Embryogenic Callus Induction Research Articles

Establishment of embryogenic cultures and determination of their bioactive properties in Rosa rugosa

Somatic embryogenesis represents a powerful in vitro plant culture system and provides high potential for the production of biomass in woody plants. Rosa rugosa is known not only for its ornamental value, but also as a source of antioxidants with the potential to be used as ingredients for industrial products such as cosmetics. The present study was conducted to investigate the effects of hormonal concentration on embryogenic callus (EC) induction from petal and leaf segments of this species. The effect of selected cell lines on the production of total phenolics and flavonoids in R. rugosa was also studied. The highest percentage of EC (14.1 ± 3.9%) and root formation (20.3 ± 6.9%) was observed in the presence of 45.0 μM of 2,4-D. Among the three studied lines, the highest total phenolics and flavonoid levels were observed in H-1 (17.71 mg·g-1 DW), while H-18 contained the lowest. Histological studies of the H-1 and H-18 cell lines revealed differences in polyphenol accumulation and polysaccharides. The effect of developmental stage on growth, shoot, and root regeneration, as well as on phenolic and flavonoid content, was also studied in H-18. The present study indicates that selection of the proper type and concentration of hormone is essential for EC induction from petals in R. rugosa. Moreover, the accumulation of bioactive compounds was found to greatly depend on the genotype. The results of this study are useful for the production of selected antioxidants by somatic embryos from R. rugosa petals.

A comparative study of three different methods of shoot meristem excision for induction of embryogenic calli in coconut

<p><strong>A protocol was standardized to maximize yields of embryogenic calli from shoot meristem culture of coconut. Three different shoot meristem excision methods were tested <em>viz</em>., excision of shoot meristem aseptically from <em>in vitro</em> germinated embryo after 10-12 days, excision of shoot meristem from <em>in vitro</em> germinated embryo subjected to GA3 treatment for five days and excision of shoot meristem from fresh embryo. The primary calli induction after 30 days of culture incubation for the three treatments were 21%, 27% and 79% respectively. Further, the primary calli formed from the shoot meristem excised from fresh embryo gave rise to 56% of embryogenic calli. The calli obtained from the shoot meristem which were excised from <em>in vitro</em> germinated embryo formed less percentage of embryogenic calli because of the presence of cotyledonary tissues which inhibited the multiplication of meristematic tissues. In the case of shoot meristem extracted from GA3-treated embryos, the percentage of non-embryogenic calli was more compared to the shoot meristem excised from fresh embryo. It was observed that the addition of GA3 in the initial stages of culture inhibited the formation of embryogenic calli and favored direct shoot development. Currently, the shoot meristem excised from fresh embryo is being employed for scaling up the planting material production from released varieties of coconut.</strong></p>

Evaluation of biochemical markers during somatic embryogenesis in Silybum marianum L.

In present report effects of explants type, basal media and plant growth regulators (PGRs) were tested for induction of indirect somatic embryogenesis in medicinally important plant Silybum marianum L. Leaf, petiole and root explants were exploited in vitro on B5 (Gamborg), SH (Schenk and Hildebrandt) and MS (Murashige and Skoog) media for induction of embryogenic callus followed by somatic embryogenesis. Highest callus induction frequency (76 ± 4.8 %) was recorded when petiole explants of in vitro derived plantlets were cultured on B5 medium supplemented with 1.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4_D) in combination with 1.5 mg l−1 Thidiazuron (TDZ). Induction and multiplication of somatic embryos were observed, when the embryogenic calluses were sub-cultured on to B5 medium containing 0.5 mg l−1 2,4-D plus 1.5 mg l−1 TDZ. At this PGRs treatment, 77 % of the cultures responded with 39.1 somatic embryos per callus. Furthermore, MS0 medium was indicated more reponsive for growth and maturation of somatic embryos. Analysis of biochemical markers during various growth phases in somatic embryogenesis revealed that somatic embryos exhibited highest level of total carbohydrate, starch, ascorbic acid and total free amino acids. However, higher protein levels were detected in non-embryogenic callus. Nevertheless, considerable amount of silymarin (4.1 mg g−1 DW) was detected in somatic embryos than other growth phases. Thus, the present study concluded that biochemical and physiological changes during embryogenesis are influenced by interplay of explants type, basal media and PGRs.

Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs.

