阿月浑子与黄连木的杂种果实发育及幼胚培养研究

 以阿月浑子与黄连木的杂种果实和杂种幼胚为试材,对果实生长发育规律及幼胚培养进行了研究,探讨了胚龄、培养基类型和6-BA浓度等对杂种胚培苗再生的影响。结果表明：（1）杂种果实的纵、横径生长呈“S”型曲线变化,果实鲜质量生长呈双“S”型曲线变化。（2）杂种胚的发育始于授粉后80 d,80 ~ 100 d是杂种胚败育的关键时期。（3）授粉后80 d的杂种幼胚难以成苗,授粉后100和120 d的杂种幼胚成苗率分别为37.04%和83.33%。（4）适宜杂种胚培苗增殖的培养基为：DKW + 2.0 mg · L^-1 6-BA + 0.05 g · L^-1 IBA + 0.3 g · L^-1 LH + 1.5 g · L^-1 PVP,增殖系数为3.47,苗高为3.32 cm。（5）适宜杂种胚培苗生根的培养基为：1/2WPM + 2.0 mg · L^-1 IBA + 0.05 mg · L^-1 NAA,生根率为80%。     

‘奥迪亚’葡萄杂交后代胚挽救及无核性状分子鉴定

【目的】为了研究影响杂交后代胚挽救萌发率的主要因素,并对其无核性状进行早期鉴定,【方法】以无核、抗病葡萄品种‘奥迪亚’为母本,‘玫瑰香’、‘摩尔多瓦’和‘红地球’3个主栽品种为父本进行杂交,通过L9(33)正交试验,建立简单高效的胚挽救体系,并用分子标记对杂交苗无核性状进行鉴定。【结果】结果表明,影响胚挽救萌发率的最关键因素为接种时期,其次为培养基种类和NAA浓度;以‘奥迪亚’为母本的杂交胚其适宜接种程序为授粉后49～56 d剥取胚珠,接种在B5或NN69培养基上充分发育70 d,再转入1/2 B5培养基上萌芽,‘奥迪亚’ב摩尔多瓦’杂交胚通过此程序萌发率达到了50.48%;经分子标记鉴定,各组合后代的无核株系均超过50%。【结论】利用胚挽救方法以‘奥迪亚’为杂交母本可高效创新葡萄无核种质。     

Assessing the influence of subcultures and liquid medium during somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis Jacq.)

The current analysis describes an improved protocol for somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis) through liquid medium, and assesses the influence of successive subcultures during induction of calluses in three Brazilian oil palm varieties. Calluses were induced in a Murashige and Skoog (MS) medium with 450 Picloram, 0.5 g L^?1 glutamine, 2.5 g L^?1 activated charcoal, 30 g L^?1 sucrose, and solidified with 2.5 g L^?1 Phytagel. In a first experiment, the effect of continued subculture of explants every 30 days to fresh culture medium was determined. During a second experiment, part of the embryogenic calluses obtained were transferred to a liquid medium under agitation, consisting of MS with 5 µM picloram or 2,4-dichlorophenoxyacetic acid (2,4-D). After 210 days, the calluses were transferred to semi-solid media for differentiating somatic embryos. It was observed that continued subculture of explants monthly was a determinant in stimulating and improving the formation of embryogenic calluses. Embryogenic calluses in liquid medium with 2,4-D significantly improved the percentage of differentiated somatic embryos (up to 80.2%), with the largest amount of torpedo embryos (8.3 per callus). Regenerated plants with roots were individualised and transferred to a greenhouse, with close to 95% survival.     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

Effects of IAA and BA on development of globular,heart- and torpedo-stage embryos from cell suspensions of three grape genotypes

In order to initiate cell and embryo suspensions, embryogenic calluses derived on NN solid medium with 2,4-D and BA from petioles of in vitro grown plants of three interspecific grapevine hybrids were cultured in three versions of liquid NN medium: (1) without growth regulators, (2) 0.1 mg l⁻¹ IAA and (3) 0.5 mg l⁻¹ BA. Cell and embryo suspensions were incubated two and three times in these versions of liquid media in various combinations. Incubating suspensions two times in hormone-free media and/or with 0.1 mg l⁻¹ IAA led to formation of globular embryos in the three cultivars studied and small numbers of heart-stage embryos in ‘Podarok Magaracha’ and ‘Intervitis Magaracha’. Numerous heart-stage embryos developed in ‘Intervitis Magaracha’ and ‘Podarok Magaracha’ when the suspensions had been initiated in medium with 0.5 mg l⁻¹ BA, and in ‘Bianca’ this was achieved after two incubations in the above medium. Torpedo-stage embryos were formed after subculturing heart-stage embryo suspensions in medium with 0.1 mg l⁻¹ IAA in all study cultivars. If only small numbers of embryos of a certain developmental heart- or torpedo- stage were formed, such cell and embryo suspensions need to be repeatedly subcultured in liquid medium with specific growth regulators to enable this process.     

