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1.
Gaurav Krishna P. Sairam Reddy Pramod W. Ramteke Pogiri Rambabu Sayed S. Sohrab Debashis Rana Parthasarathi Bhattacharya 《Physiology and Molecular Biology of Plants》2011,17(4):375-385
In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L−1 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L−1 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds. 相似文献
2.
Seyed Ali Ravanfar Vladimir Orbovic Mahdi Moradpour Maheran Abdul Aziz Ratna Karan Simon Wallace 《Biotechnology & genetic engineering reviews》2017,33(1):1-25
Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed. 相似文献
3.
Summary St John’s wort (Hypericum perforatum) is a valuable plant used as a herbal remedy or in phytopharmaceutical drugs to treat a variety of physical ailments. Much
research has been performed to study the biochemical production of secondary metabolites of in vitro cultured plants or organs. However, all of these studies have looked at the regeneration of plants from explants in only
one genotype. In addition, no study has revealed the mechanism of plant regeneration in H. perforatum, i.e. organogenesis or somatic embryogenesis. We found that different genotypes Helos, Topas, Elixir, and Numi responded
similarly to regeneration medium. The regeneration responses (i.e. callus, root, or shool production) of identical explants
from different genotypes were similar. However, the source of explant material (leaves, hypocotyls, and roots) from the same
genotype had significant effects on the response to media and plant regeneration frequency. Using scanning electron microscopy
and light microscopy, the progress of organogenesis and embryogenesis under similar culture conditions was recorded. Root
segments were the most responsive explants, producing the maximum number of shoots per explant of all the genotypes. 相似文献
4.
A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. 相似文献
5.
Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants. 相似文献
6.
Mithila Jugulam Murch Susan J. KrishnaRaj Sankaran Saxena Praveen K. 《Plant Cell, Tissue and Organ Culture》2001,67(1):1-9
Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation of regeneration by growth regulators, choice of explant and characterization of induction and expression phases of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium opened up the possibilities for developing genotypes with novel characters, including resistance to some of the major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional crop improvement programs, thereby providing a valuable resource to the horticultural industry. 相似文献
7.
S. J. Ochatt C. Pontécaille M. Rancillac 《In vitro cellular & developmental biology. Plant》2000,36(3):188-193
Summary The production of whole plants from explants of protein pea (Pisum sativum L.) using an efficient, reliable and rapid strategy, while maintaining trueness to type, will be required before regeneration
can be exploited for genetic transformation. Seeds of the pea genotypes Terese, Solara, Frisson and P64 (a hypernodulating
mutant line of Frisson) were surface-sterilized and imbibed overnight, whereafter embryo axes were dissected and germinated
on hormone-free medium for 7–10 d. Hypocotyl sections lacking pre-existing meristems were harvested and cultured on a range
of media with various concentrations and combinations of growth regulators in order to induce either caulogenesis or somatic
embryogenesis. Differences in responsiveness were apparent between genotypes, but regeneration via caulogenesis was consistently
more reliable than via the induction of somatic embryos. Few explants underwent somatic embryo production and their conversion
into plants has remained elusive so far, irrespective of the genotype studied. Conversely, large numbers of buds were produced
within 10 d by organogenesis, and healthy, rootable shoots were obtained. A clear relationship was observed between the growth
regulators employed for bud regeneration and shoot rooting phases and the subsequent competence of the regenerated plants
for flowering, pod formation and viable seed production. 相似文献
8.
Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum
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Chouaib Meziadi Sophie Blanchet Manon M.S. Richard Marie‐Laure Pilet‐Nayel Valérie Geffroy Stéphanie Pflieger 《Plant biotechnology journal》2016,14(8):1777-1787
Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus‐induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)‐derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4‐β‐D‐glucanase, respectively. The features of the ‘one‐step’ BPMV‐derived VIGS vector include (i) the ease of rub‐inoculation, without any need for biolistic or agro‐inoculation procedures, (ii) simple cost‐effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low‐ to high‐throughput VIGS studies. 相似文献
9.
Guava (Psidium guajava L.), an important fruit crop of several tropical and sub-tropical countries, is facing several agronomic and horticultural
problems such as susceptibility to many pathogens, particularly guava wilting caused by Fusarium oxysporium psidii, low fruit growth, short shelf life of fruits, high seed content, and stress sensitivity. Conventional breeding techniques
have limited scope in improvement of guava owing to long juvenile period, self incompatibility, and heterozygous nature. Conventional
propagation methods, i.e., cutting, grafting or stool layering, for improvement of guava already exist, but the long juvenile
period has made them time consuming and cumbersome. Several biotechnological approaches such as genetic transformation may
be effective practical solutions for such problems and improvement of guava. The improvement of fruit trees through genetic
transformation requires an efficient regeneration system. During the past 2–3 decades, different approaches have been made
for in vitro propagation of guava. An overview on the in vitro regeneration of guava via organogenesis, somatic embryogenesis,
and synthetic seeds is presented. Organogenesis in several different genotypes through various explant selection from mature
tree and seedling plants has been achieved. Factors affecting somatic embryogenesis in guava have been reviewed. Production
of synthetic seeds using embryogenic propagules, i.e., somatic embryos and non-embryogenic vegetative propagules, i.e., shoot
tips and nodal segments have also been achieved. Development of synthetic seed in guava may be applicable for propagation,
short-term storage, and germplasm exchange, and distribution. An initial attempt for genetic transformation has also been
reported. The purpose of this review is to focus upon the current information on in vitro propagation and biotechnological
advances made in guava. 相似文献
10.
