Plant Regeneration from the Hybridization of Brassica juncea and B. napus Through Embryo Culture

Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocals with the aid of embryo culture techniques. A better response of hybrid embryo culture was obtained from two cross combinations of B. juncea × B. napus (Ames 24521 × Huyou 15 and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg l^?1 naphthylacetic acid (NAA) + 1.5 mg l^?1 6‐benzylaminopurine (BAP) and with 0.3 mg l^?1 NAA + 2.0 mg l^?1 BAP. Callus formation and plant regeneration on these two media reached 55.43 and 26.65 %, and 66.98 and 24.61 %, respectively.     

Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.     

High frequency of plant regeneration from anther culture in flax, Linum usitatissimum L.

The effects of induction medium compositions on flax anther culture were investigated in order to improve the efficiency of callus induction and plant regeneration. Anthers were inoculated onto the modified MS medium supplemented with five different combinations of plant growth regulators. The medium containing the combination of 2mg/l 2,4- dichlorophenoxy-acetic acid (2,4-D) and 1 mg/1 6-benzylaminopurine (BAP) produced a significantly higher number of calli forming shoots/100 responded anthers and a significant increase in overall efficiency of regeneration than the same basal medium containing 1 mg/1 a-naphthalene-acetic acid (NAA) and 2 mg/1 BAP (CK). Among the five levels of thiamin hydrochloride tested, the modified MS medium containing 10 mg/1 thiamin hydrochloride significantly increased the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration compared with the same basal medium containing 0.1 mg/1 thiamin hydrochloride. Maltose concentration had a significant effect on the percentage of anthers producing call, the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration. The medium containing 6% or 9% maltose produced the highest overall efficiency of regeneration among the five levels of maltose evaluated. Sucrose concentration significantly affected the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration, and dramatically affected the frequency of microspore-derived plants and the frequency of spontaneous chromosome doubling in microspore-derived plants. The efficiency of doubled haploid line production obtained in this study appears adequate for applied breeding programmes.     

Plant Regeneration Through Anther Culture of Three Wild Species of Hordeum (H. murinum, H. marinum and H. bulbosum)

Plant regeneration was achieved through anther culture of three wild species of Hordeum (H. murinum, H. marinum and H, bulbosum). Calli or embryoids were formed from microspores in anthers cultured on a medium containing 6-benzylammopurine (BAP) and ficoll. These calli or embryoids regenerated green or albino shoots and roots after transfer to regeneration media. Green plantlets which developed on regeneration media were transferred to soil where they showed further growth.     

Intergeneric hybridization between Brassica species and Crambe abyssinica

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N₆-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of genotype and culture medium on callus formation and plant regeneration from immature embryos of Triticum turgidum Desf. cultivars

Twelve durum wheat cultivars were evaluated for their response to in vitro tissue culture. Zygotic immature embryos were used to induce callus formation using four different Murashige and Skoog‐based media. Each contained 9.05 μM 2,4‐dichlorophenoxy acetic acid but differed in their carbon source (sucrose or maltose) and the presence of NaCl (0 mM or 40 mM). The influence of both genotype and medium on the type and percentage of callus produced was observed. Calli were either compact and frequently embryogenic, or soft and watery. Percentages ranged from 54 to 100%, depending upon genotype and induction medium. All calli were then plated on a regeneration medium containing 20 g/l sucrose, 2.68 μM 1‐naphthaleneacetic acid and 2.22 μ 6‐benzylaminopurine. The regeneration of plantlets was higher from compact than from soft calli, with a strong dependence on genotype and type of induction medium used. MSm induction medium (30 g/l maltose) and MS40s (30 g/l sucrose plus 40 mM NaCl) were best for inducing compact calli, and gave the highest proportion of regenerated plants. The in vitro response (number of total shoots from a compact callus/number of embryos plated) was higher for immature embryos of ‘Baztan’, ‘Bradano’ and ‘Don Pedro’. These cultivars are a good starting material for experiments involving transformation of calli from zygotic immature embryos.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

