<i>In vitro</i> Germination and Micropropagation of <i>Aconitum vilmorinianum</i>: An Important Medicinal Plant in China

Aconitum vilmorinianum, a well-known traditional Chinese herb, is recently being threatened by overexploitation and environment disturbance. This study was conducted to provide propagation methods through in vitro germination and explant cultivation. Germination was stimulated up to 66.00% on Murashige and Skoog (MS) medium containing 2.0 mg L⁻¹ 6-benzylaminopurine (BAP), 0.1 mg L⁻¹ 1-napthaleneacetic acid (NAA), and 30 g L⁻¹ sucrose. Three bacteria (Pantoea agglomerans, Erwinia persicina, and Pseudomonas tolaasii) would be responsible for consistent contamination during germination. The latter two were effectively eradicated after disinfected. The influence of explant types and hormone combinations on direct and indirect organogenesis was evaluated in the present work. The frequency of shoot induction from axillary bud explants was 100% on the MS fortified with 2.0 mg L⁻¹ BAP and 0.3 mg L⁻¹ NAA. Shoots multiplication was optimized on MS medium supplemented with 0.1 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA. High callus induction percentage (96.67%) was obtained from stem segments on MS medium with 2.0 mg L⁻¹ 2,4-D, then successfully regenerated into shoots on MS medium in the presence of 0.1 mg L⁻¹ TDZ and 0.2 mg L⁻¹ NAA. The present work could be useful for the utilization and conservation of this valuable species.     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

An Efficient Plant Regeneration System of <i>Hydrangea bretschneideri</i> Dipp via Stem Segments as Explants

Hydrangea bretschneideri Dipp is a highly popular ornamental plant for garden decoration. Genetic engineering technology has been successfully used in many plant species, but it is limited in Hydrangea. Here we established an efficient regeneration system by using stem segments as explants for the first time. In our study, the plant growth regulators (PGRs) were evaluated at the different regeneration processes, including axillary shoots regeneration and root induction. We found that the optimal concentration for axillary buds’ induction was 2.0 mgL⁻¹ 6-BA and 0.5 mgL⁻¹ 1 IAA, its highest induction rate was 70%. Moreover, the highest axillary shoots proliferation coefficient was 10.7 on the Murashige and Skoog (MS) medium with 2.0 mgL⁻¹ 6-benzyladenine (BA), 0.2 mgL⁻¹ indole-3-butyric acid (IBA), and 1.0 mgL⁻¹ gibberellin A3 (GA3). The highest frequency of root induction was 80.0 ± 0.06% by culturing the elongated shoots in 1/2 MS medium containing 0.1 mgL⁻¹ IBA. In summary, our study will provide an effective technology for large-scale propagation and important pathway for promoting the popularization and application of Hydrangea bretschneideri Dipp.     

Rapid Propagation of <i>Rhynchostylis retusa in Vitro</i>

An efficient regeneration system of Rhynchostylis retusa was established to provide technical reference for the application of tissue culture tube seedlings in production. The mixtures of callus and protocorm from aseptic germination were used as explants. The optimal media of each stage was selected for callus proliferation, protocorm occurrence and growth, rejuvenation and rooting via a single, complete combination and orthogonal experiment. The results showed that the optimal medium for callus proliferation, protocorms occurrence and growth was 1/2 Murashige and Skoog (MS) medium adding 50 g·L⁻¹ banana puree, 0.1 mg·L⁻¹ α-naphthaleneacetic acid (NAA), 1.5 mg·L⁻¹ 6-benzylaminopurine (6-BA) and 1.0 mg·L⁻¹ kinetin (KT) with 17.33 proliferation coefficient of callus and 19.63 occurrence coefficient of buds after 90 days. Then the buds occurred from protocorm were cultured on 1/2 MS medium including 100 g·L⁻¹ banana puree, 1.0 mg·L⁻¹ NAA, 2.0 mg·L⁻¹ 6-BA and 0.05 mg·L⁻¹ KT, in which the proliferation coefficient of callus was 10.32 and occurrence coefficient of buds reached 17.87. In the further subculture, the same medium was simultaneously used for callus proliferation, protocorm occurrence and bud growth. The plantlets developed roots in 1/2 MS medium containing 70 mL·L⁻¹ coconut water and 1.5 mg·L⁻¹ NAA with 100% rooting rates after 90 days. The survival rate was more than 90% after domestication and transplantation. This regeneration protocol will provide technique foundation for protecting wild resource and developing artificial cultivation.     

