Plant Regeneration Studies Research Articles

In vitro Mutations for Biotic and Abiotic Stresses: A Review

Plants are subjected to a wide range of environmental stresses which reduces and limits the productivity of agricultural crops. Two types of environmental stresses are encountered to plants which can be categorized as (1) Abiotic stress and (2) Biotic stress. The abiotic stress causes the loss of major crop plants worldwide and includes radiation, salinity, floods, drought, extremes in temperature, heavy metals, etc. On the other hand, attacks by various pathogens such as fungi, bacteria, oomycetes, nematodes and herbivores are included in biotic stresses. 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 including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies 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

Phenotypically abnormal cotyledonary Vitis vinifera embryos differ in anatomy, endogenous hormone levels and transcriptome profiles.

In many perennial fruit species, including grapevine (Vitis vinifera L.), the highly complex process of somatic embryogenesis (SE) can result in the formation of a deformed embryo, although the underlying reasons are still poorly understood. Here, V. vinifera cv. 'Chardonnay' cotyledonary embryos with distinct morphologies were used to address this issue. Normal cotyledonary embryos (NCEs) and elongated cotyledonary embryos (ECEs) were observed to have better-developed vasculature and shoot meristems than the vitrified cotyledonary embryos (VCEs) and fused cotyledonary embryos (FCEs), but ECEs were less developed. We determined that the morphological differences in these phenotypically abnormal embryos were likely associated with endogenous hormone levels, since concentrations of the phytohormones indoleacetic acid (IAA) and abscisic acid (ABA) in NCEs were higher than in the other three types. Comparative transcriptome analysis revealed large differences in gene expression of the hormone signaling pathways in normal and abnormal cotyledonary embryos. Weighted gene co-expression network analysis of the different cotyledonary types allowed the identification of co-regulated gene modules associated with SE, suggesting a role for ERF family genes and other transcription factors (TFs) in regulating morphology. Moreover, an analysis of morphology-specific gene expression indicated that the activation of a specific protein kinase, small heat shock proteins (sHSPs) and certain TFs was closely associated with the formation of normal cotyledonary embryos. Our comparative analyses provide insights into the gene networks regulating somatic cotyledon development and open new avenues for research into plant regeneration and functional genomic studies of malformed embryos.

New Insights Into Tissue Culture Plant-Regeneration Mechanisms.

Plant regeneration occurs when plants repair or replace damaged structures based on the totipotency and pluripotency of their cells. Tissue culture is one of the most widely used regenerative technologies. Recently, a series of breakthroughs were made in the study of plant regeneration. This review summarizes two regenerative pathways in tissue culture: somatic embryogenesis and de novo organogenesis. Furthermore, we review the environmental factors influencing plant regeneration from explant sources, basal culture medium, plant growth regulators, and light/dark treatment. Additionally, we analyse the molecular mechanisms underlying two pathways. This knowledge will promote an understanding of the fundamental principles of plant regeneration from precursor cells and lay a solid foundation for applying plant micropropagation and genetic modification.

Study of hairy root formation and plant regeneration in Nicotiana tabaccum

Study of hairy root formation and plant regeneration in Nicotiana tabaccum

In vitro plant regeneration studies of switchgrass cultivars by somatic embryogenesis

Sucrose phosphorylase (SPase) is capable of specifically catalysing transglucosylation reactions and can be employed in the enzymatic synthesis of α-D-glycosides. In the present study, a putative Thermobacillus SPase gene (TSPase) was synthesised with optimised codons and overexpressed in Escherichia coli. The 1467 bp gene encodes a 488-amino acid protein with a calculated molecular mass of 55.8 kDa. The specific activity of the recombinant TSPase (rTSPase) was 6.42 U/mg for sucrose, and the optimum temperature and pH were 65 °C and pH 7.0. The T1/2 value of the rTSPase was 212 h at 50 °C and 98 h at 60 °C. A stimulating effect on the activity of the rTSPase was observed in the presence of 5 mM Co2+. The rTSPase showed increased stability against DMSO as organic co-solvent at 50 °C. The Km and kcat of the rTSPase with sucrose were determined as 6.24 mM and 5.73 s−1 respectively. The rTSPase produced 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2 G) from ascorbic acid in both crude extract and whole-cell forms. A maximum yield of 19.7% (39.94 ± 0.17 g/L) was achieved after incubation of ascorbic acid sodium salt and sucrose (1:2) with 19.76 U/mL of the rTSPase at pH 7.0 and 50 °C for 24 h.

