Embryo Rescue and Moleclar Marker-Assisted Selection of Hybrid Seedless Grape

Seedless grapes play an important role in fresh food and dry production. New varieties breeding by hybridization with seedless varieties as female parents is the most effective way to cultivate seedless varieties. However, the embryos of Seedless varieties can not develop normally, so it is difficult to obtain hybrid offspring as hybrid female parent. Moreover, grape is a perennial tree species with highly heterozygous genes, with long breeding cycle and low efficiency. In this study, embryo rescue technology was used to cultivate hybrid offspring by crossing with ‘Ruby Seedless’ as female parent and ‘Hongqitezao’ as male parent, so as to solve the problem that seedless varieties can not be female parent; and molecular technology was used to carry out assisted breeding research to solve the problems of long cycle and low efficiency. TP-M13-SSR technique was used to carry out authenticity breeding. SCAR marker SCF27-2000 was used to detect the seedless traits of hybrid plants, phenotypic traits was used to verify the results of molecular markers, and Seedless trait-related SSR markers VMC7F2, VrSD10 and P3_VvAGL11 was used to detect and verify the genotypes of individual plants with inconsistent detection results by the two methods. In this study, a total of 384 hybrid offspring were finally obtained, and the hybridization rate was 84.43%. A total of 163 fruit-bearing plants were identified, and the phenotypes of their seeds were identified. The coincidence rate of genotypic and phenotypic analyses was 93.88%. Additionally, 305 F1 plants were detected using the SCF27-2000 marker, and the abortion rate was 64.92%. We speculate that the inconsistent results were caused by parthenocarpy, SCF27 marker limitation, among other factors. Overall, this study shows that embryo rescue is an effective method for breeding seedless grape cultivars, and the application of molecular markers could facilitate the early identification of hybrid traits,and improve breeding efficiency.

Albinism in F1 Hybrids Hinders Geneflow Between Cultivated Chickpea (Cicer arietinum L.) and the Tertiary Gene Pool Species, Cicer Pinnatifidum

Wild Cicer species, especially those in the tertiary gene pool, carry useful alleles for chickpea improvement. The aim of this study was to evaluate the crossability and geneflow between three chickpea cultivars (as female parents) and four cross-incompatible Cicer pinnatifidum accessions (as pollen parents) from the tertiary gene pool. Ten crosses were conducted. One fully developed healthy F1 seed was harvested in vivo from the ICC 4958 × ICC 17269 cross, but the seedling developed an albino phenotype at 4–5 days after germination. Unlike other crosses, those involving the cultivar ICCV 96030 generated a large number of pods with comparatively large ovules. One albino plantlet was obtained from the ICCV 96030 × ICC 17269 cross by embryo rescue. Crosses involving ICCV 10 resulted in flower drop and poor pod set. These variable genotype-specific responses of pod, ovule, and seed development indicate that genetic factors affect the formation of interspecific hybrids. Although pod and seed formation in these interspecific crosses can be improved, geneflow between these materials is hindered by a strong genetic factor conferring albinism in the F1 hybrids.

Shattering tolerance in Brassica napus L.

Brassica is the second-largest oilseed crop after Soybean. The total production of Brassica in the overall world is 71 million tons. In Pakistan, its total production per unit area is very low. Biotic and abiotic stresses mainly affect the brassica crop. In agriculture, shattering is the dispersal of crops seeds before their ripening. The pod wall shatters and breaks apart when it loses its hydration and cells split in a dehiscence zone organized at a suture between the edge of the lignified pod and the vascular tissue replum. The degeneration of middle lamella and loss of cellular cohesion in the dehiscence zone are the main reasons for pod shattering and seed losses. Grain yield losses in Brassica vary from 10 to 25 percent due to shattering. More than 400 kg has-1 or 12% seed losses can be occurred due to pod shattering under unfavorable conditions. Insect pest and disease damage also accelerate ripening and pod shattering. The main breeding techniques for developing rapeseed grain yield potential are a good knowledge and application of the morphological, physiological, and genetic basis of grain yield. Modern technologies, such as embryo rescue, marker-assisted breeding, and novel variation (mutation), may make it much simpler to introduce new rapeseed types having shattering tolerance than traditional methods. Thus, an overview of anatomical and physiological aspects and genetics of shattering is presented in the context of recent advances in molecular genetics and several agronomic managements to avoid shattering in Brassica.

Embryo Rescue of Cold-hardy Table Grapes

Seedlessness is one of the most desirable traits for table and raisin grapes. Stenospermocarpic cultivars are desirable because they have large berries with superior quality. Embryo rescue techniques have been widely used to get progeny seedling populations from crosses using seedless mother plants. Selection of the female parent, sampling time, and the growth medium are the most crucial to the success of this technique. This study investigated the effect of best sampling time and media composition on embryo rescue efficiency in a cold-hardy hybrid grape breeding program. We sampled ovules 5 to 9 weeks after flowering, and we tested four media compositions. The greatest percentages of embryo germination and normal seedlings were obtained when ovules were harvest at 8 weeks after flowering, indicating that it is suitable to harvest ovules at veraison, when the extraction of ovules is easier as a result of softer berry flesh. For the media composition experiment, all ovules were harvested at 8 weeks after flowering. Nitsch & Nitsch culture medium had very low germination, and the resulting seedlings performed the lowest compared with the other treatments. Lloyd & McCown Woody Plant Basal Medium (WPM) increased the number of embryos germinated significantly, and a number of normal seedlings and plantlets developed. Although there was no significant difference among the other three media containing WPM supplemented with different doses of plant regulators, the WPM Plus medium [with cytokinin (6-benzlaminopurine), indole-3-butyric acid, gibberellin, and casein hydrolysate] promoted the greatest percentage of established plants (46.98%). Therefore, the 8-weeks-after-flowering harvest time and the WPM Plus medium were selected for use in the embryo rescue protocol at the University of Minnesota grape breeding program.

