Bushra Sadia

Biodiversity in the sorghum (Sorghum bicolor L. Moench) germplasm of Pakistan.

Sorghum ranks fifth in worldwide economic importance among cereal crops and is one of the most important summer annual grasses of Pakistan. As it is a very diverse crop, sorghum genetic fingerprinting requires an efficient marker system. We estimated genetic divergence among 29 sorghum (Sorghum bicolor) genotypes, including approved varieties and local and exotic lines collected from different ecological regions of Pakistan, using random amplified polymorphic DNA (RAPD) markers. A total of 125 RAPD loci, with an average of 66 loci per genotype, were used to calculate genetic divergence among these genotypes, of which 119 were polymorphic, showing 95% overall polymorphism. Genetic similarity ranged from 0.36 to 0.92, indicating a relatively broad genetic base. RAPD analysis revealed maximum similarity between the Indian III and K-A-113 sorghum genotypes (both exotic lines), while the F-601 and F-606 were observed to be the most diverse genotypes. Mean band frequency revealed by these RAPD primers ranged from 0.17 to 0.56, with an average of 0.36. The data presented here support the findings that RAPDs can be effectively used for studying genetic diversity in sorghum.

Haemoglobin-enhanced mitosis in cultured plant protoplasts.

Potato (Solanum tuberosum) is the world’s fourth most economically important food crop, following wheat, rice and maize. Potato breeding is slow compared to that of other major crops, making it an important candidate for in vitro genetic manipulation involving direct gene transfer into isolated protoplasts (‘naked cells’, from which the walls have been removed by enzymatic digestion), and somatic hybridisation or cybridisation. Both of the latter techniques are based on the fusion of protoplasts from potato with those of other Solanum species. A pre-requisite for such investigations is reproducible growth in culture of protoplasts to cells, followed by the differentiation of protoplast-derived tissues into fertile plants. An adequate and sustainable oxygen supply is a fundamental requirement to maximise growth of protoplasts and protoplast-derived cells. Previous studies have demonstrated the beneficial effects of medium supplementation with the chemically-modified haemoglobin (Hb), Erythrogen TM,on mitotic division of protoplastderived cells and subsequent plant regeneration in Oryza sativa (Azhakanandam et al., 1997; Al-Forkan et al., 2001), Passiflora giberti and Petunia hybrida cv. Comanche (Anthony et al., 1997). However, there have been no corresponding studies with major dicotyledonous crops, such as potato.

Alleviating effect of exogenous application of ascorbic acid on growth and mineral nutrients in cadmium stressed barley (Hordeum vulgare) seedlings.

An experiment was carried out in sand-filled pots under normal temperature (28±2oC) to assess the role of exogenously applied ascorbic acid in alleviating the effect of cadmium (Cd) stress on four barley (Hordeum vulgare L.) genotypes (Jau-83, Jau-87, Paidar 91 and Haider 93). After germination, seedlings were exposed to different Cd concentrations (0, 100, 300, 500 and 700 μM CdCl2) along with AsA (200 mg L) and grown for 15 days. The results suggested that exposure to increased Cd levels caused a significant reduction in growth and mineral nutrients contents of barley seedlings. However, there was a noticeable difference in the effect of Cd on mineral concentrations among genotypes and the difference mainly coincided with differential accumulation of Cd in the shoot and root tissues. When AsA was applied to Cd-stressed plants, it decreased Cd accumulation in shoots and roots and also showed partial reversal of Cd stress effects. It was also observed that at the same Cd concentrations Cd tolerance index of Jau-83 was the highest among the four barley genotypes, indicating that Jau-83 had lower Cd contents in roots may be more tolerant to Cd stress. The application of AsA was effective in reducing the toxicity of increased Cd by reducing the root or shoots Cd contents, as well as by improving the seedling growth attributes and the mineral nutrients in barley.

Estimation of genetic diversity using SSR markers in sunflower.