Cultivated groundnut (Arachis hypogaea L.) is considered as one of the primary oilseed crops and a major fodder for cattle industry in most of the developing countries, owing to its rich source of protein. It is due to its geocarpic nature of growth that the overall yield performance of groundnut is hindered by several biotic and abiotic stress factors. Multidimensional attempts were undertaken to combat these factors by developing superior groundnut varieties, modified with integral mechanism of tolerance/resistance; however this approach proved to be futile, owing to inferior pod and kernel quality. As a superior alternative, biotechnological intervention like transformation of foreign genes, either directly (biolistic) or via Agrobacterium, significantly aided in the development of advanced groundnut genotypes equipped with integral resistance against stresses and enhanced yield attributing traits. Several genes triggered by biotic and abiotic stresses, were detected and some of them were cloned and transformed as major parts of transgenic programmes. Application of modern molecular biological techniques, in designing biotic and abiotic stress tolerant/resistant groundnut varieties that exhibited mechanisms of resistance, relied on the expression of specific genes associated to particular stress. The genetically transformed stress tolerant groundnut varieties possess the potential to be employed as donor parents in traditional breeding programmes for developing varieties that are resilient to fungal, bacterial, and viral diseases, as well as to draught and salinity. The present review emphasizes on the retrospect and prospect of genetic transformation tools, implemented for the enhancement of groundnut varieties against key biotic and abiotic stress factors. 相似文献
11.
Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation
efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature
zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons
exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons,
at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a
higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic
potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant
survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival
of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement
of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and
subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from
initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments.
Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron
gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited
as a single Mendelian locus.
Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000 相似文献
12.
Cell wall biochemical alterations during Agrobacterium‐mediated expression of haemagglutinin‐based influenza virus‐like vaccine particles in tobacco
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François Le Mauff Corinne Loutelier‐Bourhis Muriel Bardor Caroline Berard Alain Doucet Marc‐André D'Aoust Louis‐Philippe Vezina Azeddine Driouich Manon M.‐J. Couture Patrice Lerouge 《Plant biotechnology journal》2017,15(3):285-296
Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing. 相似文献
13.
Summary This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya. Root-derived callus showed only rhizogenesis, whereas
leaf-derived callus showed simutaneous organogenesis and somatic embryogenesis. Organogenesis was optimal (12.2 shoots per
culture) in 1 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine and induction of somatic embryogenesis (16.3 embryos per culture) occurred in 2.5 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine. Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 2.0 μM indole-3-butyric acid. Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 60.6% germination in MS medium. Rooted shoots transferred to a mixture of sterile soil, sand, and peat
(1∶1∶1 by volume) showed 72% survival ex vitro. Application of these protocols would be helpful in reducing pressure in natural populations, in genetic transformation studies,
and in long-term storage of elite genotypes through synthetic seed production. 相似文献
14.
P. Jha C. B. Yadav V. Anjaiah V. Bhat 《In vitro cellular & developmental biology. Plant》2009,45(2):145-154
An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet
(Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences,
and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium
supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the
type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed
somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos
developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined
with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and
shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and
direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip
explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4,
8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin)
and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred
to soil in pots, where they exhibited normal growth. 相似文献
15.
Chao Yang Tuanjie Zhao Deyue Yu Junyi Gai 《In vitro cellular & developmental biology. Plant》2009,45(2):180-188
From a preliminary experiment on 98 Chinese soybean varieties, 12 varieties with somatic embryogenesis frequency ranging from
0.0% to 85.7% were selected for further study in order to enhance the efficiency of somatic embryogenesis and plant regeneration.
The effects of different mannitol concentrations, abscisic acid (ABA) concentrations, and embryo explant ages (sizes) were
investigated. Significant differences in somatic embryogenesis were found among the 12 soybean varieties, with initiation
frequencies varying from 22.1% to 89.0% under suitable mannitol concentration, and with N25281, N25263, and N06499 having
the highest somatic embryogenic capacity. The results showed that all three factors were relevant for raising rates of callus
initiation and somatic embryogenesis, but with differential responses among the genotypes. The treatment of 3.0% (w/v) mannitol, 5 mg l−1 ABA, and a 4- to 5-mm-sized explant was found to be optimal for somatic embryogenesis, generating the highest explant-based
regeneration rate at 83.0%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281.
