Technical Advances in Chloroplast Biotechnology

Chloroplasts are highly organized cellular organelles after master organelle nucleus. They not only play a central role in photosynthesis but are also involved in several crucial cellular activities. Advancements in molecular biology and transgenic technology have further groomed importance of the organelle, and they are the most ideal ones for the expression of transgene. No doubt, limitations are there, but still research is advancing to resolve those. Certain valuable traits have been engineered for improved agronomic performance of crop plants. Industrial enzymes and therapeutic proteins have been expressed using plastid transformation system. Synthetic biology has been explored to play a key role in engineering metabolic pathways. Further, producing dsRNA in a plant’s chloroplast rather than in its cellular cytoplasm is more effective way to address desired traits. In this chapter, we highlight technological advancements in chloroplast biotechnology and its implication to develop biosafe engineered plants

Effect of High Temperature Stress on Pollen Grains in Sunflower (Helianthus annuus L.) Inbred Lines

Abstract High temperature at reproductive stage causes a decrease in seed set that ultimately results in yield loss in important crops. Limited research is available regarding the effect of high temperature on viability attributes of pollen grain in sunflower. This study was planned to reveal the effects of high temperature stress on pollen grains in sunflower. An experiment was laid out in a triplicated Completely Randomized Design with split-split plot arrangements at the Oilseeds Research Institute, Ayub Agricultural Research Institute Faisalabad during spring 2021. Two temperature treatments T1 = 45° C for 10, 20 and 30 minutes, and T2 = 50° C for 3, 5 and 10 minutes were used on 10 sunflower inbred lines. Normal (untreated) pollen grains were used as control (T0). Data were collected on pollen viability, pollen germination, and pollen tube length. Recorded data were subjected to analysis of variance, Tukey’s HSD mean comparison test, and correlation. According to the results, ORI-1 showed the highest mean for pollen viability, whereas, RL-86 had highest mean for pollen germination and pollen tube length under normal conditions. RL-86 and ORI-73 revealed their ability to withstand both heat stresses as all pollen traits were least affected under high temperature stresses. It is advised to incorporate these inbred lines in future breeding programs aimed at the development of heat tolerant sunflower hybrids. Moreover, correlation studies displayed a positive, strong and highly significant relation of pollen germination with pollen tube length under normal and heat stress conditions revealing their importance in pollen study and reproduction

Biotechnological Interventions for the Improvement of Sugarcane Crop and Sugar Production

Sugarcane, not only fulfills 70% of world sugar needs but is also a prime potential source of bioethanol. It is majorly grown in tropical and subtropical regions. Researchers have improved this grass to great extent and have developed energy cane with ability to accumulate up to 18% sucrose in its Culm. Improvement of this crop is impeded by its complex genome, low fertility, long production cycle and susceptibility to various biotic and abiotic stresses. Biotechnological interventions hold great promise to address these impediments paving way to get improved sugarcane crop. Further, being vegetatively propagated in most of the agroecological regions, it has become more attractive plant to work with. This chapter highlights, how advanced knowledge of omics (genomics, transcriptomics, proteomics and metabolomics) can be employed to improve sugarcane crop. In addition, potential role of in vitro techniques and transgenic technology has also been discussed for developing improved sugarcane clones with enhanced sugar recovery

Citrus Biotechnology: Current Innovations and Future Prospects

Citrus is a valuable fruit crop worldwide. It not only provides essential minerals and vitamins but is also of great commercial importance. Conventional research has contributed a lot to the improvement of this fruit plant. Numerous improved varieties have been developed through conventional breeding, mutational breeding, polyploidization and tissue culture yet pathogens continue to emerge at a consistent pace over a wide range of citrus species. Citriculture is vulnerable to various biotic and abiotic stresses which are quite difficult to be controlled through conventional research. Biotechnological intervention including transgenesis, genome editing, and OMICS offers several innovative options to resolve existing issues in this fruit crop. Genetic transformation has been established in many citrus species and transgenic plants have been developed having the ability to tolerate bacterial, viral, and fungal pathogens. Genome editing has also been worked out to develop disease-resistant plants. Likewise, advancement in OMICS has helped to improve citrus fruit through the knowledge of genomics, transcriptomics, proteomics, metabolomics, interactomics, and phenomics. This chapter highlights not only the milestones achieved through conventional research but also briefs about the achievements attained through advanced molecular biology research

