Regeneration and genetic transformation in cowpea

Over the last three decades, sporadic efforts have been made to develop regeneration and transformation systems in cowpea (Vigna unguiculata L. Walp). This paper reviews the progress made to date, including highlights of culture media and explants used for regeneration and chimeric gene constructs employed in transformations. Progress has been slow, mainly due to limited resources, since very few laboratories have been involved. There is an urgent need for more focused and consistent efforts to develop genotype, and tissue-culture dependent and independent approaches for obtaining stable genetic transformation in cowpea

Use of somatic embryogenesis as a vehicle for cotton transformation

Cotton has been aptly described as the prosperity plant owing to its unrivalled economic importance as a source of feedstock, food and oil, as well as raw material for diverse industrial applications, ranging from textile and footwear to automobiles, energy, medical and pharmaceutical. As such, over 180 million people of the world depend on its production for livelihood. However, cotton production is grossly hampered, and has long been peaked in many regions where it is being grown. Without prejudice to the genetic improvement already made by conventional breeding with respect to yield and quality over the years, genetic transformation is arguably the last recourse for further development of cotton, especially with respect to the prevailing production constraints of insect pests, weeds, environmental stresses and diseases. This review therefore focuses on the use of somatic embryogenesis as a vehicle for cotton genetic transformation. It indeed attempts to overview the challenges of cotton transformation with respect to narrow genetic base coupled with the recalcitrant nature of the crop species, as well as the research success achieved so far. It then discusses the underlying mechanisms of somatic embryogenesis as well as the current constraints and various strategies being used to overcome them; all with the aim of motivating interest groups to initiate research activities in cotton biotechnology and to strive for its optimization for further genetic improvement

Irregular deposition of cell wall polymers resulting from defective cellulose synthase complexes

The crystalline cellulose microfibril is formed by the spontaneous association of about 36 β-D-glucan chains, which are simultaneously synthesised by a large membrane-localised multi-enzyme cellulose synthase complex. Antisense technology has been previously employed separately on two of the constituent cellulose synthase catalytic subunits (CesA) of the complex in potato (Solanum tuberosum), namely CesA2 and CesA4, to generate potato tuber cell walls with reduced cellulose content. Genetic crossing of two transgenic potato lines csr2-1 and csr4-8 was carried out to investigate the effects of two defective CesAs in the same genetic background, with respect to cellulose deposition in the potato tuber cell walls. It was striking to observe, through fluorescence microscopy with calcofluor white, a strong fluorescence in the cell corners and less prominent and uneven fluorescence around the cells of the csr2 tubers as compared to others. It was also noted that these phenotypes were not pronounced in the csr2/csr4 double transformants as expected

Endogenous levels of reducing sugars, free amino acids and phenols during various stages of in vitro culture of cotton (Gossypium Spp.)

Somatic embryogenesis is widely preferred as the regeneration route for in vitro studies in cotton. However, the regeneration efficiency through this approach is low; a problem that is believed to be as a result of the biochemical properties of the plant. The objective of this study was to investigate possible relationships between three biochemical factors (reducing sugars, phenols, and free amino acids) and somatic embryogenesis. In vitro cultures of the different embryogenic and non-embryogenic cultivals were established. The levels of reducing sugars, phenols and free amino acids were determined at different developmental stages of the cultures. Higher levels of reducing sugars and lower level of phenol were observed in embryogenic cultivars compared to their non-embryogenic counterparts. There was a general increase in the levels of free amino acids, which decreased with time in the highly embryogenic cultivars, whereas the levels remained high in the poorly embryogenic and non-embryogenic cultivars. The higher content of phenols and free amino acids may be implicated in the poor somatic embryogenic response. The data show that there are factors that may serve as markers of somatic embryogenesis in cotton, which need to be empirically determined for any particular cultivar chosen for genetic improvement through embryogenesis

Molecular analysis and phenotype characterization of the progeny of two antisense potato plants