The above results provide a basis for efficient regeneration of soybean and are informative for the development of genetic
transformation systems in Chinese soybean germplasm. 相似文献
16.
Chun-Lai Zhang Dong-Fang Chen Malcolm C. Elliott Adrian Slater 《In vitro cellular & developmental biology. Plant》2001,37(2):305-310
Summary Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar
beet germplasm. The effects of N
6-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated
in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious
shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion
of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced
by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf
explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production
of transgenic sugar beet following Agrobacterium-mediated transformation. 相似文献
17.
Applications of biotechnology in eggplant 总被引:10,自引:0,他引:10
Collonnier C. Fock I. Kashyap V. Rotino G.L. Daunay M.C. Lian Y. Mariska I.K. Rajam M.V. Servaes A. Ducreux G. Sihachakr D. 《Plant Cell, Tissue and Organ Culture》2001,65(2):91-107
Eggplant (Solanum melongena L.), an economically important vegetable crop in many countries in Asia and Africa, often has insufficient levels of resistance
to biotic and abiotic stresses. Genetic resources of eggplant have been assessed for resistance against its most serious diseases
and pests (bacterial and fungal wilts, nematodes and shoot and fruit borer). Attempts at crossing eggplant with its wild relatives
resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to respond well in tissue culture,
notably plant regeneration, has allowed the application of biotechnology, particularly the exploitation of somaclonal variation,
haploidisation, somatic hybridisation and genetic transformation for gene transfer. Somaclonal variation has been used to
obtain lines with increased resistance to salt and little leaf disease. Traits of resistance against bacterial and fungal
wilts have successfully been introduced into the cultivated eggplant through somatic hybridisation. However, most somatic
hybrids were sterile when the parental lines were distantly related. In contrast, the use of close relatives as fusion partners
or highly asymmetric fusion resulted in the production of fertile hybrids with resistance traits and a morphology close to
the cultivated eggplant, thus avoiding the series of backcrosses necessary for introgression of desired traits into eggplant.
As far as molecular markers and genetic engineering are concerned, the information available for eggplant is very scanty.
Two genetic linkage maps have been established by using RAPD and RFLP markers. In order to analyse the genetic relationships
between eggplant and its relatives, some studies based on AFLP and ctDNA analyses have also been conducted. So far only resistance
against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation. However, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently
been initiated.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
18.
Ping Che Shujun Chang Marissa K. Simon Zhifen Zhang Ahmed Shaharyar Jesse Ourada Dennis O’Neill Mijael Torres-Mendoza Yinping Guo Kathleen M. Marasigan Jean-Philippe Vielle-Calzada Peggy Ozias-Akins Marc C. Albertsen Todd J. Jones 《The Plant journal : for cell and molecular biology》2021,106(3):817-830
Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies. 相似文献
19.
Vegetatively propagated material offers many advantages over seed material in forest tree breeding research and in reforestation programmes. Evidence is accumulating to suggest that using somatic embryos in forestry is a viable option. However, before somatic embryos can be used optimally in forestry, basic research aimed at increasing the number of responsive genotypes as well as the age of the primary explant is needed. This in turn requires the establishment of a basic understanding of the physiological and molecular processes that underlie the development of somatic embryos. The functions of genes and their developmental and tissue specific regulation are studied using transient and stable transformation techniques.The process of somatic embryogenesis can be divided into different steps: (1) initiation of somatic embryos from the primary explant, (2) proliferation of somatic embryos, (3) maturation of somatic embryos and (4) plant regeneration. Cortical cells in the primary explant are stimulated to go through repeated divisions so that dense nodules are formed from which somatic embryos differentiate. The first formed somatic embryos continue to proliferate and give rise to embryogenic cell lines. Embryogenic cell lines of Picea abies can be divided into two main groups A and B, based on morphology, growth pattern and secretion of proteins. Our results suggest that extracellular proteins play a crucial role in embryogenesis of Picea abies. Somatic embryos from group A can be stimulated to go through a maturation process when treated with abscisic acid. Mature somatic embryos can develop into plants.Abbreviations ABA
abscisic acid
- BA
N6-benzyladenine
- 2,4-D
dichlorophenoxy acetic acid 相似文献
20.
Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic
potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic
embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from
the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation
temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium
with 90.5 μM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any
of the cultures which were in secondary medium with 13.6 μM 2,4-D. Development of somatic embryos directly from axillary meristems
was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to
obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically
on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two
parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo
system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant. 相似文献