Encapsulation of Immature Somatic Embryos of <i>Coffea arabica</i> L. for <i>in Vitro</i> Preservation

The present study aimed to develop a protocol for somatic embryogenesis and encapsulation of coffee embryos (Coffea arabica L.), for the conservation of genotypes with characteristics of commercial interest. Somatic embryos were induced from leaf explants in Murashige and Skoog medium (MS) supplemented with 1 mg · L⁻¹ of 2,4-dichlorophenoxiacetic acid (2,4-D) combined with 2 mg · L⁻¹ of benzyladenine (BA). Somatic embryos (SE) at the globular stage were encapsulated in a sodium alginate matrix; two treatments were tested: MS + 5 mg · L⁻¹ BA + 1 mg · L⁻¹ NAA + 3% (w/v) alginate, and MS + 7 mg · L⁻¹ BA + 5.7 mg · L⁻¹ indoleacetic acid (IAA) + 3% (w/v) alginate. Alginate was complexed with 100 mM calcium chloride (CaCl₂). Viability of the encapsulated SE was determined by staining with 0.01% fluorescein diacetate (FDA) after 0, 15, 30, and 45 days of storage at 4°C. Embryo viability was 100% in both treatments.     

Optimization of <i>Agrobacterium tumefaciens</i>-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize (Zea mays) lines (H8183, H8184, and H8185) were used for Agrobacterium infection. We used the β-glucuronidase gene (GUS) as the target gene and the glufosinate resistance gene (bar) as the selection marker. We conducted research on several aspects, such as different genotypes, coculture conditions, screening agent concentrations, and concentrations of indole-3-butytric acid (IBA), 6-benzylaminopurine (6-BA), and ascorbic acid (Vc) in the differentiation medium. We optimized the genetic transformation system, and the obtained results indicated that among the three lines studied, the induction rate of H8185 was the highest at 93.2%, followed by H8184, with H8183 having the lowest induction rate (80.1%). The best coculture method was that using the N6 coculture medium layered with a sterile filter paper. Using orthogonal analysis, we found that the optimal combination of the three factors in the differentiation medium was A₃ (1 mg mL⁻¹ IBA), B₃C₁ (1.6 mg mL⁻¹ 6-BA), and D₃ (1.5 mg mL⁻¹ Vc). Through GUS staining analysis, Bar test-strip analysis, and polymerase chain reaction, five transgenic plants were finally obtained. This study established the optimal conditions for genetic transformation in maize.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Tests for the presence of gametoclonal variation in barley and wheat doubled haploids produced using the Hordeum bulbosum system

Summary To investigate whether the Hordeum bulbosum system of doubled haploid production generates gametoclonal variation, populations of second generation doubled haploid lines were developed from first generation doubled haploid lines of two barley varieties and three wheat genotypes. In barley, no variation between doubled haploids from doubled haploids was detected for a range of quantitative characters, suggesting the absence of any gametoclonal effects. However, the original selfed-seed stocks were shown to contain cryptic allelic variation for some of the characters investigated. In wheat, gametoclonal variation was detected for ear emergence time, plant height and yield, and its components for two out of the three genotypes investigated. The type and range of variation was similar to that reported from studies of somaclonal variation from immature embryos and gametoclonal variation from anther culture. Generally, the effects appeared to reduce the yield performance of individual lines. The difference in response between the two species and the consequences for the use of the doubled haploid system in breeding programmes are discussed.     

