One of the most commonly used methods to produce doubled haploid plants is MICROSPORE culture. In this research work, the isolated MICROSPORE culture system in two rose cultivars i.e. Apollo and Amarosa was investigated. Important factors including isolation media (AB, B), AT3 induction medium with different carbohydrate sources (sucrose, maltose or glucose) and amino acid source (lactalbumin hydrolysate) were studied. Carbon starvation and temperature (heat and cold) treatments as two important stresses alone or in combination with each other for various periods were evaluated on the induction of symmetrical (sporophytic) divisions. A mixture of different developmental stages of MICROSPOREs was used to initiate the cultures but the majority of them were at late uni-cellular stage. For eliminating bacterial or fungal contaminants, buds were surface-sterilized by immersion in 70% ethanol for 15, 30, 60 Sec. or 3.5% (w/v) sodium hypochlorite solution for 5, 10, 15 min prior to MICROSPORE isolation. The best sterilization procedure was observed when MICROSPOREs were sterilized with sodium hypochlorite (%3.5) for 10 minutes. Two isolation media did not show a significant difference on the viability of MICROSPOREs. Among induction media tested, in cv. Amarosa, the highest viability was observed in AT3 medium supplemented by glucose. Induction media in Apollo cultivar did not show a significant difference on viability of MICROSPOREs. Combination of starvation (B medium) and cold (4oC) treatments for 3 days induced formation of pro-embryos in cv. Amarosa. Present investigation reports a protocol for induction of embryogenic developement in rose (Rosa hybrida) MICROSPOREs.

In many crops, androgenesis is one of the most common methods for in vitro haploid induction. The most effective technique in this system is the isolated-MICROSPORE culture, in which haploid cells are reprogrammed to follow the sporophytic pathway. But this pathway has been stopped at the globular shape stage of embryogenesis in eggplant, and efforts are still being made to overcome this problem. In this study, the effects of different concentrations of gum arabic, sucrose, and plant growth regulators (6-Benzylaminopurine, BAP, and 1-Naphthaleneacetic acid, NAA) were evaluated on isolated MICROSPORE cultures from two cultivars of eggplant. In cultivar Ricarda, the highest number of MICROSPORE-derived calli (711.4 per Petri dish) was produced when 2000 mg/l gum arabic, 2% sucrose, 0.5 mg/l BAP, and 0.5 mg/l NAA were used together. By combining 2600 mg/l gum arabic, 2% sucrose, and 0.5 mg/l BAP and NAA, the cultivar Chantale produced the most calli (230.33 per Petri dish; 5.27-fold higher than the control (43.73 per Petri dish). In addition, the results showed that heart-shaped embryos could be produced in eggplant. The culture of MICROSPOREs of cultivar Ricarda in NLN medium supplemented with 2600 mg/l of gum arabic, 2% sucrose, 0.5 mg/l BAP, and 0.5 mg/l NAA led to the developmental progression of some of the globular structures. In fact, the globular embryos were induced to develop into heart-shaped embryos, which is a promising step forward in the process of eggplant MICROSPORE embryogenesis.

The production of haploid/doubled haploid plants allows one to speed up breeding programs, improve selection efficiency, detect linkage and gene interactions, estimate genetic variance and the number of genes for quantitative characteristics, produce genetic translocations, substitutions and chromosome addition lines and facilitate genetic transformation. In breeding programs, besides doubled haploids production, the MICROSPORE culture can be used to produce and maintain malesterile plants via MICROSPORE embryogenesis, possibility to restore male-fertility via in vitro MICROSPORE maturation, to overcome self-incompatibility and to induce and select for mutants. In genetic engineering, Male Germ Line Transformation (MAGELITR) in which DNA is transferred into MICROSPOREs and cultured in vitro to form mature pollen and then using the transformed pollen for pollination in vivo, is very efficient method. In basic studies, the MICROSPORE culture can be used to investigate pollination, pollen development, embryogenesis, totipotency, cell cycle, phase change and the role of stress in development. In this paper, various aspects of in vitro MICROSPORE culture are discussed.

Three spring cultivars of rapeseed (Global, PF and Option) were chosen as plant materials for this experiment. Donor plants were grown in a growth chamber at a day/night temperature of 15/10°C (16/8 hours). MICROSPOREs at late-uninucleate to eariy- bionucleate were isolated from buds 2.5-3.5 mm in length and cultured in modified Lichter medium (NLN-13) containing 13% sucrose. Cultures incubated at 30°C and darkness for 14 days, then transferred to shaker in the growth chamber at 25°C. Results showed that there were significant differences between cultivars at 1% level. The comparison of means showed that PF and Option, with 3412/25 and 3079/5 embryos were the best cultivars for embryogenesis respectively.

