Multiple Buds Research Articles

Chemical transformation of the multibudding yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.

Breeding Maize Hybrids with Improved Drought Tolerance Using Genetic Transformation

Drought is considered the main agricultural menace, limiting the successful realization of land potential, and thereby reducing crop productivity worldwide. Therefore, breeding maize hybrids with improved drought tolerance via genetic manipulation is necessary. Herein, the multiple bud clumps of elite inbred maize lines, DH4866, Qi319, Y478 and DH9938, widely used in China, were transformed with the Escherichia coli betA gene encoding choline dehydrogenase (EC 1.1.99.1), a key enzyme in the biosynthesis of glycine betaine from choline, using Agrobacterium to generate betA transgenic lines. After 3–4 consecutive generations of self-pollination in these transgenic plants, progenies with a uniform appearance, excellent drought tolerance, and useful agricultural traits were obtained. We evaluated the drought tolerance of T4 progenies derived from these transgenic plants in the field under reduced irrigation. We found that a few lines exhibited much higher drought tolerance than the non-transformed control plants. Transgenic plants accumulated higher levels of glycine betaine and were relatively more tolerant to drought stress than the controls at both the germination and early seedling stages. The grain yield of the transgenic plants was significantly higher than that of the control plants after drought treatment. Drought-tolerant inbred lines were mated and crossed to create hybrids, and the drought tolerance of these transgenic hybrids was found to be enhanced under field conditions compared with those of the non-transgenic (control) plants and two other commercial hybrids in China. High yield and drought tolerance were achieved concurrently. These transgenic inbred lines and hybrids were useful in marginal and submarginal lands in semiarid and arid regions. The betA transgene can improve the viability of crops grown in soils with sufficient or insufficient water.

In Vitro Micropropagation of Eria Lasiopetala (Willd.) Ormerod- A Native Medicinal Orchid in Bangladesh

The present study focuses on the asymbiotic germination of seeds through in vitro culture of Eria lasiopetala, a native medicinal orchid in Bangladesh. Out of the four basal media, MS was found to be the most effective medium, where 93.58±0.374% seed germinations were recorded. Different plant growth regulators (PGRs), e.g. BAP, Kin, NAA and were used for secondary protocorm proliferation, plantlet development, and multiplication of shoot buds through in vitro conditions. The maximum number of secondary protocorms (12.80±0.115) were developed that were derived from primary protocorms on MS medium fortified with 0.5 mg/l BAP and 1.0 mg/l NAA. Data revealed that 0.5 mg/l BAP + 0.25 mg/l NAA showed the best combination for height elongation of plantlets (3.12±0.012 cm) in MS medium. While for multiple shoot buds (MSBs) 1.0 mg/l BAP + 0.5 mg/l NAA showed better (6.8±0.127) performance than other combinations of PGRs. The single shoots were isolated from the multiple shoots and sub-cultured on ½MS medium supplemented with NAA (0.5 - 2.0 mg/l), IAA (0.5 - 2.0 mg/l) and IBA (0.5 - 2.0 mg/l) for root induction. The maximum number of roots (7.53±0.176) and its growth (4.61±0.01 cm) were found in ½MS medium supplemented with 1.0 mg/l IAA. The well rooted plantlets were hardened and successfully transferred after acclimatization in pots containing the mixture of coconut husk, charcoal and brick pieces in the ratio of 2:1:1 and eventually established under natural condition. J. Bio-Sci. 32(1): 45-60, 2024

An effective in vitro propagation of Coelogyne mossiae Rolfe: An endangered endemic orchid of Western Ghats, India

