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Cell and Tissue Culture Approaches in Relation to Crop Improvement
Abstract
Plant cell and tissue culture involves the growing of cells, tissues and organs on synthetic medium under closely controlled and aseptic conditions. Plant cell and tissue culture methods offer a rich scope for the creation, conservation and utilization of genetic variability for the improvement of field, horticultural and forest plant species. Micropropagation of selected plant species is one of the best and most successful examples of the commercial application of tissue culture technology. Micropropagation ensures true-to-type, rapid and large-scale multiplication. Now scores of multimillion-dollar industries around the world propagate a variety of plant species through micropropagation. Tissue culture technology offers environmental-friendly industries to flourish. It is likely that automation of multiplication systems will be commercially feasible within the next few years for several species including potato microtubers, lily bulblets and gladiolus corms. Meristem culturing and in vitro grafting help in developing disease-free plants. Improvement of somatic embryogenesis, coupled with embryo desiccation and encapsulation technology, may lead to the utilization of ‘artificial seeds’ for mass cloning of plants. Further induction of somatic embryogenesis in plants helps in cloning and transformation. Somaclonal variation is a potent emerging aspect for broadening the genetic base and thus obtaining incremental improvement in the commercial cultivars, more particularly, in the vegetatively propagated plant species. Using the technique of in vitro selection, many million cells/protoplasts can be screened against various biotic and abiotic stress factors in a single Petri dish which is more efficient as compared to the screening of similar number of plants in the field which requires more time and space as well. Production of haploids through bulbosum, anther/pollen culture and embryo rescue from wide hybrids has been exploited for the production of haploids/doubled haploids for early release of varieties. These methods ensure true-breeding (doubled haploids) plants in less than 1 year, which are otherwise obtained after seven to eight generations through conventional methods. Since the possibility of producing useful secondary products in plant cell cultures was first recognized in the 1970s, considerable progress has been made, and a number of plant species have been found to produce secondary products such as shikonin, diosgenin, caffeine, glutathione and anthraquinone. Embryo culture is the practical approach to obtain interspecific and intergeneric hybrids among otherwise difficult to cross parents. It has been successfully used to transfer desirable genes from wild relatives into cultivated varieties of several field and vegetable crops. Somatic cell hybridization helps in combining characteristics even from otherwise sexually incompatible species and to obtain cybrids and organelle recombination not possible through conventional methods. In vitro freeze storage and cryopreservation are very important techniques for germplasm conservation especially of the vegetatively propagated crops. Plants have been successfully regenerated from tissues cryopreserved at –196 °C in liquid N2 for several months to years in several crops. During the past 25 years, the combined use of recombinant DNA technology, gene transfer methods and cell and tissue culture techniques has led to the efficient transformation and production of transgenics in a wide variety of crop plants. In fact, transgenesis has emerged as an additional tool to carry out single-gene breeding or transgenic breeding of crops.
... It is an innovative and dynamic strategy for increasing genetic variety, broadening the genetic base, and, consequently, genetic enrichment to achieve significant and incremental crop improvement. [56,57]. It includes DNA-related genetic or epigenetic variations, which induce phenotypic changes distinguishable from the original parent. ...
... Generally, the older callus and cell suspension cultures produce a higher number of calliclonal variants than fresh tissues because of the augmented mutation rate [8]. The plants regenerated from callus/cell suspension cultures are transferred to field/glasshouse conditions and can be directly screened for desirable traits/variation, whereas in vitro selection at a cellular level can be carried out by their ability to grow in the presence of an inhibitor for some specific traits such as antibiotic resistance or biotic/abiotic tolerance [56,59]. However, phenotypic variation in selfed progenies of a tissue culture originated a stable line of rice named TC-reg-2008 compared to its wild type under several abiotic stresses [59]. ...
Sources of new genetic variability have been limited to existing germplasm in the past. Wheat has been studied extensively for various agronomic traits located throughout the genome. The large size of the chromosomes and the ability of its polyploid genome to tolerate the addition or loss of chromosomes facilitated rapid progress in the early study of wheat genetics using cytogenetic techniques. At the same time, its large genome size has limited the progress in genetic characterization studies focused on diploid species, with a small genome and genetic engineering procedures already developed. Today, the genetic transformation and gene editing procedures offer attractive alternatives to conventional techniques for breeding wheat because they allow one or more of the genes to be introduced or altered into an elite cultivar without affecting its genetic background. Recently, significant advances have been made in regenerating various plant tissues, providing the essential basis for regenerating transgenic plants. In addition, Agrobacterium-mediated, biolistic, and in planta particle bombardment (iPB) gene delivery procedures have been developed for wheat transformation and advanced transgenic wheat development. As a result, several useful genes are now available that have been transferred or would be helpful to be transferred to wheat in addition to the current traditional effort to improve trait values, such as resistance to abiotic and biotic factors, grain quality, and plant architecture. Furthermore, the in planta genome editing method will significantly contribute to the social implementation of genome-edited crops to innovate the breeding pipeline and leverage unique climate adaptations.
... There is some strong evidence in the model conifer species Picea abies that environmental conditions during embryogenesis can establish an epigenetic memory that modulates different developmental traits , Kvaalen and Johnsen 2008, Yakovlev et al. 2010. These epigenetic marks can be inherited as a pre-adaption to environmental conditions by subsequent generations as a form of maternal effect (Zas et al. 2013, Gosal andWani 2018). This middle-term memory, together with short-term (developmental plasticity) and longterm (local adaptation) epigenetic developmental responses, is responsible for the great phenotypic plasticity and adaptation capacity observed in plants ( Le Gac et al. 2018), which pave the way for the production of plants pre-adapted to different environmental conditions (Pascual et al. 2014). ...
Vegetative propagation through somatic embryogenesis is an effective method to produce elite varieties and can be applied as a tool to study the response of plants to different stresses. Several studies show that environmental changes during embryogenesis could determine future plant development. Moreover, we previously reported that physical and chemical conditions during somatic embryogenesis can determine the protein, hormone and metabolite profiles, as well as the micromorphological and ultrastructural organization of embryonal masses and somatic embryos. In this sense, phytohormones are key players throughout the somatic embryogenesis process as well as during numerous stress-adaptation responses. In this work we first applied different high-temperature regimes (30°C, 4 weeks; 40°C, 4 days; 50°C, 5 minutes) during induction of Pinus radiata somatic embryogenesis, together with control temperature (23°C). Then, the somatic plants regenerated from initiated embryogenic cell lines and cultivated in greenhouse conditions were subjected to drought stress and control treatments to evaluate survival, growth and several physiological traits (relative water content, water potential, photosynthesis, stomatal conductance and transpiration). Based on those preliminary results even more extreme high-temperature regimes were applied during induction (40°C, 4 hours; 50°C, 30 minutes; 60°C, 5 minutes) and the corresponding cytokinin profiles of initiated embryonal masses from different lines were analysed. Results showed that the temperature regime during induction had delayed negative effects on drought resilience of somatic plants as indicated by survival, photosynthetic activity and water use efficiency. However, high temperatures for extended periods of time enhanced subsequent plant growth in well-watered conditions. High-temperature regime treatments induced significant differences in the profile of total cytokinin bases, N6-isopentenyladenine, cis-zeatin riboside and trans-zeatin riboside. We concluded that phytohormones could be potential regulators of stress-response processes during initial steps of somatic embryogenesis and that they may have delayed implications in further developmental processes, determining the performance of the generated plants.
