Evaluation of carbon sources,gelling agents,growth hormones and additives for efficient callus induction and plant regeneration in Indian wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) genotypes using mature embryos

Various factors affecting in vitro regeneration like different carbon sources, different gelling agents, and growth additives were assessed comprehensively for callus induction and plant regeneration for five Indian wheat cultivars using mature embryos as the explants for the first time. The tissue culture responses of cultivars WH-1105, HD-2967, and PBW-343 have not been reported earlier. Besides, the effect of different concentrations of the cytokinin, zeatin has also been optimized. Using the optimized factors, the efficiency of five different varieties, i.e., HD 2967, C 306, RAJ 3765, WH 1105, and PBW 343 was evaluated for regeneration. Modified MS basal medium containing dicamba reduced precocious germination of the embryo and induced embryogenic callus more efficiently. Removal of embryogenic calli from non-regenerable structures during early callus phase improved plant regeneration. These calli on zeatin (1.0 mgl^-1) and dicamba (0.1 mgl^-1) containing medium showed the highest regeneration frequency (98%) with a maximum of 8-9 shoots per calli. Maltose had the maximum callusing and regeneration percentage than other carbon sources. Various gelling agents did not have any significant difference on the regeneration. Of all the varieties, C-306 and HD-2967 were found to be more regenerative and can be used in transformation experiments.     

Regeneration potential of CIMMYT durum wheat and triticale varieties from immature embryos

Twenty‐five durum wheat elite advanced lines and released varieties, and five triticale varieties were evaluated for their ability to produce embryogenic callus using three different media. For callus initiation and maintenance there were basal Murashige and Skoog (MS) medium containing double strains of macroelements and 2.5 mg/l 2,4D (DW1), basal MS medium containing 2.0 mg/l 2,4D (DW2), or basal MS medium supplemented with 1.0 mg/l 2,4 D and coconut milk (DW3). Plant regeneration was achieved on basal MS medium with indoleacetic acid and 6‐benzylaminopurine, and plants rooted on MS with 1‐naphthale‐neacetic acid. DW3 medium proved better than the other media tested for embryogenic callus initiation and maintenance. Regeneration rates varied widely with both genotype and initiation medium, with values ranging from no regeneration to 100% regeneration; the plantlets produced per embryo ranged from five to 20. Fourteen of the durum wheat genotypes showed 63–100% regeneration from DW3 callus formation medium, four lines from DW1 medium, and two lines from DW2. Four of the triticale varieties had regeneration of 48–100% from DW3 medium. After six subcultures, over a 6‐month period, genotypes lost their ability to regenerate plants. Only 10 lines retained some plant regeneration potential but regeneration was at reduced levels. Successful regeneration of durum wheat and triticale varieties will be used as an integral part of the transformation process.     

Optimization of mature embryo-based high frequency callus induction and plant regeneration from elite wheat cultivars grown in China

We have developed an efficient procedure for plant regeneration of elite wheat cultivars using mature embryos. Firstly, we established the optimal combination of basal media, inoculation method and pretreatment method using biostatistical methods. The results indicated that the combination of MS medium and longitudinally bisected mature embryos showed the highest culture efficiency, whereas the pretreatment method had no significant effects on callus induction or plant regeneration. A 70% primary callus induction rate was achieved on MS medium containing 2 mg/l 2,4‐d for all tested cultivars. Primary calli were then transferred onto the subculture medium to initiate embryogenic calli. Supplementation of the subculture medium with the appropriate combination of phytohormones (2.0 mg/l 2,4‐d , 0.5 mg/l BA and 0.1 mg/l NAA) significantly enhanced embryogenic callus production. The addition of AgNO₃ (10 mg/l) in regeneration medium promoted plant regeneration, whereas CuSO₄ stimulated root formation. The use of this protocol achieved successful plant regeneration in eight tested cultivars. The culture efficiency ranged from 15.3% to 36.8%, suggesting this regeneration system may be an effective alternative for wheat genetic transformation.     