Coelogyne mossiae Rolfe, an unexplored taxon given its red-listed status by IUCN was selected for in vitro propagation studies due to its conservational significance. The mature pods harboring viable seeds were subjected to inoculation on eight different nutrient media, revealing full-strength Murashige and Skoog (MS) media as highly efficient for seed germination (96.65 %) and subsequent stages of seedling development. Protocorm explants are used for micropropagation on MS media supplemented with various plant growth regulators (PGRs). α- Naphthaleneacetic acid (NAA) in the concentration of 0.5 mg/L facilitated optimal shoot induction resulting in an average of 2.39 multiple buds and 2.46 cm shoot length while 1.0 mg/L 6-Benzyl amino purine (BAP) augmented cultures provided 2.32 multiple shoot buds with an average 1.92 cm height. Half macro-MS augmented with 0.5 mg/L BAP promoted pseudobulb elongation (0.73 cm) while 0.5 mg/L NAA supported root formation (8.33 roots with an average length of 1.12 cm). The resultant plants were acclimatized in pots containing coco peat and brick pieces, exhibiting a survival rate of 63.23 %. The anatomical characteristics of the hardened plants provided insight into factors conducive to their adaptability. Also, the 3-month-old hardened plants demonstrated significantly greater total photosynthetic pigment (0.4915±0.00 mg g-1), total carbohydrate content (72.33±1.69 mg SSE/g), and total protein content (40.26±0.53 mg BSAE/g) when compared to in vitro plants. These findings underscore the potential of in vitro propagation techniques in the conservation and sustainable utilization of Coelogyne mossiae.

In vitro Propagation of Leucas biflora (Vahl) Sm. - A Vulnerable Medicinal Herb

A rapidly well-developed in vitro regeneration system has been established for Leucas biflora using nodal segments as explants and MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRS). MS medium containing 2.0 mg/l BAP and 0.5 mg/l NAA showed the highest response (95.99%) in induction of shoot buds. This media combination also produced the maximum number (11.20 ± 0.49) of multiple shoot bud per explant after 3 subcultures at an interval of 14-days. Microshoots showed the maximum elongation (4.70 ± 0.07 cm) in the same medium along with subcultures. These elongated shoots were further grown on rooting media, producing vigorous and healthy root systems. The best response (95.99 ± 1.63%) for rooting was obtained when half-strength MS medium was supplemented with 0.5 mg/l IAA + 0.5 mg/l IBA in 21 days of culture. In this combination, the average number and length of roots were 5.20 ± 0.33 and 4.80 ± 0.30 cm, respectively. The seedlings were removed from culture tubes and transplanted into the pots via several stages of acclimatization. Finally, 98% of the seedlings were found to survive under natural condition. Plant Tissue Cult. & Biotech. 34(1): 49-54, 2024 (June)

Establishment of Efficient Protocols for In Vitro Regeneration of Hyptis Suaveolens (L.) Poit. Using Apical Shoots and Nodal Segments

The present work was undertaken with a view to standardize an efficient protocol for in vitro rapid propagation of Hyptis suaveolens. Shoot apices and nodal segments of H. suaveolens produced multiple shoot buds when cultured on MS medium supplemented with 1.5-3.0 mg/l BAP or Kn in the combinations with 0.5-1.0 mg/l IAA or NAA. Nodal explants exhibited highest proliferation (90%) and gave maximum number of shoots per culture (8.40 ± 0.18) on MS medium fortified with 1.5 mg/I BAP and 1.0 mg/l IAA. The shoot buds were elongated on MS medium fortified with different concentrations and combinations of auxins and cytokinins. Elongated shoot buds were further cultured in rooting media for root induction. One fourth MS medium -without PGR was - proved ·the most effective for - induction and proliferation of roots. The--rooted plantlets ·were 'established in the field through successive phases of acclimation and the survival rate was 70%. The Chittagong Univ. J B. Sci. 8(1&2): 99-105, 2013