This book is a comprehensive review of secondary metabolite production from plant tissue culture. The editors have compiled 12 meticulously organized chapters that provide the relevant theoretical and practical frameworks in this subject using empirical research findings. The goal of the book is to explain the rationale behind in vitro production of secondary metabolites from some important medicinal plants. Biotechnological strategies like metabolic engineering and the biosynthesis, transport and modulation of important secondary metabolites are explained along with research studies on specific plants. In addition to the benefits of secondary metabolites, the book also aims to highlight the commercial value of medicinal plants for pharmaceutical and healthcare ventures.
Topics covered in this part include:
1. In vitro propagation and tissue culture for several plants including Withania somnifera (L.) Dunal, Aloe vera, Oroxylum indicum (L) Kurz, Ocimum basilicum L, Rhubarb, Tea, and many others (including plants in Northern India).
2. Genetic Improvement of Pelargonium
3. Bioactive Components in Senna alata L. Roxb
4. Plant tissue culture techniques
The book caters to a wide readership. It primarily prepares graduate students, researchers, biotechnologists, giving them a grasp of the key methodologies in the secondary metabolite production. It is a secondary reference for support executives, industry professionals, and policymakers at corporate and government levels to understand the importance of plant tissue culture and maximizing its impact in the herbal industry.
Industrial crops serve as vital components of global economic systems, fulfilling multifaceted roles in various sectors. However, they encounter challenges, such as the impacts of climate change and susceptibility to diseases. In the face of climate change and a growing global population, ensuring food security entails the development of climate-resilient crop varieties with superior nutritional quality. Traditional breeding methods, involving the selection and introduction of desirable traits, are time-consuming. However, in recent years, modern biotechnological tools have expedited the development and release of new cultivars by enhancing our understanding of genetic traits. By integrating these tools with conventional breeding, opportunities arise to cultivate industrial crops with improved quality, quantity, and tolerance to environmental stresses. Additionally, local adaptation can be achieved through methods such as plant domestication and farmer-led selection. This chapter delves into conventional breeding techniques and modern biotechnological tools specially in industrial crops, including plant tissue culture, genetic transformation, targeting induced local lesions in genome (TILLING), RNAi, and genome editing, emphasizing their role in knowledge transfer from scientists to farmers.
Sugarcane is an important crop as the main raw material producing sugar and as an energy source in the world. In Indonesia, sugarcane is one of the most crucial industrial plants due to sugar being regarded as a strategic food commodity. The slow supply of conventional sugarcane seeds often hinders the spread of the new superior varieties program, positioning tissue culture technique as an option to accelerate that program. However, somaclonal variation was reported in sugarcane seeds from callus culture. This study aims to determine the genetic variation in sugarcane seeds from cuttings and callus culture using molecular and morphological markers. The PS 864 variety was used to assess genetic variation of seeds cane from callus culture and cuttings. The results show no genetic variation in seeds cane from cuttings, while in seeds cane from callus culture there is a tendency for genetic variation of 4.6-11.6% based on the polymorphic RAPD bands. However, somaclonal variations are found in the phenotype of sugarcane seedlings from cuttings and callus culture, in leaves, stems and buds. The results of this study indicated that the more callus subcultures represent the greater tendency of genetic variation in sugarcane seeds.
Triticale (× Triticosecale Wittmack) is a hybridized grain crop developed from wheat and rye crossings. Today, triticale is a multipurpose commercial grain crop with great potential as a human food and animal feed. The sown area of the new grain crop in the world reached 4 million hectares in 2018, grain production was about 14 million tons. The current climate change, the rapid evolution of pathogens, as well as the requirements of the modern market dictate the necessity for accelerated development of varieties while reducing the cost of their development. The production of double haploids makes it possible to reduce the time required for the development of homozygous lines by an average of 5–7 years. For the mass production of haploid triticale plants in vitro, there are used two methods, namely anther/microspore culture and distant hybridization followed by selective chromosome elimination of the pollinator. The most critical factors for the success of developing haploids in anther culture are a genotype, growing conditions of donor plants, a microspore development stage, stress effects on heads or anthers, and a nutrient media. Among the unresolved problems of the method are a genotypic dependence, a high incidence of albinism and a presence of aneuploids in the androgenic plant progeny. The rye genome is more often involved in chromosomal transformations than the wheat genome. Most aneuploids are nullisomics, most often on the 2R and 5R chromosomes. Nullisomic plants for 2R and 5R chromosomes have fewer number of spikelets per head and fewer number of kernels per head. In order to develop haploids by the method of selective chromosome elimination during distant hybridization, there have been successfully used such grain crops whose pollen is insensitive to Kr-genes, as maize (Zea mays L.) and wild cereal grass ‘Imperata Cylindrical’ (Imperata cylindrical L.). The advantages of the method are less genotypic dependence, absence of albino plants, genetic stability of regenerants, and reduced costs for developing haploid plants. The length of flowering period of ‘Imperata Cylindrical’ and the absence of the need to combine the timing of flowering period of the parents ensure the economy and efficiency of using this species when developing haploid triticale plants. The purpose of the current review was to characterize the methods of mass development of haploid triticale plants, to describe their advantages and disadvantages when being used in the breeding process.
The current paper presents the estimation efficiency results of in vitro fabric culture methods used to develop the initial material for triticale breeding in the Povolzhie. A culture of immature embryos (embryo rescue) was used to obtain wheatrye amphigaploids when cross breeding durum wheat with rye. The hybrid kernels formation varied from 10.4 to 47.7%. When polluting 2223 flowers of durum wheat there were formed 768 kernels, 604 of which had embryos with different degrees of differentiation. The largest frequency of hybrid kernels formation was found in the hybrids where the mother forms were the durum wheat varieties “Amazonka” (47.7%), “Diona” (37.5%), and “Aksinit” (37.6%). Fertile amphidiploids (primary hexaploid triticale) were obtained after doubling the number of chromosomes. In vitro immature embryo cultivation was also used to obtain triticale hybrids with soft wheat in those cases when it served as the maternal parent in cross breeding. The efficiency of haploid production in the triticale anthers depended on the genotype. The frequency of embryogenic anthers formation varied from 6.7 to 17.4%, plant regeneration from the new forms was 11.4–31.4%. The 68 obtained of the 338 plants were green and 270 were albinos. In vitro microclonal propagation using segments of young heads as explants allowed preserving and propagating sterile plants for further work to restore their fertility.
Elimination of chromosomes is a phenomenon widespread in distant hybrids. It ranges from the loss of one or two chromosomes to elimination of whole chromosome complement of one of the parents. Such elimination leads to the production of haploid plants, which then are treated with colchicine to double the chromosome number and to develop DH‑lines. Homozygosity of doubled haploids serves as a basis for their wide use in plant genetics and breeding. The use of this approach reduces the time required for obtaining homozygous lines by 5 years on the average. It leads to savings in human resources, energy and acreage. The development of the “ bulbosum ” method for haploid barley production had a strong influence on the chromosome engineering in cereals and its implementation in plant breeding. However, the method developed on that basis could not be used effectively for producing haploids of wheat, triticale, etc. because of Hordeum bulbosum L. pollen sensitivity to genes inhibiting wheat crossability ( Kr genes). The crosses with Imperata cylindrica (L.) Raeusch. is an efficient alternative to the widely used wheat × maize and triticale × maize crosses due to abundant pollen supply within a longer time period, significantly higher frequency of embryos formation and haploid plants regeneration.