高羊茅高效组织培养再生体系的建立

植物分子育种与基因功能鉴定是建立在高效的组织培养再生体系基础上的。本研究以高羊茅(Festuca arundinacea Schreb)为材料,探讨了不同外植体状态、基本培养基类型、外源激素组合与固化剂种类与浓度对愈伤组织诱导的影响,并进行了愈伤组织分化长芽与生根研究。结果表明:愈伤组织诱导过程中,离体成熟胚比完整种子作外植体愈伤组织出现早、质量优、诱导率高;基本培养基的选择上以N6诱导率最高,MS最低;激素组合研究中高羊茅三个品种(SAFari, FirewarⅡ, Barleduc)在无6-BA培养基中,生长激素选择上都是2,4-D优于IAA,2~2.5 mg/L 2,4-D在三个品种中诱导率都在50%以上,而在添加0.5 mg/L 6-BA时诱导率都偏低;固化剂选择上则以Phytagel与Agarose二都最优,Phytagel固化剂最佳诱导浓度为2~4 g/L。分化长芽最佳培养基为MS+0.1 mg/L 2,4-D+0.1 mg/L 6-BA+0.5 mg/L NAA+20 mg/L AgNO3+2%蔗糖+0.6%琼脂,生根壮苗最佳培养基为1/2MS+0.5 mg/L NAA+3%蔗糖+0.3%Phytagel。本研究为高羊茅的高效转基因体系建立与CRISPR基因功能研究提供准备。     

普通小麦(Triticum aestivum)和毛穗赖草(Leymus paboanus)的杂交,杂种细胞无性系的建立及植株再生

以3个普通小麦品种富可(Fuhuko)、中国春(Chinese Spring)及小偃759和毛穗赖草杂交,发现三个品种都可与毛穗赖草杂交,其中Fuhuko×L.paboanus平均结实率高达17.6％,杂种只有发育不全的幼胚而无胚乳。对杂种幼胚在N_6+1—2mg/11BA+0.5mgGA_3或MS(其中NH_4NO_3含量降低一半)附加1mg/1IBA的培养基上进行保姆培养,部分幼胚发育成     

春小麦成熟胚愈伤组织诱导及再生体系的优化

为优化小麦成熟胚再生体系,以CB037和中国春(CS) 2个春小麦品系的成熟胚为外植体,在培养基中添加不同浓度的TDZ (Thidiazuron, N-苯基-N’-1,2,3-噻二唑-5-脲)。结果显示,CB037和CS成熟胚胚性愈伤组织诱导率最适TDZ浓度为0.5 mg/L,在培养14 d时分别为90.43%和87.88%,均高于对照,达到极显著差异水平。CB037愈伤组织分化率和绿芽诱导率在0.5 mg/L TDZ时达到最高值,分别为48.94%和60.64%,与对照相比具有极显著性差异和显著性差异;CS愈伤组织分化率和绿芽诱导率的最适TDZ浓度为1.0 mg/L,与对照相比,二者均达到极显著性差异。TDZ最适响应浓度存在品种间差异,适量添加可提高成熟胚再生效率。本研究通过优化培养基成分提高成熟胚再生能力,为高频小麦遗传转化体系的建立提供了依据。     

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.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.     

Long-term callus cultures of diploid Barley (Hordeum vulgare). I. Auxin effects on culture initiation and maintenance

Summary Use of 2,4-D was superior to NAA or IAA for embryogenic callus initiation or maintenance in barley cultivar Bruce. A concentration of at least 2.0 mg/l 2,4-D was desirable for culture initiation. The developmental size of the embryo was more important than embryo age for obtaining embryogenic calli. Even brief exposures (20–40 days) of calli to concentrations of higher than 5.0 mg/l 2,4-D or 10.0 mg/l NAA resulted in inhibition of subsequent plant regeneration and therefore, concentrations above these could not be used for maintenance cultures. In the long-term maintenance cultures, the best production of embryogenic calli was with 0.1 mg/l and 1.0 mg/l 2,4-D.     