Microsatellites or simple sequence repeats (SSRs) were used for the estimation of genetic diversity among a group of 40 sunflower lines developed at the research area of Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad. Total numbers of alleles amplified by 22 polymorphic primers were 135 with an average of 6.13 alleles per locus, suggesting that SSR is a powerful technique for assessment of genetic diversity at molecular level. The expected heterozygosity (PIC) ranged from 0.17 to 0.89. The highest PIC value was observed at the locus C1779. The genetic distances ranged from 9% to 37%. The highest genetic distance was observed between the lines L50 and V3. Genetic distances were low showing lesser amount of genetic diversity among the sunflower lines.

Development of a species-specific sequence-characterized amplified region marker for roses.

DNA fingerprints of four rose species, Rosa centifolia, R. Gruss-an-Teplitz, R. bourboniana, and R. damascena, were developed using RAPD-PCR. We identified a unique polymorphic band in R. centifolia. This 762-bp fragment was produced by the random primer GLI-2. The fragment was eluted and directly cloned in a TA cloning vector, pTZ57R/T. Digestion of the plasmid with EcoRI confirmed the cloning of GLI-2(762) in pTZ57R/T. A second enzyme, PstI, used in combination with EcoRI, gave complete digestion of the plasmid, and the 762-bp fragment was confirmed on the gel. Subsequently, the polymorphic amplicon was sequenced with an AB1 373 DNA sequencer system using the PRISM(TM) Ready Reaction DyeDeoxy(TM) Terminator Cycle Sequencing kit. After sequencing, specific primers (23 bp long) were designed based on the sequence of the flanking regions of the original RAPD fragment. These primers will effectively allow fingerprinting for the identification of R. centifolia species. In essence, we developed an SCAR marker to authenticate the identity of R. centifolia species and to distinguish it from its substitutes. Such techniques are required not only to complement conventional parameters in creating the passport data of commercial and medicinal products of rose, but also for routine quality control in commercial and government rosaries and rose nurseries.

Use of TALEs and TALEN Technology for Genetic Improvement of Plants

Genome editing with engineered nucleases has become a powerful tool of targeted genome modifications providing unprecedented control over animal and plant genetic material for precise, robust and highly specific genome engineering. Precise genome editing has been a long standing goal in the field of biology which has been achieved with the help of engineered nucleases like zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated system. These engineered nucleases consist of a binding and a nuclease domain which are generally used in the form of a pair. The binding domain binds specifically to a DNA sequence whilst the nuclease domain creates double-strand breaks (DSBs) which are further used for non-homologous end joining or homologous recombination repair. Creation of DSBs is the principle of this technology which can be further used for gene addition, deletion and modification in the targeted DNA. Besides nuclease activity, TALE (transcription activator-like effector) proteins have also been used along with other effector domains for different purposes like gene activation, gene repression, epigenetic modifications, etc. The use of TALEs and TALENs for precise genome modifications of plants is now a common practice. So far, tens of crop plants have been modified using engineered nucleases like rice, wheat, tomato, potato, tobacco, maize, barley, cotton, etc. The TALE and TALEN technology is being used for development of biotic and abiotic stress-resistant plants as well as yield and quality improvement. In this article, we will briefly review and discuss TALEs and TALENs, their discovery, binding specificity, designing, functional domains, delivery and use for genome editing specifically in plants.