Thesugarcane crop stands out in the world mainly because it is a renewable energy source for producing ethanol and contributing to produce 65% of world sugar. In vitro micropropogation techniqueisemerging as a powerful tool for fast multiplication at larger scale and production of disease freeplanting material in a number of crops. Induction of directorganogenesis leading to reduced duration in vitro can lead toproduction of genetically stable plantlets. The present work describes early assessment of clonal fidelity in Sugarcane plants regenerated through direct organogenesis using RAPD markers. Somaclones of sugarcane (SCI-SC12) were obtained by above micropropagation. Twelve samples were collected at random from regenerated plantlets (SCI-SC12) being transferred for hardening and form field grown mother plant. Analysis of RAPD banding patterns generated by PCR amplification using Thirty RAPD primers were screened in 12 somaclones samples but only 15 primers were selected to compare genetic similarity among somaclones of sugar-cane with mother plant. Analysis of RAPD banding patterns gave no evidences for somaclonal variation and RAPD patterns of the plantlets were identical to the original mother plant. This indicated that direct regeneration from eye-bud did not induce any somaclonal variation in micropropagated plants during seven cycles. RAPD banding pattern confirmed the strong genetic purity of all micropropagated plants under study resembling to the parental genotype. Early assessment of genetic fidelity of micropropagated plants supports the acceptable modifications in protocol parameters and measure suit-ability of regeneration protocol for large scale applications .
Artificial seeds are most commonly described as encapsulated somatic embryos. They are product of somatic cells, so can be used for large scale clonal propagation. Apart from somatic embryos, other explants such as shoot tips, axillary buds have also been used in preparation of artificial seeds. Artificial seeds have a variety of applications in plant biotechnology such as large scale clonal propagation, germplasm conservation, breeding of plants in which propagation trough normal seeds is not possible, genetic uniformity, easy storage and transportation etc. For some plants such as ornamental plants, propagation trough somatic embryogenesis and artificial seeds is the only way out. In the present paper-the types, advantages, production methods and various applications of artificial seeds have been reviewed.
The most successful technique used to obtain haploid plant in pepper is anther culture. The chromosome content of haploid plants can be doubled spontaneously or using colchicine. In this study, we compared the rate of spontaneous doubled haploidy of different pepper types. Seven charleston, six bell, eight capia, and seven green pepper genotypes were used as plant material. Murashige and Skoog (MS) nutrient medium with 4 mg·L ⁻¹ naphthaleneacetic acid (NAA), 0.5 mg·L ⁻¹ 6-benzylaminopurine (BAP), 0.25% activated charcoal, 30 g·L ⁻¹ sucrose, and 15 mg·L ⁻¹ silver nitrate (AgNO 3 ) was used. Ploidy levels of plants obtained through anther culture were detected using both flow cytometry and simple sequence repeats (SSR) markers. The results showed that different spontaneous doubled haploidy rates were obtained from different pepper types. The highest rate was observed in bell pepper type with 53.4% (mean of six genotypes) of haploid plants undergoing spontaneous chromosome doubling. This was followed by charleston and capia types with 31.9% and 30.4% doubling, respectively. Green pepper type gave the lowest spontaneous doubled haploidy rate with 22.2% doubling. The results obtained from this study will be useful both for future work on haploidy in pepper and for breeding programs.
An experiment was conducted to steady the effect of L-Tryptophan and salicylic acid on callus induced on leaf explants of Catharanthus roseus. Callus induction was achieved by culturing true leaves of the plant on Murashige and Skoog (MS) medium supplemented with 0.5 mg /L 2,4-D and 1mg / L Kin. The best medium to maintain callus was MS medium supplemented with 0.5 mg /L 2,4-D and 1.5 mg / L Kin. Different levels added to MS medium for each L-Tryptophan 0,200,300or400 mg /L and salicylic acid 0, 0.5,1or1.5mg /L. The medium supplemented with 30 g/ L sucrose was used as a control. Results showed the medium supplemented with 200 mg\L of L-Tryptophan gave the highest quantity of Vincristine reached 48.66 μg/100 fresh weight of callus. MS medium content at the concentration 1 mg \L of salicylic acid gave the highest level of Vinblastine recording 50.98 μg/100 fresh weight of callus. While MS medium supplemented with 0.5 mg /L of salicylic acid gave the highest level of Serpentine 24.76 μg/100 fresh weight of callus. The concentrations of active compound derived from plant leaves were much less than the concentrations produced by the callus grown in vitro. The concentration of Serpentine was 0.059 while Vinblastine was 0.183 and the concentration of Vincristine was 0.064.
We report comprehensive information about genetics of diverse characters in a set of somaclones developedfrom a salt tolerant indica cultivar – Pokkali for the first time, which are novel, unique and deemed to be usefulto devise breeding strategies to exploit somaclonal variation. 2nd degree statistics, correlation and pathcoefficient analyses were performed involving 31 most productive somaclones, evaluated for 3 consecutiveyears (SC6-SC8 generation). By adjudging the results appropriate breeding strategies are envisaged to improvisePokkali rice. A wide range of variations for all major agronomic characters was observed. Phenotypiccoefficient of variation (PCV) was higher than Genetic coefficient of variation (GCV) for all characters,indicating substantial influence of the environment for major characters. Plant height, flag leaf area, leaf dryweight; harvest index had high-moderate heritability with high genetic advance, indicating predominance of additive gene actions. Plant height, panicle length, flag leaf area, biomass yield and harvest index had significantpositive associations with grain yield at genetic level convey their predominant role in governing grain yield.Leaf area, leaf dry weight, panicle length and number of productive tillers per plant had direct effect on grain yield. Direct selection is advocated for improving panicle length and restriction selection is recommended to enhance leaf area, leaf dry weight, and number of tillers per plant. Biomass yield bore maximum negative effecton grain yield. Maximum 81.08% of the variability was contributed by grain yield/plant. Amongst allsomaclones, BTS 24 was found to produce maximum of 3.7 tonnes per hectare, which is recommended forlarge-scale cultivation
Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25 °C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4 °C. The viability of synthetic seeds was higher when stored at 25 °C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.
Teak (Tectona grandis L.f) is a tropical tree of commercial value due to the high demand of its high-quality wood and rapid growth. Genetic improvement programs for this species have resulted in seeds of better quality and, at the same time, improvement in the quality of plantations. To preserve genetic diversity and guarantee the raw material for improvement programs and for future reproduction, seeds are kept under conventional seed banks conditions with temperature ranging between 4 and -20ºC. However, there are other means to conserve this valuable germplasm. Cryopreservation is the storage of plant material in liquid nitrogen (NL, -196ºC) and its major advantage is the conservation for indefinite periods of time, under high genetic stability conditions. Survival and regeneration of plants after seed freezing in liquid nitrogen (LN) were evaluated in this work, using the dessiccation and rapid freezing technique. The methodology was tested both, in seeds isolated from endocarps (seeds) asin seeds inside endocarps (seeds with endocarps), with germination rates, after thawing and 28 days in culture, of 84% and 70% respectively.
