Nabil Ahmad

This study assessed genetic diversity and population structure among 23 citrus genotypes comprising 14 grapefruit ( Citrus paradisi ), seven pummelo ( Citrus maxima ) and two sweet orange ( Citrus sinensis ) genotypes using inter‐primer binding site (iPBS) markers. Twenty primers produced 425 amplified bands, of which 396 were polymorphic (93.18% overall). Primer‐specific polymorphism ranged from 86% to 100% and polymorphism information content (PIC) ranged from 0.21 to 0.35, indicating moderate marker informativeness. Species‐group diversity estimates were highest for C. paradisi (97.44% polymorphic loci), followed by C. maxima (84.62%); estimates for C. sinensis were substantially lower (25.65%) but should be interpreted cautiously because only two genotypes were sampled. The first two principal coordinates explained 32.06% of the molecular variation and showed partial, overlapping species‐associated structuring. Analysis of molecular variance (AMOVA) attributed 92% of the variation to differences within species groups and 8% to differences among groups (PhiPT = 0.080, p = 0.020). Bayesian analysis supported three major genetic components at K = 3; some genotypes were strongly assigned to one component, whereas others had mixed membership. These results provide a preliminary molecular baseline for citrus germplasm characterisation in Punjab and require confirmation through larger, balanced sampling and codominant or sequence‐based markers.

The Australian tomato Solanum orbiculatum ssp. orbiculatum is an edible bush tomato endemic to the more arid areas of Western Australia, South Australia, and the Northern Territory. Breeding system data indicate that the plants are potentially self-compatible but are unable to carry out spontaneous autogamy or agamospermy. The flower is protogynous, as the stigma become receptive to pollen germination while still in bud condition and the anthers do not release pollen immediately after anthesis. This arrangement is a simple and common way to avoid too much self-pollination, favours cross pollination, and would allow forced bud pollination for hybrid development. The floral structure and morphology of this species can also encourage cross pollination, as the stigma is mostly exserted above the anther’s tips. In an attempt to examine the hypothesis of a positive correlation between pollen grain size and style length, we found a statistically significant difference between the pollen size of the long-styled and short-styled flowers. Pollen in vitro germination and viability tests have been optimised to facilitate effective breeding work on this species. A modified Brewbake and Kwack (BK) medium supplemented with 20% sucrose and 2.5% PEG 4000 has been found to be the most efficient media components for the in vitro germination of viable pollen grains. Alternatively, Alexander’s and acetocarmine (1%) stains have shown the highest positive correlation with the in vitro pollen germination test and, therefore, can be used as quick tests for checking pollen viability. Moreover, pollen grains stored for three months under 4 °C and dry conditions can be used efficiently to effect fertilisation in breeding programs, as it can maintain more than 50% of the original viability. This study will contribute to understanding the evolution and systematic relationships of species and for founding effective conservation programs. Furthermore, understanding the reproductive biology of this species is also of interest because of its potential for tomato breeding.

Okra is a vegetable crop adapted to summer temperatures, but heat stress has been shown to reduce its growth and productivity. We measured physiological traits of 104 okra genotypes in response to high temperature, augmented by the molecular characterization of selected genotypes to identify parents for crossing. Genotypes were exposed to a short heat shock (45 °C, 4 h) in a controlled environment, followed by the assessment of chlorophyll fluorescence (F_v/F_m, F_v′/F_m′) and stomatal conductance (g_s). DNA was isolated from all genotypes using a modified CTAB method with additional PVP and RNase, and the amplification of 8 polymorphic SSR markers was used to generate a dendrogram. This preliminary screening identified 33 polymorphic genotypes with less than 50% genetic similarity and contrasting F_v′/F_m′ and g_s responses. More detailed physiological measurements (F_v/F_mF_v′/F_m′, g_s, photosynthesis (A), efficiency of the open reaction centre (Φ_PSII), and electrolyte leakage (EL)) were conducted after exposure to 45 °C for 6 h and compared to the control (30 °C). EL did not significantly increase in the heat treatment; in contrast, there were significant genotype and treatment effects observed for fluorescence (F_v/F_m, F_v′/F_m′) and photosynthetic parameters (A, Φ_PSII, g_s). In conclusion, cell membranes in okra remained unaffected after short periods of heat stress, whereas the ranking of differences of measured physiological traits (∆) between control and heat-treated plants (∆F_v′/F_m′, ∆F_v′/F_m′, ∆A, ∆Φ_PSII, ∆g_s) was indicative of genotype sensitivity to heat.