Indirect Organogenesis through Seedling-Derived Leaf Segments of <Emphasis Type="Italic">Ficus Religiosa</Emphasis> - a Multipurpose Woody Medicinal Plant

The aim of this study is to introduce the suitable protocol for indirect regeneration from seedling-derived leaf segment of Ficus religiosa. The leaf explant successfully produced callus on MS medium containing various concentrations of auxin in combination with BAP. The maximum callus induction (100%) was achieved in MS medium containing 0.5 mg/l 2,4-D plus 0.05 mg/l BAP and MS medium containing 1.5 mg/l NAA plus 0.15 mg/l BAP as well. MS medium consisting of 2,4-D produced yellow-brownish and friable callus (type I) while the yellowish and compact calli (type II) were obtained in MS medium consisting of NAA. On the other hand, MS medium supplemented with IBA formed greenish and compact calli (type Ш). The regeneration rate in type II callus was less than the type I, and there was no shoot induction observed on type Ш calli. MS medium supplemented with 1.5 mg/l BAP in combination with 0.15 mg/l IBA had the highest regeneration frequency (100%) and maximum shoot numbers (5.16) as well as shoot length (2.56 cm) in type I callus. A maximum of 93.33% root induction was observed in MS medium supplemented with 2.0 mg/l IBA plus 0.1mg/l NAA. The plantlets were successfully transferred to the greenhouse. This system could be utilized for large-scale multiplication of Ficus religiosa.     

杨树叶片高效离体再生系统的构建

利用速生杨树（Populus.tomentosa Car）叶片为外植体建立离体培养体系,结果表明,叶脉处和叶柄处,极易发生不定芽;芽大多是从叶片上直接产生;培养基中BA浓度是影响不定芽形成的关键;BA浓度在05~2.0mg/L范围内均有不定芽发生,BA 1.0mg/L与NAA 0.05mg/L搭配有利于不定芽的形成,培养基中添加GA3 0.1~0.2mg/L可以提高不定芽的发生频率,增殖培养基采用MS+BA 0.5mg/L+NAA 0.05mg/L+GA3 0.05mg/L,不仅能够快速增殖,而且芽苗粗壮,玻化率降低。生根过程采用过渡培养效果较好,ABT3生根粉的加入,使试管苗质量大为提高,在此基础上建立起杨树叶片离体再生系统,利用该体系能够获得高的芽苗再生频率,但是不同品种间略有差异。     

In vitro selection and plant regeneration of copper-tolerant plants from leaf explants of Nicotiana tabacum L. cv. 'Xanthi'

Copper tolerance of Nicotiana tabacum L. var. Xanthi in vitro was achieved through plant regeneration from leaf explants on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/l BA, 0.1–0.25 mg/l IAA and 60  μ m Cu. Tolerant organogenic calli showed more vigorous growth in medium containing 60  μ m Cu than the non-tolerant calli. Standard growth parameters such as fresh and dry weight of organogenic callus, growth tolerance index (GTI), enzyme activity (peroxidase and catalase) and copper accumulation were used as indicators of copper tolerance. The activities of peroxidase and catalase as well as estimation of protein, total amino acid and chlorophyll were greater in tolerant calli than non-tolerant ones. The GTI in the 4 weeks after the beginning of treatments yielded significant differences among the tolerant and non-tolerant organogenic callus cultures. The accumulation of copper in the tolerant calli increased significantly with an increase in copper concentration in the medium. Shoot bud regeneration was achieved in both tolerant and non-tolerant organogenic calli on MS medium containing 0.5 mg/l BA and 0.1 mg/l IAA. The tolerant regenerated shoots were rooted on half-strength basal MS medium with 60  μ m Cu for selection of tolerant clones. This study may help in the selection and characterization of Cu-tolerant lines of N. tabacum cv. 'Xanthi' for building conservation strategies and also for phytoremediation programmes.     