Rapid Propagation of Virus-free Sugarcane (Saccharum officinarum) by Somatic Embryogenesis

Abstract Elimination Sugarcane Mozaic Virus ( SCMV ) in sugarcane plantlets and propagation via somatic embryogenesis was assessed. The aim of this study was to obtain a virus-free sugarcane and determine methods for propagation via somatic embryogenesis. Explants derived from sugarcane SUT-02 plantlets. The explants were cultured on ribavirin and acyclovir medium at 0, 20 and 40 mgl -1 concentrations. Virus-free plantlets detected by DAS-ELISA. Rapid propagation virus-free plantlets through somatic embryogenesis consists of 3 stages. Stage 1: callus induction on the media (a) MSo, (b) MSo + 3 mgl -1 2,4-D, (c) MSo + 4 mgl -1 2,4-D, (d) MSo + 3 mgl -1 2,4-D + 1.5 mgl -1 BAP, (e) MSo + 4 mgl -1 2,4-D + 1.5 mgl -1 BAP, stage 2: proliferation on media MSo + 1.5 mgl -1 2,4-D and stage 3: regeneration on MSo media. The results showed that the media ribavirin and acyclovir at 20 mgl -1 and 40 mgl -1 concentration produces 100% virus-free sugarcane plantlets. Best embryogenic callus induction media is MSo + 3 mgl -1 2,4-D and virus free plantlets have produced as much as ± 35 plantlets within 9 weeks.

Plantlets Regeneration via Somatic Embryogenesis from the Nucellus Tissues of Kinnow Mandarin (<i>Citrus reticulata</i> L.)

Studies were initiated to explore the role of nucellus tissues and growth regulators in plantlets regeneration via somatic embryogenesis of Kinnow mandarin [Citrus reticulata L. (Blanco)]. Nucellus tissues were cultured on MS medium supplemented with different concentrations and combinations of auxins, cytokinins and malt extract for primary callus induction. The best response for primary callus induction (90%) was obtained when MS medium was supplemented with 5 mg/l 2,4-D and 500 mg/l malt extract. Best results for embryogenic callus induction (80%) were obtained in C8 medium. The induction of somatic embryos was highest when MS medium was supplemented with 1 mg/l BAP and maturation of somatic embryos occurred when MS medium was supplemented with 5 mg/l 2,4-D and 1 mg/l BAP. Maximum plantlets were regenerated (92%) from the somatic embryos on half strength MS medium with no hormones. The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (100%) was achieved in potting mixture containing sand and peat moss (2:1).

Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion

ABSTRACT: The study was undertaken to examine the effect of two synthetic auxins on somatic embryogenesis and plant regeneration from mature embryo explants of 16 onion cultivars. Cotyledons were removed from the embryos and remaining portions were cultured on MS medium supplemented with 1.0 mg/L 2,4-D and 5.0 mg/L picloram alone or in combination (1.0 mg/L 2,4-D + 2.5 mg/L picloram) to produce embryogenic callus. MS medium supplemented with 5.0 mg/L picloram was found to be the best one for both embryogenic callus induction (85%) and callus diameter (3.8 mm). Of the 16 cultivars studied, Yeoeuijuhwang exhibited the lowest frequency of embryogenic callus induction (50.5%), whereas all the other 15 cultivars showed more than 60% embryogenic callus induction. Scanning electron micrograph (SEM) analysis of embryogenic callus showed all stages of somatic embryos such as globular, scuetellar and coleoptilar. Plant regeneration was significantly affected by the composition of embryogenic callus induction medium. The greatest frequency of somatic embryo conversion was obtained from embryogenic callus developed in MS medium with 2,4-D (70.1%) followed by picloram (38.9%) and 2,4-D + picloram (34.5%). The germinated plantlets were further developed on the half-strength MS medium containing 3% sucrose and were acclimatized in the culture room with 98% survival rate.

