Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine-supplemented media

Plating rice anthers on a semisolid induction medium containing 250 or 500 mg/l colchicine for 24 or 48 h-incubations followed by transfer to colchicine-free medium and standard anther culture procedures resulted in overall 1.5- to 2.5- fold increases in doubled haploid green plant productions compared to control anther cultures. The addition of colchicine had no detrimental effects on the different anther culture efficiency parameters, but in some treatments led to significant enhancement of anther callusing frequency or callus green plant regenerating ability. The most efficient treatment raised doubled haploid plant recovery from 31% to 65.5%. These results suggest that post-plating colchicine treatment of anthers, since it was found to improve both anther culture efficiency and doubled haploid plant recovery frequency, could be integrated into rice doubled haploid plant production programmes.Abbreviations DH doubled haploid - NAA naphthalenacetic acid - PAS periodic acid Schiff     

Rapid attainment of a doubled haploid line from transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants by means of anther culture

Summary We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.     

Comparative mapping of the barley genome with male and female recombination-derived, doubled haploid populations

Male (anther culture) and female (Hordeum bulbosum) derived, doubled haploid populations were used to map the barley genome and thus determine the different recombination rates occurring during meiosis in the F1 hybrid donor plants. The anther culture-derived (male recombination) population showed an 18% overall increase in recombination rate. This increased recombination rate was observed for every chromosome and most of the chromosome arms. Examination of linkage distances between individual markers revealed eight segments with significantly higher recombination in the anther culture-derived population, and one in the Hordeum bulbosum-derived population. Very strong distortions of single locus segregations were observed in the anther culture-derived population, but map distances were not affected significantly by these distortions. There were 1.047 and 0.912 recombinations per chromosome in the anther culture and Hordeum bulbosum-derived doubled haploid populations, respectively.     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Anther culture in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) in vitro

Pepper (Capsicum annuum L.) is an important vegetable crop that can be improved using plant tissue culture and biotechnology. However, it is difficult to develop appropriate breeding material by in vitro cultivation in this species. Haploid plant production is useful in the breeding programs to facilitate recovery of recessive mutations and unique genetic recombinations. In embryogenesis, haploid formation from pollen in anther culture is a scientifically advanced, but controversial system. Various techniques for haploid plant regeneration are used to establish an efficient double haploid production method. The purpose of this article is to summarize, through comparison, results in pepper anther culture, problems associated with work in this field, and the influence of critical factors for successful embryo formation and plantlet development.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH doubled haploid - LS Linsmaier and Skoog basal medium - BAP benzylaminopurine - GLM Generalized Linear Model - SAS Statistical Analysis System     

Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) anther cultures

The generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative to the classical inbreeding and selection programs. However, this technology is poorly developed in tomato, where doubled haploid tomato plants have only been obtained through anther culture. Despite the fact that anther culture is routinely used in a number of economically interesting crops, there are still many drawbacks that prevent tomato breeders from adopting this technique, and improvements in methodology are required. One key issue is the correct identification of the optimal stage for anther excision and culture. In this paper we characterise in vivo microsporogenesis in tomato, defining the different microspore stages and relating them to the length of the donor flower bud. In parallel, we cultured anthers of these stages to obtain embryogenic callus, and followed the microscopic development of the callus contained within the anther. Our data suggest that the stage with the highest response, in terms of callus generation, is meiosis. In particular, we propose the window from metaphase I to telophase II, including tetrad cellularisation, as the timeframe where induction can be accomplished in tomato anther cultures.     

Anther culture of kale (Brassica oleracea L. convar.acephala (DC.) Alef.)

The pollen development and androgenic ability of 18 kale (Brassica oleracea convar.acephala) genotypes was observed during an anther culture study. Anther culture was successful in 6 of the genotypes and the highest yield obtained was 17 embryos per 100 anthers plated. Two stages of anther development were identified as being responsive to anther culture. The first and most responsive was that corresponding to the late uninucleated stage and the second to the late binucleated stage. These stages correspond with the onset of mitotic events in the microspores. Pollen viability was studied and low viability was noted which declined to zero after 9 days of anther culture. The initial viability level however was not clearly related to androgenic ability. The significance of the production of haploid and dihaploid kale genotypes in the study and breeding of resistance to clubroot is discussed.     

The statistical analysis of potato anther culture data

Methods for the production and utilisation of haploids have been developed for a range of crop plants. Genetical and environmental factors are known to influence both the rate of haploid induction and the mode of regeneration. Investigations designed to examine the parameters that influence haploid production from anthers are usually based on the overall percentage of responding anthers. Previous studies have rarely taken into account the frequency or distribution of anther culture response within a potato flower. In this paper the binomial, poisson and multiplicative binomial distributions are used for the first time to describe the distribution of anther culture response within a Solanum tuberosum flower.     