Effects of Growth Regulators and Genotypes on Callus and Embryoid Induction from Maize Anther Culture *

Fifty genotypes and ten growth-regulator combinations were used in two experiments (I and II) to investigate genotype and hormonal effects on production of callus and embryoids via anther culture in maize (Zea mays L.). Hormonal effects across all genotypes were not significant in either experiment. However, highest callus-induction frequencies in both experiments occurred on YP basal medium plus 2,4-D at 2.0 mg 1^?1, and kinetin at 1.5 mg 1^?1 indicating that this combination was more effective than others. Genotypic differences in callus or embryoid induction across all media were significant. The most responsive genotypes in experiment I were single-cross hybrids Yuanwu × 592 and K727 × K305, which produced 18.3 and 6.7 % calli, respectively, with their appropriate media. The most responsive entry in experiment II was CIMMYT Pool 29, which produced 15.0 % calli on appropriate medium and an average of 10.0 % calli across 10 media. Twenty-three plantlets was regenerated from this study. Most of them developed embryogenically.     

Callus Production and Plant Regeneration from Mature Embryos of Poa pratensis L.

Plant regeneration from callus cultures may provide a source ot somaclonal variation for the improvement ot the apomictic grass Poa pratensis L. It is first necessary to be able to induce callus and regenerate plants in this species at a high frequency. Variation was observed between 50 cutivars of Poa pratensis for callus induction and plant regeneration. Using the cultivars ‘Merion’ and ‘Victa’, three basal media were tested along with various media additives. Murashige and Skoog's basal medium with 0.2 mg 1^?1 2,4-dichlorophenoxyacetic acid, 0.1 mg 1^?1 6-benzylamanopurine, 100 mg 1^?1 casein hydrolysate and 25 g 1^?1 sucrose is considered to be a good medium for callus growth and plant regeneration. Embryo-like structures were observed in the callus of some cultivars but plant regeneration appeared to be predominantly from shoot meristems on the callus surface. The majority of regenerated shoots were green, but chlorophyll deficient shoots were obtained from media containing coconut milk. Green plantlets could be transferred to soil without difficulty.     

Studies on somaclonal variants for resistance to scab in bread wheat (Triticum aestivum L.) through in vitro selection for tolerance to deoxynivalenol

This study was conducted to develop an efficient in vitro selection system for scab resistance by using in vitro screening for tolerance to deoxynivalenol (DON). Immature embryos of two wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11, and their reciprocal F₁ hybrids were cultured on MS medium supplemented with 2,4-D 2 mg/l and 0.6 × 10^-4 M DON for callus induction. The responses of callus induction and plant regeneration to 0.6 × 10^-4 M DON differed significantly between resistant and susceptible varieties, according to observed scab resistance levels at the plant level in the field. The percentage of callus formation of resistant variety Sumai 3 on induction medium containing DON was higher than that of susceptible variety Mianyang 11. Regeneration of DON-tolerant calli on DON-containing differentiation medium differed significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was decreased 3-fold on DON-containing medium. By an inoculation test with conidiospores of Fusarium graminearum Schw, we obtained several resistant lines from progenies of regenerated plants from DON-tolerant calli. These somaclonal lines had lower disease scoring (reaction index, infected spikelets and disease incidence), shorter plants and better yield components than Sumai 3, a famous Chinese resistant variety. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Effect of Sugars and Growth Regulators on Embryoid Formation and Plant Regeneration from Barley Anther Culture