Tree breeding has been developed new horizon in producing new hybrids which play basic role in supplying wood and help to prevent forest reduction. The main aim of the current project was to investigate inter-specific and inter-generic hybridization in Salicaceae with the target of increasing genetic diversity and to achieve new hybrids suitable for producing woody biomass. This is the successful attempt to perform Inter-specific crossing between Populus nigra×P. deltoides and inter-generic crossing between P. nigra×salix alba. An embryo rescue technique as an efficient method was used on ovaries isolated from unwanted pollens. After pollination, 10 and 14-days-old embryos were transferred to MS and 1/2 MS media containing 30 gr/lit sucrose. In view of the results, inter-specific and inter-generic hybridization in the willow family was possible with these species. The high rate of germination was occurred 14 days after pollination and in the MS medium. This hybridization is accomplished through an in vitro system. These results defined a successful protocol for in vitro breeding of P. nigra×P. deltoides and P. nigra×salix alba.
Analysis of somaclonal variation by molecular markers allows precise varietal characterization. In this
study, somaclonal variation of two local cultivars of proso millet was developed through callus culture and
Inter Simple Sequence Repeat (ISSR) markers were used to detect somaclonal variation within somaclones
and between somaclones and their parent cultivar since these markers are more reliable and detect the
polymorphism at individual level. Thirteen ISSR primers were used per cultivar which has generated a total
of 158 alleles in Sakhroli while 170 alleles in Asond cultivar with an average of 12 and 13 alleles in Sakhroli
and Asond cultivar, respectively. The polymorphic information content (PIC) in Sakhroli cultivar ranged
from 0.14 to 0.88 with an average of 0.50 while in Asond cultivar, it was ranged from 0.39 to 0.89 with an
average of 0.69 The unweighted pair group method of arithmetic means (UPGMA) grouped 22 somaclones
with their control parent of Sakhroli cultivar into two main clusters which were further divided into two subclusters.
Similarly, UPGMA grouped 16 somaclones with its control parent of Asond cultivar into two main
clusters which were further divided into two sub-clusters.
Tree breeding has been developed new horizon in producing new hybrids which play basic role in supplying wood and help to prevent forest reduction. The main aim of the current project was to investigate inter-specific and inter-generic hybridization in Salicaceae with the target of increasing genetic diversity and to achieve new hybrids suitable for producing woody biomass. This is the successful attempt to perform Inter-specific crossing between Populus nigra×P. deltoides and inter-generic crossing between P. nigra×salix alba. An embryo rescue technique as an efficient method was used on ovaries isolated from unwanted pollens. After pollination, 10 and 14-days-old embryos were transferred to MS and 1/2 MS media containing 30 gr/lit sucrose. In view of the results, inter-specific and inter-generic hybridization in the willow family was possible with these species. The high rate of germination was occurred 14 days after pollination and in the MS medium. This hybridization is accomplished through an in vitro system. These results defined a successful protocol for in vitro breeding of P. nigra×P. deltoides and P. nigra×salix alba.
Sugarcane (Saccharum officinarum L.) is one of the most important field crops grown in México. In 2014, 828,609 ha were sown and accounted for 3.7 % of the total national cultivated area. However, diseases caused by fungi, bacteria, viruses and phytoplasms are one of the main challenges for cultivation. This research developed a micropropagation system of three varieties to produce entire, healthy plants for pathogenicity studies. A system of clonal propagation of ITV 92-1424, Laica 82-2220 and Q28-2 varieties was developed. Plant regeneration was achieved via organogenesis from apical meristems dissected from six-months old mother plants. Shoots were induced to multiplication in MS medium supplemented with concentrations of 2.5 to 7.5 uM BA. Shoot multiplication was achieved with the same concentrations of BA in combination with 1 μM IAA. In vitro rooting of the three varieties was efficient in the MS medium with half-salt concentration. Acclimation of plants was achieved at six weeks with 95 to 98 % survival in a mixture of peat and perlite (1:1 v/v).
An improved procedure has been developed for high frequency androgenesis in an elite long duration indica rice hybrid. The effects of cold temperature pretreatment, duration of treatment and media with different plant growth regulators on callus induction and shoot regeneration were examined for generation of doubled haploids. N6 medium supplemented with 2.0 mg l⁻¹ 2,4-D, 0.5 mg l⁻¹ BAP and 30 g l⁻¹ maltose was found to be most effective for callusing when compared with MS and SK1. The N6 media grown calli showed maximum green shoot regeneration frequency in MS medium supplemented with 0.5 mg l⁻¹ NAA, 0.5 mg l⁻¹ Kinetin, 1.5 mg l⁻¹ BAP and 30 g l⁻¹ sucrose after 2 week of culture. The cold temperature treatment of spike at 10°C for 2 days alongside by 8 days was found to be most suitable conditions for callusing and green shoot regeneration producing 186 green plants from indica rice hybrid, CRHR32. The ploidy status assessed on the basis of morpho-agronomic characters revealed fertile diploids at a frequency of about 81.10%; 16.10%, 2.68% and 1.08% were polyploids, haploids and mixploids respectively. Microsatellite marker analysis showed 1: 1 ratio of the alleles of CMS and restorer lines used for development of CRHR32. Homozygosity was detected for all the marker loci in 150 DHs and only one plant was identified as heterozygote. This investigation identified the favorable media composition and condition for callus induction and green plant regeneration which would further increase the knowledge and better understanding in rice hybrids for development of DHs. © 2016 The Japanese Society for Plant Cell and Molecular Biology.
Papaya (Carica papaya L.) is an economically important fruit crop of tropics and subtropics. It has high nutritional value, as well as medicinal and industrial applications. Papaya is a polygamous species with three sex types male, female, and hermaphrodite. Conventional methods of papaya breeding are time consuming and needs advent of anther culture which may be effective for shortening of breeding cycles. The present study on in vitro androgenesis in papaya cv. Pusa Nanha observed the highest embryo induction rate (8.0%) when anthers were cultured on agar medium with 0.1 mg/L BA and 0.1 mg/L NAA after incubation in liquid MS medium with 2.0% sucrose for 7 days at 35ºC. The high temperature (35ºC) was more suitable for embryo induction in papaya than slightly low temperature (25ºC). At these both temperatures longer incubation of anthers in water reduced embryo induction rate. Sugar starvation results were ambiguous. Shoots were also developed in the media when used in liquid form. The highest rooting (75.0%) was observed at 2.0 mg/L IBA. Increasing IBA concentration reduced rooting. All well rooted plants were hardened in hardening chamber and successfully transferred to field. The present findings indicated that anther culture can be efficiently contributed for the direct micro-propagation of papaya plants. This study would also be helpful to the researchers to develop more efficient anther culture protocols for further improvement of papaya through in vitro androgenesis.
These studies were conducted to determine the most effective methods for increasing shoot elongation during the initial proliferation stage of micropropagation in two dwarfing apple, Malus × domestica (Borkh.), rootstock cultivars. Several experiments were conducted to compare explant collection date, exposure to chilling (5 ± 1 °C) temperatures, and varying concentrations of plant growth regulators in Murashige and Skoog (MS) media. Microshoot growth of ‘Geneva 41' (‘G.41') was very low and unaffected by chilling duration from 0 to 8 weeks or by gibberellic acid (GA3) concentration from 0 to 1.0 mg·L⁻¹, but was improved by an additional subculture which increased shoot length from 1 to 15 mm. In ‘Geneva 30' (‘G.30'), shoot elongation was most affected by date, chilling explants, and by optimizing cytokinin concentration and type. Explant collection date in April increased shoot growth compared with August or November. Microshoot growth of ‘G.30' was increased by chilling nodal explants for 4 and 6 weeks when explants were collected in August and November, but not in April. Eight weeks chilling was detrimental for explants collected in April, and generally had little or no effect with August and November. The cytokinin 6-benzylaminopurine (BA) increased shoot number to a greater extent than thidiazuron (TDZ) or zeatin (ZT), and was also more effective for increasing shoot elongation with concentrations of 0 to 2.0 mg·L⁻¹. In ‘G.30', GA3 increased shoot growth at the optimum concentration of BA, but not with lower concentrations. ‘G.30' microshoots were fewer and shorter with 24-epibrassinolide (EBR) at concentrations of 0.1 and 1.0 mg·L⁻¹. Chemical names: N-phenyl-N’-(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(4-hydroxy-3-methylbut-2-enylamino)purine (ZT). © 2016 American Society for Horticultural Science. All rights reserved.