Effect of polyamines on in vitro anther culture of Citrus clementina Hort. ex Tan

The improvement of the induction rate in Citrus anther culture is important for taking practical advantage of the haploid potential in breeding. The influence of polyamines on anther culture of Citrus clementina, cv Nules, with particular attention to the free, soluble and insoluble-conjugated polyamine levels, has been investigated. Putrescine, spermidine and putrescine plus spermidine, were added to the standard induction medium. Before culture, spermidine was the most abundant among the free polyamines detected in anthers. The exogenous supply of either putrescine or spermidine, either independently or combined, effected greater uptake and accumulation of polyamines. The addition of 2 mM spermidine to the medium stimulated gametic embryogenesis in clementine Nules, whereas putrescine did not influence embryo production. Regenerants were mostly tri-haploids; a few doubled-haploids and no haploid plants were obtained.     

Stimulation of embryogenesis and haploid production in Brassica campestris anther cultures by elevated temperature treatments

Summary Culture of Brassica campestris anthers at 35°C for one or three days prior to culture at 25°C significantly stimulated the yield of microspore-derived embryos. More than 100 plants were regenerated from cultured embryos and haploids were identified amongst them. The haploid frequency was greater than 70% if all small-flowered sterile plants were considered to be haploid. The yield of microspore-derived plants in B. campestris is approaching the level where anther culture may be utilized as a practical breeding tool.     

Reflexions sur nos cultures d'antheres de plantes cultivées

Abstract

Considerations about our anther cultures of cultivated plants. – One of the main activities performed at the Casaccia Nuclear Centre, in the framework of a contract between CNEN and the European Communities, centers on the induction of haploid plants by anther culture and the subsequent chromosome doubling in order to obtain completely homozygous diploid plants. In tobacco, it is now possible to obtain haploid plants from any cultivar; we perform in vitro culture of internodes from which homozygous diploid plants are regenerated, taking advantage of natural phenomenon of endopolyploidy. In order to try to generalize this method of producing haploid plants in other plant species, we are studying the mechanism involved in haploid embryogenesis which occurs in vitro in the microspores. Datura, Nicotiana and Atropa are among the genera in which a direct embryogenesis from the microspore is observed; it is interesting to note that all three genera belong to the family Solanaceae and are very rich in alkaloids. In almost all the other cases of in vitro induction of haploids, microspores produce calli from which plantlets can be differentiated, but this way of plant regeneration is less interesting because only few plantlets are obtained and it is not sure that each haploid comes from a single microspore. We examined the factors which could influence the transformation of microspores into embryoids in tobacco, namely: the developmental stage of microspore, the degeneration of tapaetal cells, the genotype of microspore, the composition of cultural media, the physiological conditions of the plant from which the anthers were taken. From a practical point of view, it would be desirable to have informations on methods giving a maximum number of haploid plants from one embryogenic anther and the greatest number of embryogenic anthers from the cultured anthers. Our recent experiments on anther culture in liquid shaken medium have yielded good results (about 7,000 embryoids from 25 embryogenic anthers). Further, we are conducting several experiments in order to synchronize the development of the microspores in the anthers; to this end, we analyse the effect of cold treatment, ionizing radiation and gravity force. Experiments are being performed with other cultivated species, beside tobacco, in order to solve some problems of plant breeding more easily and quickly through haploidy. With the aim of introducing, in cultivated tomato, some desirable characters from the wild species, Lycopersicum peruvianum, (self-incompatibility, disease resistance, simultaneous flowering), we have obtained the interspecific hybrid through in vitro culture of young embryos. Haploid production from this hybrid could allow to quickly obtain various genetic recombinations from these two species. For this purpose we are carrying out anther cultures as well as single microspore cultures. In rice, strawberry and L. peruvianum, several diploid and tetraploid plantlets were obtained from our anther cultures. Work is in progress to ascertain the mode of their origin.     

The agronomic performance of anther culture derived plants of barley produced via pollen embryogenesis

Anther culture was used to generate microspore-derived doubled haploid (DH) plants from four spring barley crosses. The culture medium used contained maltose as the sole carbohydrate source and the mode of plantlet regeneration was mainly via pollen embryogenesis. Both haploid and spontaneously doubled regenerants were produced and the doubled haploids were compared to recom-binant inbred lines generated by several rounds of selfing (single seed descent). Parental, DH and single seed descent (SSD) lines were grown in randomised, replicated field trials and the samples were scored for a range of agronomic traits. The mean performance and phenotypic distribution of the DH and SSD samples were similar and there was little evidence to support the conclusion that anther culture derived lines exhibit a reduction in vigour. Where significant differences were detected between groups these were mainly confined to crosses which were segregating for the denso dwarfing gene. The differential transmission of particular regions of the barley genome may therefore influence and confound the expression of agronomic traits in DH populations. This is the first report of the agronomic performance of anther culture lines produced via pollen embryogenesis and the results are discussed in relation to the exploitation of anther culture technology in barley breeding.     

