In vitro regeneration of plantlets from unpollinated ovary culture in sweet orange (Citrus sinensis L. Osbeck) (original) (raw)
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American Journal of Plant Sciences, 2019
In-vitro callus induction and regeneration method was developed using different plant growth regulators (PGRs), and basal media (Murashige and Skoog (MS), CHU (N6) and Gamborg (B5) media) of Citrus sinensis (L.) Osbeck. Observations of the effect of PGRs were carried out using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D),1-naphthalene acetic acid (NAA) and combinations of 2,4-D and NAA using different basal media. This study found Citrus sinensis (L.) Osbeck exhibited a high frequency of callus induction on MS medium supplemented with 3 mg/L 2,4-D and callus induction frequency was 86.7% ± 3.4% whereas N6 and B5 showed lower callus induction frequency of 83.3% ± 8.8% and 82.2% ± 1.9% respectively compared to that of MS media with supplementation of the same hormone. Among the induced calli, the morphological analysis showed only 40%-50% was embryogenic calli. Regeneration of plantlets from calli was done using different concentrations and combinations of auxin and cytokinin. The study showed that 3 mg/L 6-benzylaminopurine (BAP) supplemented medium has the maximum potential to promote regeneration of Citrus sinensis (L.) Osbeck from embryogenic calli with the frequency of 89.3% ± 8.8% but no regeneration occurred from the non-embryogenic calli. The regenerated plantlets were rooted on MS medium with supplementation of 5 mg/l NAA. These observations in Citrus sinensis (L.) Osbeck regeneration will be helpful for genetic improvement with desired traits.
Agricultural Sciences, 2019
Citrus reticulata (Mandarin Orange), commonly known as "Sweet Orange", is one of the most difficult plants to improve through traditional breeding approaches as it poses various biological limitations that greatly hinder the cultivar improvement. In the present study, using the fresh seed of native orange as explant, an efficient, reproducible, regeneration method was developed through in vitro organogenesis. Mature, healthy and dehusked seeds were treated with Murashige and Skoog, (MS) media containing 3% sucrose, 0.7% agar supplemented with different concentrations and combinations of phytohormones. The highest calli initiation (93.3% ± 0.5%) responses were observed on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 3.0 mg/L followed by 2,4-D at 3.5 mg/L (86.7% ± 1.75%) in this experiment. Maximum shoot regeneration (86.7% ± 3.35%) responses were reported using MS medium supplemented with the combination of 6-benzylaminopurine (BAP) at 3.0 mg/L and 1-naphthaleneacetic acid (NAA) at 2.0 mg/L. MS medium supplemented with NAA at 1.0 mg/L showed the best rooting (80% ± 2.89%) response in comparison to (70% ± 5.20%) indole-3-butyric acid (IBA) at 1.0 mg/L. The regenerated plantlets were acclimatized in pots containing sterile garden soil mixture to examine their response in natural conditions.
2020
The present investigation was intended to establish an effective protocol for direct shoot regeneration as well as root induction of Citrus sinensis using seed germinated shoot tip and nodal segments. Surface sterilization of explants was attributed with NaOCl (Sodium hypochloride) in shoot tip and nodal segments for successful contamination free culture. For direct shoot proliferation, nodal segments and shoot tips showed effective results when it cultured aseptically on MS (Murashige and Skoog) medium containing different concentrations and combinations of BAP (Benzylamino purine), GA3 (Gibberellic acids), NAA (Naphthalene acetic acid) and IAA (Indole acetic acid) the best result was observed in the combination of 1.5mg/l BAP+ 0.2 mg/l GA3. The explants were inoculated on MS medium supplemented with 8g/l Agar and 30g/l sucrose, different combination and concentration of plant growth regulators was used for plantlet regeneration. Root induction has also done here using various concentration of IAA hormone. The highest number of root induction was found to be MS containing 2.0mg/l IAA. Induced roots were elongated properly at the same hormonal concentration after two weeks of inoculation. From the above findings, it is suggested that different hormonal concentration and combination has significant effect on the propagation of sweet orange plant. The established protocol would be helpful for direct plantlet regeneration as well as proliferation of this valuable fruit crop plants.