不同二倍体和四倍体小麦成熟胚再生体系研究

为了研究不同基因型和培养条件对小麦成熟胚愈伤组织诱导和分化的影响,以硬粒小麦、墨西里卡、野生一粒麦Tu和野生一粒麦Tb为实验材料,对其成熟胚在不同激素配比下愈伤组织的诱导和植株分化进行研究。结果表明,不同基因型小麦成熟胚愈伤组织诱导、分化及再生均存在很大差异。其中硬粒小麦的成熟胚培养效果最佳,其愈伤组织在不同2,4-D浓度（1.0~4.0 mg/L）下诱导率均在93%以上。不同激素配比对成熟胚愈伤组织绿点率、再生率均有显著影响。硬粒小麦、墨西里卡、野生一粒麦Tb在激素配比为KT 1.5 mg/L+NAA 0.5 mg/L的分化培养基中培养效果最好,其绿点率分别为85.22%,61.67%,8.50%,成苗率分别为40.40%,32.06%,1.72%;而野生一粒麦Tu的最适分化培养基激素配比为KT 1.0 mg/L +NAA 1.0 mg/L,其绿点率和再生率分别为18.64%和8.47%。研究表明,基因型是影响二倍体和四倍体小麦成熟胚培养的主要因素,愈伤组织的诱导和植株的再生是相互独立的。     

欧李高效再生体系的建立

研究旨在利用植物组织培养技术,筛选出引进资源品种（系）SN6、SN7和自育品系YC24的最适培养基,确定3个欧李品种的再生体系,为欧李高效快速规模化繁殖提供技术性支持。以3个欧李品种（系）为材料,对外植体的消毒方式、分化、增殖和生根培养阶段的最适培养基和激素配比进行研究。结果表明,头孢霉素浓度为100 mg/L时,外植体存活率最高、污染率最低;SN6、SN7的最适分化培养基为MS+ 0.5 mg/L 6-BA+ 1.0 mg/L ZT+ 0.05 mg/L IBA,YC24的最适分化培养基为MS+ 0.5 mg/L 6-BA+ 0.5 mg/L ZT+ 0.05 mg/L IBA;SN6的最适生根培养基为1/2MS+ 0.2 mg/L NAA+ 0.1 mg/L IBA+ 1.0 g/L活性炭,SN7、YC24的最适壮苗生根一步培养基为1/2MS+ 0.05 mg/L NAA+ 0.2 mg/L IBA。本研究建立了欧李嫩枝快速繁殖体系,可为优良种质的后续练苗移栽、扩大培养和推广应用提供参考。     

不同培养条件下长寿花叶片再生体系的构建

（河南农业职业学院植物科学系，河南中牟 451450）     

NAA或潮霉素对小麦成熟胚愈伤组织分化的影响

以5个栽培小麦品种诱导而来的成熟胚愈伤组织为外植体,研究了在分化培养基中加入NAA对小麦成熟胚愈伤组织分化的效果以及潮霉素浓度对不同阶段成熟胚愈伤组织分化的影响。研究结果表明,在对照的分化培养基中(含5mg/L激动素)加入0.1mg/L萘乙酸(NAA,1-naphthlcetic acid)形成的分化培养基,可显著提高4个供试小麦基因型的成熟胚愈伤组织的分化率,提高幅度达到12%～32%。不同的潮霉素浓度在"花培1号"愈伤组织的诱导阶段进行抗性筛选的结果表明,即使是在潮霉素浓度达到250mg/L时,仍有24%左右的愈伤组织存活率,这些存活的愈伤组织仍可能分化形成绿点甚至分化成苗,说明在"花培1号"愈伤组织的诱导阶段进行潮霉素抗性筛选是不适合的。5个不同小麦品种分化阶段进行潮霉素筛选具有明显的效果,但不同品种之间存在一定差异,其中:宁麦13的适宜潮霉素浓度为20mg/L,花培1号和扬麦158的适宜潮霉素浓度约为30mg/L,宁麦9和宁麦16的适宜潮霉素浓度在30～40mg/L左右。因此,我们认为,在分化阶段进行潮霉素抗性筛选是理想的筛选时期,在分化培养基中添加20～40mg/L浓度的潮霉素即可完全抑制小麦成熟胚愈伤组织的分化。本研究结果将有助于改进和完善普通小麦成熟胚遗传转化体系。     