Investigation of callogenesis and indirect regeneration of Freesia × hybrida Bailey ‘Argenta’

Abstract An investigation was conducted to study the effects of explant sources, plant growth regulators, carbohydrates and light conditions on indirect cormlet regeneration and the induction of embryogenic callus of freesia (Freesia × hybrida Bailey ‘Argenta’). Sections of two different types of explants, corms and pupae (cold storage-produced corms), were placed on Murashige and Skoog (MS) media containing different concentrations of plant growth regulators. The results showed that the highest percentage of callus induction (100%), the highest callus growth (15 mm diameter) and the best type of calli were achieved for pupa explants grown on the medium that contained 4 mg L−1 1-naphthaleneacetic acid (NAA) and 2 mg L−1 6-benzylaminopurine (BAP) in the dark. Increasing BAP up to 3 to 4.5 mg L−1 resulted in the maximum number of regenerated cormlets from 1 cm2 calli (2 cormlets) under light conditions. Overall, the best rooting of regenerated cormlets was achieved on MS media supplemented with 1 mg L−1 indole-3-butyric acid (IBA). In the next stage, high quality calli were subcultured on MS media containing sorbitol, sucrose, maltose and mannitol (0, 5, 10 and 15 g L−1). The results indicated that 15 g L−1 maltose was able to induce the highest percentage of embryogenic callus, with an average of 88.9% on media containing 2 mg L−1 BAP and 1 mg L−1 NAA.

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Efficient plant regeneration of watermelon (Citrullus lanatus Thunb.) via somatic embryogenesis and assessment of genetic fidelity using ISSR markers

A simple and effective somatic embryogenic system was established for watermelon (Citrullus lanatus) cv. ‘Arka Manik’. Embryogenic callus was obtained from leaf explants of 20-d-old in vitro-grown seedlings cultured on embryogenic callus induction medium. The highest frequency of embryogenic callus induction (96.8%) occurred on Murashige and Skoog (MS) medium supplemented with 2.44 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.27 μM thidiazuron (TDZ). Transfer of embryogenic calluses with proembryogenic masses to embryo maturation medium led to the asynchronous development of somatic embryos (SEs), which progressed from the globular stage to the cotyledonary stage. The maximum number of SEs/explant (16.1 ± 0.24) was obtained on MS medium supplemented with 2.44 μM 2,4-D, 2.27 μM TDZ, and 30 g L−1 sucrose. Plantlet conversion from cotyledonary-stage SEs was tested on different strengths of MS medium (quarter-, half-, and full-strength) lacking plant growth regulators. The highest frequencies of germination (91.5%) and survivability (82.1%) of plantlets were achieved on full-strength MS medium. Transverse sections of embryogenic callus revealed SE development from callus cells near the epidermis. Secondary SEs occasionally formed from globular-shaped primary embryos. Genetic fidelity of mother plants and ex vitro plants was confirmed by inter-simple sequence repeat (ISSR) markers. The present study is the first report on the use of molecular markers in in vitro culture of watermelon. The developed protocol facilitates rapid production of true-to-type watermelon plants by somatic embryogenesis and thus could serve to generate effective target material for genetic transformation protocols.

Efecto de Dicamba y de ácido 2,4 diclorofenoxiacético sobre la embriogénesis somática en caña de azúcar

<p><strong>Título en ingles</strong>: Efecto de Dicamba y de ácido 2,4 diclorofenoxiacético sobre la embriogénesis somática en caña de azúcar</p><p>El cultivo <em>in vitro</em> de la caña de azúcar ha sido establecido en muchas variedades comerciales con el propósito de producir material libre de enfermedades microbianas, conservar germoplasma, detectar resistencia a enfermedades y plagas, etc. En este sentido, el objetivo de este trabajo fue analizar la efectividad de las auxinas sintéticas ácido 2,4-diclorofenoxiacético (2,4D) y ácido 3,6-dicloro-2-metoxibenzoico (Dicamba), en la inducción del proceso de embriogénesis somática y la regeneración de vitroplántulas de distintas variedades de caña de azúcar (C26670, RB855546, V99245, V756, V781, V0050, CC8592, CC8475). Para esto se cultivaron discos de hojas en fase de macollamiento, de 1 cm de diámetro y 2 mm de grosor, en medio Murashige-Skoog, 1962 (MS) suplementado con 50 ml.l-1 agua de coco, 30 g.l-1 sacarosa y dos tratamientos diferentes: 3 mg.l-1 2,4-D ó 6.63 mg.l-1 Dicamba, ambos en completa oscuridad a 25ºC, durante 1 mes. Los callos obtenidos se colocaron en medio de regeneración, conteniendo ½ sales MS, 200 ml.l-1 agua de coco y 60 g.L-1 sacarosa, incubándose bajo luz continua, 25ºC, por 2 meses. El mayor porcentaje de callo embriogénico se obtuvo en medios suplementados con Dicamba un promedio de 70,83 % de callo embriogénico por variedad ; mientras que en los medios con 2,4D se obtuvo 62,08 % de callo embriogénico por variedad. Se obtuvo un promedio de 89,00 % de plantas regeneradas a partir de los callos obtenidos en medios con Dicamba y 66,12 % de plantas a partir de callos obtenidos en medios con 2,4D. Con el uso de Dicamba se estableció un sistema eficiente de embriogénesis somática para estas variedades de caña de azúcar.</p>

Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion

We investigated the effect of two synthetic auxins on somatic embryogenesis and plant regeneration from mature embryo explants of 16 onion cultivars. Cotyledons were removed from the embryos and remaining portions were cultured on MS medium supplemented with 1.0 mg L -1 2,4-D, 5.0 mg L -1 picloram or 1.0 mg L -1 2,4-D and 2.5 mg L -1 picloram. Among the media studied, MS medium supplemented with 5.0 mg L -1 picloram was found to be best for both embryogenic callus induction (85%) and callus growth (3.8 mm). Of the 16 cultivars studied, ‘Yeoeuijuhwang’ exhibited the lowest frequency of embryogenic callus induction (50.5%), whereas all the other 15 cultivars showed more than 60% embryogenic callus induction in all studied media. SEM analysis of embryogenic callus showed all stages of somatic embryos such as globular, scuetellar and coleoptilar. Plant regeneration was significantly affected by the embryogenic callus induction media. The greatest frequency of somatic embryo conversion was obtained from embryogenic callus develop in MS medium with 2,4-D (70.1%) followed by picloram (38.9%) and 2,4-D + picloram (34.5%). The germinated plantlets further developed on the half-strength MS medium containing 3% sucrose and were acclimatized in the culture room with 98% survival rate.

Effects of cefotaxime, amino acids and carbon source on somatic embryogenesis and plant regeneration in four Indian genotypes of foxtail millet (Setaria italica L.)

The effects of cefotaxime, amino acids and carbon source on somatic embryogenesis and plant regeneration using mature seeds in four genotypes (‘CO5’, ‘CO7’, ‘TNAU43’ and ‘RS118’) of foxtail millet have been studied. The ‘CO5’ gave a superior response in callus induction, somatic embryogenesis and regeneration. The highest percentage (69.3%) of embryogenic callus induction was obtained in ‘CO5’ on Murashige and Skoog (MS) medium supplemented with 3.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg L−1 kinetin and 1 mg L−1 1-naphthaleneacetic acid (NAA). Somatic embryogenesis and shoot regeneration were influenced by amino acids, carbohydrates and cefotaxime in culture medium. Maximum response of somatic embryo induction and maturation was seen on MS medium containing 3.5 mg L−1 2,4-D, 1 mg L−1 kinetin and 1 mg L−1 NAA, 750 mg L−1 proline, 2.0 mg L−1 glycine, 150 mg L−1 arginine, 800 mg L−1 casein enzymatic hydrolyzate, 20 g L−1 each sucrose and maltose and 500 mg L−1 cefotaxime. The highest frequency of plant regeneration with 21.3 shoots was obtained in ‘CO5’ on MS medium containing 3 mg L−1 6-benzylaminopurine, 0.2 mg L−1 2,4-D, 750 mg L−1 proline, 2.0 mg L−1 glycine, 150 mg L−1 arginine and 800 mg L−1 casein enzymatic hydrolyzate. The highest response of root induction with more roots and longer roots was observed in ‘CO5’ when cultured on half-strength MS medium. The in vitro-regenerated plantlets were carefully transferred to soil cups, maintained in growth chamber for a week, hardened and grown to maturity in the field.

Indução e regeneração de embriões somáticos de cana-de-açúcar RB996961

Sugarcane is a crop widely planted in Brazil because of the economic importance of its products, like sugar and ethanol. Thus, the crop is often inserted in breeding programs. Therefor, the aim of this study was to evaluate the morphogenic response to somatic embryogenesis and somatic embryos regeneration for genotype RB996961. Transversal segments of young leave rolls with 2-3 mm size from plants with 6 to 9 months old were subcultured on solid MS medium supplemented with three concentrations of 2.4-dichlorophenoxyacetic acid (2.4-D) for embryogenic callus induction. Better results were obtained using 13.5 or 31.5 µM L -1 of 2.4-D in 45 or 60 days of incubation on induction medium, one to three subculture and 15 to 45 days of incubation on regeneration medium for genotype RB996961. This results show that the protocol used to genotype RB996961 was efficient to somatic embryos production (over 80%), nevertheless, it is necessary to adjust the regeneration approach (62.3 to 73.4%).