Successful haploid induction in loquat (Eriobotrya japonica (Thunb.) Lindl.) through in situ-induced parthenogenesis with gamma-ray irradiated pollen has been achieved. Female flowers of cultivar ‘Algerie’ were pollinated using pollen of cultivars ‘Changhong-3’, ‘Cox’ and ‘Saval Brasil’ irradiated with two doses of gamma rays, 150 and 300 Gy. The fruits were harvested 90, 105 and 120 days after pollination (dap). Four haploid plants were obtained from ‘Algerie’ pollinated with 300-Gy-treated pollen of ‘Saval Brasil’ from fruits harvested 105 dap. Haploidy was confirmed by flow cytometry and chromosome count. The haploids showed a very weak development compared to the diploid plants. This result suggests that irradiated pollen can be used to obtain parthenogenetic haploids.
The present study focused on the development of efficient in vitro regeneration protocols for six southern African bread wheat genotypes (Triticum aestivum L.). The tested wheat genotypes showed variation in their in vitro coefficiency abilities, with efficiencies ranging from 0 to 36.5%. Gamtoos and Tugela genotypes displayed the highest and lowest regeneration efficiencies, respectively on the tested growth media. The data indicated that immature embryos, isolated 12 days post-anthesis, resulted in embryogenic calli formation 4 to 6 days after initiation in the presence of picloram and/or 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoots emerged from 3 to 4 week old calli in the presence of 6-benzylaminopurine (BAP) or zeatin. Maltose as carbon source, MS vitamin mix and the addition of 50 μM silver nitrate also increased the in vitro regeneration abilities of the wheat explant material. Rooting of emerged in vitro plantlets was established on MS or half strength MS without the addition of any phytohormones. Furthermore, a novel cryopreservation protocol was successfully developed by encapsulation/vitrification/dehydration of immature wheat seeds. Immature seeds encapsulated in alginate beads, vitrified in 0.5 M sucrose, desiccated for 72 h, cryoprotected in 80% glycerol and flash frozen in liquid nitrogen resulted in the highest survival rate of immature embryos isolated after cryopreservation and regenerated in vitro. Desiccation of tissue prior to cryopreservation seems to be the singularly most important step to ensure successful preservation of wheat tissue.
Genetic improvement to develop varieties with high yield potential and resistance/tolerance to abiotic and biotic stresses with acceptable end use qualities is the most viable and environment friendly option to increase wheat yield in a sustainable fashion. In vitro haploid production followed by chromosome doubling greatly enhances the production of complete homozygous wheat lines in a single generation and increases the precision and efficiency of selection process in wheat breeding. It also enables the detection of linkage and gene interactions, estimate genetic variance and the number of genes for quantitative characteristics, produce genetic translocations, substitutions and chromosome addition lines, and facilitate genetic transformation and mutation studies. Wheat cultivars developed from doubled haploids using anther-culture and maize induction systems have been released for cultivation in both developed and developing countries. In this review, the origin and production of haploids, techniques in anther-culture and wheat x maize wide crosses, and their application in wheat breeding are summarized.
Anther culture technique offers great opportunities to accelerate breeding progress. The current study aimed to develop some good doubled haploid lines (DH) through anther culture technique and to evaluate them along with their five parents including two check varieties for yield and yield related traits. Analysis of variance revealed that varietal differences were signified and wide variability present among the genotypes with respect to all the characteristics studied. Correlation analysis revealed that grain yield was highly positive correlated with panicle length. The information on the inter association among the yield attributing characters showed the nature and extent of relationship with each other. Consequently, this will help in the improvement of different characters along with yield in breeding programmes.
Genetic devlopment to imrove genotypes with high quality is the most important approach of rice. Thus, anther culture technique is one of straight forward approaches for improvement of rice cultivars with good grain quality. Therefore, this investigation aimed to develop some doubled haploid lines (DH) through anther culture technique and evaluate them along with their five parents including two check varieties for some nutritional characteristics. The results indicated that the three mineral element contents (Zn, Mn and Fe) of rice grain were clearly different among genotypes (DH), which implied that genotypic variations might provide opportunities to select for higher mineral element content. Analysis of variance revealed that the differences among genotypes were significant for all studied traits. Highly significant positive correlations were recognized among the studied characteristics. Accordingly, Rice lines with the high nutritional values will use as donors for this trait in rice breeding programs for exploitation and in hybridization.
Authors: A.B. Molina, V.O. Sinohin, E.G. Fabregar, E.B. Ramillete, M.M. Loayan, C.P. Chao
Keywords: disease resistance, Fusarium oxysporum f. sp. cubense, Panama wilt, somaclonal variants
DOI: 10.17660/ActaHortic.2016.1114.31
Abstract:
Recent epidemics of Fusarium wilt, caused by the virulent strain tropical race 4 (TR4) of Fusarium oxysporum f. sp. cubense (Foc), pose a serious threat to the banana industry. Four Cavendish (AAA genome) somaclonal variants selections from Taiwan ('GCTCV-105', 'GCTCV-119', 'GCTCV-218' and 'GCTCV-219') and three important Philippine local cultivars ['Latundan' (AAB genome, Silk subgroup), 'Lakatan' (AAA genome, Lakatan subgroup) and 'Saba' (ABB genome, Saba subgroup)], were compared to the commercial Cavendish 'Grand Naine' (AAA genome, Cavendish subgroup) in a heavily Foc-infested soil in the southern Philippines. Experimental plots were planted with 10 tissue culture-derived seedlings, replicated 10 times, and arranged in a completely randomized design. Disease incidence was assessed weekly by monitoring early symptoms of yellowing of older leaves and/or other symptoms such as pseudostem splitting. Onset of disease symptoms was recorded. The infected plants were allowed to develop severe typical symptoms of Fusarium wilt (leaves showing clear yellowing, and marginal necroses appear on older leaves) and then checked for vascular necroses as confirmation. The commercially-grown cultivars 'Grand Naine' and 'Lakatan' showed susceptibility with a disease incidence of 64 and 76%, respectively, in the primary crop; and 79 and 92%, respectively in the ratoon crop. Disease incidence in the GCTCV cultivars ranged from 0-6% in the primary crop and 0-8% in the ratoon crop. 'Saba' showed 0% incidence in the primary as well as in the ratoon crop. Foc VCG 01213/16, the Foc strain associated with TR4, was confirmed in the infected plants using a Polymerase Chain Reaction (PCR)-based diagnostic tool. Results confirm the stability of resistant somaclones to Foc TR4, even in agro-ecosystems differing from those where these were first developed. They also show that selecting favorable somaclonal variants is a feasible approach in cultivar improvement, and they have a potential role in integrated Foc TR4 management. The study identifies the TR4-resistant cultivar 'Saba', showing that TR4 does not severely affect all cultivars.
http://www.actahort.org/books/1114/1114_31.htm
Somatic embryogenesis and plant regeneration are basic processes for the success of citrus somatic hybridization via protoplast fusion. In many cases, few embryos develop normally and only a small number of plants are recovered. The development of methodologies able to increase the recovery of plants after protoplast fusion experiments it is an important requirement to improve the efficiency of the procedure. Here, plants were regenerated at high efficiency using in vitro micrografting of shoots, roots, and embryos recovered after different somatic hybridizations. Hybridizations were performed using protoplasts isolated from Chios mandarin callus with protoplasts isolated from Clementine mandarin leaves and from Sanguinelli sweet orange callus. Recovered plants were analyzed with flow cytometry and nuclear simple sequence repeat (SSR), mitochondrial InDel, and chloroplast SSR markers to determine genomic structure. One tetraploid cybrid and numerous diploid cybrids were recovered, and these exhibited a range of mitochondrial and chloroplastic genome combinations.