CYTOLOGICAL CONSEQUENCES OF DNA AMPLIFICATION IN AN ANTHER CULTURE-DERIVED DOUBLED HAPLOID LINE OF NICOTIANA TABACUM

An increase in nuclear DNA, without an increase in chromosome number, has been found to occur as a result of anther culture in Nicotiana tabacum L. The objective of this study was to determine the cytological consequences of this DNA amplification in F₁ hybrids between a doubled haploid that had undergone a substantial increase in DNA and the cultivar from which that doubled haploid was derived. Mitotic and meiotic analyses were performed on plants obtained from reciprocal crosses of N. tabacum cv. NC95 and NC95 SCDHL 12, a doubled haploid line that has 41% more DNA than the parental cultivar. While no cytological abnormalities were observed in either parental line, numerous abnormalities were seen in both somatic and meiotic tissues of the F₁ hybrid. Chromosome losses, which appeared to result from spindle errors, were observed in these tissues. It is speculated that the spindle errors may be the result of a genetic unbalance caused by combining genomes with widely differing amounts of DNA. In addition to the spindle errors, a quadrivalent with an atypical morphology was observed in meiotic diplotene and metaphase I cells of the hybrid. The quadrivalent configuration was interpreted to represent pairing between amplified homologous regions in homeologous chromosomes. Further investigations of additional doubled haploid × cultivar lines is required before the significance of these findings to the anther culture process in N. tabacum can be fully assessed.     

Frequency of induced mutations at the haploid and diploid levels inNicotiana tabacum L.

The frequencies of chlorophyll mutants were investigated in anther cultures derived from mutagen-treated plants ofN. tabacum cv. Samsun (haploid level) and in the seed offspring from the same treated plants (diploid level). Comparison of the induced mutation frequencies at the haploid and diploid levels demonstrated that selection existed against the haploid embryoids with induced chlorophyll deficient mutations. The diploid vegetative stage with phenotypic expression of the chlorophyll mutation was more vital than the haploid one. The suitability of anther cultures for studying induced mutagenesis is discussed.     

Methodical improvements in rye anther culture

Summary The crucial problem in anther culture of rye (Secale cereale L.) is the very low regeneration capacity. Our study was conducted to overcome this restriction. The plant material used included a doubled haploid line (DH), two single crosses between DH Unes, and a tetraploid Secale cereale L. population. The factors carbohydrate source, post-plating temperature treatment, and gelling agent were investigated. Substantial progress was achieved by substituting maltose for sucrose. Top rates of 49 % responding anthers and 20 % green plants were obtained from one of the single crosses after a post-plating cold treatment on geirrte solidified medium. We consider our results a methodical step forward in rye anther culture.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

Effect of medium osmotic potential on callus induction and shoot regeneration in flax anther culture

Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is the prerequisite for the application of doubled haploid technology in a practical breeding program. Pre-culture of anthers on a medium containing 15% sucrose for 2–7 days before transfer to the same medium containing 6% sucrose for a total of 28 days culture period significantly increased shoot regeneration for all four genotypes evaluated. Moreover, pre-culture of anthers on medium containing 15% sucrose for 2–7 days was sufficient to dramatically reduce the frequency of shoot regeneration from somatic tissues and thereby to increase the frequency of microspore-derived plants in flax anther culture. Furthermore, replacing 15% sucrose with 6% sucrose and 9% polyethylene glycol (PEG), or 3% sucrose and 12% PEG, in pre-culture medium did not significantly affect callus induction and shoot regeneration. The results indicate that sucrose may act as carbon/energy source as well as an osmotic regulator in flax anther culture. Sucrose as an osmotic regulator may be replaced by a non-metabolizable osmoticum: PEG. The implication of this study in flax anther culture and breeding is discussed.     

Development and differentiation of haploid Lycopersicon esculentum (tomato)

Summary Haploid callus cultures of selected races of Lycopersicon (tomato) species can be obtained from anther culture. This is a further demonstration of a proposed general method of haploid culture developed with Arabidopsis thaliana. Differentiation of haploid callus of Lycopersicon esculentum can be controlled both in the dark and the light by hormones added to defined minimal media. Development to plantlets is achieved only in the light. Callus cells can be induced to develop into seedless pseudo-fruits. Chromosome counts on callus cells or root-tip cells establishes haploidy (n=12).Haploidy can be maintained in culture on defined minimal media for at least one year.