Pretreatment Of Buds Research Articles

Optimizing Different Medium Component Concentration and Temperature Stress Pretreatment for Gynogenesis Induction in Unpollinated Ovule Culture of Sugar Beet (Beta vulgaris L.)

The great economic importance of sugar beet determines the ongoing biotechnological studies conducted worldwide to improve the technology of obtaining doubled haploids (DHs) using the method of unpollinated ovule culture in vitro. To improve the induction of gynogenesis, we tested the effect of thidiazuron (TDZ), temperature bud pretreatment, different concentrations of sucrose, and culturing on liquid or solid medium. Three genotypes were tested in this study. The use of TDZ at a concentration of 0.4 mg/L in solid IMB (induction medium for Beta vulgaris) induction nutrient medium with 3 g/L phytagel, 50 g/L sucrose, 200 mg/L ampicillin and cultivation at 28◦C in the dark produced up to 16.7% induced ovules. The liquid nutrient medium of the same composition induced up to 8% ovules. Increasing TDZ concentration to 0.8 mg/L resulted in reduction or total inhibition of gynogenesis, depending on the genotype. Reducing the sucrose concentration to 20 g/L or increasing it to 80 g/L was not effective. In all three genotypes, the absence of temperature pretreatment of buds (5–6 °C) showed the best results. The plant regeneration with MS nutrient medium of 20 g/L sucrose, 3 g/L phytagel, 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L gibberellic acid (GA3) resulted in up to seven shoots from one induced ovule in the most responsive genotype. We showed by flow cytometry, chromosome counting and chloroplast number assessment that all regenerant plants were haploid (2n = x = 9).

Embryogenesis induction of carrot (Daucus carota L.) in isolated microspore culture

Haploid technologies are used to create homozygous lines for accelerated breeding. We aimed to optimize the technology for using the isolated microspore culture in vitro to obtain doubled haploids of the carrot (Daucus carota L.).
 We studied two carrot varieties with different responsiveness to embryogenesis, Altajskaya lakomka and Breeding line 17. Carrot microspores were isolated from buds and cultivated in liquid nutrient media supplemented with an antibiotic and activated carbon in vitro. They were exposed to different thermal treatments.
 The experiment showed the benefits of combining cold pre-treatment of buds (5°C for 1 day) with heat shock of isolated microspores in vitro (32°C for 2 days). The induction of embryogenesis on the NLN-13 medium was twice as high as on the MSm-13 medium. The use of 1% activated carbon in 0.5% agarose increased the yield of embryoids by more than 1.5 times. 100 mg/L of ampicillin was found to be the most efficient concentration. After 30 days of cultivation under optimized conditions, the yield was 161.3 and 44.0 embryoids per Petri dish for the cultivar Altajskaya lakomka and Breeding line 17, respectively.
 The induction of carrot embryogenesis is determined by the type and duration of thermal stress, the composition of the nutrient medium, the use of activated carbon as a sorbent, the addition of β-lactam antibiotics, and the type of explant exposed to thermal treatment. Our technology enabled us to obtain homozygous doubled haploid lines of carrots during a year, and these lines were included in the breeding process to create F1 hybrids.

Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.

Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)

We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research.

Review of research on haploid production in cucumber and other cucurbits

ABSTRACT This review provides a summary of haploid induction methods and factors affecting the efficacy of specific methodologies as applied to cucumber (Cucumis sativus L.), melon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), winter squash (Cucurbita maxima Duch. ex Lam.), summer squash (Cucurbita pepo L.) and other cucurbits. This report is focused on studies that were carried out during the last 20 years. The main objective of the research on the production of haploid cucurbit plants is to accelerate breeding programs through the use of homozygous double haploid lines (DHL) and to facilitate the selection of desired (e.g. disease-resistant) genotypes for breeding. Unfortunately, currently used protocols result in a low number of double haploids (DH). The most common and best-known method of obtaining haploid cucurbit plants is via pollination with irradiated pollen, which induces parthenogenetic development of haploid embryos in planta. The embryos are extracted from immature seeds and cultured in vitro to facilitate the maturation and development of plants. The studies described below were primarily aimed at the determination of an appropriate dose of radiation and the evaluation of the impact of the genotype and the time of year on the number of haploid embryos and plants obtained. A less popular method of haploid production - ovule and ovary culture - is based on in vitro gynogenesis. The studies related to this method concentrated on optimising the composition of the medium and pre-treatment conditions (primarily temperature) to which the flower buds were subjected. Recently, increasing attention has been paid to anther and microspore culture. As in the case of in vitro ovule and ovary culture, the medium composition and flower bud pre-treatment conditions were optimised. The most recent studies suggest that anther culture is comparable in effectiveness to the irradiated pollen technique.

Effect of Low Temperature Pretreatment of Buds or Inflorescence on Isolated Microspore Culture in Brassica rapa(syn. B.campestris).