Interaction of <i>Acaena elongata</i> L. with Arbuscular Mycorrhizal Fungi under Phosphorus Limitation Conditions in a Temperate Forest

The aim of this study was to analyze the performance of Acaena elongata colonized by arbuscular mycorrhizal fungi (AMF) to different phosphorus (P) concentrations, as a measure of AMF dependency. A. elongata, is a species from soils where P availability is limited, such as temperate forests. Our research questions were: 1) How do different P concentrations affect the AMF association in Acaena elongata, and 2) How does the AMF association influence A. elongata’s growth under different P concentrations? A. elongata’s growth, P content in plant tissue, AMF colonization and dependency were measured under four P concentrations: control (0 g P kg⁻¹ ), low (0.05 g P kg⁻¹ ), intermediate (0.2 g P kg⁻¹ ) and high (2 g P kg⁻¹ ) in different harvests. A complete randomized block design was applied. A. elongata’s growth was higher under -AMF in intermediate and high P concentrations, and the lowest growth corresponded to +AMF in the low and intermediate P concentration. We observed a negative effect on the root biomass under +AMF in intermediate P concentration, while the P concentration had a positive effect on the leaf area ratio. The AMF colonization in A. elongata decreased in the highest P concentration and it was favored under intermediate P concentration; while the low and the high concentrations generated a cost-benefit imbalance. Our results suggest that the performance of some plant species in soils with low P availability may not be favored by their association with AMF, but a synergy between AMF and intermediate P concentrations might drive A. elongata’s growth.     

Efficient Evergreen Plant Regeneration of <i>Cinnamomum japonicum</i> Sieb. through <i>in vitro</i> Organogenesis

Cinnamomum japonicum Sieb. is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value. In this study, we successfully developed a large-scale micropropagation protocol for C. japonicum for the first time. Sterilized shoots were excised and used as explants for shoot induction on several basal media, supplemented with different concentrations of plant growth regulators (PGRs), such as Thidiazuron (TDZ), N⁶ -Benzyladenine (6-benzylaminopurine) (BA), α-naphthaleneacetic acid (NAA) and Gibberellic acid (GA₃). After comparison, the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^–1 BA, 0.05 mg L^–1 NAA and 0.2 mg L^–1 GA₃, which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d, respectively. Then, elongated adventitious shoots were transferred to induce roots. 86.7% of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^–1 NAA and 0.1 mg L^–1 BA. The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d. Our study shows that C. japonicum can be successfully regenerated through de novo organogenesis, which lays a foundation for future transformation research on this tree.     

Improved Culture Media for Embryogenic Callus Generation in Sorghum [<i>Sorghum bicolor</i> (L.) Moench]

Many attempts on optimization of sorghum [Sorghum bicolor (L.) Moench] tissue culture induction media have been made, but the culture system remains with some bottlenecks compared to that of other crops. This study aimed at assessing the suitability of various induction media to produce embryogenic callus (yellow and friable) with high induction rates and reduced phenolic exudation. The six culture medium modifications: 3 based on Murashige and Skoog (MS) medium and one each based on Chu N6, Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes. Although there was a genotype influence on the attainment of a yellow callus, friability of the callus was determined to be dependent on the culture medium and not the genotype. Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite® as the gelling agent modified with 1.0 g/l KH₂PO₄, 1.0 g/l L-proline, 1.0 g/l L-asparagine and 0.16 mg/l CuSO₄·5H₂O (type E) was found to be the most effective resulting in about 60% yellow coloured callus induction with 25% friability. Addition of CuSO₄·5H₂O, KH₂PO₄, L-proline and L-asparagine significantly reduced the phenolic production. Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds. The half strength MS medium was also observed to suppress phenolic production. Medium 190-2 produced the highest regeneration frequency (40%) among the 3-regeneration media tested. The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.     

<i>In Vitro</i>-Propagation of <i>Agave tequilana</i> Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L⁻¹ agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.     

Factors affecting anther culturability of recalcitrant barley genotypes

One major problem encountered with cereal anther culture is that some genotypes are low or non-responders to the technique. The objective of this study was to improve anther culture efficiency of recalcitrant barley (Hordeum vulgare L.) genotypes. Reciprocal F₁s between the two low responsive cultivars, Morex and Steptoe, were used. These were chosen because doubled haploids (DH) were required from these genotypes for the North American Barley Genome Mapping project. Ficoll 400 at 200 g l^–1 in the induction medium significantly increased green plant production compared to four other media formations containing different gelling/viscosity modifying agents. Cold pretreatment of donor spikes of 28 vs 14 d resulted in an increase in embryoid, total plant and green plant production. Anther culture response in these experiments was little influenced by donor plant growth conditions. Indole-3-acetic acid (1 mg l^–1) or 1-naphthaleneacetic acid (2 mg l^–1) in the induction medium did not affect anther culturability or plant regeneration. Based on this research, the negative genotypic effect for doubled haploid production could be diminished, which is desirable for practical application.Abbreviations BAP 6-benzylaminopurine - IAA Indole-3-acetic acid - LS Linsmaier & Skoog - NAA 1-naphthaleneacetic acid - DH doubled haploid     