Mass Scale Micropropagation Of Andrographis Paniculata (Burm. F.) Wall Ex Nees

A reliable and efficient protocol was developed for in vitro propagation of Andrographis paniculata (Burm. f.) Wall ex Nees with the use of nodal and shoot tip explants. Nodal segments and shoot apex of in vitro grown seedlings were cultured on agar solidified MS medium supplemented with different concentrations and combinations of plant growth regulators (PGRs). Both the explants underwent direct organogenesis and produced multiple shoot buds. In case of shoot apex, maximum number of multiple shoot buds were formed when cultured on MS medium containing 1.5 mg/l Kn + 1.0 mg/l IAA. But nodal segments produced highest number of shoot buds on MS medium fortified with 1.5 mg/l BAP + 1.0 mg/l IAA. Multiple shoot buds underwent rapid elongation on elongation media and maximum elongation took place on MS medium fortified with 2.0 mg/l Kn + 1.5 mg/l IAA. Elongated shoot buds, on further culture in rooting media, produced strong and stout root systems. Half strength MS medium fortified with 2.0 mg/l IAA was most effective for induction and proliferation of roots. The healthy complete plantlets were successfully acclimatized and established in outside pots through successive phases of acclimatization. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &2):121- 130, 2012.

An Efficient Micropropagation Protocol For Mucuna Pruriens (L.) Dc.

An efficient protocol was developed for rapid micropropagation of Mucuna pruriens (L.) DC., an important medicinal plant species of Bangladesh. For the purpose, leaf segments, nodal segments and shoot apices of in vitro grown seedlings and cotyledon segments of immature seeds of field grown plants were cultured on agar solidified MS medium supplemented with different combinations of PGRs (Plant Growth Regulators). Only leaf and cotyledon segments produced light green to green friable/ compact callus in some of the media combinations. Combination and concentration of PGRs played important role in induction of such tissue. The induced tissues failed to differentiate on further culture in a wider spectrum of PGR supplemented media. On the other hand nodal segments and shoot apices produced multiple shoot buds when grown on a wide range of PGRs (BAP, NAA, IAA, Kn) supplemented media. Maximum number of multiple shoot buds were produced in medium containing 1.5 mg/l BAP + 1.0 mg/l IAA. The elongated shoot buds on further culture in rooting media produced strong and stout root systems. Half strength MS media fortified with either 1.0 mg/l IAA or 2.0 mg/l IAA was most effective for induction and proliferation of roots. These complete plantlets were successfully acclimatized and established in outside pots through a successive phase of acclimatization. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &2):79-89, 2012.

Cotyledonary somatic embryo is one kind of intermediate material similar to callus in the process of in vitro tissue culture from Rosa hybrida ‘John F. Kennedy’

BackgroundRose is recognized as an important ornamental plant worldwide, and it is also one of the most widely used flowers in gardens. At present, the improvement of rose traits is still difficult and uncertain, and molecular breeding can provide new ideas for the improvement of modern rose varieties. Somatic embryos are quite good receptors for genetic transformation. However, little is known about the molecular mechanisms underlying during the regeneration process of rose somatic embryos. To elucidate the molecular regulation mechanism of somatic embryo plantlet regeneration, the relationship between the differences in traits of the two different regenerated materials and the significantly differentially expressed genes (DEGs) related to phytohormone pathways in the process of regeneration were be investigated.ResultsThese representative two regenerated samples from single-piece cotyledonary somatic embryo (SPC) culture of Rosa hybrida ‘John F. Kennedy’, were harvested for transcriptome analysis, with the SPC explants at the initial culture (Day 0) as the control. The differentially expressed genes (DEGs) in the materials from two different types for regeneration approach (SBF type: the regeneration approach type of single bud formed from SPC explants; MBF type: the regeneration approach type of multiple buds formed from SPC explants) were be screened by means of the transcriptome sequencing technology. In this study, a total of about 396.24 million clean reads were obtained, of which 78.95–82.92% were localized to the reference genome, compared with the initial material (CK sample), there were 5594 specific genes in the material of SBF type and 6142 specific genes in the MBF type. The DEGs from the SBF type material were mainly concentrated in the biological processes of GO terms such as phytohormones, substance transport, cell differentiation, and redox reaction. The KEGG enrichment analysis revealed these DEGs were more active in ubiquinone and other terpenoid-quinone biosynthesis, fatty acid elongation, steroid biosynthesis, and glycosphingolipid biosynthesis-globo and isoglobo series. In contrast, the DEGs induced by the MBF type material were mainly associated with the biological processes such as phytohormones, phosphorylation, photosynthesis and signal transduction. According to KEGG analysis, these DEGs of MBF type were significantly enriched in the porphyrin and chlorophyll metabolism, brassinosteroid biosynthesis, carotenoid biosynthesis, and peroxisome. Furthermore, the results from the phytohormone pathways analysis showed that the auxin-responsive factor SAUR and the cell wall modifying enzyme gene XTH were upregulated for expression but the protein phosphatase gene PP2C was downregulated for expression in SBF type; the higher expression of the ethylene receptor ETR, the ethylene transduction genes EBF1/2, the transcription factor EIN3, and the ethylene-responsive transcription factor ERF1/2 were induced by MBF type.ConclusionsAccording to the GO and KEGG analysis, it indicated the DEGs between two different regenerated materials from somatic embryos were significantly different which might be causing morphological differences. That was somatic embryos from Rosa hybrida ‘John F. Kennedy’ could regenerate plantlet via both classic somatic embryogenesis (seed-like germination) and organogenesis, cotyledonary somatic embryos should be considered as one kind of intermediate materials similiar to callus, rather than the indicator materials for somatic embryogenesis.