Study of Plant Regeneration by Tissue Culture from Immature Embryos of the Lacquer Tree, <i>Toxicodendron vernicifluum</i>

Study of Plant Regeneration by Tissue Culture from Immature Embryos of the Lacquer Tree, <i>Toxicodendron vernicifluum</i>

Mesophyll protoplast isolation technique and flow cytometry analysis of ancient Platycladus orientalis (Cupressaceae)

Platycladus orientalis (Cupressaceae) is a native tree species used widely for landscaping and afforestation in arid and semiarid areas in Asia. Ancient P. orientalis trees not only have an important humanistic and historical value but also have a significant scientific value in aging mechanism research. Mesophyll protoplasts are an important material for studies of plant regeneration, transient gene expression, and senescence. Although an easy and effective technique of mesophyll protoplast isolation from mature trees is urgently needed, the isolation parameters have great material specificity. The young tissue of plants is an ideal material for protoplast preparation. In this study, we employed an orthogonal experiment and several single-variable experiments to determine the main factors influencing the successful isolation of mesophyll protoplasts and established an efficient technique for isolating mesophyll protoplasts from the young scale leaves of ancient P. orientalis. The optimal parameters for mesophyll protoplast isolation are as follows: mechanical homogenization of the leaf tissue, 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, 1.0% ligninase, 0.7 M mannitol (pH 5.8), and 16 h of incubation. Two centrifugations (100 × g for 3 min and 500 × g for 5 min) were repeated 2 times to obtain purified protoplast suspension. The yield and viability of protoplasts under optimal conditions were 4.8 × 106 g FW-1 (per gram fresh weight) and 82.5%, respectively. The results of flow cytometry analysis showed that the isolated protoplasts had ideal viability to meet the demands of further protoplast-based research.

Multiple shoot bud induction and plant regeneration studies of Pongamia pinnata.

Pongamia pinnata is a legume plant which has great potential to be used as a biofuel feedstock. Conventional propagation of P. pinnata was found to be inefficient for mass propagation. Employing plant tissue culture techniques for micropropagation and further plant improvement of P. pinnata will be the right path to fulfill future challenges in biofuel production. This study aimed to establish a plant regeneration system for potential micropropagation and genetic manipulation of P. pinnata in future. In vitro nodal explants were used and Woody Plant Medium (WPM) containing 30 µM 6-benzylaminopurine (BAP) and 1 mM phloroglucinol (PG) was able to induce higher frequency of multiple shoot buds compared to other media investigated in this study. For shoot regeneration study, WPM containing 15 µM of zeatin and 1 mM PG was able to induce longer shoots while rooting of the regenerated shoots was enhanced by WPM supplemented with indole-3-butyric acid (IBA) in combination with silver thiosulphate (STS). A simple and effective acclimatisation protocol was established with very high survival frequency of regenerated plantlets. Root nodulation of the successfully acclimatised plants was also observed. In short, multiple shoot buds were successfully induced, regenerated and rooted in vitro. The rooted plantlets were successfully acclimatised and grown healthily. It was concluded that a successful plant regeneration protocol of P. pinnata was achieved for potential application in micropropagation and genetic manipulation.

A Conceptual Framework for Cell Identity Transitions in Plants.

Multicellular organisms develop from a single cell that proliferates to form different cell types with specialized functions. Sixty years ago, Waddington suggested the 'epigenetic landscape' as a useful metaphor for the process. According to this view, cells move through a rugged identity space along genetically encoded trajectories, until arriving at one of the possible final fates. In plants in particular, these trajectories have strong spatial correlates, as cell identity is intimately linked to its relative position within the plant. During regeneration, however, positional signals are severely disrupted and differentiated cells are able to undergo rapid non-canonical identity changes. Moreover, while pluripotent properties have long been ascribed to plant cells, the introduction of induced pluripotent stem cells in animal studies suggests such plasticity may not be unique to plants. As a result, current concepts of differentiation as a gradual and hierarchical process are being reformulated across biological fields. Traditional studies of plant regeneration have placed strong emphasis on the emergence of patterns and tissue organization, and information regarding the events occurring at the level of individual cells is only now beginning to emerge. Here, I review the historical and current concepts of cell identity and identity transitions, and discuss how new views and tools may instruct the future understanding of differentiation and plant regeneration.

