Regeneration ofSolanum tuberosum cv. Katahdin from leaf expiantsin vitro

A previously published medium has been found to be effective for plant regeneration from leaf expiants ofSolanum tuberosum cv. Katahdin. A number of combinations of thidiazuron and naphthaleneacetic acid, along with a medium previously published for shoot regeneration from protoplast-derived calli ofSolanum phureja, which contained zeatin, indoleacetic acid and gibberellic acid (ZIG), were tested for callus induction, and all calli were transferred to ZIG for shoot induction. We conclude that leaf expiants cultured on ZIG medium at all stages of culture were the most effective at shoot production. A mean of over six shoots were produced per expiant after four months in culture.     

籼稻绿芽悬浮细胞原生质体再生成株

采用籼稻Hu-18绿芽为外植体,在N6附加2 mg/L 2,4-D的培养基上诱导愈伤组织。加20 d后转人AA 培养基进行悬浮培养。继代培养45 d左右形成了分裂旺盛的胚性细胞悬浮系。即从绿芽诱导至胚性细胞悬浮系的建立仅用了65 d左右。继代后前6 d,细胞干重几乎每2 d增加1倍,而培养液的渗透压及pH 值迅速下降。取继代后4 d的悬浮细胞游离原生质体,产率为8.74X10⁶/g鲜重。纯化后的原生质体在KPR培养基中进行琼脂糖包埋培养,原生质体植扳率为12.0％ 。将20 d后的小愈伤组织(0.1 mm)转入 N6附加0.5 mg/L 2,4- D、1 mg/ L BA、1 mg/L KT、0.3 mg/L ZT的培养基上增殖,待长成直径2～3 mm 左右时,用N₆附加1 mg/L BA,1 mg/L KT,0.3 mg/L ZT的培养基进行分化培养, 5 d后同时出现芽根生长,最终再生成绿色植株,绿苗分化率为3.5株/10000个原生质体。     

金花茶松散型愈伤组织诱导与悬浮细胞系建立

采用组织培养及显微观察技术,以金花茶植株茎段、叶片、花药及离体培养的金花茶体细胞胚为试验材料,对松散型愈伤组织的获得及悬浮细胞系的建立进行了研究。结果表明:在黑暗条件下,以金花茶体细胞胚切块为外植体,在MS+KT 0.5 mg/L+NAA 8.0 mg/L+硝酸银5mg/L+蔗糖30 g/L培养基上诱导出愈伤组织后,将其转接到该诱导培养基上连续培养3个月,最后从培养物中挑选状态良好的松散型愈伤组织在分别含有肌醇与硝酸银的2种培养基上交替培养,可以获得金花茶松散型愈伤组织;方差分析表明:蔗糖添加量、肌醇添加量及硝酸银添加量均对金花茶悬浮培养液细胞密度有显著影响且后两者分别与光照条件存在相互作用;将松散型愈伤组织在分别添加100 mg/L肌醇与2.5 mg/L硝酸银的液体增殖培养基上交替培养,可以建立并保持金花茶悬浮细胞系。该研究结果可为金花茶大规模细胞培养提供科学依据。     

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.     

农杆菌介导将雪莲凝集素(GNA)基因转入籼稻单倍体微芽的初步研究

 以含有双元载体(携带GNA基因,nptⅡ基因)的根癌农杆菌菌系LBA4404转化籼稻单倍体无性系微芽Hu18,经共培养后在含G418的保存培养基中连续筛选G418抗性芽,抗性芽经生根培养基中壮苗获得单倍体转化植株。PCR分析证明GNA基因已进入到Hu18细胞中,抗虫鉴定表明转基因植株具有白背飞虱抗性。并探明了G418对单倍体微芽的致死浓度和时间,共培养时间对G418抗性芽产生的影响。      