Long-term callus cultures of diploid barley (Hordeum vulgare). II. Effect of auxins on chromosomal status of cultures and regeneration of plants

Summary Chromosomal abnormalities increased with time on the relatively high concentrations (2.0mg/l or more) of 2,4-D. Prolonged exposures of cell cultures to high concentrations of 2,4-D significantly increased the percent polyploid mitoses and were also associated with increased abnormalities of the spindle apparatus. Cultures maintained in the presence of NAA had a large number of cells with structural alterations of the chromosomes. The best embryogenic callus production (those maintained on 0.1 and 1.0 mg/l 2.4-D) occurred in cultures with least detectable genetic variation. Plants could not be regenerated from cultures that had been on the maintenance media for seven months or more. The loss of regeneration potential was uniform across the concentrations and auxins tested, and only correlated with time. This loss could not be associated directly with detectable genetic variation.     

紫花苜蓿愈伤组织诱导和分化研究

以紫花苜蓿SANDITI无菌苗下胚轴、茎、叶3种不同外植体,在附加不同激素浓度的MS培养基上诱导愈伤组织。结果表明,SANDIT最佳外植体为下胚轴,最佳下胚轴愈伤组织诱导培养基为MS+2,4-D 2.0 mg/L+6-BA 0.5mg/L。15 d继代1次,或适当降低2,4-D浓度,提高6-BA或KT浓度,可以降低愈伤组织褐化率。愈伤组织在6-BA0.5 mg/L,NAA 0.1 mg/L,GA31.0 mg/L,YE 250 mg/L,CH 250 mg/L的MS培养基上,可获得较高的分化率。     

“一步法”诱导三角紫叶酢浆草再生体系的形成

本研究以三角紫叶酢浆草叶片为材料,在现有研究的基础上,采用固定配比的6-BA（0.5 mg/L）和NAA（0.5 mg/L）,研究了KT、2,4-D对“一步法”诱导三角紫叶酢浆草再生体系的影响,研究表明MS+ 0.5 mg/L 6-BA + 0.5mg/L NAA + 1.0mg/L KT + 0.1mg/L 2,4-D的激素组合更能促进再生体系的形成,该激素组合可以诱导愈伤组织的形成,并诱导根及不定芽的发生及其增殖,三角紫叶酢浆草再生体系分化效率高,每块1cm2的叶片外植体平均可诱导成苗21.8棵。“一步法”诱导形成三角紫叶酢浆草再生体系方法能有效的降低工作量,而且具有再生苗诱导效率高的特点,有利于规模化繁育三角紫叶酢浆草。     

小麦和土壤中辛硫磷残留量的液相色谱分析方法研究

研究了小麦茎叶、籽粒和土壤中辛硫磷残留物的液相色谱分析方法。该方法仪器最小检出量为0.5 ng;当小麦籽粒和土壤中辛硫磷的添加浓度为0.02,0.2,2 mg/kg时,样品的平均回收率为89.8%~98.9%,变异系数为0.8%~5.7%,最低检测浓度为0.02 mg/kg;当茎叶中辛硫磷的添加浓度为0.04,0.4,2 mg/kg时,样品的平均回收率为89.7%~95.8%,变异系数为0.5%~5.9%,最低检测浓度为0.04 mg/kg。方法的灵敏度、精密度和检测限都符合农药残留分析的要求。