The use of biotechnology for the genetic improvement of wheat (Triticum aestivum L.) has been hampered by its recalcitrance to standard transformation and regeneration protocols. Male gametes present a potentially useful option for introducing gene edits via clustered regularly interspaced short palindromic repeats (CRISPR). However, the utility of male gametes for introducing genetic improvements would be dependent on the retention of viability after treatment to introduce the CRISPR components. We have studied wheat pollen morphology and its viability in a range of germination media to identify conditions that optimize the viability of in vitro hydrated pollen. The size, shape, and aperture from seven different wheat genotypes were compared using scanning electron microscope (SEM). The SEM results revealed that the pollen of all of the wheat genotypes examined in this study were monoporate; however, a significant variation in the size of the mature pollen grains was observed. The hydrated pollen of the wheat genotypes remained viable for up to five hours at 20 ± 2 °C. Of all of the germination media tested, the medium containing 5% sucrose, 10% PEG4000, 100 mg/L boric acid, 200 mg/L calcium nitrate, 100 mg/L potassium nitrate, and 100 mg/L magnesium sulphate at pH 6.5 achieved the highest percentage of pollen germination after 5 h of hydration. Impedance Flow Cytometry (IFC) provided similar results to the in vitro germination study. This work elucidates important factors that can form the basis for a pollen-based non-genetically modified system for gene editing in wheat.

The utility of CRISPR in plants has remained limited by the dual difficulties of delivering the molecular machinery to target cells and the use of somatic cell techniques that require tissue culture-based de novo organogenesis. We developed 5-10 nm isodiametric polyplex nanoassemblies, comprising poly [2-(dimethylamino)ethylmethacrylate] PDMAEMA (PD) polycationic linear homopolymers and CRISPR/Cas9 ribonucleoproteins (RNPs), that enable endocytosis-driven RNP uptake into pollen grains. Pollen from wheat plants (genotype Gladius+Sr50), homozygous for monogenic Sr50 -mediated resistance to stem rust ( Puccinia graminis f. sp. tritici -Pgt), were incubated with RNP/PD nanoassemblies targeting the dominant, Sr50 rust resistance gene. The treated pollen grains were then used to fertilize Gladius+Sr50 florets and the resulting M1 plants were tested for loss of Sr50 function via rust resistance screens. The identification of fully susceptible M1 seedlings indicated that the Sr50 RNPs acted on both alleles, indicating they were transferred via the treated pollen to the zygote. The ability to readily deliver CRISPR RNPs to reproductive cells via biodegradable, polymeric nanocomplexes has significant implications for the efficiency of gene editing in plants.

Motherwort (Leonurus cardiaca L.) is an annual species indigenous to central Europe and Scandinavia and has been used in traditional medicine because of its health benefits. The reproductive biology of L. cardiaca was investigated to provide a basis for the genetic improvement of secondary metabolites or extracts that could have human health benefits. L. cardiaca is self-compatible but bears protandrous flowers, which promote cross-pollination. The stigma becomes receptive 2 or 3 days after anthesis and anther dehiscence. Phenological observations revealed that the mean flowering duration was approximately 2 weeks within each inflorescence and 2 months within a plant. The timing of anthesis of flowers within each inflorescence and flowers of different inflorescences is usually synchronous, leading to the possibility of geitonogamous pollination. A high degree of synchronisation in flowering was observed among the plants within each of the populations studied. Among the populations, Khansar was the first to flower, whereas Dargaz was the last. A modified Brewbaker and Kwack (BK) medium optimised for in vitro germination of L. cardiaca pollen was used to establish a rapid and simple test that generally correlated with seed set. The optimised medium comprised 15% sucrose and 2.5% polyethylene glycol 4000. Data from staining with iodine–potassium iodide solution (IKI) and Alexander’s stain were positively correlated with in vitro germination and, therefore, could be used in rapid pollen-viability assays for L. cardiaca.