High frequency regeneration of plants from cotyledon and hypocotyl cultures in Brassica oleracea cv. Pride of India

Morphogenic potential of cabbage cv. Pride of India, for multiple shoot induction was tested under in vitro conditions using cotyledon and hypocotyl explants. Aseptically grown seven to nine days old seedlings of cabbage were used as source of explants for reproducible plant regeneration studies. Forty different concentrations and combinations of TDZ (alone), TDZ with adenine, TDZ with NAA and TDZ with IAA were tried. Maximum shoot regeneration response from cotyledon explants (91.11%) and hypocotyl (94.40%) was obtained on MS medium containing 0.330mg/l TDZ+79.70mg/l Adenine and 0.220mg/l TDZ+0.088mg/l IAA, respectively. Rooting was achieved within two to three weeks on all the rooting media, but MS medium containing 0.10mg/l NAA produced the maximum number of strong and healthy roots (100%). The regenerated complete plantlets with healthy roots and shoot system were transferred to pots containing sterilized cocopeat and successfully acclimatized and no phenotypic variations were observed among regenerated plants. Highly efficient, reproducible plant regeneration protocol has been standardized in cabbage cv. Pride of India, which would be valuable for Agrobacterium-mediated gene transfer studies in cabbage.

Modeling and predicting solar radiation transmittance in mixed forests at a within-stand scale from tree species basal area

Light under tree canopy cover is essential for the study and understanding of plant diversity, regeneration, plant growth and many other forest ecosystem processes; however, quantifying light is difficult and requires specialized equipment. That is why proxies or models predicting light availability can help scientists to obtain estimates of transmittance and forest managers to better assess and adjust silvicultural practices at a reasonable cost. The main objective of our research was to develop a model to predict local solar radiation transmittance from species basal area in mono-specific and mixed stands of sessile oak (Quercus petraea) and Scots pine (Pinus sylvestris). Models based on the Beer-Lambert law were fitted and compared using 163 measures of solar radiation transmittance obtained from hemispherical photographs and light sensors. In mono-specific stands, local transmittance was predicted by local basal area considered on a radius equaling approximately tree height. In mixed stands, the same model broken down into its mono-specific components (local basal area of each species multiplied by their own extinction coefficients) predicted transmittance well. The extinction coefficients we obtained were very close to those previously established for these species and did not differ between mono-specific and mixed stands. Our model explained 77% of the variation in transmittance when random effects were included and 64% of the variance without taking into account these random effects. The predictive value of the model was good with high accuracy (mean signed deviation not significantly different from zero) and a fairly high precision (relative mean absolute error=20%). The fact that tree canopy transmittance in mixed stands can be predicted by extinction coefficients obtained from mono-specific stands indicates that modifications in crown structure and leaf distribution are only slight, or even non-existent, when the two species grow in a mixture.

Composition, Population Structure and Regeneration of Rhododendron arboreum Sm. Temperate Broad-Leaved Evergreen Forest in Garhwal Himalaya, Uttarakhand, India

The present studied was carried out in four districts of Garhwal Himalaya. Survey and sampling of vegetation were completed used standard ecological method with aim to study of plant composition, population structure and regeneration at different site in the four districts viz. Pauri, Rudrapryag, Tehri and Chamoli of Garhwal Himalaya. Each district represents two sites, falls an altitude in Pauri (1850-2070 m), Rudrapryag (1775-2147 m), Tehri (1938–2459 m) and Chamoli (1711–1960 m). Among the altitudes over all range is (1711-2459 m) altitudes in all eight sites, total 16 tree species has been recorded. Total tree density varies from 670-1510 Ind ha-1, total seedling density from 220-1540 Ind ha-1 and total sapling density 165-1300 Ind ha-1 was recorded in all sites among the attitudes. Species diversity (H-) was observed between 0.12-0.19 for trees, 0.04-0.40 for seedling and 0.07-0.15 for sapling in all sites among the attitudes.