In order to improve drought resistance of sunflower in the context of the global warming and to accelerate this improvement, introgression of resistance genes from wild species was attempted, using embryo rescue techniques, combined with classical procedures to improve crossing, self-pollination and backcrossing. Thirty four approved or pending approval lines (B and C) were tested for combination capacity with the wild Helianthus argophyllus. After crossing between H. annuus and H. argophyllus, 6 generations in two years (2008-2009) were obtained, as follows: F1 (interspecific crossing - harvest immature hybrid embryos), BC1F1; BC2F1; BC3F1; BC4F1; BC4F2. In case of the crossing between species H. argophyllus and H. annuus only four generations (F1; BC1F1; BC1F2; BC2F1) in 2 years were obtained, due to the long vegetation period of the wild species, the long flowering period, the high degree of branching and not finally higher incompatibility between the two species.
Key message:
Using DArT analysis, we demonstrated that all Solanum × michoacanum (+) S. tuberosum somatic hybrids contained all parental chromosomes. However, from 13.9 to 29.6 % of the markers from both parents were lost in the hybrids. Somatic hybrids are an interesting material for research of nucleus-cytoplasm interaction and sources of new nuclear and cytoplasmic combinations. Analyses of genomes of somatic hybrids are essential for studies on genome compatibility between species, its evolution and are important for their efficient exploitation. Diversity array technology (DArT) permits analysis of the composition of nuclear DNA of somatic hybrids. The nuclear genome compositions of 97 Solanum × michoacanum (+) S. tuberosum [mch (+) tbr] somatic hybrids from five fusion combinations and 11 autofused 4x mch were analyzed for the first time based on DArT markers. Out of 5358 DArT markers generated in a single assay, greater than 2000 markers were polymorphic between parents, of which more than 1500 have a known chromosomal location on potato genetic or physical map. DArT markers were distributed along the entire length of 12 chromosomes. We noticed elimination of markers of wild and tbr fusion components. The nuclear genome of individual somatic hybrids was diversified. Mch is a source of resistance to Phytophthora infestans. From 97 mch (+) tbr somatic hybrids, two hybrids and all 11 autofused 4x mch were resistant to P. infestans. The analysis of the structure of particular hybrids' chromosomes indicated the presence of markers from both parental genomes as well as missing markers spread along the full length of the chromosome. Markers specific to chloroplast DNA and mitochondrial DNA were used for analysis of changes within the organellar genomes of somatic hybrids. Random and non-random segregations of organellar DNA were noted.
In vitro micropropagation of banana ( Musa spp.) cv.virupakshi (Hillbanana) was studied. Suckers were collected from the germ plasm block of Jain R&D (originally established from the suckers from Palani Hills, Tamil Nadu) during summer. The sucker surface sterilized with 1% NaOCl for 30 min gave 100% survival without any contamination. Apical meristems that were isolated and cultured on MS based media supplemented with benzylaminopurine (BAP) 10.0 mg/l and IAA1.0 mg/l gave higher number of shoots (134.3 shoots/explant) within168 days (24 weeks). Kinetin 2.0 mg/l and NAA0.5 mg/l gave early rooting in just five days with 6.6 roots per plant. Observations were recorded after every four weeks up to six sub-culturing. Acclimatization was done in poly house, followed by shade house under 50% light conditions. The hardened plants when shifted to the field showed luxurious growth. The regenerated micro propagated banana plants were tested for genetic uniformity through 13 inter simple sequence repeat (ISSR) markers recommended by NCS-TCP, DBT. Profiles obtained by all the three ISSR primers namely, 834, 840 and 850, respectively exhibited similar banding patterns, which revealed the existence of genetic uniformity in micro- propagated plants. Keywords: Micropropagation, Virupakshi, hill banana, banana bunchy top virus
The cultivated apple (Malus domestica) is important fruit crops cultivated in world. For production and breeding of high quality apple, inducing and enhancing new genetic diversity and suitable traits are necessary. In Iran, different local and imported apple genotypes are cultivated and common apple root stocks (Malling 7) M7 and (Malling 9) M9 are mostly used root stocks in the country. Therefore, we studied genetic diversity of M7 an M9 tissue culture regenerated plants produced by different treatments by using twenty ISSR markers. In total 51 randomly selected plants were studied for the occurrence of somaclonal variation in apple mother plants and tissue culture regenerated plants. Four different treatments were used for tissue culture. Genetic diversity parameters, genetic distance and polymorphism percentage were studied in regenerated plants. Variations in loci frequency and combination were checked by STRUCTURE and the presence of similar loci in the plants was studied by reticulation NJ tree. Genetic relationship versus distinctness was determined by principal coordinate analysis. The results showed the occurrence of genetic variation among mother plants and tissue culture regenerated plants of each subculture due to somaclonal variation. Significant difference in the genome size among some of the regenerated plants indicates that change in genetic structure of plants during tissue culture is also accompanied with quantitative change in DNA. However, degree of genetic variation differed among apple rootstocks and also among different treatments used.
The efficiency of immature embryo-derived in vitro culture of G genome wheats is significantly influenced by various auxins and sugars which are used for induction of embryogenic response, and by regeneration media composition for promotion of plant development from subcultured embryogenic calli. The embryogenic calli of Triticum timopheevii has demonstrated the highest regeneration ability when the initial explants were cultured on the media supplemented with 4 mg l−1 of Picloram (29.0 %), 4 mg l−1 of Dicamba (28.7 %) or 3 mg l−1 of 2,4-D (29.1 %). The media supplemented with 5–6 mg l−1 of Picloram were considered to be the most effective for promotion of embryogenic/regenerable callus production in Triticum
kiharae cultures (73.7–75.0 %). Both T. timopheevii and T. kiharae embryogenic structures were characterized by the formation of green and albino plantlets. Generally the medium that was initially supplemented with Picloram promoted the formation of lower albino plants fraction rather than 2,4-D and Dicamba. As it was measured by the total green plant production per initial explant, the overall efficiency has been reduced when sucrose was substituted by glucose or maltose. The regeneration medium supplemented with 0.25 mg l−1 TDZ significantly enhanced the regeneration capacity of embryogenic callus in T. kiharae. In culture of T. timopheevii the difference between the medium lack of growth regulators and the medium supplemented with TDZ was not prominent, though both of the media have demonstrated the greater efficacy as compared to those supplemented with BA and Zeatin.
The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed.
