A. M. R. Ferrie

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

An isolated microspore culture provides an excellent system for the study of microspore induction and embryogenesis, provides a platform for an ever-increasing array of molecular studies, and can produce doubled haploid (DH) plants, which are used to accelerate plant-breeding programs. Moreover, isolated microspore cultures have several advantages over anther culture, wherein presence of the anther walls can lead to the development of diploid, somatic calli and plants. Although protocols for isolated microspore culture vary from laboratory to laboratory, the basic steps of growing donor plants, harvesting floral organs, isolating microspores, culturing and inducing microspores, regenerating embryos, and doubling the chromosomes, remain the same. Over the past few years, a large proportion of the research reports on isolated microspore culture have focused on cereal and Brassica species. For some of these species, isolated microspore culture protocols are well established and routinely used in laboratories around the world for developing new varieties, as well as for basic research in areas such as genomics, gene expression, and genetic mapping. Although these species are considered highly responsive to microspore culture, improvements in efficiency are still being made. However, with many species, isolated microspore culture is simply not yet efficient enough at producing DH plants to be cost-effective for breeding programs. There has been a recent resurgence of haploidy research with response being reported in some species once considered recalcitrant. Future research programs aimed at elucidating pathways involved in microspore induction and embryogenesis will be of benefit, as will novel approaches to improve the efficiency of microspore culture for DH production. With many species, anther culture has proven to be more effective than isolated microspore culture, necessitating more research to clarify the contribution of the anther wall to embryogenesis. The development of molecular markers for use in determining the gametic origin of regenerated plants, irrespective of their ploidy, would also be beneficial. In this review, we aim to provide an overview of the basic isolated microspore culture protocol with an emphasis on recent progress in several crop species.

Transcript Profiling and Identification of Molecular Markers for Early Microspore Embryogenesis in Brassica napus

Isolated microspores of Brassica napus are developmentally programmed to form gametes; however, microspores can be reprogrammed through stress treatments to undergo appropriate divisions and form embryos. We are interested in the identification and isolation of factors and genes associated with the induction and establishment of embryogenesis in isolated microspores. Standard and normalized cDNA libraries, as well as subtractive cDNA libraries, were constructed from freshly isolated microspores (0 h) and microspores cultured for 3, 5, or 7 d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time course. Detailed expressed sequence tag analyses of 3 and 5 d cultures indicate that most sequences are related to pollen-specific genes. However, semiquantitative and real-time reverse transcription-polymerase chain reaction analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BABYBOOM1, LEAFY COTYLEDON1 (LEC1), and LEC2 as early as 2 to 3 d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7 d of culture and that this time point is optimal for isolation of embryo-specific expressed sequence tags such as ABSCISIC ACID INSENSITIVE3, ATS1, LEC1, LEC2, and FUSCA3. Following extensive polymerase chain reaction-based expression profiling, 16 genes were identified as unequivocal molecular markers for microspore embryogenesis in B. napus. These molecular marker genes also show expression during zygotic embryogenesis, underscoring the common developmental pathways that function in zygotic and gametic embryogenesis. The quantitative expression values of several of these molecular marker genes are shown to be predictive of embryogenic potential in B. napus cultivars (e.g. ‘Topas’ DH4079, ‘Allons,’ ‘Westar,’ ‘Garrison’).

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

The availability of a highly efficient and reliable microspore culture protocol for many Brassica species makes this system useful for studying basic and applied research questions. Microspores and microspore-derived embryos are ideal targets for modification by mutagenesis and transformation. Regenerated doubled haploid plants are widely used in breeding programs and in genetic studies. Furthermore, the Brassica microspore culture system allows the identification of genomic regions and genes involved in the microspore embryogenic response, spontaneous diploidization and direct embryo to plant conversion. This review summarizes current achievements and discusses future perspectives.

Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis

Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7 d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10 642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered.

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position.

An overview of preliminary studies on the development of doubled haploid protocols for nutraceutical species

There is very little activity underway to improve the genetics of herbs, spices, and nutraceutical crops. Much of the industry relies on the harvest of “wild” plants; therefore, the potential for variability in performance and active ingredients is high. This presents significant challenges for an industry that is striving to achieve market credibility and meet current regulatory standards. Uniform varieties would also be beneficial for use in clinical trials. The development of plants performing consistently in cultivation under various environmental conditions and producing a stable quality and quantity of desired active ingredients cannot solely rely on traditional plant breeding, but must be supported by the development of tissue culture methods targeted to the species of interest.We have screened over 80 herb, spice, and nutraceutical species for microspore culture response using the Brassica napus microspore culture protocol. The majority of the species did not respond. Swelling and initial divisions of the microspores were observed in some species. Embryogenesis, however, was observed in the Apiaceae and the Caryophyllaceae. Species within these families were selected for further optimization.Improvements in embryogenic frequency were observed in both families. Haploid and doubled haploid plants have been regenerated in anise (Pimpinella anisum), carrot (Daucus carota), caraway (Carum carvi), dill (Anethum graveolens), fennel (Foeniculum vulgare), lovage (Levisticum officinale), laceflower (Amni majus), parsnip (Petroselinum crispum), and cow cockle (Saponaria vaccaria).