Efficient Callus Initiation and Plantlet Regeneration of Citrus japonica Margarita
IOSR Journal of Pharmacy and Biological Sciences, 2016
In vitro approaches has become necessary for overcoming the hurdles of cultivation of Citrus, a commercially important fruit. The present study deals with establishment of protocol for micro propagation of Citrus Japonica Margarita through callus induction and regeneration. Mature seeds of this variety of citrus are used as explant. The Explants were cultured on Murashige and Skoog medium (MS) containing 30 g/L sucrose and 7 g/L agar supplemented with different concentrations and combinations of different phyto-hormones; 6benzylaminopurine (BA), naphthalene acetic acid (NAA), 2,4-dichloro phenoxy acetic acid (2,4 D). The maximum callus induction (88%) was observed from the mature seeds of Citrus japonica observed on MS medium supplemented with 2,4 D 16 μM. Maximum shoot regeneration response (70%) was observed on MS medium supplemented with BA 13.0 μM. Maximum root regeneration response (80%) was observed on MS medium supplemented with IBA 10 μM and when it is supplemented with NAA 5 μM it is 70%. The regenerated plantlets were successfully acclimatized in pots containing sterile soil mixture to study their response in in vivo conditions.
International Journal of Sciences: Basic and Applied Research, 2016
A method for in-vitro propagation of wild type Indian orange ( Citrus chrysocarpa L.) was developed by shoot organogenesis from seed. Mature seed embryos were used as explants and treated with different hormones and plant growth regulators on MS medium for callus, shoots and roots induction. For callus induction seed embryos were treated with different concentration of 2, 4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) and a-naphthalene acetic acid (NAA) and maximum 90.90% callus was observed on MS medium supplemented with 2, 4 -D at 16.0 µM concentration. Approximately 85% of the callus was granular, while 15% was smooth and compact. Maximum 83.33% shoot regeneration were observed on MS medium supplemented with BA at 13µM concentration. Regenerated shoots were rooted on MS medium supplemented with different hormones and maximum 80% roots were observed in MS medium supplemented with 10 µM Indole-3-butyric acid (IBA). The regenerated plantlets were successfully acclim...
Plantlet Regeneration from Unfertilized Ovule of Mandarin (Citrus reticulata)
Annual Research & Review in Biology, 2018
The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide, many genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches of cultivar improvement. in this objective We aime at studying the effects of various culture media on the induction and the development of citrus somatic embryos.Callus cultures were initiated from the infertlized ovules of six varieties of mandarin (Anana, Lee, Murcott, Ortanique, Temple, and Wilking) within 3 media: MT (Murashig and Tuker, 1969) without hormones, MT + 1 mg/l BAP, MT + 1 mg/l Kinetin, the experiments show a highly significant effect (P < 0.001) of the culture media and genotype. No reactivity was observed on the MT environment in the absence of growth regulator, while the culture media MT in addition to 1 mg/l BAP gave the best results of induction of embryogenic callus induction. The induction of somatic embryogenesis was obtained on MT media without hormones. For the plantlets regeneration the favorable media was MT without hormones or added to ANA and active coal.
In vitro Propagation of Citrus Species through Callus Induction and Regeneration: A Review
International Journal of Current Microbiology and Applied Sciences
Citrus, one of the most important group of fruit crops around the world, are propagated at large scale with many difficulties. Propagation through seeds is challenging because of Phytophthora foot rot together with recalcitrance of citrus seeds. Vegetative propagation of Citrus species is mainly performed now-a-days by budding on seedling rootstocks. As heavy losses are experienced among the susceptible seedlings due to Phytophthora and Citrus tristeza virus (CTV), the interest in resistant rootstocks has greatly increased. The potential of conventional methods of citrus plant breeding of rootstocks are limited by physiological factors such as heterozygosity, inbreeding depression, nucellar polyembryony and juvenility. Under such conditions advanced tissue culture techniques provide best possible alternative for producing large number of resistant progenies from elite citrus genotypes. Plant tissue culture provides reliable and economical method of maintaining pathogen free plants that allows rapid multiplication and international exchange of germplasm. Generally, when in vitro propagation protocols are developed for any specific plant species, specialized conditions for individual genotypes, elite species and even various developmental stages of the explants plants are selected via error-andtrial experiments. Because large diversity is observed in Citrus plant family, it takes many months to develop protocols for most suitable culture medium, best concentrations and combinations of plant growth regulators and other supplements for better development of explant cultures. Therefore, in this review, we tried to put together results from difficultto-find literatures and listed all the identified findings, in which callus induction or somatic organogenesis was used to develop citrus plants. Successful protocols of surface sterilization method, culture establishment, shoot regeneration, in vitro rooting and acclimatization are presented systematically.