Tools for live-cell imaging of cytoskeletal and nuclear behavior in the unconventional yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

The molecular mechanisms underlying the regeneration process in the earthworm, Perionyx excavatus exhibit indications of apoptosis-induced compensatory proliferation (AICP).

Regeneration is a multifaceted biological phenomenon that necessitates the intricate orchestration of apoptosis, stem cells, and immune responses, culminating in the regulation of apoptosis-induced compensatory proliferation (AICP). The AICP context of research is observed in many animal models like in Hydra, Xenopus, newt, Drosophila, and mouse but so far not reported in earthworm. The earthworm Perionyx excavatus is used in the present study to understand the relationship between AICP-related protein expression and regeneration success in different conditions (normal regeneration and abnormal multiple bud formation). Initially, the worms are amputated into five equal portions and it is revealed that regeneration in P. excavatus is clitellum independent and it gives more preference for anterior regeneration (regrowth of head portion) than for posterior regeneration (regrowth of tail portion). The posterior segments of the worm possess enormous regeneration ability but this is lacking in anterior segments. Alkaline phosphate, a stem cell marker, shows strong signals throughout all the posterior segments but it decreases in the initial 1st to 15th anterior segments which lack the regeneration ability. While regenerating normally, it was suggested that the worm follow AICP principles. This is because there was increased expression of apoptosis signals throughout the regeneration process along with constant expression of stem cell proliferation response together with cellular proliferation. In amputated posterior segments maintained in vitro, the apoptosis signals were extensively detected on the 1st day. However, on the 4th and 6th days, caspase-3 and H2AX expression are significantly suppressed, which may eventually alter the Wnt3a and histone H3 patterns that impair the AICP and result in multiple bud formation. Our results suggest that AICP-related protein expression pattern is crucial for initiating proper regeneration.

Response of various carbon sources for in vitro culture establishment and shoot proliferation of banana cv. Chinichampa (AAB) through shoot tip

Bananas are one of the most important tropical fruits in the world market, a significant staple food and a major export commodity. In vitro propagated banana plants are steadily becoming the planting material of choice because of disease free, uniformity and the possibility of rapid multiplication. The present investigation was carried out at the tissue culture laboratory, Department of Horticulture, School of Agricultural Sciences, Nagaland University, Medziphema campus during the year 2019-2022. Data recorded for different parameters viz. days to greening, days required for multiple buds’ initiation, number of multiple buds produced per explant and length of multiple buds after 30 days were subjected to completely randomized design (CRD). The present study revealed the importance of various carbon sources and its role in micropropagation of banana through shoot tip culture. The minimum day to greening, multiple buds initiation and maximum length of multiple buds was observed in shoot proliferation medium supplemented (SPM S) with 30g sucrose. The highest number of multiple buds was recorded in shoot proliferation medium supplemented with 30g table sugar. The results indicate the significance of sucrose as carbon sources in micropropagation of banana. They improved plant regeneration and facilitated rapid multiplication of disease and virus free banana plantlets.