In vitro plant regeneration and transformation studies in millets: current status and future prospects

Millets are a group of small seeded cereals and forage grasses grown in arid and semi-arid regions of Asia and Africa, where majority of cereals cannot be relied upon to provide sustainable yield. While major cereals such as wheat, rice and maize provide only food security, millets provide multiple securities, viz., food, fodder, health, nutrition, livelihood and ecological. Each of the millets is a reservoir of several nutrients in large quantity. They are nutritionally rich in minerals, dietary fiber, phenolics, vitamins, and are gluten-free. In the present review recent advances in tissue culture and genetic transformation studies conducted in millets to date have been summarized. Although there is a vast amount of literature available on millet tissue culture, only a limited number of transformation experiments have been conducted so far. Millets have been transformed primarily by particle bombardment, whereas Agrobacterium-mediated transformation is still lagging behind. Efforts need to be made to genetically improve millets by incorporating certain agronomically important traits, such as resistance to biotic and abiotic stresses, resistance to lodging, increased seed size and palatability along with softness of grain to make these crops more desirable for consumer.

REVIEW: In vitro plant regeneration studies and their potential applications in Populus spp.: a review

Genus Populus comprises about 25–35 species of deciduous flowering plants in the family Salicaceae which are widely distributed in temperate climates of the Northern Hemisphere. Populus species are important resources in certain branches of industry and have a special role for the scientific study of biological and agricultural systems. The poplar is known for its remarkable significance among the commercially propagated tree species such as teak, eucalyptus, wild cherry, red wood, and radiata pine. In vitro regeneration refers to growing and multiplications of cells, tissues and organs on defined liquid/solid media under aseptic and controlled environments. In vitro clonal propagation of forest trees, due to the high multiplication rate, is an attractive alternative for rapid propagation of elite genotypes of those species that could not easily be propagated through conventional methods. Owing to their widespread uses at the industrial level and for meeting the ever-increasing global demand for biomass production and wood industry, tissue culture techniques can be exploited for rapid cloning and large-scale production of planting material of various poplar species. Recent progress in the field of plant tissue culture determined this area to be one of the most dynamic and promising for experimental biology. Much work has been carried out on in vitro plant regeneration studies in Populus spp. including direct organogenesis, indirect organogenesis and somatic embryogenesis. These reviews provide an insight for in vitro plant regeneration studies in poplar species and their potential in its improvement.

Factors affecting in vitro propagation of Dracaena sanderiana Sander ex Mast. cultivars. I. Sterilization, explant browning, and shoot proliferation

Dracaena sanderiana Sander ex Mast. (lucky bomboo) is an ornamental plant belonging to the family Agavaceae. The factors affecting sterilization, explant browning, and shoot proliferation of two cultivars Green and Variegated of D. sanderiana were studied. Micropropagation of D. sanderiana is very important because of the limitations in its conventional propagation by classical vegetative propagation methods which give rise to several bacterial, fungal, viral and mycoplasma diseases. The in vitro condition can overcome these problems. Half-strength Murashige and Skoog’s (MS) media supplemented with different concentrations of 6- benzyl-amino-purine (BA mg l-1) and naphthalene acetic acid (NAA) (0.25 and 0.5 mg l-1) were used for shoot proliferation and plant regeneration studies. The effect of explant positioning on the culture media, whether horizontal or vertical, was also assessed on proliferation and growth of shoots produced. Explants of the ‘Green’ cultivar, cultured horizontally on media, were successful in yielding proliferated shoots. The highest mean value (4.8) was recorded on the medium supplemented with 2 mg l-1 BA and 0.25 mg l-1 NAA. Explants of the ‘Variegated’ cultivar, cultured horizontally on media, were also successful in yielding proliferated shoots. The highest mean value (3.66) was recorded on the medium supplemented with 2 mg l-1 BA and 0.25 mg l-1 NAA.

Biotechnological applications in in vitro plant regeneration studies of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

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.