In vitro regeneration of sour orange (Citrus aurantium L.) via direct organogenesis
2013
Low cell competency for regeneration and transformation is the main cause of so-called recalcitrance to transform a species or a genotype. A research was conducted to determine the optimum conditions for in vitro plant regeneration involving organogenesis in Citrus aurantium, which is an important rootstock worldwide. Seeds with peeled teguments were germinated in vitro, either kept in dark for 6 weeks or maintained in the absolute dark for 4 weeks followed by 10 days in 16-h photoperiod (56 µmol m-2 s-1) at 27 ± 2°C. Epicotyl-originated explants were cultured in MS medium supplemented with 6-benzylaminopurine (BAP) (0, 1, 2 and 3 mg L-1) and Naphthaleneacetic acid (NAA) (0, 0.05, 0.1 and 0.2 mg L-1) to induce organogenesis. Effects of Pre-culture in liquid MS medium (0, 1 and 2 days) on the number of responsive explants (RE) have been also evaluated. In the next step, explants having buds were transferred to MS medium containing Gibberellic Acid (GA 3) (0, 0.5 and 1 mg L-1) and the size and number of shoots, which have been produced by RE are then measured. The highest percentage of responsive explants (90%) obtained by using 2.5 mg L-1 BAP in combination with the 0.05 mg L-1 NAA which had 2 days pre-culture period of epicotyls for allowing to grow in the absolute darkness for 4 weeks, followed by 10 days in 16-h photoperiod (56 µmol m-2 s-1). The highest number of well-developed shoots was 4.2 shoots per explant and obtained with medium containing 0.5 mg L-1 GA 3. These protocols are suitable in association with Citrus aurantium Agrobacterium-mediated genetic transformation.
2018
Studies were initiated to explore the role of plant growth regulators and explant types on efficient plantlets regeneration via direct and indirect organogenesis in Kinnow mandarin [Citrus reticulata L. (Blanco)]. Explants were cultured on MS medium containing varying concentrations of phytohormones. The best callus induction response was obtained in MS medium containing 5 mg/l 2,4-D and 1 mg/l BAP where 90% from nucellus tissue, 58% from shoot apical meristem and 56% from nodal segments explants showed callogenic response after 2 weeks of inoculation.Best shoot induction medium via indirect organogenesis was found for nucellus tissue when MS medium was supplemented with 1.5 mg/l Kin and 500 mg/l malt extract which was 46% whereas for SAM and nodal segments, best response was obtained when MS medium was supplemented with BAP and NAA (3.0 + 0.5) mg/l which was 40% and 48% from SAM and nodal segments respectively after 11 weeks of inoculation.The shoot induction as well as multiplicat...
2010
In order to evaluate the formation of adventitious buds and in vitro regeneration of sour orange plants (Citrus aurantium L.) two organogenesis-inducing experiments were conducted. In the first experiment, the induction and in vitro regeneration of adventitious buds were tested on epicotyl and internodal segments under the influence of BAP or KIN associated with NAA. The second experiment evaluated the in vitro regeneration of sour orange plants related to different explant types (epicotyl segments, internodal segments of in vitro germinated plantlets and internodal segments of greenhouse cultivated plants). Data collected on both experiments included the percentage of responsive explants (explants that formed buds), and the number of buds per explant. The addition of BAP showed the best organogenic response. In vitro germinated epicotyl segments and internodal segments are recommended as explants for sour orange in vitro organogenesis. Rooting of regenerated shoots was achieved without the need for auxin in the medium. Index terms: Citrus aurantium, organogenesis, epicotyl segment, internodal segment Regeneração de gemas de laranja-azeda e desenvolvimento in vitro de plantas em função da composição do meio de cultura e tipo de explante Resumo -Com o objetivo de avaliar a formação de gemas adventícias e regeneração in vitro de plantas de laranja-azeda (Citrus aurantium L.), foram realizados dois experimentos de indução à organogênese. No primeiro experimento, a indução e a regeneração in vitro de gemas adventícias foram investigadas a partir de segmentos internodais e segmentos de epicótilo sob o efeito de BAP ou CIN associados com ANA. O segundo experimento avaliou a regeneração in vitro de plantas de laranja-azeda em função do tipo de explante (segmentos de epicótilo, segmentos internodais de plantas germinadas in vitro e segmentos internodais de plantas 1 (Trabalho cultivadas em casa de vegetação). Os dados coletados em ambos os experimentos incluíram a porcentagem de explantes responsivos (explantes que formaram gemas) e número de gemas por explante. A adição de BAP revelou a melhor resposta organogenética. Segmentos de epicótilo e segmentos internodais são explantes recomendados para a indução de organogênese in vitro de laranja-azeda. Enraizamento das brotações foi alcançado sem a adição de auxinas ao meio de cultura.