The influence of carbon source and growth regulator composition in induction medium on anther culture response was investigated using spring barley genotypes. Anthers were cultured on BAC3, Ficoll-containing medium, supplemented with one of the following carbohydrates: sucrose, maltose, cellobiosc and melibiose (6 % w/v). The use of either maltose or cellobiose resulted in a significantly higher anther response, calli and/or embryoid production and green plant regeneration compared to the incubation of anthers on a medium containing sucrose. Contrary to these results, the replacement of sucrose by melibiose in BAO medium, drastically reduced the efficiency of anther culture. As an average for the three genotypes tested, the frequency of green plants per 100 anthers plated was 9- to 22-fold higher on medium supplemented with sucrose or cellobiose than on medium containing melibiose as a sole carbohydrate. Among the growth regulators tested, the combination of auxin NAA (2 mg/l) and cytokinin BAP (1 mg/1) performed much better than the employment of auxin 2,4-D combined either with zeatin riboside or BAP as cytokinins. The beneficial effect of medium supplemented with NAA and BAP was associated with better embryoid formation compared to the other growth regulator combinations tested. The hormone-free combination gave a similar anther response and production of calli as any medium supplemented with growth regulators, but the regeneration capacity of calli produced on hormone-free medium was much lower, resulting in the drastic reduction of the number of both total and green regenerants.     

马齿苋组织培养的优化

用野生马齿苋的种子得到无菌苗。以无菌苗的下胚轴和子叶为外植体,在18个不同浓度6_BA,2,4-D,NAA和KT组合的MS培养基上进行愈伤组织诱导、不定芽分化和增殖及生根的培养。得到诱导马齿苋愈伤组织的最佳条件是:初代培养基:MS 3%蔗糖 6_BA 1 mg/L 2,4_D 1 mg/L;丛生芽增殖培养基:MS 3%蔗糖 KT 3 mg/L NAA 0.5 mg/L;生根培养基:MS 3%蔗糖。     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic Embryogenesis in Hypocotyl Protoplast Culture of Rapeseed (Brassica napus L.)

By using a system of agirose plating and agarose bead culture, it was possible to induce efficient somatic embryogenesis in protoplast-derived calli of two rapeseed varieties, ‘Ceres’ and ‘Duplo’. Protoplasts were isolated from hypocotyls. For the initial protoplast culture a modified 8P medium was employed containing 2,4D (1.0 mg/l), NAA (0.1 mg/ 1), BAP (0.4 mg/l) and mannitol (7 %). After microcalli were obtained in four weeks, somatic embryos were induced by a two-step method. This involved a modified MS medium containing 2,4D (3.0 mg/l) in the first step and no 2,4D, but BAP (3.0 mg/l) and GA₃ (0.1 mg/l) in the second. This procedure also secured plant regeneration.     

Investigations on the Anther Culturability of Four German Spring Wheat Cultivars and the Influence of Light on Regeneration of Green vs. Albino Plants

Homozygous doubled-haploid plantlets derived from anther culture of wheat (Triticum aestivum L.) and triticale (×Triticosecale Wittmack) are useful breeding materials. However, efficiency of an-drogenesis needs improvement. We used media (basic components, are the same as 85DI2) each containing one of the seven auxins [2,4,5-trichlorophenoxyacetic acid (2,4,5–T), P-chloraphenoxyacetic acid (pCPA), 3,6-dichloro-o-anisic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-butrytic acid (IBA), indole-3-acctic acid (IAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) as a control] in combination with 6-furturyl-aminopurine (kinetin). In addition, each of the four cytokinins [6-benzylaminopurine (6-HA), 2-isopenlylnyl adenine (2-ip), 6-(4-hydroxy-3-meihylbut-2-enylamino) purinc (zeatin), and kinetin as a control] was tested in combination with 1-naphthalene acetic acid (NAA). Anthers containing microsporcs at miduninucleatc stage from live wheat cultivars (Angus, Centurk, Chris, K.itt, and Pavon 76) and two octoploid trilicale lines (T81, T82) were tested mainly for callus induction and polyhaploid production on each of the 11 media. The cultivar × medium interaction was not significant, When averaged over all growth regulators, Pavon was (he best cultivar which produced 14.4 % calli and 23 % polyhaploid plantlets. Averaged over all cultivars, the medium containing 2, 4-U produced the highest calli (13.9 %). Undifferentiated calli were regenerated on 87T1 medium, which contained IAA (1 mg/1) and kinetin (2 mg/1).     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

Diploid, tetraploid, and octoploid plants from anther culture of tetraploid orchard grass, Dactylis glomerata L.