Evaluation of Pre-Emergence and Post-Emergence Herbicides for Weed Management in <i>Miscanthus sacchariflorus</i> and <i>Miscanthus sinensis</i>

Miscanthus, is a promising bioenergy crop, considered superior to other bioenergy crops because of its higher water and nutrient use efficiency, cold tolerance, and higher production of biomass. Broadleaf weeds and grass weeds, cause major problems in the Miscanthus field. A field experiment was conducted in 2018 and 2019, to assess the effects of pre-emergence (alachlor and napropamide) and post-emergence herbicides (nicosulfuron, dicamba, bentazon, and glufosinate ammonium) on broadleaf and grass weeds in M. sinensis and M. sacchariflorus fields. The weed control efficiency and phytotoxicity of pre- and post-emergence herbicides were evaluated at 30 days after treatment (DAT) and compared to those of the control plots. The results showed wide variations in the susceptibility of the weed species to the treated herbicides. Treatment with nicosulfuron 40 g.a.i.ha⁻¹ provided the most effective overall weed control (with 10% visual injury), without affecting the height and biomass of neither Miscanthus species in the field. Post-emergence herbicides such as glufosinate ammonium 400 g.a.i.ha⁻¹ and dicamba 482 g.a.i.ha⁻¹ were effective and inhibited the growth and density of the majority of weeds to a 100%; however, they showed significant phytotoxicity (toxicity scale of 1–10) to both species of Miscanthus. The application of glufosinate ammonium caused severe injuries to the foliar region (90% visual injury) of both Miscanthus sps. Comparatively, M. sinensis showed a slightly higher tolerance to the herbicides nicosulfuron, bentazon and napropamide with 10% visual injury at the recommended dose than M. sacchariflorus. The present study clearly showed that infestation of broadleaf and grass weeds in Miscanthus fields can cause significant damage to the growth and biomass of Miscanthus and applying pre-emergence and post-emergence herbicides effectively controls the high infestation of these weeds.     

Spatial Heterogeneity of Selected Soil Nutrients Related to <i>Torreya grandis</i> cv. <i>Merrillii</i> Plantation in Southeastern China

Chinese Torreya grandis (Torreya grandis cv. Merrillii) is a unique economic tree species in China. Intensive management related to application of chemical fertilizer and herbicides caused serious soil quality degradation of Chinese Torreya grandis plantations. Totally, 120 soil samples were collected from the main disbtributed areas of Chinese Torreya grandis in Southeastern China. In this area, soil pH values varied from 3.68 to 6.78, with a median value of 4.91, implying a trend of acidification. The average concentrations of organic matter, available nitrogen, available phosphorus and available potassium were 27.52 g kg⁻¹, 135.77 mg kg⁻¹, 15.12 mg kg⁻¹, and 153.43 mg kg⁻¹, respectively. The results of spatial analysis revealed that target variables had clear spatial patterns. The soil fertility was relevantly high in most of the study area, with soil nutrient imbalances existing. The stand age had a large effect on soil properties, which were also mainly influenced by human activities. Therefore, it is necessary to change the fertilization method for a sustainable management of Torreya grandis plantation.     

Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L⁻¹) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L⁻¹ sodium nitrophenol and 0.01 ~ 0.2 mg· L⁻¹ of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L⁻¹ sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L⁻¹ significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

     

Integrated Nutrient Management Improves Productivity and Quality of Sugarcane (<i>Saccharum Officinarum</i> L.)