Two transgenic potato lines csr2-1 and csr4-8, containing two different antisense constructs, csr2 and csr4, respectively, were crossed to investigate the possibility of achieving double transformants with combined effects of the two antisense transgenes on plant phenotypes and cellulose deposition. Molecular analysis revealed an expected segregation ratio of 1:1:1:1 of the four classes. Phenotype characterization revealed that offspring containing either one or both transgenes produced more tubers than the control plants but individual tubers were mostly smaller and had lesser weight than the control tubers

Exciting Times for Cowpea Genetic Transformation Research

Cowpea represents a major food crop for the poor in Asia and especially in Africa. However, its production is constrained principally by insect pests as well as diseases. Attempts to improve cowpea insect resistance have not yielded significant result till date. This paper reviews biotechnological approaches that have been employed to transfer foreign genes into cowpea with a view to conferring desirable traits on it. The recent advances made in generating cowpea transformants with stable inheritance of trangenes by the progenies heralds exciting times for the genetic transformation research on this erstwhile recalcitrant food crop. As such, this genetic transformation approach could be used to transfer insect resistance traits to the crop species

STUDIES ON KOLA TISSUE CULTURE II: Effect of plant growth regulators on callus induction

Callus induction was studied on leaf and single node cutting explants obtained from Cola nitida (Vent.) Schott and Endlicher seedlings. Callus was induced successfully from cut surfaces (periphery) ofyoung leaves in vitro on the medium supplemented with 0.5-5.0 mgl-1 naphtheneacetic acid (NAA), combined with 0.23 mgt- I 6-benzyl-aminopurine (BAP) and on medium supplemented with 0.2-0.6 mgl-1 BAP, combined with 1.0 mgl-1 NAA. Successful callus induction was al so obtained from the buds of single nodal explants cultured on MS medium supplemented with 0.1-1 .0 mgl-1 NAA, combined with 2.3 mgl-1 BAP

Expression of the C-terminal family 22 carbohydratebinding module of xylanase 10B of Clostridium themocellum in tobacco plant

Carbohydrate-binding modules have been shown to alter plant cell wall structural architecture. Hence, they have the potential application of being used to engineer the plant to produce tailor-made natural fibers in the cell wall. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The C-terminal CBM22-2 of the glycoside hydrolase (GH) 10 had been characterized to interact with xylan, a major hemicellulosic component in the secondary cell wall of plants. In this work, the expression of the CBM22-2 in transgenic tobacco plants was evaluated. Histological examinations of the transgenic stems did not reveal marked cell wall phenotype. In addition, there were no observable changes in the height or the appearance of the transgenic plants expressing the CBM22-2 module. The results indicate that the family 22 carbohydrate binding module is not a potential candidate for use in in planta modification of the cell wall

HIGH FREQUENCY MULTIPLE SHOOTS INDUCTION AND PLANT REGENERATION IN SIX ELITE INDIAN COTTON CULTIVARS

Direct multiple shoot induction and regeneration from the cotyledonary nodal explants of two Indian cultivars of upland cotton, G. hirsutum (hybrid H8 and Khandwa-2) and four cultivars of G. arboretum (BD-1, BD-6, Sarvottam and Jawahar Tapti BD) were investigated, using varying concentrations of BAP. An overall average of 5.5 shoots per explant was achieved in the study. The best multiple shoots formation (9 shoots per explant) was obtained from the two G. hirsutum cultivars cultured on 3.0 mg L-1 BAP. Shoots were harvested and elongated in the presence of 0.5 mg L-1 GA3. Root formation was achieved on hormone-free MS medium

STUDIES ON KOLA TISSUE CULTURE I: Protocols for Establishing Kola Tissues in vitro

The micropropagation of Cola nitida (Vent.) Schott and Endlicher by means of tissue culture was investigated to provide baseline infonnation on the requ irements for the survival of kola tissues and organs in vitro. Investigations were conducted on development of sterilization protocols, and medium selection and modification. The best sterilization procedure was established with the step-wise treatment of 70% ethanol, for 20 seconds and 10% (w/v) CaOCl, for I 0 minutes .. The use of modified Murashige-Skoog (MS) medium (without Zn and Cu elements) as basal medium was found as appropriate as the original MS medium, for explants' survival. The appropriate antioxidant technique was also established with 10mgl-1 ascorbic acid