In this study, eight commercial Iranian wheat cultivars were tested for their MICROSPORE culture response. Spikes aseptically pretreated in strile inducer formulation [0.1g L-1 of 2-hydroxynicotinic acid, 10-6 mol L -1 2,4-D and 10-6 mol L -1 BAP] and placed in incubator at 33oC for a required period of time, ranging between about 48 and 72 h. MICROSPOREs were isolated through blending of spikes in MICROSPORE is olation soluting (0.3 mol L-1 mannitol). Isolated MICROSPOREs were cocultured with immature ovaries in liquid NPB99 medium at a density of approximately 4x103 MICROSPOREs mL-1 and incubated in the dark and at 27oC. After 3-4 weeks, response of wheat cultivars for embryogenesis was evaluated. Cytological investigations using inverted microscope showed that multicellular structures, were formed in approximately one week after culture. During the second week, the MICROSPORE wall ruptured and immature embryoids emerged which grew into mature embryos within about 10 to 14 days. Analysis of variance indicated significant differences among cultivars for embryogenesis that demonstrates the genetic dependence of this trait. Cultivars of Falat and Moghan1 with means of 1427±81.58 and 1108±54.55 embryos per spike respectively, were identified as the most superior cultivars in MICROSPORE culture.

Introduction: Homozygous doubled haploid lines production through induction of androgenesis is a promising method to accelerate the classical breeding program. However, this technology is relatively under -developed in tomato so that improvements in methodology are required. Tomato (Lycopersicon esculentum Mill) is one of the most important vegetables which in addition of it is importance as a food, is utilized as a model plant for cytological and cytogenetic studies. Tomato breeding programs are often based on the production and selection of hybrid plants. To produce hybrid plants and application of features that is needed to breed pure lines with high specific combining abilities, new technologies such as doubled haploid production through induction of androgenesis can be an effective strategy to provide pure lines in tomato. One of the critical factors for induction of androgenesis in tomato is to use of MICROSPOREs being in appropriate developmental stage. Cytological examination is one of the most accurate methods for determining the correct stage of MICROSPORE development. In this study, a number of characteristics were evaluated including the cytological properties of normal MICROSPOREs development and pollen grains as well as the relationship between length of flower bud and anther length…

Application of doubled haploid method can be used for breeding objectives such ac reduction time of breeding periods. In this research, the effects of photoperiod on embryo production from MICROSPORE culture of three rapeseed cultivars; Global, Option and PF7045/91 and plantlet regeneration in embryo-derived from MICROSPOREs were studied in two separate experiments. In the first experiment, embryo production were analyzed from MICROSPOREs cultured in NLN-13 medium influenced by photoperiod treatments were; darkness, 16/8 (light/dark) and alternative daily darkness and 16/8 (light/dark) in a factorial experiment based on completely randomized design with two factors in 5 replications. The results showed that there were significant differences between two factors and interaction between them on embryogenesis. The numbers of embryos were increased significantly in Global and PF7045/91 cultivars under darkness condition and embryogenesis were laid in group A for this two cultivar. However, the effect of photoperiod was not significant on embryogenesis in Option cultivar. In the second experiment, the plant regeneration in embryos-derived from MICROSPOREs was evaluated in a factorial experiment based on completely randomized design with two factors in 10 replications. Cultivar contain Global, Option and PF7045/91 and embryo-derived from MICROSPOREs under darkness and 16/8 (light/dark) were first and second factor, respectively. The results indicated that only the cultivars had significant differences. The results also indicated PF7045/91 cultivar had the highest plantlet regeneration and was set in group A.

The effect of some factors on embryogenesis from isolated MICROSPORE and plantlet regeneration from MICROSPORE-derived embryos was studied separately in Brassica napus. Experiments carried out in factorial based on completely randomized design. The first factor in all of the experiments was cultivars contained Global, Option and PF7045.91. The MICROSPOREs were isolated from 3-4 mm buds and cultured on NLN-13 medium. Cultures incubated at 30oC and darkness for 14 days, and then transferred to shaker in the growth chamber at 25oC. In regeneration experiment, embryos with 20-25 days old were transferred to B5 medium and nearly 20-25 days after transferring embryos, normal regenerated plantlets, abnormal regenerated plantlets, rooted embryos and non differentiated embryos were counted. In the first embryogenesis experiments, the effects of medium volume, cultivar and interaction effects of two factors were significant. In Global cultivar, the highest values of embryos (695.5 per Petri) were observed in 12.5 ml medium volume. In the second embryogenesis experiment, the interaction between activated charcoal and cultivar on embryogenesis was significant. In the third embryogenesis experiment, the form of carbohydrate had a significant effect on embryo yield. In the first experiment of plantlet regeneration, GA3 had significant effect on normal regenerated plantlets, abnormal regenerated plantlets, rooted embryos and non differentiated embryos and the interaction between GA3 and cultivar was significant for normal regenerated plantlets. In the second experiment of plantlet regeneration, the interaction between gelling agent and cultivar was significant on normal regenerated plantlets.