The effect of low temperature pretreatment of buds or inflorescence on microspore culture for the production of haploids of Brassica rapa (syn. B.campestris) was examined. Incubation of the buds or the inflorescence at 4°C for 3 or 10 days before the culture of microspores induced efficient microspore embryogenesis. Pretreatment of flower buds was more effective than that of the inflorescence. Prolonged pretreatment up to 20 days promoted embryo induction. Microspores in the buds were examined for developmental stage before and after the pretreatment. Buds with a petal length to anther length ratio of about 0.5-0.7, which had microspores were at the late unicellular stage, were collected and were used. All microspores were at the late unicellular stage before the pretreatment. The percentage of the microspores at the stage of late unicellular decreased during the pretreatment while the percentage of bicellular stage microspores with two unequal size nuclei increased. At the same time, a small but notable number of bicellular stage microspores with equal size nuclei, which is the first step of microspore embryogenesis, was observed after the pretreatment.

Callus induction in culture of Oenothera hookeri and Oenothera picensis anthers

In the present work we try to determine optimum conditions for callus induction in anther culture of Oenothera hookeri and O. picensis. The anther callus yield was increased when the anthers were cultured on modified MS medium supplied with 2 mg dm-3 2,4-D and 2 mg dm-3 NAA, in both species. In O. hookeri, best results were obtained when anthers were excised from 7.2 - 9 mm buds at the stage of vacuolated microspores, then pretreated at 4 °C for 2 d and grown under 16-h photoperiod. The response to anther culture of O. picensis was generally very poor compared with that of O. hookeri. The higher yield of calli was obtained when anthers were excised from 6.2 - 8 mm buds at the stage of vacuolated microspores and grown under continuous light. The cold pretreatment of buds decreased anther response in this species.

Influence of Genotype, Plant Growth Temperature and Anther Incubation Temperature on Microspore Embryo Production inBrassica napus ssp. oleifera

Eleven Fx hybrid genotypes of winter rape (Brassica napus ssp. oleifera) were used in a study of induction and growth of microspore-derived embryos. Plants of each genotype were grown in controlled environments at either a constant 15 CC or a constant 20 °C, both with a 16 h photoperiod. Equal numbers of buds, approximately 2-5 mm in length, containing uninucleate microspores were harvested from each genotype and either pretreated (14 d at 4°C) or dissected immediately after harvest. Anthers were cultured on liquid medium based upon that of Murashige and Skoog (1962) and containing 8% sucrose, 0-5 mg dm-3 naphthylacetic acid and 0-05 mg dm-3 benzylaminopurine. Anthers from equal samples of buds were incubated at 35 °C for 0,1, 2 or 3 d before transfer to 30 °C (21 d) and then 25 °C. After a total of 42 d incubation, cultures were scored for the presence of macroscopic embryos (1-2 mm in length) and for the presence of anthers containing aborted embryoids which had not developed further. The results showed first that bud pretreatment completely inhibited induction and secondly that anthers of all genotypes had an absolute requirement for a 35 °C treatment (optimal duration 2 d) in order to induce embryoid formation. In the great majority of genotypes plants grown at 15 °C provided ore productive anthers than plants grown at 20 °C. However, within each treatment there were great differences both in the frequency of anthers showing induced embryoids and of the final yield of embryos. There was evidence that hybrids with a common parent responded similarly under certain treatments. This confirmed the importance of genotypic control for some components of embryo yield.

The Production of Haploids from Brassica hirta Moench (Sinapis alba L.) Anther Cultures

Summary Both embryogenesis and callus proliferation were induced in anther cultures of Brassica hirta Moench ( Sinapis alba L.). Culture temperature was significant with embryogenesis occurring only when anthers were cultured at 35°C for two days prior to maintenance at 25°C. Pretreatment of buds at 40°C (2 h) prior to anther culture failed to increase the frequency of embryogenesis and a 50°C (10 min) pretreatment inhibited embryogenesis. Bud pretreatment under reduced atmospheric pressure (500 mm of mercury) for 0.5 h prior to culture at 25°C stimulated embryogenesis. Plants were regenerated more easily from embryos than from callus. The cytological examination of 14 embryo-derived plants revealed the presence of two haploids, 10 diploids and two triploids whereas the single callus-derived plant examined was triploid.

A note on the pretreatment of excised flower buds in float culture of Hyoscyamus anthers

As a means of enhancing the embryogenic response of Hyoscyamus pollen in anther culture, excised bud pretreatment is shown to be more effective at 15°C than at 7.5°C. Pretreatment at 5°C is ineffectual. The pretreatment optimum at 15°C is 5 days compared to 15 days at 7.5°C. A greater response is invoked by bud pretreatment than by excised anther pretreatment. Without pretreatment, the anthers are most responsive when cultured just before the first mitotic division of the pollen, but with pretreatment, they become most responsive at or just after the first mitotic division. The pretreatment-induced response in Hyoscyamus is compared with that in Nicotiana tabacum.

Mode of Action of IAA and GA3on Root and Shoot Growth of Epiphyllous Buds ofBryophyllum tubiflorum

Darkness and GA3 stimulate the elongation of the first internode but inhibit the production of roots while IAA inhibits internodal elongation but promotes the production of roots on epiphyllous buds of Bryophyllum tubiflorum. Cycloheximide inhibits both, implicating the synthesis of proteins in the growth of both root and shoot. Even pre-treatment of buds with cycloheximide for 4 h inhibits rooting as well as internodal elongation when these are subsequently transferred to IAA or GA3. On the other hand, pre-treatment with IAA or GA3 even for 8 h does not alleviate the inhibitory effect of cycloheximide suggesting that there is a lag period between the application of these regulators and the synthesis of proteins caused by them.