Response of carbon sources and their various concentrations on in vitro rooting of banana cv. Chinichampa (AAB) through shoot tip ​

In vitro propagated banana plants are steadily becoming the planting material of choice because of disease free, uniformity and the possibility of rapid multiplication. The present investigation was carried out at the tissue culture laboratory, Department of Horticulture, School of Agricultural Sciences, Nagaland University, Medziphema campus during the year 2019-2022. Data recorded for different parameters viz. days to greening, days required for multiple buds’ initiation, number of multiple buds produced per explant and length of multiple buds after 30 days were subjected to completely randomized design (CRD). The present study revealed the importance of various carbon sources and their role in micropropagation of banana through shoot tip culture. The minimum days required for root initiation were observed in root induction media (RIM) supplemented with 30g glucose. Rooting percentage (%), number of roots per shoot and length of longest roots (cm) after 30 days was seen in RIM supplemented with table sugar at 40g, 30g and 20g respectively. Meanwhile maximum average length of shoot (cm) after 30 days was observed in RIM supplemented with 40g glucose and highest number of leaves per plantlets was recorded in RIM supplemented with 30g sucrose. The results indicate the significance of carbon sources in micropropagation of banana. They improved plant regeneration and facilitated rapid multiplication of disease and virus free banana plantlets.

The tongue of the red-eared slider (Trachemys scripta elegans): morphological characterization through gross, light, scanning electron, and immunofluorescence microscopic examination

The red-eared slider (Trachemys scripta elegans) is renowned for its remarkable adaptations, yet much of its complex biology remains unknown. In this pioneering study, we utilized a combination of gross anatomy, scanning electron microscopy (SEM), light microscopy, and immunofluorescence techniques to examine the tongue’s omnivorous adaptation in this species. This research bridges a critical knowledge gap, enhancing our understanding of this intriguing reptile. Gross examination revealed a unique arrowhead-shaped tongue with a median lingual fissure and puzzle-piece-shaped tongue papillae. SEM unveiled rectangular filiform, conical, and fungiform papillae, with taste pores predominantly on the dorsal surface and mucous cells on the lateral surface of the papillae. Histologically, the tongue’s apex featured short rectangular filiform and fungiform papillae, while the body exhibited varying filiform shapes and multiple taste buds on fungiform papillae. The tongue’s root contained lymphatic tissue with numerous lymphocytes surrounding the central crypt, alongside lingual skeletal musculature, blood and lymph vessels, and Raffin corpuscles in the submucosa. The lingual striated muscle bundles had different orientations, and the lingual hyaline cartilage displayed a bluish coloration of the ground substance, along with a characteristic isogenous group of chondrocytes. Our research represents the first comprehensive application of immunofluorescence techniques to investigate the cellular intricacies of the red-eared slider’s tongue by employing seven distinct antibodies, revealing a wide array of compelling and significant findings. Vimentin revealed the presence of taste bud cells, while synaptophysin provided insights into taste bud and nerve bundle characteristics. CD34 and PDGFRα illuminated lingual stromal cells, and SOX9 and PDGFRα shed light on chondrocytes within the tongue’s cartilage. CD20 mapped B-cell lymphocyte distribution in the lingual tonsil, while alpha smooth actin (α-SMA) exposed the intricate myofibroblast and smooth muscle network surrounding the lingual blood vessels and salivary glands. In conclusion, our comprehensive study advances our knowledge of the red-eared slider’s tongue anatomy and physiology, addressing a significant research gap. These findings not only contribute to the field of turtle biology but also deepen our appreciation for the species’ remarkable adaptations in their specific ecological niches.

