlet regeneration in pakchoi (Brassica rapa var. multiceps)

Hybrids are often superior to conventional varieties because of their high yield and strong stress resistance. The production of homozygous inbred lines is an important prerequisite for hybrid seed production; however, they may require more than six years by traditional breeding methods. Microspore culture is an attractive approach to obtain homozygous doubled haploids (DHs) in a short period. However, low embryogenesis frequency and high rates of embryo callus or mortality remain the main problems of this method in pakchoi. In this study, four different concentrations (0.01, 0.02, 0.04, 0.08 mg L−1) of brassinolide (BR) were added into NLN liquid medium and used to examine the effect of microspore embryogenesis frequency and plant regeneration rate in three genotypes of pakchoi. The results showed that 0.01 mg L−1 BR was the optimum concentration for genotype ‘XM2’, significantly increasing the frequency of embryogenesis induction and plant regeneration by 2.05-fold and 1.25-fold, respectively, compared with those in the control treatment; there was also an observed 0.56-fold decrease in the rate of callus formation. For both ‘XM1’ and ‘XM3’, 0.02 mg L−1 BR was the most optimum concentration, resulting in a 3.41-fold and 6.39-fold increase in embryogenesis frequency compared with that in the control treatment; the frequency of plant regeneration was more than 74%. Meanwhile the rate of callus formation decreased 0.28-fold and 0.59-fold, respectively. The average spontaneous doubling rate of the three genotypes was 68.50%, whereas the lowest spontaneous doubling rate of the genotype ‘XM2’ was 65.8%. For ‘XM1’, ‘XM2’ and ‘XM3’, we obtained 202, 144 and 85 DHs, respectively. Excellent horticultural traits were identified in DH lines from XM2, including increased leaf number and bright leaf color. All DH lines derived in this study were self-incompatible lines and showed high stability and consistency, which will effectively accelerate the application of microspore technology in hybrid breeding.

Efficient doubled haploid production in microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee)

Microspore culture has been implemented in breeding programs to produce doubled haploids (DH) and thus accelerate the breeding process. This study aimed to establish an efficient microspore culture protocol for Zengcheng flowering Chinese cabbage, a highly nutritious and valued vegetable, that would be suitable for heterosis breeding. Microspores of three genotypes, 17AY010, 17AY011, and 17AY012 were successfully induced to produce embryos in NLN-13 medium. Two genotypes, 17AY011 and 17AY012 were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.2, 1, 5 or 25 μM) of L-ascorbic acid sodium salt (Vitamin C Sodium Salt, VcNa) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. In both genotypes, the addition of 1 μM and 0.2 μM VcNa significantly increased the frequency of embryogenesis, 6.55-fold and 10.33-fold as compared with the control, respectively. The optimum concentration of VcNa application that would improve the rates of direct plant regeneration in the two tested genotypes was 1 μM. Moreover, the doubled haploid rates of regenerated plants of the three genotypes were above 60%. Five DH lines of 17AY012 with high yield were obtained. According to the measurement of the affinity index, they were prospective self-incompatible lines for hybrid breeding.

High Frequency of Plant Regeneration through Adventitious Shoots Proliferation from Leaf Explant of Patchouli–Pogostemon cablin (Blanco) Benth

Plant regeneration potential through adventitious shoots proliferation was studied from leaf explant of evergreen perennial aromatic herb, patchouli plant, or Pogostemon cablin. Leaf explant showed differentiation of embryogenic callus on Murashige and Skoog’s medium (MS) supplemented with 2,4-D 0.5 mg L-1, 3% sucrose and 0.9% agar. Embryogenic callus was further transfer to same medium with BA 0.5 mg L-1 produced somatic embryos and adventitious shoots having dark green colour after four weeks. The maximum range of embryogenesis frequency was 89%, depending on the BAP concentration in combination with 2,4-D. The best response was observed on MS medium containing 0.5 mg L-1 BAP along with 0.5 mg L-1 2,4-D. The embryos or adventitious shoots matured and developed on fresh MS medium with growth regulators. Maximum numbers of 74 adventitious shoots germination were observed and developed further for root induction on the medium containing 0.5 mg/l BAP and 0.5 mg/l NAA after six weeks of culture. Regenerated shoots were separated and rooted on same half strength MS medium supplemented with 1.0 mg/l of NAA alone for three weeks. Approximately 30-105 plantlets were regenerated from each leaf explant. Well-regenerated plants grew vigorously after transplanting to a soil-less container in shade house.

In vitro Direct Regeneration from Node and Leaf Explants of Phalaenopsis cv. ‘Surabaya’

