水田七的组织培养和植株再生

用水田七成熟种子为材料进行无菌播种,得到无菌苗再用幼叶、叶柄为材料进行组织培养和植株再生。经试验得出各阶段适宜的培养基分别为：(1)诱导愈伤组织：MS＋2.0 mg/L TDZ;(2)诱导芽分化：MS＋1.5 mg/L 6-BA＋0.5 mg/L NAA;(3)生根培养：1/2MS＋0.5 mg /L IBA＋0.1 mg/L NAA。     

基因型对油菜子叶外植体再生植株的影响

为了了解细胞核和细胞质基因在油菜再生中的作用，采用MS培养基附加不同的激素，对6个基因型相关材料的子叶外格体进行了愈伤组织诱导及植株再生研究。愈伤组织诱导频率、生长速度以及不定芽的生根频率，主要受细胞核基因的影响，细胞质施加的影响不明显。细胞核基因极显著地影响着不定芽的再生，在此过程中，细胞质也起了显著的作用。陕2A与PolimaA的胞质效应表现一致，两种不育材料可能属同种类型来源的细胞质。     

Plant regeneration from leaf tissues of four North Dakota genotypes of potato (Solanum tuberosum L.)

An efficient plant regeneration system was developed fromin vitro leaf tissues of four North Dakota potato genotypes. The best medium for genotype ND860-2 was Murashige and Skoog medium with 20.0 μM IAA and 11.4 μM zeatin riboside. Two to three weeks of initial dark treatments had a significant effect in reducing the time for shoot regeneration and increasing the number of regenerated shoots. Four antibiotics commonly used inAgrobacterium- mediated transformation were tested for their effects on shoot regeneration. Shoot induction was completely inhibited by kanamycin at 15 mg/L and hygromycin at 4 mg/L or higher, but not by carbenicillin (except at 1000 mg/L) and cefotaxime. Hygromycin significantly stimulated shoot induction at 1 mg/L.     

In vitro shoot regeneration and chromosome doubling in 2X and 3X potato clones

The possibility of scaling ploidy levels up and down offers several advantages for genetic studies and breeding work in potato. Shoot regeneration and chromosome doubling in plants regenerated fromin vitro cultures were investigated in 4 diploid (2n=2x=24) and 2 triploid (2n=3x=36) clones. Internode and leaf expiants were taken from plants propagated eitherin vitro as shoot cultures, orin vivo in a greenhouse. Two regeneration procedures were compared. Regeneration frequencies were significantly higher using a two-step regeneration protocol and from leaf explants, while doubling was more efficient starting fromin vivo grown plants. No doubling was observed in triploid clones. Considering altogether the percentage of regeneration and doubling, and the number of shoots regenerated per explant, the most efficient conditions were considered leaf explants taken fromin vivo grown plants, and cultured according to a two-step regeneration protocol.     

Production of somatic hybrids between frost tolerantSolanum commersonii andS. Tuberosum: Protoplast fusion,regeneration and isozyme analysis

Mesophyll protoplasts ofSolanum commersonii, a frost tolerant wild species not crossable with the cultivated potato, were fused with either dihaploid or tetraploid S.tuberosum. Protoplasts were aggregated by means of alternating current (AC) or polyethylene glycol (PEG), and electrofused with three direct current (DC) pulses. The treatments with PEG/DC generally resulted in very low heterofusion frequency and protoplast viabiity. On the other hand, AC/DC fusion conditions were optimized by increasing the fusion density of protoplasts and adding CaCl₂ to fusion medium. When a density of 4.8 × 10⁵ protoplasts ml^?1 was used in the fusion medium containing 0.2 mM Ca⁺⁺, AC/DC treated protoplasts showed heterofusion frequencies and plating efficiencies of about 10 and 3%, respectively. Fast growing calli from AC/DC fusion experiments were further cultured for regeneration. Fifty-seven plants were regenerated and clonedin vitro as shoot cultures. Compared to parents they showed heterotic vigor and could be identified as hybrids, based on isozyme analysis.     

Aluminium-tolerant regenrants from potato cell cultures

Summary Cell cultures were developed from dihaploid clones ofSolanum tuberosum L. Selection in cell suspensions as well as plating of cells on selective medium supplemented with 5 mmol Al produced tolerant cell lines. The constancy of Al-tolerance of cell lines was confirmed by culturing the calli for 3 months in Al-free medium and then transferring them back to selective medium. 4 tolerant regenerant clones were obtained which maintained Al-tolerance also after subculture in control medium. Two of the 4 clones that constantly maintained Al-tolerance, originated from cell lines subcultured for 5 months under stress conditions. However, the regeneration rate of these cell lines was low compared with that of lines obtained after a shorter selection period.     