Inducing mutation and ascertaining lethal dosage of in vitro cultures of banana cv. Ney Poovan to ethyl methane sulfonate

In vitro mutation breeding in vegetatively propagated crops like banana offers a benefit in screening for beneficial variants in plant cells or cultured tissues. An attempt was made to induce mutants and determine the lethal dose, as it is the prerequisite to optimize the concentration and duration of the mutagen used to recover a larger population in mutation research. Shoot tip cultures were treated for 2 and 4 h at six different EMS concentrations ranging from 80 mM to 160 mM, whereas proliferating multiple shoots were exposed for 30 and 60 min at six different EMS concentrations ranging from 8 mM to 40 mM. Survival percentage, shoot length, and number of shoots reduced linearly and significantly as concentration and duration increased in both shoot tips and proliferating multiple buds. The probit curve-based analysis of mortality of treated explants revealed that the LD50 was 155.83 mM for 2 h and 113.72 mM for 4 h, respectively for shoot tip cultures, whereas for proliferating multiple buds, the LD50 value was adjusted to 39.11 mM for 30 min and 30.41 mM for 60 min. 160 mM EMS for 4 h resulted in a shorter shoot, a longer rooting duration, a lesser number of roots, and decreased root development. In proliferating multiple shoots, the smallest shoot, longest rooting duration, least number of roots, and shortest root were observed in 40 mM EMS for 60 min. Similar reductions in growth parameters were observed in proliferating multiple shoots at higher exposure to EMS for a longer duration.

Characterization of phytochemical profile of rhizome of artificial cultured Polygonatum sibiricum with multiple rhizome buds

Rhizome of Polygonatum sibiricum is both a renowned traditional Chinese remedy and a commonly consumed delicacy. Due to the escalating demand and excessive overexploitation, there has been a growing interest in the artificial cultivation of this plant in recent years. To assess the therapeutic benefits of artificially cultivated P. sibiricum, it is crucial to identify and classify its phytochemical components, which are the primary bioactive compounds found in its rhizome. In this study, the phytochemical profile of an artificially cultivated P. sibiricum rhizomes with multiple rhizome buds (ACM) was characterized by using untargeted UHPLC-Q-Orbitrap-MS based approach. In addition, two-wild-types P. sibiricum rhizomes, namely the wild-type with multiple rhizome buds (WTM) and the wild-type with single rhizome bud (WTS), were used for comparison. A total of 183 phytochemicals, including 20 alkaloids, 48 flavonoids, 33 phenolic acids, and 82 terpenoids, were tentatively identified. Generally, the phytochemical profile of ACM was comparable to that of WTM and WTS. In specific, most of the identified alkaloids and phenolic acids, and approximately half of the identified terpenoids, were not significantly different. Notably, several phytochemicals with potent therapeutic properties, such as epiberberine, laetanine, sinapic acid, curcumenol, were present in ACM. Additionally, artificial cultivation increased the abundance of geniposide and naringenin, which have been linked to cardioprotective effects. These findings provide valuable insights for the future utilization of artificially cultivated P. sibiricum.

Multiple bud clumps induction of banana cv. Barangan Merah (Musa acuminate Colla.) under in vitro treatment of benzyl amino purine and indole-3 acetic acid

