Guide Plant Improvement and Somatic Cell Genetics

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In general, guava propagates through seeds but seedlings are variable in both plant and fruit characteristics due to heterozygous nature of crop Saw et al. Conventional guava propagation employed through cutting, grafting, stooling or air layering met with limited success in development of disease resistant cultivars due to the narrow genetic variability present in the guava germplasm Chandra et al. Therefore, the best alternative on the road to guava genetic improvement is to resort somatic embryogenesis that help in mass propagation of elite and new genotypes in shorter periods.

Somatic embryogenesis SE is a perfect confrontational system in which every somatic cells under special circumstances build up into embryos and passing through various developmental stages ultimately give rise to a complete plantlet formation Arnold et al. SE is one form of asexual reproduction by which somatic cells, under suitable induction conditions, undergo a complete genome shift and embark into a new developmental pathway ending in the formation of asexual embryos morphologically identical to their zygotic counterparts Zimmerman, ; Schmidt et al. During this unique developmental process, cells have to dedifferentiate, activate cell division, and reprogram their physiology, metabolism, and gene expression patterns Yang and Zhang, Thus, SE is an ideal demonstration of plant totipotency, which represents how a somatic cells contain the essential genetic draft for complete plant development without fertilization Zimmerman, In SE, direct root and shoot induction occurs, thus the plantlet could be multiplied and acclimatize fast.

The formation of an embryogenic cell is related to nuclear DNA methylation in the presence of 2, 4-D Leljak- Levanic et al. Thus, the dynamic changes occurs in chromatin structure induced by DNA methylation under the influence of 2, 4-D lead to genomic reprogramming in somatic cells. This modification might affect hormone responsive genes required for acquisition of embryogenic competence. The somatic cells within a plant contain the genetic information necessary to form a complete and functional plant Feher et al. Even though they are not exactly the same as zygotic embryos Dodeman et al.

There are several reports on regeneration of normal plants from somatic embryos in different species. In SE chances of the occurrence of chimera is less thus a more efficient way of regeneration of the transformed plants. Besides, plants in which somatic embryogenesis was obtained, secondary or recurrent embryogenesis was often present, a phenomenon in which new embryos emerges from somatic embryos Raemarkers et al. Secondary somatic embryogenesis has many uses in biotechnology, such as the mass propagation of new plants, cryopreservation, genetic transformation and the induction of mutation Litz and Gray, ; Raemarkers et al.

Somatic embryogenesis in guava for biotechnological improvement programme has been reported by a various researchers Akhtar et al. Numerous problems encountered during the optimization of a high-efficiency protocol for induction of somatic embryogenesis from zygotic embryo explants of various cultivars of guava. Immature zygotic embryos utilized as the primary explants Jaiswal and Litz, for the induction of somatic embryogenesis.

The induction of somatic embryogenesis starting from mature and immature zygotic embryos has shown good results and efficiency in several plants of family Myrtaceae Cruz et al. The advancement of efficient protocols for plant regeneration is one of the significant essentials for starting genetic transformation system in guava. In this research paper, we report an efficient recurrent regeneration via somatic embryogenesis in guava that can potentially pave the way in development of genetically modified guava with improved traits like seedlessness, increased shelf life and Fusarium wilt disease.

To ensure self-fertilization, un-opened flower buds were bagged and the 70 days post-anthesis immature fruits were collected. Immature fruits were brought to the laboratory and surface sterilized with water and then soaked in HgCl 2 0. The sterilized fruit was bisected followed by the removal of immature seeds for zygotic embryos excision under aseptic condition.

Immature zygotic embryos were chose as starter explants for inducing somatic embryogenesis. Dissection of zygotic embryos was isolated as described by Akhtar The pH of the medium adjusts to 5. Globular embryos appeared after weeks of inoculation, which formed bipolar-shape Fig 1. Somatic embryos subculture at every 4 weeks up to 12 weeks incubation in dark. Subculturing on induction medium devoid of hormone resulted in repetitive embryogenesis.