Using cotyledons as explants a new method of peanut regeneration was established via somatic embryogenesis (SE), involving a kind of first-observed SE structure that was shaped like bulbil and therefore named bulbil-like body (BLB). On Murashige and Skoog (MS) media containing a higher concentration of 2,4-D (20.0 mg/L) and incubated in the dark, all the inoculated cotyledon explants formed BLBs with a high frequency of average 26 BLBs per cotyledon explant, and 100.00 % of the formed BLBs spontaneously developed into multiple shoots (3–5 shoots per BLB) without changing induction and incubation conditions. On average, about 103 shoots can be induced from an individual cotyledon explant. On MS media supplemented with 1.0 mg/L NAA all the separated shoots formed normal roots to develop into plantlets. The morphological and histological analyses of BLBs at different development stages revealed that BLB was a special kind of SE structure originated from cortex cells of peanut cotyledon explants, and each individual BLB was composed of multiple embryoids further developing into multiple shoots. To our knowledge, BLB is a novel type of SE structure in peanut different from the known SE structures because of its special shape, induction conditions, somatic embryogenic nature, and multiple-embryoid characteristics.
An experiment was conducted during rainy season (kharif) 2003 and 2004 to evaluate 31 somaclones of a salt-tolerant traditional 'Pokkali' rice (Oryza sativa L.) at advanced generation (SC12, SC13). The crop duration of the somaclones ranged between 120 and 140 days. Leaf growth and related parameters were analyzed at various growth stages, viz tillering, flowering and physiological maturity. Leaf area and leaf area index were found to be maximum at flowering stage. The flag leaf area too was maximum during flowering stage. The leaf area duration and leaf area index duration were high between tillering and flowering stages among the somaclones than between the flowering and maturity stages. Specific leaf area was maximum during flowering and specific leaf weight was higher at physiological maturity. Grain yield was significantly high in somaclones, viz 'BTS 11-1', 'BTS 28', 'BTS 24', 'BTS 9-2(S)', 'BTS 17(S)', 'BTS 10-2' and 'BTS 11-7 ' which exhibited higher flag leaf area and moderate leaf area index. These somaclones could be effectively used in future crop improvement programme especially for enhancing salinity tolerance or abiotic stress tolerances.
Production of interspecific and intergeneric hybrids is useful for transfer of desirable genes from wild species into cultivated species. In many instances, progeny from wide crosses is difficult to produce owing to several barriers. Post-zygotic barriers such as endosperm abortion and, at later stages, embryo degeneration are of common occurrence, leading to low fertility; but these have been overcome through the use of embryo rescue and several hybrids have been developed. This approach is especially useful in horticultural crops, more so in fruit crops. In our laboratory, we have developed protocols for hybrid embryo rescue in several top-of-the-line fruit crops that suffer from an inability to cross naturally (e.g., distant crosses, use of seedless parent/s) or instances where initial fruit drop is very high. Thus, interspecific, intergeneric and intervarietal hybrids have been generated in mango, banana, seedless grape, papaya and seedless citrus using embryo rescue. Culture of embryos has also been demonstrated in rose, capsicum, hot pepper, onion and tomato. Among the very important strategies under non-GM biotechnologies figure techniques of hybrid embryo rescue, and related applications like ovule/ovary/placental cultures through sequential embryo culture. Embryo culture applied to practical problems is a tissue culture technique that has proven to be of greatest value to breeders.
A rapid micro propagation and acclimatization response of two different varieties of sugarcane Co86032 and CoN 04131(Saccharum officinarum L.) was obtained in this study. The shoot apical meristem of different sizes wascultured on Murashige and Skoog medium supplemented with different concentrations and combinations of ben-zylaminopurine and kinetin either alone or in combination with each other alongwith GA3. Best shoot formation response in Co 86032 was obtained on MS medium containing 1.5mg/l BAP while in CoN 04131 the combination of 0.5 mg/l BAP with 0.25 mg/l Kinetin showed best shoot formation response from apical meristem. Meristem of 3.0 mm size proved to be the best size for micropropagation of sugarcane. Excellent multiplication response of In vitro formed shoots was obtained when the concentration of BAP was decreased to 1.0 mg/l in Co 86032and 0.25 mg/l BAP and Kin in CoN 04131 (i.e. 0.25 mg/lBAP + 0.25 mg/l Kinetin. MS medium containing 1.0 mg/l NAA and 2.0 mg/l IBA showed 100% rooting response of In vitro regenerated shoots of both the varieties of sugarcane within eight days of inoculation. Best hardening response was obtained in sand+ soil + pressmud (1:1:1) media.
For several years it has been recognized that introduction of plant cells into culture results in genetic changes. These genetic alterations have been recovered in the plants regenerated from cell cultures. More recently it has been recognized that this method of introducing genetic changes into crop plants could be used to develop new breeding lines. The technology of introducing genetic variation by using cell culture has been termed somaclonal and gametoclonal variation. This paper reviews the history of this technology and offers genetic documentation of somaclonal variation in tomato. As this variation represents a new tool for the plant breeder, breeding strategies for the use of this variation are presented and discussed. Somaclonal and gametoclonal variation are new tools for the geneticist and plant breeder that permit reduction in the time period for new variety development and that permit access to new classes of genetic variation.
In vitro axillary shoot proliferation can be used to increase availability of citrus (Citrus) types in high demand, while limiting somaclonal variation. However, established protocols could be improved to increase efficiency. Therefore, this study investigated some factors [plant growth regulators (PGRs), basal media, and successive subculturing] which affect the in vitro axillary shoot proliferation of mature ‘Washington Navel’ orange (Citrus sinensis) and juvenile ‘Carrizo’ citrange (C. sinensis × Poncirus trifoliata). In ‘Washington Navel’ orange, maximum axillary shoot induction (66.9% explants producing axillary shoots with a mean of 2.45 shoots per explant) was obtained in Driver and Kuniyuki walnut (DKW) medium supplemented with 0.1 mg·L−1 6-benzylaminopurine (BA), 0.05 mg·L−1 naphthalene acetic acid (NAA) along with 1 mg·L−1 6-furfurylaminopurine [kinetin (kin)], whereas in ‘Carrizo’ citrange, axillary shoot production was greatest (82.6% and 87.5% of explants producing axillary shoots with a mean of 4.3 and 4.1 shoots per explant) at 1.0 or 2.0 mg·L−1 BA in DKW medium. The initial nodal propagules (with basal tissue remaining from removed shoots) were repeatedly subcultured for six times every 4 weeks onto DKW medium with the same levels of PGRs used for initial culturing. Woody plant medium (WPM), Murashige and Skoog medium (MS), and DKW were also compared for rooting at quarter to full strength for salt components, all amended with 2.0 mg·L−1 indolebutyric acid (IBA) and 0.5 mg·L−1 NAA. MS at full strength provided the highest rooting in ‘Carrizo’ citrange (93%) and longest root length (58 mm), whereas half-strength MS provided the highest rooting in ‘Washington Navel’ orange (60% to 61%) and the longest roots (26 mm). Addition of 1 μM spermidine to the rooting medium enhanced root length only for ‘Washington Navel’ orange on full-strength MS, but accelerated rooting for both cultivars on all media. The plantlets were successfully transferred to greenhouse conditions, exhibiting normal development, with high uniformity, and no evidence of somaclonal variation. © 2016, American Society for Horticultural Science. All rights reserved.