Cultivar Barangan is one of the famous banana plants that has good taste and price. The disadvantages of this banana is susceptible to Fusarium disease. Tissue culture techniques is one ways of plants propagation for this plant. This study is aimed to determine the effective concentration of benzyl amino purines (BAP) and indole-3 acetic acid (IAA) for optimal multiple bud clumps (MBC) induction in explants of Barangan Merah banana. The Research was carried out at the Laboratory of Tissue Culture, Faculty of Agriculture, Universitas Syiah Kuala. The research design was a completely randomized design, 3x2 factorial. BAP concentration was the first factor, i.e. 5, 10 and 15 mg L−1. The second factor was the concentration of IAA, i.e. 0.1 and 0.5 mg L−1. The results of this research showed that 96,29% explants of all treatments survived. Explant contamination was 11,11% occurred by bacteria and brownish exudate that spread to the medium. MBC are significantly increased at 16 days after induction at concentration of BAP 5 mg L−1 + IAA 0.5 mg L−1. All combinations of BAP and IAA concentrations have produced MBC explants (100%) at eighth weeks after induction. The concentration of 5 mg L−1 BAP + 0.5 mg L−1 IAA resulted the highest shoots number. Analysis of variance and Least Significant Different further test showed that the longest of shoot length was founding the concentration of 5 mg L−1 BAP compared to other treatments. Increasing concentrations of BAP 10 mg L−1and 15 mg L−1 resulted in significantly lower shoot length. It can be concluded that taken together the concentration of 5 mg L−1 BAP + 0.5 mg L−1 IAA is the best to effectively multiple bud clumps induction in explants of Barangan Merah banana through tissue culture technique.

In vitro plantlet regeneration from cotyledon explants of Capsicum annuum L

An efficient In vitro plant regeneration was established in Capsicum annuum L. from cotyledon explants. The best performance i.e., 80% of the explants responded for multiple shoot bud proliferation on MS medium containing 10 mg/l Zeatin. The maximum number of buds per explants was 50.28 ± 2.69 and the bud length of 0.5 ± 0.22 cm was obtained in this medium after 30 days. The average number of 80.32 ± 0.16 multiple shoot bud and the average shoot bud length of 1.0 ± 0.19 cm was attained on the same medium after 90 days. These shoot buds on transfer to MS + 10 mg/l Zeatin + 2.0 mg/l GA3 yielded 6.3 ± 0.35 number of multiple shoots with an average shoot length of 4.6 ± 0.62 cm after 30 days. Shoots rooted well (80% of induced shoots) on half strength MS medium supplemented with 0.5 mg/l IBA. The average number of roots per shoot was 8.2 ± 0.79 and the average root length of 4.5 ± 0.58 cm was recorded. About 80% of In vitro derived plantlets were survived under open field conditions.

Commercial scale genetic transformation of mature seed embryo explants in maize.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

Micropropagation and cytological studies of Aole vera Linn

Aloe vera Linn. is an essential medicinal plant. In this present research work, a protocol of in vitro regeneration and karyomorphological analysis of Aloe vera was developed using different concentrations and compositions of media. Shoot apices of field-grown plants were used as explant and aseptically cultured on Murashige and Skoog (MS) medium fortified with different concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP and Kn). The highest number of multiple shoot buds (4.36 ± 0.07) was obtained from MS + 2.0 mg/l BAP + 1.0 mg/l IAA and induced shoot buds underwent rapid elongation (4.24 ± 0.06 cm) on the same medium composition. Half strength MS media with 2.0 mg/l IBA was suitable for induction and proliferation (6.31 ± 0.05) of roots and 95% of plantlets were acclimatized to field conditions successfully. Somatic chromosome numbers of mother and in vitro grown plants were confirmed to be 2n = 14. Chromosome length ranged from 4.28 - 13.74 µm in the naturally grown plants and 4.46 - 14.1 µm for in vitro grown plants. The total form percent (TF%) of mother and in vitro grown plants was 41.69% and 42.23%, respectively. The karyotype formula of in vivo grown plants was 2n = 14 = 4Lsm + 6Mm + 4Sm, whereas that of the micropropagated plants was 2n = 14 = 4Lsm + 4Mm + 6Sm. The frequency of the chromosome having arm more than 2:1 was 0.08 for mother plants and 0.15 for in vitro grown plants. Therefore, the karyotype of both plants falls into the 2B symmetrical type based on Stebbins classification (1971).