Altering male fertility by manipulating proteases involved in pollen development

Male sterility and controlling fertility are considered as valuable traits that can be exploited in agriculture to improve crop productivity by selective breeding and hybrid development. Therefore, understanding the processes of anther and pollen development will not only contribute to the understanding of this process, but also to the development of new varieties for crop production. The formation of viable pollen relies on a specific cell layer in the anther, the tapetum, which is critical for pollen wall development and subsequently goes through programmed cell death (PCD) to facilitate deposition of pollen wall materials and viable pollen formation. Tapetum PCD requires the activity of specific proteases, which are associated with proteolytic breakdown. Seven protease genes and one F-Box protein that showed altered expression patterns in Arabidopsis mutants involved in pollen development have been analysed. These target genes consisted of six Aspartyl- protease (AP) genes (AT1G25510, AT2G23945, AT4G30030, AT5G10760, AT3G20015 and AT5G19120); one cysteine proteinase gene (AT2G21430) and an F-box protein, AT3G16210 gene. Defects in pollen development were seen associated with misexpression of AT2G23945. The work presented in this thesis shows that AT2G23945 is expressed in the anther and when mutated has impaired fertility due to impaired tapetum development. AT2G23945 T-DNA insertion- SALK 135523, showed defective pollen and anther development after analysis of fertility and pollen development using DAPI, Auramine O, ethidium bromide acridine orange, Alexander’s staining and microscopy. To gain insight into the expression pattern of the AT2G23945 gene, transgenic plants carrying the AT2G23945pro:GUS and the AT2G23945pro:AT2G23945:GFP translational fusion were generated. Characterization of the AT2G23945-GUS activity in transgenic Arabidopsis plants indicated different regions of regulatory control in the upstream region. Young silique base is the only region of GUS expression for the construct directed by the short promoter. Constructs with a medium and long promoter resulted in expression in anther tissues as expected. No specific subcellular localization of pUBQ10:AT2G23945:YFP could be determined with expression in the plasma membrane and nucleus, which is identical to the control pUBQ10::YFP construct. Further analysis is needed for transgenic plants harbouring pGKGWG:AT2G23945:GFP

In vitro mutagenesis of Etlingera elatior (Jack) and early detection of mutation using RAPD markers.

Mutation breeding techniques in combination with tissue culture and molecular marker methods provide a powerful tool for improvement of vegetatively propagated plants. The aim of this study was to develop a protocol for shoot regeneration and mutation induction of Etlingera elatior. The results of irradiation on in vitro buds of E. elatior showed LD50 to be 10 Gy, with the survival of explants being sharply reduced at this dosage. All 8 selected gamma irradiated regenerants were differentiated from the untreated control based on the banding patterns obtained using 9 primers, which generated 59 reproducible bands, whereby 35 (55.31%) were found to be polymorphic. Jaccard’s coefficient of similarity values ranging from 0.537 to 0.860 were indicative of the level of genetic variation among the mutants studied. For comparison between the potential lines (PL) and the control, a maximum similarity value(0.814) was observed in PL1 mutant, while the minimum value (0.537) was observed in PL7. In summary, a combination of irradiation, regeneration, multiplication, and random amplification of polymorphic DNA (RAPD) analysis for early screening of mutants can speed up the breeding program of E. elatior

Isolation and characterization of beneficial endophytic bacteria from Oryza Sativa cultivated in acid sulfate soil

Abstract: Pyrite (FeS2) oxidation in acid sulfate soil led to high acidity and aluminium (Al) and/or iron (Fe) toxicity to the plant. This problem causes nutrient deficiency and root inhibition, which retards plant nutrient uptake efficiency for growth. Plant-microbe interaction is one of the most important determining factors influencing nutrient uptake and plant growth in acid sulfate soil. Thus, this research aims to isolate and characterize the beneficial endophytic bacteria from Oryza sativa cultivated in acid sulfate soil. The O. sativa samples were collected from a paddy field in Nenasi, Pekan, Pahang. Eight endophytic bacteria were successfully isolated using serial dilution methods after surface sterilization of plant samples was achieved. All isolated bacteria showed different capacities for plant growth-promotion features, including production of plant growth hormone, solubilization of phosphate and converting atmospheric nitrogen into a plant-usable form. This preliminary analysis found that the isolated bacteria have potential for use in integrated plant nutrient management that promotes vegetation growth in acid sulfate soils. Keywords: plant growth-promoting bacteria, indole 3-acetic acid, phosphate solubilization, biological nitrogen fixation

