黑麦草高效丛生芽的发生及离体开花的初步研究

以一年生和多年生黑麦草优良品种为材料,取无菌种子苗的茎尖生长点部分在加有不同浓度的2,4-D和6-BA的MS诱导培养基上诱导丛生芽发生,然后在加有不同浓度6-BA的增殖培养基上产生大量丛生芽,并长期继代培养.从丛生芽块上剥取单芽转移到生根培养基上诱导根的发生,生根植株移栽成活率达100%.本试验建立了一种诱导频率高、增殖速度快、试验周期短的黑麦草丛生芽离体培养体系.在附加适宜浓度6-BA和ABA的培养基上丛生芽可直接分化出大量花序,40 d后产生花序的芽块频率最高为81%.进而研究了光照、激素和继代培养时间等因素对黑麦草离体开花的影响.     

黑麦草幼穗离体培养及植株再生

以多年生黑麦草和一年生黑麦草的适宜发育期(1～3 mm)的幼穗为外植体,在附加适宜浓度2,4-D (1 mg/L)的改良MS培养基上诱导愈伤组织发生,诱导率可达95%.一些愈伤组织在诱导培养基上直接发生不定芽,另一些愈伤组织能多次继代培养并保持高分化能力.后类愈伤组织在转入含有0～0.5 mg/L 6-BA和0～0.5 mg/L 2,4-D的分化培养基后产生大量丛生芽,部分小芽发育成苗.幼穗发育时期、培养基的激素组成明显影响愈伤组织的继代培养和植株再生能力,基因型对愈伤组织诱导率和植株再生能力也有影响.该体系适用于黑麦草基因工程和细胞工程研究,且具有实验周期短、基因型制约小、植株再生率高等优点.     

草地早熟禾高效丛生芽体系的建立

本试验以草地早熟禾无菌种子苗茎尖为外植体,建立了高效的草地早熟禾丛生芽离体培养体系.结果表明:茎尖在附加0.5 mg/L 2,4-D和3.0 mg/L 6-BA的MS培养基上可诱导茎尖部位基部膨大,且诱导率最高.膨大的基部在2.0mg/L6-BA的丛生芽诱导培养基上,其诱导频率最大,然后将丛生芽转入附加3.0mg/L6-BA和0.07mg/L2,4-D的培养基上可进行继代培养,丛生芽在1/2MS培养基上生根率达100%.     

通过花粉管通道法导入红树总DNA获得耐盐紫花苜蓿T0代植株及其RAPD验证

利用花粉管通道法将盐生植物红树总DNA导入紫花苜蓿,共导入1391朵花,获得894粒T0代转化种子.T0代种子种植在含有225 mmol/L NaCl的MS培养基上,获得12株耐盐性强的植株.以供体和受体为对照,对12株耐盐植株进行RAPD分析,在55条随机引物中有8条扩增出稳定、清晰的条带,条带的差异性表现为新增条带、供体特异条带和受体条带丢失.以上结果初步证实外源DNA已经整合到受体的基因组中,而且T0代植株耐盐能力提高可能与外源基因的导入有关.     

草地早熟禾高效丛生芽体系的建立

本试验以草地早熟禾无菌种子苗茎尖为外植体,建立了高效的草地早熟禾丛生芽离体培养体系。结果表明:茎尖在附加0.5mg/L2,4-D和3.0mg/L6-BA的MS培养基上可诱导茎尖部位基部膨大,且诱导率最高。膨大的基部在2.0mg/L6-BA的丛生芽诱导培养基上,其诱导频率最大,然后将丛生芽转入附加3.0mg/L6-BA和0.07mg/L2,4-D的培养基上可进行继代培养,丛生芽在1/2MS培养基上生根率达100%。     