This report describes a study of androgenesis in Dactylis glomerata, where the main aim was to find anther culture-responsive clones. Two types of media and two sugars were compared for their effectiveness in anther culture induction and subsequent plantlet production. Embryo formation from the cultured anthers was obtained from 28 of the 108 cloned genotypes using two different substrates, R₂M and FW. Both induction media supported the formation of embryos from the cultured anthers, but around 4.5 times more embryos (0.81 embryos per 100 anthers) were obtained with R₂M compared with FW, and R₂M also gave 5.5 times more green plants (0.054 green plants per 100 anthers) than the FW substrate. In the investigation of a carbohydrate source, responsive clones from the genotype study were tested using maltose as a substitute for sucrose in R₂M. Using maltose instead of sucrose increased embryo formation so that 133 embryos per 100 anthers were obtained compared with 7.1 embryos per 100 anthers obtained with sucrose. The total number of green plants obtained was also improved with maltose compared with sucrose, resulting in 66.3 and 1.9 green plants per 100 cultured anthers, respectively.     

Trichomes and regeneration by direct organogenesis of medicinal plant Dracocephalum kotschyi L. using shoot tips (Lamiaceae)

The present study describes the procedure for micropropagation of Dracocephalum kotschyi L. using shoot tips from in vitro-germinated plants. The best response was observed for shoot tips on MS medium containing 5 mg 6-benzylaminopurine L^?1 and 0.2 mg 1-naphthaleneacetic L^?1 acid. Regeneration for other types of the explant hypocotyls and cotyledons did not show satisfactory results so that the explants did not develop into normal shoots and in turn developed into the calli after 12 days of culture. Histochemical analysis showed that only the shoot tip revealed a direct induction of more teratological protuberances that arise around the cut end of the explants. Elongation of shoot buds was obtained on MS medium containing 1 mg BAP L^?1 + 0.5 mg IBA L^?1. Regenerated shoots rooted best on the same medium of elongation. After hardening, the rooted plants were transferred to the greenhouse where they grew, matured, and flowered normally with a survival rate of 95%. We concluded that the present protocol can be efficiently used for mass propagation of Dracocephalum kotschyi.     

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l^-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l^-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l^-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l^-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l^-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of genotype and light intensity on somatic embryogenesis and plant regeneration in melon (Cucumis melo L.)

The efficiency of 14 commercial cultivars of melon (Cucumis melo L.) for callus induction, plant regeneration and somatic embryogenesis under different photosynthetic photon flux densities (PPFDs) (150 or 50μmol m^?2 s^?1) was investigated. Cotyledonal explants were cultured on a Murashige and Skoog (MS) medium supplemented either with 9.0 μM 2,4-dichlorophenoxyacetic acid and 23.2μM kinetin or with 0.05 μM 2,4-dichlorophenoxyacetic acid and 0.26 μM 6-benzyladenine for the induction of somatic embryogenesis and shoots, respectively. For embryo maturation and root induction, growing callus tissues were transferred on growth regulator-free MS medium. Both genotype and the intensity of light significantly affected the rate of somatic embryo-genesis, embryo maturation and plant regeneration. On average, 12–47 primary globular-stage embryos were produced per mm² of explant surface. Fully developed, cotyledonary-stage somatic embryos were obtained from only three cultivars. Relatively high root induction rates were observed both on the shoot induction medium (11 cultivars) and on growth regulator-free medium (seven cultivars). In contrast, only six cultivars responded positively to the shoot induction treatment. Callus growth and somatic embryogenesis were significantly improved if cultures were incubated under higher PPFD values, although plant regeneration from all cultivars was significantly reduced under the same conditions.