Anatomical, morphological and physiological leaf characters of black betel (Piper betle L. var. nigra) in varying natural and man-made habitats

Piper betle L. var. nigra or black betel (known as Sirih hitam in Indonesia) contains valuable secondary metabolites, such as alkaloids, flavonoids, saponins, tannins, phenols, carotenoids, steroids and terpenoids. Black betel leaf extract has been shown to have antimicrobial activity thus there is a prospect to be developed as a promising herbal plant. Nevertheless, little information is available about its development as a medicinal plant. This research studies the influence of different habitats on several characters of black betel leaves with the aim to understand the suitable environmental conditions for the optimum growth of black betel plants. We used a survey method and random sampling of black betel leaves in four locations in Java Island, Indonesia, namely Banyuwangi which represents natural habitat, and Karanganyar, Ngaglik and Pakem which represent man-made habitats. Measurements of temperature, humidity, soil moisture and light intensity were carried out at each location. Analyses of leaf area, leaf water content, total leaf chlorophyll content and flavonoid content were undertaken and statistically analyzed using SPSS software. Leaf transverse sections were also observed. The results showed that the environmental parameters differed in the four locations. Leaves samples from the natural habitat in Banyuwangi were significantly different (P<0.05) from the three man-made habitats for chlorophyll and flavonoid content. For water content, significant difference was only for Banyuwangi samples with those from Karanganyar and Pakem. For leaf area, significant difference was only found between the Banyuwangi samples and Karanganyar. Observations on the transverse cross section of midrib of black betel leaves from the four locations showed structures that are generally found in Piper betle species, namely the presence of an epidermal layer, trichomes in the abaxial part of the leaf, several layers of the hypodermis, visible vascular tissue and the presence of secretion cells. There were several differences in the leaf anatomy such as greater number of trichomes on the leaves from Karanganyar, the secretory cells that were more visible in the leaves from Ngaglik and Banyuwangi and the sclerenchymal tissue that was more visible in the leaves from Banyuwangi. Such differences are likely influenced by variations in environmental parameters thus showing that the man-made habitat in the Karanganyar location can affect leaves characters similar to black betel plant grown in its natural habitat in Banyuwangi

Callus induction and identification of DNA variation in callus derived from Etlingera elatior in vitro culture using ISSR marker

Etlingera elatior or torch ginger is a promising horticultural plant with various economic values that has been used for medicinal, culinary, and ornamental purposes in many countries. The extravagant and showy inflorescence has made E. elatior a valuable plant that can be used for cut flower and floral decoration. However, the plant itself lack of genetic variety with narrow genetic base due to its nature as an asexual propagated plant. To overcome this problem, somaclonal variation arises from the in vitro culture can be used to overcome the lack of variation in E. elatior. Moreover, early detection of the variation in callus stage using ISSR markers will help to examine the genetic variability of the induced callus. For this project, an optimum sterilization technique with contamination rate of 5% has been successfully developed. Furthermore, white friable calli were successfully developed from the innermost part of young closed buds. The results showed that the Murashige and Skoog medium supplemented with 30 g/L glucose, 3 mg/L 2, 4-D and 1.5 mg/L BAP has the highest percentage of callus induction (50%) after 20 weeks of culture. The calli were transferred into shoot induction media with different concentrations of BAP, NAA and TDZ. The calli from the 11 different media were evaluated for their genetic variations by seven primers of ISSR markers. A total of 72 bands were generated of which 51 were polymorphic with mean percentage of polymorphic bands was 72%. From our results, the calli were affected by various concentrations of auxin and cytokinin for callus and shoot induction treatments. In short, ISSR marker successfully revealed the occurrence of genetic variations in the induced callus. Even though, no in vitro shoots were successfully regenerated, this study revealed that there is a potential to generate new variants through in vitro culture

Optimisation of culture condition for Sacha Inchi (Plukenetia volubilis) Callus induction