Frequency of Embryogenesis FE : The proportion of explants that showed at least one somatic embryo at any stage of development, out of the total number of explants inoculated in a particular treatment. FE as a percentage were calculate as follows:. Intensity of Embryogenesis IE : The mean number of somatic embryos produced per responsive explants in a particular treatment. Samples examine after 21 days of culture for the histo-chemical analysis of somatic embryos.

Ethanol dilution varies according to the soft tissue. In greenhouse, transfer plants into pots filled with sand: soil: FYM and remove the polybags and let plants acclimatize completely too external environment. Now, transfer plants into the field. All experiments was repeat at least three-times. All cultures were observed daily and changes in the zygotic embryo explants were noted after each week. Embryogenic responses were usually record days after culture initiation.

The efficacy of the various treatments calculated using two different embryogenic parameters as described by Akhtar Data were analyzed for two-way analysis of variance SPSS version In the present study, an efficient and highly reproducible system for Psidium guajava L. Somatic embryogenesis induction: Somatic embryogenesis induction achieved using the immature zygotic embryo 70 DAP as explant on MS medium, containing 2, 4-D 2.

Direct embryogenesis observed after 4 weeks post culture induction and later pre-embryogenic masses of cells PEMC developed directly from the immature zygotic embryo explants in all treatment except in the control after 4 weeks Fig 1. The addition of 2, 4-D 2 mgL -1 with L-glutamine mgL -1 significantly increased formation of somatic embryos. Removal of auxin or decreasing concentration of auxin led to formation of repetitive embryogenesis Chandra et al. After 6 weeks of incubation in induction, medium, granular and shiny masses of cellular embryos observed Fig 1.

This process continues in subsequent sub culturing leading to the successive formation of somatic embryos that emerged from the peripheral areas of the callus mass after 8-weeks of culture incubation. Various developmental stages observed of somatic embryos first appeared as clusters of globular embryos, further into heart-shaped or cordiform shape then torpedo-shaped and finally into cotyledonary stage to form a complete plantlet. Maturation, Proliferation and Regeneration of Somatic embryos: Maturation is a key phase between embryo development and germination.

This is the most critical stage for establishment of any successful regeneration protocol depends on conversion efficiency. Percentage of conversion record after 3 weeks Fig 4. Represents maximum embryogenesis induction in terms of frequency and intensity with reference to the four genotypes. Embryogenic potential of four guava genotypes viz. Allahabad Safeda, Lalit, Sardar L and Shweta was estimated on the maximum embryogenesis induction, maturation, plantlet regeneration was recorded in Allahabad Safeda which accounts for Least plantlet regeneration was recorded in Sardar L and Shweta which accounts for only Comparative overall embryogenic potential we reported Allahabad Safeda as the most promising genotype of guava in terms of induction, maturation, proliferation via somatic embryogenesis through tissue culture as thus, could potentially use further in establishment of genetic transformation protocol.

Four different carrier substrates blend with bioagent Trichoderma harzianum was used viz. Later the genetic fidelity analysis of all plants was confirm using Molecular markers. Advancement of efficient protocol for plant regeneration is crucial for establishment of genetic improvement. An orderly series of characteristic embryogenesis system have developed in guava under present investigation.

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Immature zygotic embryos have proved to be an efficient regenerative tissue for the many recalcitrant tropical fruit species. Zygotic embryos are made up of PEDC's pre-embryogenic determined cells in which, cells have the embryogenic competence and could easily induced to follow the embryogenic pathways Sharp et al.

Our results revealed that immature zygotic embryo 70 days post-anthesis undergo rapid induction of somatic embryogenesis in terms of highest frequency and intensity over immature mesocarp. Our results are also in accordance with the results of Vilchez et al. Immature zygotic embryos have proved to be a potent regenerative tissue for the many recalcitrant tropical fruit species. Among other Myrtaceae plants in which somatic embryogenesis has been induced Feijoa sellowiana pineapple guava and Myrtle communis where somatic embryos differentiate mostly from mature zygotic embryo Canhoto and Cruz, ; Canhoto et al.