Sugarcane as Future Bioenergy Crop: Potential Genetic and Genomic Approaches

Biofuels are gaining increased scientific as well as public attention to fulfill future energy demands and can be the only potential candidates to safeguard and strengthen energy security by reducing the world’s reliance on exhausting fossil energy sources. Sugarcane is an important C4 crop with great potential to contribute to global biofuel production as sugarcane juice can be easily fermented to produce ethanol. The success of bioethanol production from sugarcane in Brazil has widened the scope of the technology and has led to increased demand of purpose-grown sugarcane for biofuel production. Scientific interventions have not only helped to improve the cane crop but industrial procedures have also been upgraded resulting in improved production of bioethanol. Likewise, advancements in omics have led to high hopes for the development of energy cane. This chapter highlights the advancements as well as potential and challenges in the production of sugarcane biofuel, focusing on genetic and genomic interventions improving the crop as energy-cane. Further, controversies in the production and usage of biofuel derived from sugarcane have also been discussed

Emerging Trends to Improve Tropical Plants: Biotechnological Interventions

Tropical plants are an integral part of the ecosystem and are of significance for the well-being of humanity. Since their domestication in 10,000 BC, conventional breeding has played a crucial role in their conservation and widespread adaptation worldwide. Advancements in multi-omics approaches, that is, genomics, metabolomics, transcriptomics, proteomics, whole genome sequencing, and annotation, have led to the identification of novel genes involved in crucial metabolic pathways, thus helping to develop tropical plant varieties with desirable traits. Information retrieved from the pan-genome, super-pan-genome, and pan-transcriptome has further uplifted marker-assisted selection and molecular breeding. Tissue culture techniques have not only helped to conserve endangered plant species but have also opened up new avenues in terms of mass-scale propagation of ornamental plants. Transgenic technology is increasingly contributing to the betterment of tropical plants, and different plant species have been engineered for valuable traits. Likewise, genome editing is appearing to be a promising tool to develop tropical plants having the potential to fulfill future needs. Hence, this chapter highlights the importance of conventional and modern scientific approaches for the conservation and improvement of tropical plant species

Enhanced Production Of Streptokinase By Chemical Mutagenesis Of Streptococcus agalactiae EBL-20

Abstract Streptokinase (SK) is an enzyme that is used for the treatment of cardiovascular diseases. The current study focused on the enhanced production of SK by inducing mutation in Streptococcus agalactiae EBL-20 and optimization of medium components and culture conditions for the maximum growth of mutant derived strain. S. agalactiae EBL-32 was selected as a potent mutant after exposure of S. agalactiae EBL-20 to EMS for 180 minutes. SK activity obtained from mutant derived strain was found to be 1.6 fold higher as compared to the activity achieved by wild strain. Nutritional requirements of the mutated strain were optimized by single factor analysis method suggesting glucose as the optimum carbon source; yeast extract and peptone as a suitable nitrogen sources and corn steep liquor (CSL) as an appropriate substrate for the maximum SK production. The culture conditions determined by response surface methodology (RSM) suggested that a temperature value of 37.5⁰C and pH 7 of the fermentation medium with 2.50 mL inoculum size for 36 hours of incubation was optimum for maximum yield of SK. Hence the optimization studies resulted into 1.92 fold increase in the yield of SK suggesting the new isolate suitable for commercial scale production of SK

In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols

Rapid and efficient in vitro regeneration of transplastomic potato (Solanum tuberosum L.) plants after particle bombardment

In contrast with traditional nuclear gene transformation, transplastomic technology has opened a new horizon for the transgenic plant research that offers several beneficial aspects including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. However, this technology has been well adopted and established in tobacco, the introduction and adoption of cost-effective, swift, and reproducible protocol for in vitro regeneration of transplastomic potato is challenging and laborious. The present research aimed to develop such prompt and efficient protocol to instigate and revive the regeneration potential with the combinations of different plant growth regulators (PGRs). Leaves and internodal explants from four potato cultivars were transformed with chloroplast transformation vector via particle bombardment and cultured on MS media supplemented with suitable PGRs and selection agents. Leaf explants of cultivar Kuroda induced highest (92%) number of calli where cultivar Sante produced the highest (85.7%) transplastomic shoots. Thidiazuron was found more proficient (41%) for shoot regeneration. Finally, within only seven weeks, we got 21 spectinomycin resistant shoot, and 16 of those showed integration of target genes into the plastome in PCR screening