An efficient and reproducible procedure for the direct regeneration of phalaenopsis cv. ‘Surabaya’ using of nodal explants and leaf segments derived from in vitro flower stalk was conducted. Three experiments were carried out for shoot development and subsequent plant regeneration: Direct shoot regeneration from nodal explants of Phalaenopsis cv. ‘Surabaya’ flower stalks on MS added with different combination of NAA and BAP, direct regeneration of protocormlike bodies (PLBs) from leaf explants in a MS with different concentrations of the TDZ, acclimatization of regenerated plantlets in different mixture of components and nutrients. The results showed that 5 mg/l BAP and 2 mg/l NAA were most effective concentration for shoot regeneration, regenerated shoots were cultured on half strength of MS containing activated charcoal, IAA and NAA at various concentrations, highest number of root (6.7) was obtained in higher concentration of NAA (2 mg/l). TDZ induced a higher frequency of embryogenesis from leaf explants than BAP, the highest number of embryos per explant was 22.45 at 3 mg/l TDZ. Altogether, BAP at higher concentration (10 mg/l) with 1 mg/l NAA had the highest enhancement on the amount of direct embryogenesis. In our investigation 87.20% plantlets via nodal explants survived acclimatization process in medium containing cocopeat and coal (1 : 1). The survival rate of regenerated plantlets via nodal explants (82.07%) was more than of regenerated plantlets via leaf explants (70.47). This protocol provides the basis for further investigation on micropropagation and breeding programs in Phalaenopsis cv. ‘Surabaya’.Plant Tissue Cult. & Biotech. 25(2): 193-205, 2015 (December)

Construction of a high-density linkage map and mapping quantitative trait loci for somatic embryogenesis using leaf petioles as explants in upland cotton (Gossypium hirsutum L.).

The first high-density linkage map was constructed to identify quantitative trait loci (QTLs) for somatic embryogenesis (SE) in cotton ( Gossypium hirsutum L.) using leaf petioles as explants. Cotton transformation is highly limited by only a few regenerable genotypes and the lack of understanding of the genetic and molecular basis of somatic embryogenesis (SE) in cotton (Gossypium hirsutum L.). To construct a more saturated linkage map and further identify quantitative trait loci (QTLs) for SE using leaf petioles as explants, a high embryogenesis frequency line (W10) from the commercial Chinese cotton cultivar CRI24 was crossed with TM-1, a genetic standard upland cotton with no embryogenesis frequency. The genetic map spanned 2300.41 cM in genetic distance and contained 411 polymorphic simple sequence repeat (SSR) loci. Of the 411 mapped loci, 25 were developed from unigenes identified for SE in our previous study. Six QTLs for SE were detected by composite interval mapping method, each explaining 6.88-37.07% of the phenotypic variance. Single marker analysis was also performed to verify the reliability of QTLs detection, and the SSR markers NAU3325 and DPL0209 were detected by the two methods. Further studies on the relatively stable and anchoring QTLs/markers for SE in an advanced population of W10 × TM-1 and other cross combinations with different SE abilities may shed light on the genetic and molecular mechanism of SE in cotton.

Low Sucrose and High Gibberellic Acid Levels Positively Regulates Relative Growth Rate and Organogenesis in D. Membranaceus Munro

Dendrocalamus membranaceus Munro., an economically important edible bamboo has been listed as a priority species requiring conservation by International Union for Conservation of Nature (IUCN) and hence selected for the present investigations. It is opined that the cells of bamboo are undoubtedly totipotent but some vital hormonal and/or nutritional factor or their combination could prove vital for differentiation as the frequency of embryogenesis is lower than that of organogenesis.

English

An efficient in vitro protocol has been developed for the multiplication of shoots and conservation of a rare succulent medicinal plant Caralluma stalagmifera var. longipetala growing wildly in Karnataka State. Proliferation of multiple shoots was achieved on Murashige and Skoog’s (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), Kinetin (Kn), indole acetic acid (IAA), α- naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) alone or with various combinations from the nodal explants. The nodal explants cultured on medium containing BAP (2.0 mg/L) along with 0.5 mg/L Kn and 0.3 mg/L IAA produced the highest number of shoot sprouting (2.60±0.16) and maximum shoot length (3.96±0.20). The considerable frequency of callus induction and embryogenesis was noticed both in 1.0 mg/L NAA and 0.5-2 mg/L, 2, 4-D. The calli transferred to shoot induction medium containing the combination of hormones BAP (1.0 mg/L) plus IAA (0.2 mg/L) and NAA (0.1 mg/L) successfully regenerated in vitro shootlets. The in vitro rooting was achieved from both direct shoots regenerated from nodal explants and callus derived shootlets with NAA (0.2 mg/L). The in vitro rooted plantlets were successfully acclimatized (75%) in the greenhouse and gradually transferred to open field conditions. Key word: Micro propagation, medicinal plant, Caralluma stalagmifera var. longipetala.

Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra

Brassica nigra is generally regarded as a recalcitrant species for microspore culture among Brassica crops. Conditions for reliable induction of microspore embryogenesis of B. nigra were studied in this context. Flower bud length and microspore developmental stage were correlated with further embryogenesis. The optimal bud size range was 2.0–2.5 mm for the highest proportion of totipotent, late uninucleate microspore and the highest frequency of microspore embryogenesis. Treatment of a short heat shock by incubating the microspore culture at 32°C for 24 h was suitable for the microspore survival, sustained cell divisions, and further induced embryogenesis. Subsequently, the use of NLN medium with the addition of 13% sucrose and 0.1% activated charcoal (AC) provided the optimal conditions for the development of microspore-derived embryos (MDEs). The early cotyledonary (EC) stage embryos cultured on MS medium fortified with 4.6 μM zeatin (ZT) and 0.12 μM indole-3-acetic acid (IAA) resulted in the most efficient rates of plantlet regeneration. The ploidy levels of regenerated plants of B. nigra were determined by flow cytometry, revealing that 50.6% were diploid. The results enable the advancement of breeding programs and genetic studies in B. nigra.