Our results revealed that 2 mgL -1 2, 4 - D was the best concentration inducing somatic embryogenesis in Psidium guajava L. Conversely, Akhtar, and Rai et al. The lower doses of 2, 4-D 1. In case of guava Psidium guajava L. Similarly, Moura and Motoike, reported that 2, 4-D alone cannot induce somatic embryogenesis in guava cv. The addition of 2, 4-D 2. Furthermore, combination of 2, 4-D and glutamine induced high production of SEs Chandra et al. Fortification of an organic nitrogen form has a positive influence on somatic embryogenesis Khlifi and Tremblay, ; Carman et al.

L-Glutamine had shown improved somatic embryogenesis in both monocotyledons and dicotyledons Dhir et al. The addition of L-glutamine mgL -1 in media influenced efficient somatic embryogenesis formation in P. Similarly, several reports confirmed that formation of direct SEs was enhanced by glutamine Baskaran and Jayabalan ; Deo et al.

Somatic Cell Genetics and Its Application in Potato Breeding - Dimensions

After weeks in induction medium small clusters of pre-embryogenic mass cells PEMC of cytoplasm rich cells were visible Fig. Since direct somatic embryogenesis had often seen in hypocotyls region in several species including Myrtaceae, it is likely that PEMC originate from hypocotyls region.

In vitro growth and development of tissues requires a carbon source because of heterotrophic mode of nutrition. Sucrose is the most conjoint carbohydrate source used in the plant tissue culture and present dominantly in the phloem Zimmerman and Zeigler, Research claimed that carbohydrate concentration affects greatly in the formation of somatic embryos Lou and Kako, ; Luo et al. Similarly, enhanced induction of somatic embryos achieve by adding high concentration of sucrose to the culture medium in cucumber Luo et al.

High carbohydrate content in culture medium found increased somatic embryogenesis in many plant species Luo et al. However, it is a common fact that the role of high sucrose concentration in somatic embryogenesis may influence the cell osmolarity May and Trigiano, Therefore, the role of sucrose in the present study interpreted as both nutritional and osmotic regulatory functions of the carbohydrate which creates a stress that can lead to embryogenesis induction.

Therefore, it is recommended that, osmotic effect of sucrose may cause normal development of somatic embryos. Genotype is the most important factor influencing embryogenic response and SE is highly genotype dependent process. Some genotypes are highly regenerative where as others are recalcitrant especially in case woody tree species. Variability in the induction and frequency of the obtained embryos observed among different species of genera and within the cultivars.

Considerable variations in embryogenic capacity observed between individuals of one cultivar or species. Genotype-dependent embryogenic capability was widely reported. In some genotypes, genes involved in plant regeneration may be suppressed due to inappropriate culture medium and culture condition. According to the present findings, we claimed Allahabad Safeda as found best genotype for establishing successful somatic embryogenesis.

Individual genotypes may also have unique requirements for optimal regeneration capacity. Such genotypic differences in embryogenic capacity might reflect current differences in the ability to activate key elements in the embryogenic pathway Merkle et al. The use of PEG as an osmoticum in maturation media for somatic embryos has proven effective in increasing germination and conversion Capuana and Debergh, Polyethylene glycol reported to improve the quality of somatic embryos by promoting normal differentiation of the embryonic shoot and root Stasolla et al.

PEG molecules are too large to move through the cell wall and do not cause plasmolysis. Non-plasmolyzing osmotic are more effective in promoting somatic embryo maturation Linossier et al. Similarly, Rai et al. Carrier substrate plays a major role in hardening and acclimatization of tissue culture raised plantlets.

This is the most critical stage to look after the tissue-cultured plantlets before planting into field as the maximum plants dies during hardening stage. This indicated that coco peat was effective carrier substrate for acclimatization. June This article needs additional citations for verification.

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Somatic hybrid plants of Nicotiana glauca and Nicotiana tabacum obtained by protoplast fusion

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