Establishment of efficient in vitro culture protocol for wheat land races of Pakistan

The reliability of the production and presence of disease resistance especially rust has sparked a renewed interest in improving landraces and exploiting these in wheat variety development programs. In vitro culture is a pre-requisite for most of the tools of biotechnology. In this context, three Pakistani wheat ( Triticum aestivum L.) land races viz., LLR-13, LLR-15 and LLR-16, having leaf rust resistance were assessed for in vitro plant regeneration response. Immature embryos were cultured on Murashige and Skoog medium supplemented with 2, 4, 6 and 8 mg/L of 2,4-Dichlorophenoxyacetic acid (2,4-D) for callogenesis and MS + zeatin riboside (1.0 mg/L) medium for regeneration of these calli. All three land races produced callus on all 2,4-D concentrations; higher doses (8 mg/L) being the most effective for callus fresh weight. However, there was a strong carry over effect of 2,4-D on regeneration capacity. Calli induced at 2 mg/L of 2,4-D showed the highest regeneration frequency for LLR-16, LLR-13 and calli of LLR-15 induced on 4mg/L were more regenerative. Overall, LLR-16 produced the maximum regenerants. Rooted plantlets were transferred to glasshouse for further evaluation. Key words: Leaf rust, immature embryos, wheat land races, regeneration, Murashige and Skoog (MS) medium.

Screening of sunflower (Helianthus annus L.) accessions under drought stress conditions, an experimental assay

Drought is the major abiotic stress that limits the crop production at drastic level. Screening of tolerant accessions from available germplasm is the basic step in plant breeding. Sunflower is becoming popular and major oilseed crop in world but unfortunately it is drought sensitive. Screening in field has uncertainties due to the uncontrolled conditions, interaction of biotic and abiotic stresses and variability in environmental factors. Response of the sixty sunflower accessions to drought stress at germination and seedling stage was examined by using polyethylene glycol (PEG-6000) as drought simulator under laboratory. Normal and drought stress treatments i.e. T1= zero (control), T2= -1.33 MPa and T3= -1.62 MPa were developed by dissolving 0, 15 g and 20 g Polyethylene Glycol (PEG-6000) in 100 mL distilled water and a completely randomized design with three replications were used. Promptness, germination, seedling height, fresh weight, dry weight and stress indexes were determined to evaluate the response of sunflower accessions under normal and PEG simulated drought stress treatments. Principal component analysis was used to select the drought tolerant and sensitive accessions. The accessions 017583, A-75, A-79, 017592, G-33, A-48, A-23, G-61, HBRS-1 and 017566 were selected as drought tolerant while, CM-621, 017577, HA-124, HA-133, HA-342 and HA-341 were as drought sensitive. This study may be helpful for the comparison of drought indexes in a controlled experimental assay and for the identification of drought tolerant sunflower cultivars to be used in further breeding programs.

Gender Identification in Date Palm Using Molecular Markers

Breeding of date palm is complicated because of its long life cycle and heterozygous nature. Sexual propagation of date palm does not produce true-to-type plants. Sex of date palms cannot be identified until the first flowering stage. Molecular markers such as random amplified polymorphic DNA (RAPD), sequence-characterized amplified regions (SCAR), and simple sequence repeats (SSR) have successfully been used to identify the sex-linked loci in the plant genome and to isolate the corresponding genes. This chapter highlights the use of three molecular markers including RAPD, SCAR, and SSR to identify the gender of date palm seedlings.

Initiation and Maintenance of Cell Suspension Cultures of Two Citrus Species for Protoplast Isolation

Cell suspension cultures were initiated from six month old ovule-derived embryogenic calli of two Citrus species named Citrus aurantifolia (Christm.) Swingle cultivar Sweet lime and Citrus reticulata Blanco cultivar Kinnow. The growth rate of cells was observed on two different media MT basal medium + 0.5 g L −1 malt extract (EME) and MT containing half strength NH4NO3 and KNO3, supplemented with 1.55 g L -1 glutamine and 0.75 g L -1 KCl (H+H). Kinnow and Sweet lime cells showed a sigmoidal growth pattern on (H+H) and (EME) liquid media, when PCV was plotted against time. The logarithmic phase of cells started from 4 th to 10 th day and after that a stationary phase observed in cells with elongated vacuoles. This indicated that citrus cells active phase started soon after subculture and ends after two weeks. These embryogenic suspension cultures were later used for protoplast isolation which is a key step towards somatic hybridization.