‘过山香’香蕉原生质体培养及植株再生

 以'过山香'香蕉的胚性悬浮细胞（Embryogenic cell suspensions,ECS）为起始材料分离原生质体,酶解液的组成为：3.5%纤维素酶R-10、1%离析酶R-10、0.15%果胶酶Y-23、204 mmol/L KCl、67 mmol/L CaCl2和0.41 mol/L甘露醇,原生质体产量为3.1×107个/mL PCV ECS （PCV：packed cell volume,细胞密实体积）。分别以培养基‘A’和‘B’为培养成分在液体浅层培养和看护培养两种培养系统中进行原生质体培养。结果表明：在液体浅层培养系统中,采用培养基‘B’比采用培养基‘A’效果好,原生质体的细胞分裂频率和细胞团形成频率分别约是采用培养基‘A'的3倍和10倍;所获得的培养物为只能增殖而不能进一步分化的非胚性细胞团。在看护培养系统中,采用培养基‘A’与采用培养基‘B’时,细胞分裂频率和细胞团形成频率没有显著地差异;所获得的细胞团具有典型的胚性细胞特征。将从看护培养中获得的细胞团转移到体胚诱导培养基上,培养45 d后,从105个原生质体获得1550个体胚。继续在体胚诱导培养基上培养30 d,7.8%的体胚能萌发。萌发的体胚在MS+0.1%活性炭的培养基中发育成健壮植株,移栽后成活良好。     

Breeding of disease-resistant seedless grapes using Chinese wild Vitis spp.: I. In vitro embryo rescue and plant development

Seedless grape cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Fungal diseases are a great threat of them. Several wild Chinese Vitis species showed high resistance to many fungal diseases. Therefore, an investigation was conducted to assess the potential to incorporate these species in a breeding project for development of the disease-resistant seedless cultivars. Hybridization was conducted using V. vinifera as female parents and the wild Chinese Vitis spp. as male parents. In-ovulo embryo rescue was used to develop hybrid plants from the seedless females. An efficient protocol is reported here for the in vitro embryo rescue and plant development from the cross Emerald Seedless × Beichun. Ovules were excised from immature fruits 7 weeks after pollination (WAP) and cultured in the double-phase ER medium supplemented with 6.0% sucrose and 0.3% activated charcoal (AC). Following 8–12 weeks of culture, embryos were removed from the ovules and transferred onto WPM supplemented with 1.0 μM 6-benzyladenine (6-BA), 2.0% sucrose and 0.2% AC and solidified with 0.6% agar. After 8 weeks of culture, the embryos germinated and subsequently grew into whole plantlets. With the optimized parameters developed in the present study, about 34.0%, 91.2% and 77.4% of embryo formation, embryo germination and plant development were obtained, respectively. When this protocol was applied to 11 other cross combinations, genotype was found to significantly influence embryo formation, embryo germination and plant development, with different frequencies of hybrid plants successfully obtained in all crosses.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.     

Induced androgenesis as a biotechnology method for obtaining DH plants in Daucus carota L.

The influence of various factors on the efficiency of obtaining double haploid lines in anther cultures was examined in Daucus carota L. The impact of the genotype, the formation of donor plants and the growth conditions was investigated. The effects of various regeneration media were analysed. Acclimatisation of androgenic plants and their evaluation were also conducted. Androgenesis was induced on B₅ medium containing 2,4-D and NAA at 0.1 g·L^?1. Depending on the genotype, 1.2–305.3 embryos per 100 anthers were obtained. The highest number of embryos (5.5 per 100 anthers) was obtained from donor plants with the first-order shoots containing one umbel. Cultivation of donor plants under controlled conditions improved the number of embryos from 305 in the open field to 1764 in the growth chamber. Regeneration of plants from androgenetic embryos proceeded most effectively on B₅ medium without hormones and amino acids. Regenerated plants adapted in over 60%. Ploidy analysis showed the presence of 92% of plants with a doubled chromosome set and 8% with a tetraploid number of chromosomes. Plants with a doubled chromosome set were 73% homozygous for the glucose phosphate isomerase isoenzyme and 100% homozygous for the aspartate aminotransferase isoenzyme, which confirms their gametic origin.     

辐照花粉诱导西葫芦单倍体

摘　要：利用不同剂量的60Co γ射线辐照西葫芦花粉后授粉，剥取不同发育阶段的胚接种在E20A培养基上进行培养，研究不 同基因型、辐照剂量、剂量率对西葫芦单倍体诱导的影响。结果发现：单倍体的产生和植株再生率受基因型、胚发育阶段和 辐照剂量的影响明显。281基因型平均单倍体诱导率最高，为1.30 %；点状胚和箭形胚易发育成单倍体苗；辐照剂量80~100 Gy时获得点状胚和箭形胚及胚总数最多，杆状胚的植株再生率和植株成活率最高，分别是83.33 %和35.71 %；适宜的剂量率 为45.19 Gy·min^-1。