利用组织培养技术研究枣和酸枣耐盐性

以‘金丝小枣’、‘泗洪大枣’和酸枣枣头枝为试材,研究了基本培养基含0、25、75mmol/L NaCl对它们初代培养、继代培养、生根培养效果的影响及多代转接培养后的耐盐性变化。结果表明,培养基中NaCl含量为25mmol/L对3种试材微茎段组培繁殖效果影响甚微,但NaCl含量达75mmol/L则产生显著抑制作用;抑制作用的主要表现在不同阶段有所不同,总体上对根系生长发育的影响显著大于对地上部分的影响;无论在何种培养阶段,3种试材的耐盐性均为酸枣>金丝小枣>泗洪大枣;随盐胁迫下培养代数增多,3种试材的耐盐性均表现出增强趋势,枣的提升幅度大于酸枣,耐盐性弱的泗洪大枣的提升幅度大于耐性强的金丝小枣。     

通过花粉管通道法导入红树总DNA获得耐盐紫花苜蓿T0代植株及其RAPD验证

利用花粉管通道法将盐生植物红树总DNA导入紫花苜蓿,共导入1 391朵花,获得894粒T₀代转化种子。T₀代种子种植在含有225 mmol/L NaCl的MS培养基上,获得12株耐盐性强的植株。以供体和受体为对照,对12株耐盐植株进行RAPD分析,在55条随机引物中有8条扩增出稳定、清晰的条带,条带的差异性表现为新增条带、供体特异条带和受体条带丢失。以上结果初步证实外源DNA已经整合到受体的基因组中,而且T₀代植株耐盐能力提高可能与外源基因的导入有关。     

高羊茅胚性愈伤组织的高效诱导及其耐盐突变体筛选

以高羊茅Fine-lawn种子和下胚轴为外植体,分别在诱导培养基D1、D3、D5和D9上培养.下胚轴愈伤组织的出愈率除了在D1培养基上为51.3%外,在其他诱导培养基上均为100%.在D5培养基上继代3个月左右,从下胚轴获得了胚性愈伤组织.取在NaCl浓度为0.5%～3.0%的培养基上生长13 d的高羊茅胚性愈伤组织,分别测定其存活率、相对生长量、鲜干重比和脯氨酸含量.利用直接筛选的方法,将胚性愈伤组织分别放在含1%, 2%和3% NaCl的选择培养基上连续筛选60 d,在1% NaCl浓度下获得了耐盐的胚性愈伤组织并且获得了再生植株.再生植株能够在含有1% NaCl的Hoagland培养液中生长.     

红豆草耐盐突变体的研究

以普通红豆草子叶为外植体诱导出的愈伤组织置于含1%氯化钠的附加0.5mg/LKT和1mg/L 2,4-D的MS培养基中,连续继代筛选12代之后转入不含氯化钠的分化培养基MS+0.5mg/L 6BA+0.5mg/L IBA中,分化出完整再生植株.经田间栽培观察和染色体鉴定,再生株外部形态特征和染色体数目皆发生显著变异,获得红豆草耐盐突变体.     

黑麦冬牧70幼穗和未成熟胚愈伤组织诱导及植株再生

将黑麦冬牧70幼穗切段和未成熟胚接种在附加不同激素浓度的改良MS培养基上培养,获得了能多次继代培养的愈伤组织,后者在分化培养基上再生植株.2,4-D和6-BA浓度明显影响愈伤组织质量和诱导率,幼嫩幼穗和较小的未成熟胚愈伤组织诱导率高.幼穗切段在改良MS 1.0 mg/L 2,4-D 200 mg/L谷氨酰胺的培养基上愈伤组织诱导率最高,质量较好;未成熟胚在改良MS 2.0 mg/L 2,4-D 0.1 mg/L 6-BA 200 mg/L谷氨酰胺的培养基上愈伤组织诱导率最高,质量最好.继代培养后的愈伤组织转移到附加200 mg/L谷氨酰胺、1.0 mg/L 2,4-D和0.1 mg/L TDZ的培养基上可分化出大量小芽.该体系可用于黑麦冬牧70遗传转化和细胞工程育种研究.     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.

     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.