Plukenetia volubilis or commonly known as sacha inchi produces wide range of health‐promoting bioactive compounds. The plant has large edible seeds that are rich in phenolics, minerals and essential fatty acid, such as omega 3, omega 6, omega 7 and omega 9. In vitro cultures could serve as alternative in producing many essential sacha inchi bioactive compounds. In this study, as an initial step towards initiating in vitro cultures, the effect of 2,4-D and TDZ on callus induction using leaves and male flower as explants were investigated. Surface sterilization of the sacha inchi explants were done by using 70% ethanol (30 seconds) and 0.5% sodium chloride (8 minutes) to overcome culture contamination. The sterilization method resulted in 82.5% and 95% survival rate for leaf and flower explants, respectively. Next, for callus induction the explants were cultured on MS medium supplemented with different concentrations of 2,4-D and TDZ, either alone or in combination and grown in 24 hours dark photoperiods. The morphology and size of callus obtained varied according to the treatment. Callus produced are either friable or compact with either creamy white, pure white or brownish colour. For both explants, the best response in term of callus size, friability and creamy white callus was obtained when cultured on MS medium supplemented with 3% (w/v) sucrose in combination with 1.0 mg/L 2,4-D and 0.005 mg/L TDZ

Altering male fertility by manipulating proteases involved in pollen development

Male sterility and controlling fertility are considered as valuable traits that can be exploited in agriculture to improve crop productivity by selective breeding and hybrid development. Therefore, understanding the processes of anther and pollen development will not only contribute to the understanding of this process, but also to the development of new varieties for crop production. The formation of viable pollen relies on a specific cell layer in the anther, the tapetum, which is critical for pollen wall development and subsequently goes through programmed cell death (PCD) to facilitate deposition of pollen wall materials and viable pollen formation. Tapetum PCD requires the activity of specific proteases, which are associated with proteolytic breakdown. Seven protease genes and one F-Box protein that showed altered expression patterns in Arabidopsis mutants involved in pollen development have been analysed. These target genes consisted of six Aspartyl- protease (AP) genes (AT1G25510, AT2G23945, AT4G30030, AT5G10760, AT3G20015 and AT5G19120); one cysteine proteinase gene (AT2G21430) and an F-box protein, AT3G16210 gene. Defects in pollen development were seen associated with misexpression of AT2G23945. The work presented in this thesis shows that AT2G23945 is expressed in the anther and when mutated has impaired fertility due to impaired tapetum development. AT2G23945 T-DNA insertion- SALK 135523, showed defective pollen and anther development after analysis of fertility and pollen development using DAPI, Auramine O, ethidium bromide acridine orange, Alexander’s staining and microscopy. To gain insight into the expression pattern of the AT2G23945 gene, transgenic plants carrying the AT2G23945pro:GUS and the AT2G23945pro:AT2G23945:GFP translational fusion were generated. Characterization of the AT2G23945-GUS activity in transgenic Arabidopsis plants indicated different regions of regulatory control in the upstream region. Young silique base is the only region of GUS expression for the construct directed by the short promoter. Constructs with a medium and long promoter resulted in expression in anther tissues as expected. No specific subcellular localization of pUBQ10:AT2G23945:YFP could be determined with expression in the plasma membrane and nucleus, which is identical to the control pUBQ10::YFP construct. Further analysis is needed for transgenic plants harbouring pGKGWG:AT2G23945:GFP

Exploring the potential of torch ginger (Etlingera elatior) as ornamental, medicinal plant, and food products

Torch ginger or Etlingera elatior which belongs to the Zingiberaceae family is one of the most commonly known species of Etlingera. Torch ginger is popular in Southeast Asia where its inflorescences have various purposes in ornamental, medicinal and culinary. Due to its beautiful appearance, it is widely marketed as a promising ornamental plant. For ornamental industry, it is always necessary to produce new and different cultivars with appeal for cut flower. This could be achieved through different techniques such as intensive germplasm collection, hybridization programme and plant biotechnology. Besides, every part of torch ginger can be used as a material source in preparing traditional medicines. For instance, the extract from its stem is used to reduce swelling, the leaves are used by post-partum women and the fruits are used as treatment for earache, diarrhoea, coughs, and mouth sores. In addition, torch ginger has been reported to have high nutritional and phytochemicals properties. Findings of phytochemicals has led to the research for pharmacological activities, cosmetic products, and green nanotechnology of torch ginger. In Thailand, the flower and leaf extracts has been successfully tested for whitening cream. Interestingly, the product is biodegradable and environmentally friendly, which make it a good choice compared to the synthetic ingredients. Meanwhile, researchers in Malaysia have developed an eco-friendly and economical reducing agent from aqueous extract of torch ginger inflorescence, which can be used in green synthesis of gold nanoparticles (AuNPs). It is suggested that torch ginger can be planted into industrial scale and abundance of potentials make this plant as a perfect candidate for the plant of the future

In vitro propagation and mutation induction of torch ginger (Etlingera elatior J.)