Sugarcane is one of the major important sugar yielding crops in Bangladesh. As an exhaustive crop, sugarcane removes a huge amount of plant nutrients from the soil. However, the combined use of organic and inorganic fertilizers can be a good approach to deal with nutrient depletion and promote sustainable crop production as well as improve soil health. Therefore, an attempt was made to identify the most fruitful and profitable integrated nutrient management on the aspects of growth, yield and quality of sugarcane in two consecutive growing seasons. Seven treatments: T₁=Control, T₂=165:55:120:30:10:2.5:4 kg N:P:K:S:Mg:Zn:B ha⁻¹, T₃=Poultry Litter (PL) at 5 t ha⁻¹+95:51:87:9:10:2.5:4 kg N:P:K:S:Mg:Zn:B ha⁻¹, T₄=Cow Dung (CD) at 15 t ha⁻¹+ 36:52:60:17:10:2.5:4 kg N:P:K:S:Mg:Zn:B ha⁻¹, T₅=Press Mud (PM) at 15 t ha⁻¹+10:50:43:0:10:2.5:4 kg N:P:K:S:Mg:Zn:B ha⁻¹, T₆=Mustard Oil Cake (MOC) at 0.5 t ha⁻¹+140:54:115:25:10:2.5:4 kg N:P:K:S:Mg:Zn:B ha⁻¹ and T₇=GM (Green Manure) at 5 t ha⁻¹+140:53:100:28:10:2.5:4 kg N:P:K:S:Mg:Zn:B ha⁻¹ were used in this experiment. Two years data showed that treatment T₃ produced the maximum amount of tillers, total dry matter yield, millable sugarcane, cane yield and sugar yield, followed by the T₄ treatment. The highest stalk heights were recorded in the T₃ treatment, which was statistically similar to all other treatments except T₁ and T₂. The juice quality parameters viz., brix and pol in cane were found significant in treatment T₃ while the highest purity was obtained in the T₇ treatment. All the data of Jaggery (goor) quality parameters, the highest sucrose content, color transmittance, Jaggery (goor) recovery and the lowest ash content of Jaggery (goor) were observed in the T₃ treatment, which was statistically similar to the T₄ treatment in both seasons. The highest cost of production was obtained from the T₆ treatment while the highest gross return, net return and BCR were recorded in the T₃ treatment. No significant changes were found in one cycle of sugarcane in initial and post-harvest soil characteristics viz., pH, organic carbon, total N, and available P, K and S contents due to integrated use of different fertilizer packages. From the experimental findings, it was concluded that treatment T₃ followed by T₄ treatment would be the better productive and profitable integrated nutrient management technology for ensuring higher yields and quality of sugarcane without soil fertility degradation in the High Ganges River Floodplain soils.     

Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

Effect of Water Stress Induced by Polyethylene Glycol on Growth,Proline Accumulation in <i>Agave americana</i> L.

The effect of water deficit was determined on both in vitro and soil seedling as well as in cells in suspension of Agave americana L. In order to do the establishment of cells, the formation of callus was induced; for it two auxins were evaluated: 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-mino-3,5,6-trichloropicolinic acid (picloram) at three concentrations (0.25, 0.5 and 0.75 mg L⁻¹) in three explants (leaf, root and meristems) cultured in MS semisolid medium. The callogenesis response was related to the type and section of the explant, as well as the regulator used, and a cell suspension was established using 0.5 mg L⁻¹ naphthaleneacetic acid (NAA) + 0.5 mg L⁻¹ Benzylaminopurine (BAP). Seedlings were exposed to polyethyleneglycol (15% and 30% w/v) with a water potential of −0.87 and −2.67 MPa, respectively, under soil conditions. Water stress was applied through restricted irrigation. Fresh weight, root system growth, and chlorophyll concentration were some of the parameters that were affected by the effect of water deficit on A. americana L. Chlorophyll concentration values were significantly decreased by 15 at 30% PEG (19.6 SPAD units) compared to the control treatment. In in vitro plants, the highest concentration of proline was found in the roots, being the treatment with 30% polyethylene glycol where the highest concentration of this osmoregulator was obtained (62.5 mg g⁻¹ DW). Under restricted irrigation conditions, an increase in proline concentration was observed both in the aerial part (2.2 µg 100 g⁻¹ DW) and in the root system (1.8 µg 100 g⁻¹ DW). However, the concentrations found were approximately ten times greater, less than those found under in vitro conditions. Therefore, the accumulation of proline can be considered an indicator of stress in Agave Americana L. growth in vitro.     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.