Advances in Agricultural Biotechnology

Agricultural biotechnology is becoming the major sector in crop improvement through the use of scientific techniques for the modification of genes conferring resistance to biotic, abiotic stress and improving the quality of crops. With the evolvement from Mendelian genetics to molecular biotechnology, there have been several developments in the field of crop improvement. Recent biotechnological advances have aimed towards removing the physiological constraints of the crops and increasing crop yield potential. With the use of different tools of agricultural biotechnologies like genetic engineering, tissue culture, embryo rescue, somatic hybridization, molecular marker-assisted selection, genome doubling, and omics technologies, various transgenic crops have been developed over the decades and have been approved for commercialization. This development and adoption of transgenic technology have been shown to increase crop yields, reduce CO2 emission, reduce pesticide and insecticide use and decrease the costs of crop production. Even though the biotechnological approach and transgenic organisms have immense potential to contribute to the world’s food security, several concerns of genetically modified crops being a threat to the environment and human health have developed. This review will address applications and concerns of biotechnology in crop improvement considering health hazards and ecological risks.

Joint action of Pb1 and Pb2 provide dominant complementary resistance against new races of Peronospora belbahrii (Basil Downy Mildew)

Sweet basil (Ocimum basilicum, 2n=4x=48) is susceptible to downy mildew caused by Peronospora belbahrii. Pb1 gene exhibit complete resistance to the disease. However, Pb1 became prone to disease due to occurrence of a new virulent races. Here we show that Zambian accession PI 500950 (O. americanum var pilosum) is highly resistant to the new races. From an interspecies backcross between PI 500950 and the susceptible cv ‘Sweet Basil’ we obtained, by embryo rescue, a population of 131 BC1F1 plants. This population segregated 73 Resistant: 58 Susceptible (1: 1, P=0.22), suggesting the resistance is controlled by one incompletely dominant gene called Pb2. To determine whether allelic relationship is existing between Pb1 and Pb2, we used two differential races, race-0, avirulent to both PI 500945 (Pb1) and PI 500950 (Pb2) and race-1, virulent to PI 500945 but avirulent to PI 500950. F1 plants obtained from ‘12-4-6’ (BC6F3 derived from PI 500945) and ‘56’ (BC3F3 derived from PI 500950) showed resistant superiority to both races due to dominant complementary interaction. F2 plants segregated to race-0 as follow; 12:3:1, immune: incomplete resistant: susceptible, as against to 9:3:4 to race-1, indicating Pb1 and Pb2 are not alleles. Since joint action is contributed in F1 plants and in advanced (BC3F3(56) x BC6F3(12-4-6) F4) populations who carrying both genes, it can be assumed that both accessions carry two unlinked genes but share a common signal transduction pathway which leading to dominant complementation superiority of the resistance against different races of BDM.

Development and Verification of an Interspecific Hybridization Protocol for Asclepias

Milkweed (Asclepias sp.) is an important pollinator genus across North America and is a host plant for many butterfly species, notably the monarch butterfly (Danaus plexippus). Commercial production of Asclepias is limited to a few species, because most species lack commercial traits, with minimal branching habit, excessive height, and minimal color variation. This study used a commercially viable Asclepias species, butterfly weed (Asclepias tuberosa L.), as a maternal parent and trialed three different pollination methods in an attempt to create interspecific hybrids. Pollination methods included a traditional method, a pollen–solution-based method, and a novel inverted pollinia method. The inverted pollinia method increased pollination success rates 4-fold among intraspecific crosses of A. tuberosa. When pollination methods were optimized, A. tuberosa was used as the maternal parent, and one-way crosses were made to seven other Asclepias species using the inverted pollinia method. Of the seven species used as pollen donors, four developed hybrid seed successfully: green milkweed (Asclepias hirtella Woodson), purple milkweed (Asclepias purpurascens L.), showy milkweed (Asclepias speciosa Torr.), and common milkweed (Asclepias syriaca L.). As germination methods vary significantly among Asclepias species, three methods of germination were trialed on seed developed via interspecific hybridizations: direct seeded, cold–moist stratification, and embryo rescue. Of the three methods, cold–moist stratification was superior to direct seeding and embryo rescue. This research is the first documented case of a controlled interspecific hybridization event among these species.

Hydropriming Improves Germination and Plant Recovery During Embryo Rescue of Wild Banana Musa acuminata var. tomentosa

Wild bananas are believed to have genes for resistance to biotic and abiotic stress in nature, making them potential genetic resources for creating superior varieties. Wild banana seeds, such as Musa acuminata var. tomentosa are generally difficult to germinate in vivo, so that in vitro embryo culture technique is needed. This study aimed to increase embryo germination and regeneration of wild banana M. acuminata var. tomentosa by soaking the seeds as hydropriming. The treatment comprised of soaking the seeds in sterile distilled water for four periods of time: 0 (control), 1, 4, and 7 days. A total of 45 embryos for each treatment were planted on petri dishes containing MS + 0.5 mg/L BA + 1 mg/L biotin + 1 mg/L proline. The results showed that hydropriming increased the rate of embryo germination and regeneration. Seeds soaked for 1, 4, and 7 days successfully resulted in embryo germination percentages of 87%, 62%, and 62%, respectively, while the control unsoaked seeds germinated with a lower percentage of 42%. One-day soaking treatment was the most optimal treatment to increase the rate of germination and regeneration as well as obtained the best vigor as demonstrated by the highest average height of plantlets, number of leaves, and roots than other treatments. Thus, 1-day seed hydropriming is the best treatment for embryo rescue and regeneration of wild banana M. acuminata var. tomentosa