Induction Somatic Embryogenesis Used 2,4-Dichlorophenoxyacetic Acid (2,4-D) and Kinetin in Spindle Leaf Explant Sugarcane

Induction of somatic embryogenesis in sugarcane requires the composition Plant Growth Hormone (PGH) appropriate. Utilizing of PGH (2,4-D and kinetin) is expected to induce sugarcane somatic embryogenesis. The purpose of this study was to obtain the concentration of 2,4-D and kinetin that effective for the multiplication of sugarcane var. NXI 1-3 through somatic embryogenesis. This study consists of four stages: callus induction, callus proliferation, regeneration of shoots and encapsulation. The plant material used is a spindle leaf sugarcane var. NXI 1-3. Callus induction used 2,4-D with different concentration (2 ppm, 3 ppm and 4 ppm). Callus proliferation used 2,4-D with concentration 1 ppm and 2 ppm. Regeneration of shoots used kinetin 0,5 ppm. The results are showed that the optimal induction of embryogenic callus on medium MS + sucrose 30 g / L + CH 300 ppm + 300 ppm PVP + 2,4-D 4 ppm as indicated by the high percentage of explants forming embryogenic callus that is equal to 40% and the respective concentration 2 ppm and 3 ppm is 33,3% and 37,5%. In proliferation stage, the development callus optimal on medium MS + sucrose 30 g / L + CH 300pm + PVP 300 ppm + 2,4-D 2 ppm and formulations for regeneration shoot on medium MS + sucrose 30 g / L + kinetin 0.5 ppm. The result of encapsulation can be shaped 100 sythetic seed. Keywords: Somatic embryogenesis, spindle leaf, kinetin, 2,4-D

TDZ and 4-CPPU Induce Embryogenic Response on Scalps of Recalcitrant East African Highland Banana

Breeding of banana conventionally is very difficult due to high levels of sterility arising from aneuploidy. Transformation of East African highland banana (EA-AAA banana) has been limited by difficulties in induction of embryogenic callus and/or embryogenic cell suspension (ECS), a requirement for efficient genetic transformation. In this study N-phenyl-N’-1,2,3-thidiazol-5-ylurea (TDZ) and N-(2-chloro-4-pyridyl)-N’-phenylurea (4-CPPU) that are highly active in regulating morphogenesis in tissue culture of many plant species were tested for enhancing embryogenic callus induction in recalcitrant EA-AAA banana through scalps. In vitro derived shoot-tips of banana cultivars Mbwazirume, Mpologoma, Nakabululu, Nakinyika and Nfuuka were subcultured on various combinations of TDZ and 4-CPPU till scalps were formed. In all the cultivars, ideal scalps for callus induction were achieved in over 50% of the shoot-tip cultures within four subculture cycles in the medium containing equal proportions of 9–13 µM of TDZ and 4-CPPU, and 26 µM of each. Embryogenic callus was formed on 2.5–20% of the scalp cultures depending on cultivar and, TDZ and 4-CPPU combination. Cultivars Nakinyika, Nakabululu, Mbwazirume and Nfuuka developed regenerable embryogenic callus. Of the plantlets regenerated 5 out of 12 and 4 out of 5 plantlets of cultivars Nakinyika and Mbwazirume, respectively, developed normal roots and were weaned onto soil. These results suggest that TDZ and 4-CPPU increases embryogenic response from scalps of EA-AAA banana which can enhance their genetic transformation.