蒲公英的组织培养研究

以盆栽蒲公英的叶柄和叶片为试材,对蒲公英进行组织培养。结果表明：300mg/L的抗坏血酸能够较好地防止褐化,直接诱导再生植株的最佳组合是MS＋0.5mg/L6-BA＋0.1mg/LNAA;诱导愈伤组织和继代的最佳组合是6-BA0.5mg/L＋2,4-D0.5～1.0mg/L;1/2MS＋15g/L蔗糖＋NAA0.1mg/L是诱导生根的理想培养基。     

An evaluation of callus induction and plant regeneration in twenty-five turf-type tall fescue (Festuca arundinacea Schreb.) cultivars

In order to investigate the genetic variation in tissue culture response and to find the cultivars with high regeneration ability for genetic transformation, twenty-five turf-type tall fescue ( Festuca arundinacea Schreb.) cultivars, including many elite ones released recently, were evaluated for their callus induction and plant regeneration responses. Callus induction was initiated from mature seeds on a Murashige and Skoog (MS) medium containing 9·0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). Induced calli were subcultured on the same medium with 2·0 mg l^–1 2,4-D and then transferred to a MS medium supplemented with 2·5 mg l^–1 6-benzylaminopurine (BAP) for plant regeneration. Significant differences were observed among the twenty-five cultivars in both callus induction and plant regeneration ( P  < 0·001). Callus induction rate of viable seeds varied from 4·4% to 51·9%. Callus regeneration rates ranged from 16·7% to 58·8%. Overall regeneration rates (number of regenerated calli over number of cultured viable seeds) ranged from 1% to 22%. Approximately 94% of the regenerants were green plantlets.     

Growth optimization and organogenesis of Gerbera jamesonii Bolus ex. Hook f. in vitro

Regeneration potentials in Gerbera jamesonii Bolus ex. Hook f. from tissues culture system was studied using leaf, petiole and root explants. In vitro regeneration, callus induction and root formation were optimized by manipulation of growth regulators during organogenesis. Various kinds of plant growth regulators such as 6-Benzylaminopurine (BAP), alpha-Naphthalene acetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2,4-D), Indole-3-acetic acid (IAA), Indole-3-Butyric acid (IBA), N6-[2-Isopentenyl]adenine (2iP), Kinetin and Zeatin were used to initiate cultures. These plant growth regulators were added to Murashige and Skoog medium in different combinations and concentrations. Adventitious shoots were obtained from petiole explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L(-1) BAP and 0.5 mg L(-1) NAA. Effectiveness of shoot regeneration medium, type of growth regulator used and duration of induction period were investigated. Leaf explants cultured on MS medium supplemented with 1.0 mg L(-1) BAP and 2.0 mg L(-1) 2, 4-D showed the best results for callus induction. Root explants were found to be non-regenerative in all experiments conducted. Petiole segment was identified as the best explant for regeneration of this species. Regenerated plants were rooted on Murashige and Skoog basal medium. Plantlets were then transferred to field with 75% survival rate.     

Isolation,culture, and regeneration of leaf mesophyll protoplasts of selected clones ofSolanum

Summary Protoplast production and regeneration of nine cultivars ofSolanum tuberosum spp.tuberosum and a hybridSolanum phureja × S. chacoense (JV-2) were compared by using methods based on Grun & Chu (1978) and Shepard (1980). The yield of protoplasts differed significantly among clones. Small leaf samples produced more protoplast per gram than large ones. Significant environmental effects on protoplast production when only the light that the plants received was controlled became non-significant when both light and temperature were controlled by use of a growth chamber. JV-2 was successfully cultured by the method based on Grun & Chu (1978), but the cultivars were not. Protoplasts of six clones developed calli, when the method based on Shepard (1980) was used, but cvs. Atlantic and Kennebec usually failed to form cell walls. Success in growth and development during each stage of protoplast and callus culture was clone specific. Calli of JV-2, Russet Burbank, and Lemhi developed shoots while those of Butte, Superior, and Green Mountain did not. Authorized for publication as paper No 7121 in the Journal Series of The Pennsylvania Agricultural Experiment Station.     