The aim of this study was to develop a protocol for in vitro propagation and mutation induction of Etlingera elatior by using gamma ray irradiation. The study included establishment an efficient in vitro plant propagation system in E. elatior,investigation of the optimum dose for radio sensitivity test, to determine the effects of various doses of gamma irradiation on multiple bud induction and also to determine the variation in genomic DNA of regenerated shoots by using random amplification of polymorphic DNA (RAPD) technique. In this study, an efficient and systematic protocol for complete plant regeneration from suckers of Etlingera elatior (J.) has been developed. The addition of N6-benzyl amino-purine (BAP) (0, 3, 5, 7 and 10 mg L-1) to the culture medium comprising of Murashige and Skoog (MS) basal salts, 3% sucrose, 0.4% gelrite did not show any significant effects on percentage of shoot induction and mean number of shoots produced. However, BAP at 3 mg L-1 was chosen as the best medium for shoot induction due to economic feasibility and it gave the highest result in all four parameters recorded. Various concentrations of BAP, 6-furfurylaminopurine (kinetin) and N6-(2-isopentenyl) adenine (2-iP) alone at 0, 3, 5, 7 and 10 mg L-1 were tested for shoot multiplication. BAP at all levels were found suitable for the multiplication of shoot. However, the low level of 3 mg L-1 BAP was chosen as the best concentration of BAP due to economic feasibility. The best root proliferation was observed on MS medium without plant growth regulator (PGR). Assessment of various potting media for acclimatization showed medium containing soil: sand: peat moss (1:1:1) produced high survival of plantlets, number of leaves produced per plant and the plant height. Mutation breeding techniques in combination with tissue culture and molecular marker methods provide a powerful tool for improvement of vegetatively propagated plants. The results of irradiation on in vitro buds of E. elatior showed that LD50 to be 10 Gy with the survival of explants being sharply reduced after this dosage. The gamma irradiated shoots were subcultured for three cycles (M1V1 to M1V3) to obtain potential mutant lines. This study showed that RAPD marker was efficient in differentiating the induced mutants from the untreated control of E. elatior. All eight selected gamma irradiated regenerants were differentiated from the untreated control based on the banding patterns obtained using 9 primers which generated 59 reproducible bands, whereby 35 (55.31%) were found to be polymorphic. The Jaccard’s coefficient of similarity values ranging from 0.537 to 0.860 were indicative of the level of genetic variation among the mutants studied. For comparison between the potential lines (PL) and the control, a maximum similarity value (0.814) was observed in PL1 mutant while the minimum value (0.537) was observed in PL7. The presence of polymorphic bands in 8 potential lines suggested that genetic variation occurred in all the treatments as compared to the control. In summary, the combination of techniques of in vitro propagation, multiplication, gamma irradiation, and RAPD analysis for early screening of mutants can facilitate breeding programme of E. elatior

Assessment of genetic relationships within Bouea (Anacardiaceae) accessions in Peninsular Malaysia using inter simple sequence repeats (ISSR) markers

Malaysia is a genetically diverse hotspot for wild relatives of Mangifera and other Anacardiaceae members. The genus Bouea is interesting in that it has small sweet-sour edible fruits. This genus consists of two species, namely Kundang (Bouea macrophylla) and Remia (B. oppositifolia) which are of economic value. In this study, we highlighted the molecular evidences using inter simple sequence repeats (ISSR) markers to understand its genetic relatedness among accessions. A total of 52 ISSR have been screened on selected accession of Kundang and Remia from Peninsular Malaysia. Ten of the 52 primers for Kundang generated 89 scorable bands, where 45 bands (50.2%) were polymorphic. The simple matching coefficient of similarity provided similarity values ranging from 0.659 to 0.955 while relationship among the different accessions was distributed among three main divergent clusters. For Remia, 10 of the 52 primers tested gave 89 scorable bands, where 54 bands (59.5%) were polymorphic. The simple matching coefficient of similarity values ranged from 0.591 to 0.977 while the cluster analysis separated 23 accessions into two well-defined clusters. These studies indicate that there was considerable ISSR variation among the accessions reflecting variation among the accessions, which were morphologically indistinguishable. DNA polymorphism detected by ISSR analysis offers a useful molecular marker for the identification of different accessions of Kundang and Remia