Induction of somatic embryogenesis and biochemical characterization of Cordyline australis (G. Forst.) Endl. ‘Red Star’ callus

The current study presents a method for the plant regeneration from stem segments of Cordyline australis (G. Forst.) Endl. ‘Red Star’ through somatic embryogenesis. We tested the effect of 3 kinds of auxin 4-amino-3,5,6-trichloropicolinic acid (picloram), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2-methoxy-3,6-dichlorobenzoic acid (dicamba), with a combination of 2 kinds of cytokinin, 6-benzyladenine (BA) and thidiazuron (TDZ), on the induction of embryogenic callus and its capacity to induce somatic embryogenesis under dark culture condition. The highest percentage of explants forming callus (nodular and friable) was observed on medium containing 25μM picloram and 0.5μM BA. The embryogenic callus, with white-yellowish nodular structures, was proliferated in the initial media and finally produced somatic embryos on all media with picloram and 2,4-D. Somatic globular embryos were obtained with a higher frequency from explants cultured on MS medium supplemented with 25μM picloram and 0.5μM BA. On this medium, a mean number of 23.0 embryos developed per gram of callus. Globular embryos developed most efficiently into cotyledonary stage on MS medium supplemented with 5μM picloram and 1μM BA. The addition of zeatin (5 and 2.5μM) and NAA (0.5 and 0.25μM) to the medium was found to be essential for the highest conversion of somatic embryos into plants. Plantlets were successfully acclimatized in the greenhouse with 90% survival rate. Callus was tested for phenolic compounds, sugars, peroxidase activity and protein contents in order to find if these compounds might be the indicators of further embryos development. The embryogenic callus of C. australis with various stages of embryo development had higher level of protein than the non-regenerable one, whereas level of sugars increased only in callus with embryos in cotyledonary stage. Callus with globular embryos contained high concentration of phenolics and low activity of peroxidase, comparing to other types of callus.

Study of Oil Palm (Elaeis guineensis Jacq) In Vitro Embryogenesis using Young Leaves Explants

This study reported in vitro embryogenesis of oil palm using young leaves as explants. Explants were grown in solid modified MS or Eeuwens medium containing different concentrations of NAA and 2,4-D, i.e. media C1, C2, C3, C4 and C5, M1, M2, M3 and M4, to induce embryogenic calli. Compact and pearly-white, globular calli were obtained from the youngest leaf explants 28 weeks after culture.C1 media (MS medium + 107.41 µM of NAA + 100 mg.L-1 of asparagine + 100 mg.L of glutamine-1) produced the highest percentage of calli formation (30.56%), whereas C4 media (C1 supplemented with 67.86 µM of 2,-D ) was the optimal media for embryogenic callus induction. Direct embryoids were obtained from slightly older leaf explants on the C3 media containing NAA after 36 weeks of culture. However, four subcultures using the same medium with gradual reduction of auxin concentration were not successful to develop embryogenic callus and embryoid cells during the course of this study.

Regeneration of Indian maize genotypes (Zea mays L.) from immature embryo culture through callus induction

Callus induction and regeneration ability of five maize genotypes UMI 757, UMI 615, UMI 112, UMI 285 and CO 1 and one promising maize hybrids COH(M) 5 were investigated using 14 days old immature embryos as explants. Callus induction depends on genotype, explants (age and size of explants), medium, type of auxin and their concentration. Explants grown on Murashige and Skoog (MS) medium supplemented with 1.5 mg/l 2, 4 - D (2,4 – dichloro phenoxy acetic acid), 0.3 mg/l kinetin with 30 g/l maltose showed the highest percentage of embryogenic callus induction. Among the six genotypes tested, COH(M) 5 maize hybrids have highest percentage of embryogenic calli. The embryogenic calli incubated on MS medium supplemented with 1.5 mg/l BAP (Benzyl Amino Purine), 0.2 mg/lNAA (Naphthalene Acetic Acid) with 1.0 mg/l kinetin was found to give the highest organogenesis response and regeneration of plantlets.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins (AGPs) and hydrogen peroxide (H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 (KN199) and Xinchun 9 (XC9), together with low-frequency regeneration wheat line Chinese Spring (CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L−1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L−1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22 (JM22), Jingdong 18 (JD18) and Yangmai 18 (YM18), respectively; whereas at 100 mg L−1 AGP level, CS (105.44%), Chuannong 16 (CN16) (80.60%) and Ningchun 4 (NC4) (62.87%) acted the best. Moreover CS (79.05%), JM22 (7.55%), CN16 (101.87%), YM18 (365.56%), Yangmai 20 (YM20) (10.48%), and CB301 (187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4 (35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP (or H2O2) application, can be further applied to wheat transgenic research.