High-efficiency regnerationin vitro from potato petioles with intact leaflets

The shoot/plantlet regenerationin vitro of seven potato (Solanum tuberosum L.) cultivars from petioles with intact leaflets was assessed using six treatment combinations-a basal medium with or without silver thiosul-phate (STS) or thidiazuron (TDZ) at two concentrations (2 or 0.5 mg/l) of the indoleacetic acid (IAA). The basal medium consisted of Murashige and Skoog (MS) salts and vitamins supplemented with 3 mg/1 6-benzylaminopurine, and 1 mg/1 gibberellic acid, 30 g/l sucrose, and 7.0 g/l PHYTOAGAR. Two full sets repeats and one partial set repeat of independent experiments were conducted and all produced similar results. Silver thiosulphate decreased the regeneration frequency and number of shoots per callus. No significant changes were observed with thidiazuron. Regeneration rates of (100% ) with up to 20 shoots/plantlets per callus were achieved at 2 mg/1 IAA with cultivars Désirée, Kennebec, Niska, and Lenape. These cultivars still showed high regeneration rates (87%–98% ) on media with 0.5 mg/1 IAA, and good regeneration rates were also achieved by the other three cultivars (48%, 94%, and 50% for Chieftain, Russet Burbank, and Shepody, respectively). Even with the single medium protocol (0.5% IAA without thiosulphate or thidiazuron), Désirée, Lenape, and Niska exhibited a regeneration rate of 98%. The use of petiole-with-leaflet explants could be ideal for the regeneration step of potato genetic transformation protocol because of their high regeneration efficiency and their small cut surface area forAgrobacterium elimination after co-incubation.     

东方百合高效离体再生体系的建立

采用东方百合鳞片叶切块为初始外植体,以从初始外植体上分化的丛生芽切段为次级外植体的两步外植体法,成功建立了东方百合的离体再生体系。研究了不同的2,4-D浓度及次级外植体的不同部位对愈伤组织诱导效果的影响,并确定了G418选择的临界浓度。结果表明:不同部位的次级外植体中,以短缩茎切片出愈率高、出愈快、愈伤组织致密;以MS附加2.0 mg/L 2,4-D和0.1 mg/L BA的培养基最适于东方百合愈伤组织的诱导;G418选择的临界浓度为50 mg/L。一个中等大小已脱春化的鳞茎通过愈伤组织再分化植株一代就能扩繁出27 000株左右的新植株,从鳞片叶开始至开花需9个月。     

TDZ和暗培养对蝴蝶兰叶片不定芽发生的影响

以蝴蝶兰幼嫩花梗诱导的无菌苗叶片为外植体,以MS为基本培养基,研究不同暗培养时间和TDZ浓度对蝴蝶兰叶片诱导不定芽的影响。结果表明：暗培养和TDZ对接种的蝴蝶兰叶片的成活率均具有显著影响,暗培养60 d时,叶片的存活率在90%以上,而在同一暗培养时间条件下,叶片的存活率随着TDZ浓度的增加而上升;在TDZ浓度为3.0 mg/L,暗培养60 d时,蝴蝶兰存活叶片不定芽的诱导率最高,达到93.45%;诱导的不定芽数最多,平均每个外植体诱导的不定芽数为13.22个。综合考虑外植体的成活率、不定芽诱导率和诱导的不定芽数,蝴蝶兰叶片诱导不定芽的最佳条件为：MS＋TDZ 3.0 mg/L,暗培养60 d。     

Morphogenic Response of Leaf and Petiole Explants of Broccoli Using Thidiazuron

Broccoli (Brassica oleracea var. italica) is an important nutritionally rich vegetable cole crop grown in the world. Environmental stress, pests, and diseases cause enormous yield losses because of a limited gene pool. Genetic manipulation is becoming an important method for broccoli improvement. The objective of present study was to evaluate the potency of thidiazuron (TDZ) as a plant growth regulator in evoking morphogenic responses in leaf and petiole explants of broccoli. An efficient, reproducible, and high frequency plant regeneration protocol has been standardized in broccoli cv. Solan green head. Leaf and petiole explants were cultured on Murashige-Skoog (MS) medium, supplemented with a wide range of TDZ concentrations. The following treatments were designed for efficient in vitro shoot regeneration: TDZ alone, TDZ with adenine, TDZ with naphthalene acetic acid (NAA), and TDZ with indole acetic acid (IAA). Among the 36 combinations of growth regulators used, the highest percentage of leaf explants producing shoot (89.25%) was recorded on MS medium containing 1.0 μM TDZ and 0.107 μM NAA. The multiple shoot regeneration response of petiole explant producing shoots (91.55%) was obtained on MS medium containing 2.0 μM TDZ and 0.107 μM NAA. Shoot multiplication and elongation were obtained on the same medium. For root regeneration in in vitro regenerated shoots, different concentrations of NAA were applied. High frequency (100%) root regeneration response with healthy and vigorous roots was observed on MS medium supplemented with 0.54 μM NAA. The regenerated plantlets with well-developed shoots and root system were transferred to pots containing cocopeat and successfully acclimatized. We recommend 1.0 μM TDZ with 0.107 μM NAA and 2.0 μM TDZ and 0.107 μM NAA combinations for adventitious shoot regeneration from leaf and petiole explants in broccoli cv. Solan green head respectively. This is the first report on high frequency organogenesis from leaf and petiole explants of broccoli cv. Solan green head using thidiazuron.     