Effect of a Potent Cytokinin Thidiazuron (TDH) on the Regener-ation Potential of Leaf and Petiole Explants of Cauliflower(Brassica oleraceaL var. botrytis)

Plant regeneration studies were carried out using leaf and petiole explants on forty dif-ferent combinations and concentrations of TDZ i. e. TDZ, TDZ+IAA, TDZ+NAA and TDZ+Adenine. Among the forty combinations tried, the high percent shoot regeneration was observed in peti-ole explants (76%) on MS medium supplemented with 0.22mg/l TDZ whereas in case of leaf ex-plants only 44% shoot regeneration was achieved on MS medium supplemented with 0.77mg/l TDZ+ 0.087 mg/l IAA. For root regener-ation the in vitro regenerated shoots were cul-tured on MS medium containing different con-centration of IAA and NAA. Hundred percent root regeneration was observed on the MS medi-um supplemented with 0.05mg/l and 0.20mg/l NAA respectively. The regenerated plantlets were transferred to the pots containing cocopeat and acclimatized. The regenerated plantlets were morphologically similar. An efficient and repro-ducible plant regeneration protocol has been standardized in cauliflower cv. Pusa Snowball K-1 and is being used for Agrobacterium- mediated cry1Aa gene transfer studies.

Incipient stem cell niche conversion in tissue culture: using a systems approach to probe early events in WUSCHEL‐dependent conversion of lateral root primordia into shoot meristems

Adventitious shoot organogenesis contributes to the fitness of diverse plant species, and control of this process is a vital step in plant transformation and invitro propagation. New shoot meristems (SMs) can be induced by the conversion of lateral root primorida/meristems (LRP/LRMs) or callus expressing markers for this identity. To study this important and fascinating process we developed a high-throughput methodology for the synchronous initiation of LRP by auxin, and subsequent cytokinin-induced conversion of these LRP to SMs. Cytokinin treatment induces the expression of the shoot meristematic gene WUSCHEL (WUS) in converting LRP (cLRP) within 24-30h, and WUS is required for LRP→SM conversion. Subsequently, a transcriptional reporter for CLAVATA3 (CLV3) appeared 32-48h after transfer to cytokinin, marking presumptive shoot stem cells at the apex of cLRP. Thus the spatial expression of these two components (WUS and CLV3) of a regulatory network maintaining SM stem cells already resembles that seen in a vegetative shoot apical meristem (SAM), suggesting the very rapid initiation and establishment of the new SMs. Our high-throughput methodology enabled us to successfully apply a systems approach to the study of plant regeneration. Herein we characterize transcriptional reporter expression and global gene expression changes during LRP→SM conversion, elaborate the role of WUS and WUS-responsive genes in the conversion process, identify and test putative functional targets, perform a comparative analysis of domain-specific expression in cLRP and SM tissue, and develop a bioinformatic tool for examining gene expression in diverse regeneration systems.

Growth, alkaloid production, rol genes integration, bioreactor up-scaling and plant regeneration studies in hairy root lines of Catharanthus roseus

Fifteen hairy root lines of Catharanthus roseus were compared for growth, alkaloid production and shoot bud regeneration potential in relation to their morphology and pattern of rol gene(s) integration. These lines could be classified into 10 distinct morphotypes on the basis of their thickness and branching behaviour. Twelve root lines showed the presence of both rolB and rolC and two indicated the integration of only rolC. Genes rolA, B and C were only detected in N5 line. The root lines did not differ significantly for their total alkaloids content (1.10–1.30% dry wt.) but showed variations in relative concentration of ajmalicine (0.007–0.08% dry wt.), serpentine (0.01–0.08% dry wt.) and catharanthine (0.01–0.04% dry wt.). Line PG2 with highest biomass accumulation (GI = 326.73) and line PI3 with highest catharanthine content were up-scaled in 5-l bioreactor wherein a 9–11 folds increment in biomass accumulation was obtained with two folds enhancement in ajmalicine (0.029 % dry wt.) in PI3 line and appearance of a major unknown alkaloid in PG2 line. Shoot buds were induced only in line N5 on a MS + 3.0 mg/l BAP and 2.0 mg/l NAA which in turn showed excessive rooting and frequent in vitro flowering.

In vitro regeneration of Eucalyptus camaldulensis.

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2mgl(-1) benzyl aminopurine (BAP) and 0.1mgl(-1) naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5mgl(-1) BAP, while root induction and elongation was superior in 1/2 MS + 1mgl(-1) Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100% coco peat resulted in maximum survival (80%) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60-148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.