Microspore embryogenesis in Apiaceae

Haploid technology is used to develop uniform, true-breeding lines, as well as to accelerate crop improvement programs. Among 20 Apiaceae species screened for response to doubled haploidy, 11 generated microspore-derived embryos, and all but one of the latter yielded doubled haploid plants. Donor plant conditions, basal media, and culture conditions were evaluated for their efficacy on inducing microspore-derived embryos. Growing donor plants at temperature conditions of 10/5 or 15/10°C promoted microspore embryogenesis in fennel (Foeniculum vulgare Mill.), whereas, growing donor plants at a temperature regime of 10/5°C, along with the use of a cold extraction method, enhanced embryogenesis in dill (Anethum graveolens L.) and anise (Pimpinella anisum L.). The culture of fennel and dill microspores in an NLN basal medium and caraway (Carum carvi L.) in AT-3 basal medium promoted the highest frequencies of embryo induction.

Field evaluation of doubled haploid plants in the Apiaceae: dill (Anethum graveolens L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.)

The Apiaceae family includes vegetables, as well as herb and spice crops. Compared to major crops, there have been few breeding or genetic improvement programs for any of the Apiaceae, especially the herb and spice species. Haploidy technology can be used to develop uniform, true-breeding lines, as well as to accelerate breeding programs. Field trials of dill (Anethum graveolens L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.) doubled haploid (DH) lines were conducted over 2–5 cropping seasons. Several of the DH dill lines had desirable agronomic characteristics such as short uniform stature along with early maturity that would be useful for crop improvement. Seed yields and the essential oil content of the seed harvested from the best performing DH dill lines were either equal to or higher than the parental line. A DH annual caraway line was identified that produced higher seed yields than the industry standard. The main constituents of the essential oil for the DH lines of both dill and caraway were similar to the parental lines. Fennel DH lines exhibited differences in height but were too late in maturity for seed production under prairie conditions. The results indicate that not only were we able to generate DH lines that could be used in a crop improvement program, but we developed DH lines that could be used directly as cultivars as these lines performed better than the industry standard (parental line).

Methods and Role of Embryo Rescue Technique in Alien Gene Transfer

Embryo abortion occurs frequently in wide crosses, and thus embryo rescue is required for survival of the next generation. Rescues are performed by either directly transferring the excised embryo to an artificial medium or indirectly through flower (ovary), immature seed (fertilized ovule), or pod (silique) culture. Various techniques used for oil crops, cereals, legumes, and horticultural crops are presented. Altering medium components were the major routes for developing protocols for each species with adaptations to the base medium, sucrose concentration, or vitamin and growth regulator content. Monocot culture tended to be more direct than dicot culture, where many protocols required a multi-step approach from pod to ovule culture to embryo rescue, shoot regeneration, and root induction. Each step required a specific medium and growth conditions. Hybrid embryos as young as 2 days after pollination have been recovered. However, many species such as soybean and chickpea still need procedures for rescue of very young embryos. In other species hurdles such as poor rooting have been overcome by using grafting techniques. Embryo rescue remains a useful component in any breeding program where wide or interspecific crosses are preformed, where rapid cycling through generations is used, and where germplasm preservation is required.

Advances in Microspore Culture Technology: A Biotechnological Tool for the Improvement of Medicinal Plants

Doubled haploidy techniques are well established for some species and are routinely used for practical application and basic research. Haploid/doubled haploid plants can be produced via a number of ways although the in vitro methods of androgenesis (culture of the male gametophyte) or gynogenesis (culture of the female gametophyte) are the most common. For medicinal plants, very little genetic improvement has been undertaken when compared to major crop species. However, callus, embryos, and doubled haploid plants have been produced in several medicinal species using a range of media formulations and culture conditions. This chapter focuses on doubled haploid production from the male gametophyte and the factors influencing this response.Doubled haploidy methodology would be beneficial for medicinal species as uniform, homozygous, true breeding lines would be available for growers. Uniformity in plants and in the active ingredient is very important for regulatory standards and for those conducting clinical trials.