An improved and efficient micropropagation of Eclipta alba through transverse thin cell layer culture and assessment of clonal fidelity using RAPD analysis

An improved and efficient in vitro regeneration system has been developed for Eclipta alba, a medicinally important plant, through transverse thin cell layer culture (tTCL). The transverse section of the nodal segment of field grown plants was used as tTCL explants for plant regeneration. Shoot multiplication from tTCL nodal explants was influenced by BAP and their interaction with Kin or NAA. MS medium containing 13.2 μM BAP and 4.6 μM Kin was most effective for shoot multiplication from tTCL nodal explants. Upon this medium, percent response for shoot proliferation was 100% with an average of 32.6 shoot buds per tTCL nodal explant. Regenerated shoots from tTCL nodal explants were rooted on the growth regulator free MS medium. The rooted plantlets were successfully acclimatized and established in soil with a survival frequency of 90-100%. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic fidelity of the micropropagated plants. RAPD profile analysis indicated that micropropagated plants were genetically similar to mother plant.     

A Histological Evaluation of Adventitious Bud Formation in Cotyledons in Crotalaria juncea L.

Abstract

In Crotalaria juncea L., adventitious buds were formed in cotyledonary expiants cultured on 0.8% agar-solidified 1/2 MS basal medium containing B5 vitamins, 1.0 or 0.5 mg L^–1 NAA, 5.0 or 10.0 mg L^–1 BA and 3% sucrose. The frequency of adventitious bud formation was 30-45% in all combinations of NAA and BA. In histological observations, prominent mitotic figures were observed in several cells of the subepidermal palisade layers on the adaxial side of the expiants in contact with medium after 3 days of culture. Calli were formed within 6 days of culture. After 10 days of culture, numerous mature tracheary elements were produced at random in the proliferated regions, and cell divisions at the superficial region led to the formation of the meristematic structure. The shoot apex of the seedling produced numerous trichomes from superficial cells, but the adventitious bud formed on the cotyledon produced no trichomes. Initiation of the meristematic region in the expiant could be used as a target site for gene transfer experiments.     

第一例栽培种花生花药培养单倍体植株的创制

以栽培种花生为材料,开展花生单倍体培养的前沿性探索,包括离体花药诱导形成愈伤组织、愈伤组织分化形成幼芽、再生苗培养、再生植株倍性鉴定和嫁接移植等研究。通过比较外植体灭菌时间、诱导培养基、分化培养基、再生培养基和再生植株创制培养条件等试验,筛选出适合花生花药培养的方法：1% NaClO消毒灭菌9 min,愈伤组织的诱导培养采用B5N1、B3N1培养基,再分化培养采用B5N1-2培养基,再生苗培养采用SG培养基。本研究获得1株花药培养的再生植株,编号为15B8-8。荧光原位杂交技术（FISH）鉴定结果表明,该植株具有20条染色体,其中9条为A染色体、11条为B染色体,是第一例来自栽培种花生花药培养的单倍体植株;研究还表明,汕油52花生品种具有较强的花药培养力,有潜力作为花生花药培养的“桥梁品种”。     

短光低温不育水稻宜D S成熟胚培养的研究

对短光低温不育水稻宜D S成熟胚在组织培养中的愈伤组织诱导和分化进行了研究,结果表明:愈伤组织诱导率与绿苗分化率之间无对应关系;诱导培养基的成分对转分化后的愈伤组织有后效作用,2,4-D与6-BA和NAA搭配使用比单独使用2,4-D诱导的愈伤组织更易于分化;同时还发现,不同诱导培养基诱导的愈伤组织在转分化后对分化培养基的要求不同.     

Shoot inhibition inSolanum tuberosum. I. Response to ammonium and nitrate nitrogen sources

Solanum tuberosum Group Tuberosum plants grown in a non-nutritive media exhibit a shoot inhibition which follows the normal tuber bud dormancy. The shoot inhibition is characterized by very slow growth of the aerial portion of the plant with little leaf development. An external source of nitrogen, either nitrate-N or ammonium-N, applied to the roots of Iopride plants overcame the inhibition while only a nitrate-N source was effective in overcoming the inhibition of Kennebec plants. Group Phureja and Stenotomum and otherSolanum species evaluated did not show the shoot inhibition phenomenon.     

In vitro regeneration from petiole explants of non-toxic Jatropha curcas

Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.