Three tetraploid somatic hybrid lines produced by protoplast fusion between a dihaploid potato, Solanum tuberosum, cultivar BF15 and the wild potato species Solanum berthaultii were evaluated here for their response to different soil-borne pathogens, that is Fusarium solani, Pythium aphanidermatum and Rhizoctonia solani as well as to infection by potato virus Y (PVY). Both hybrid and BF15 plants grown in vitro were inoculated with the tested pathogen strains, that is R. solani, P. aphanidermatum, or F. solani. The growth level and disease severity index of these plants were compared to the susceptible commercial cultivar Spunta. A better growth of inoculated hybrid plants and restricted disease symptoms were observed in comparison with the commercial plants. Under glasshouse conditions and after inoculation with R. solani and P. aphanidermatum, improved resistance of the hybrid plants to these pathogens was confirmed. Indeed, these plants showed no significant damage following inoculation and a better development in R. solani-infected plants. The susceptibility of the hybrid tubers to R. solani, P. aphanidermatum, and to F. solani infection was also determined. A significant reduction of tissue colonisation was observed in all the hybrid lines compared to the cultivated cultivars. The STBc and STBd hybrids also showed improved resistance to the PVY ordinary strain (PVYo) under glasshouse conditions.
Anther culture technique offers great opportunities to accelerate breeding progress. The current study aimed to develop some good doubled haploid lines (DH) through anther culture technique and to evaluate them along with their five parents including two check varieties for yield and yield related traits. Analysis of variance revealed that varietal differences were signified and wide variability present among the genotypes with respect to all the characteristics studied. Correlation analysis revealed that grain yield was highly positive correlated with panicle length. The information on the inter association among the yield attributing characters showed the nature and extent of relationship with each other. Consequently, this will help in the improvement of different characters along with yield in breeding programmes.
Citrus is one of the few commodities where somatic hybridization is reaching its predicted potential (Grosser and Gmitter, 1990). Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 125 at the CREC (Grosser et al., 2000). Applications of somatic hybridization towards the development of improved seedless scions for the fresh market, and improved rootstocks with potential for tree size control will be discussed. Extensive field research on citrus somatic hybrids combined with emerging molecular analyses of citrus (Nicolosi et al., 2000) has allowed for the development of additional strategies for cultivar improvement. These include targeted cybridization to achieve seedlessness, resynthesizing a better sour orange rootstock at the tetraploid level, and the breeding and selection of new rootstocks at the tetraploid level using somatic hybrids as parents. Ongoing examples of each strategy will be provided.
Interspecific hybridization in Arachis is restricted by early embryo abortion for many cross-combinations. Rescue of young embryos in vitro within a week after fertilization is necessary to recover these embryos before they abort. Peg tips, with the ovule and embryo tissues, of A. hypogaea L. cv. 'NC 6', were cultured to compare ovule growth, callus production and peg elongation. Tissues were collected 1, 2, 3 and 4 d after self-pollination, after which peg meristems were removed from half the pegs and cultured on five media combinations. One-day-old pegs had significantly (P = 0.01) more ovule growth than older tissues. Presence of the meristem had a greater inhibition to ovule growth for 2- to 4-d pegs than for 1-d-old pegs. Significantly more callus was produced on 4-d pegs than younger tissues, and kinetin had the greatest stimulatory effect on callus. Elongation of pegs with the meristem attached was observed most often in media with high sucrose levels. The observations indicate that very young ovules can be grown in vitro, and techniques may be applicable to rescue of young embryonic tissues of Arachis.
Isolated microspores of rice (Oryza saliva L.) cultivars, IR36 and IR43, belonging to the recalcitrant indica subspecies were cultured. Two types of microspores were observed after isolation from the fresh anthers and from pre-cultured anthers-one type consisted of vacuolated, larger-sized grains, while the other was composed of microspores of smaller sizes with dense cytoplasm. Within few days in culture, all the smaller sized grains were dead, and only the large grains were viable and produced pollen embryos. After 30 days from culture, microcalli were transferred to semisolid modified Murashige and Skoog medium containing 1 mg/liter each of kinetin and naphthaleneacetic acid and kept under continuous light at 25°C. IR36 showed only cell division while IR43 gave 32 green plants from these experiments.
After inoculation of glasshouse-grown somaclones regenerated from apple leaf discs (cv. Greensleeves) 33% of 270 showed an increase in resistance to the fire blight pathogen Erwinia amylovora. In contrast only 21% produced less severe symptoms than parental material after inoculation of plants held in culture in vitro. Sixteen somaclones which showed the highest levels of fire blight resistance were tested intensively as both glasshouse-grown plants and as micropropagated plants in culture. In tests conducted with glasshouse-grown plants up to 60% of plants of the most promising somaclones exhibited minimum symptoms after inoculation compared with 4% of ‘Greensleeves’ parental plants. The comparable figures for inoculations of in vitro cultured plants were 70% and 8%. The growth of E. amylovora was reduced in resistant somaclones compared with parental plants. Pretreating leaf discs with virulent E. amylovora cells prior to somaclone regeneration had no effect on the frequency of regenerated somaclones exhibiting increased resistance to the pathogen.
The citrus group of plants belongs to family Rutaceae and is comprised of six genera including Citrus, Clymenia, Eremocitrus, Fortunella, Microcitrus and Poncirus. Among these, the genus Citrus is of much economic importance for bearing edible fruits and serving as rootstocks. Swingle and Reece (1967) recognized mandarin (Citrus reticulata Blanco), pummelo (C. grandis Osbeck), lime (C. aurantifolia Christm.), citron (C. medica L.), lemon (C. limon L. Burn. f.) and grapefruit (C. paradisi Osbeck) as important Citrus species. The true citrus fruits are native to Asiatic area extending from the Himalayan foothills of India to North Central China and Philippines in the East, Burma, Thailand, New Guinea, Northern Australia and New Caledonia in the South-East. Now Citrus is cultivated in the tropical and sub-tropical regions of the world extending from equator to latitudes 40° North and South. World production of citrus fruits continues on an upward trend. Total world Citrus production in 1991 amounted to 8551 thousand metric tons (FAO, 1992). Highest production has been in Asia (4036 thousand metric tons) followed by Europe (2052 TMT), South America (1087 TMT), Africa (750 TMT) and N. America (575 TMT). Citrus fruits are consumed in different forms such as fresh fruits, juice squashes and jams.
Seventeen winter and spring wheat F 1 hybrids were crossed with five maize composite genotypes in line x tester fashion for developing winter x spring wheat derived doubled haploid lines. Developed ovaries (seeds without endosperm) were formed in all the crosses, highlighting the potentiality of wheat x maize system as a simple and genotype non-specific approach of haploid production as compared to androgenesis and the bulbosum technique which are highly genotype specific thus limiting their extensive applicability. Although, haploid embryos were obtained from crosses of all 85 wheat x maize combinations, yet, low regeneration rate of haploid embryos was achieved. The maize genotype which interacted to yield high haploid embryo formation gave very low regeneration frequency and the reverse was also observed indicating the independent genetic control of the two parameters. Line x tester analysis for percent developed ovaries and embryo formation revealed significant influence of wheat genotypes for the former parameter and that of maize genotypes for the latter. Wheat x maize interaction was found to be significant for both the traits under study. High estimates of GCA effects were obtained for percent developed ovaries in three wheat genotypes viz., Aurora x HPW 89, Sentry x HPW 147 and Virtue x HPW 143, whereas, amongst the testers "Early Composite" was the best combiner with respect to percent embryo formation. Both additive and dominance gene action with the preponderance of the latter were found to be influencing the inheritance of the two traits. The possible role of wheat x maize interaction is suggested as the key component, determining viability of the haploid embryo and its regeneration.