海马齿再生体系的优化及GUS基因的转化

以海马齿无菌实生小苗的叶片为外植体,对不同激素浓度和组合以及不同pH值等培养条件进行实验比较.结果表明:适于愈伤组织诱导的培养基为MS+2,4-D 2.0 mg/L+6-BA 0.5 mg/L+蔗糖3%,诱导率达100%;适于芽分化的培养基为MS+NAA 3.0 mg/L+6-BA 0.5 mg/L,分化率达到77.4%;诱导愈伤组织形成培养基的最适pH值为4.5,而诱导愈伤组织分化培养基的最适pH值为5.0.以小苗叶片为外植体材料和农杆菌介导的方法进行GUS基因的转化,组织化学染色检测发现共培养2 d后的外植体中有蓝色斑点产生,在转化2个月后的外植体上形成的愈伤组织也存在点状的蓝色斑点.说明GUS基因已转入海马齿外植体中.     

罗勒(Ocimum basilicum)悬浮细胞系的建立与保持

通过罗勒茎尖培养获得疏松型(CA)、泥状型(CB)和绿色块状型(CC)愈伤组织;以这3种愈伤组织为起始材料,研究了建立罗勒悬浮细胞系的方法、影响因素及继代保持的方法。结果表明:疏松型、泥状型愈伤组织均能建立悬浮细胞系;以泥状型愈伤组织为起始材料,建立悬浮细胞系时间仅需7￣8d。     

Increased induction of embryogenesis and regeneration in anther cultures ofSolanum tuberosum L.

Summary A new method is presented for anther culture. Anthers are cultivated on cubes of solid medium, surrounded by liquid medium. This allows changes of media composition at any time. When embryos or callus were produced, the liquid initiation medium was removed and replaced with regeneration medium. The best yield of embryos and the best regeneration frequency were obtained when the initiation medium contained 2,4-dichlorophenoxyacetic acid (2,4-D). Regeneration of shoots from callus was stimulated on double layer medium, with the callus placed on top of a solid medium, partly submerged in the overlaying liquid medium. The best gelling agent for shoot multiplication media was agarose, but gellan gum was a good alternative. Both the production of flower buds and embryogenesis in anther cultures was inhibited by an increased concentration of CO₂ in the air surrounding the donor plants.     

野生稻原生质体培养与植株再生

三个野生稻（O. rufipogon,O. glumaepatula 和O. latifolia）成熟胚,经愈伤组织诱导和悬浮细胞培养,分离原生质体,利用简化原生质体培养基（SPCM）,结合琼脂糖包埋,并附加看护细胞培养液,这三个野生稻原生质体均得到了成功培养,其中两个（O. rufipogon和O. glumaepatula ）分化了绿色植株。看护细胞液在野生稻原生质体培养中,可促进细胞分裂,提早细胞分裂始期,细胞分裂频率（3.2％）和植板率（19.0%）分别比未加看护细胞液的培养方法高出13.3%和6.2%。看护细胞液可大大减少野生稻愈伤组织在液体中培养始期的死亡率,提高建立其悬浮细胞的成功率。原生质体克隆在适当的后培养基上进行培养,可以显著改良其结构,明显促进绿苗分化。尤其是L3培养基诱导原生质体克隆形成胚性愈伤组织或类似胚状体结构的频率达32.1%,显著高于MS和N6培养基,从而导致较高绿苗分化频率。     

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.     

Plant regeneration from tuber discs of potato (Solanum tuberosum L.) using 6-benzylaminopurine (BAP)

Summary Shoots, roots and callus were formed from tuber discs of potato, cultivar Désirée, when grown in vitro on the basal medium of Murashige & Skoog (1962) (MS) supplemented with 2,4-D and/or BAP. Callus was formed in MS medium with 1 mg l⁻¹ BAP plus 0.5 mg l⁻¹ 2,4-D, callus and roots were formed in MS with 1 mg l⁻¹ BAP plus more than 0.5 mg l⁻¹ 2,4-D and shoots were formed directly on tuber discs cultured on MS medium with 1 mg l⁻¹ BAP without the addition of 2,4-D. Nodules produced at the explant surface after the 4th week increased in size following subculture onto the same medium (MS+BAP alone), and 2 to 6 shoots developed from each nodule. After 9 weeks total time in culture, these shoots were excised and transferred as cuttings to MS medium without growth regulators, after which roots developed and plantlets were formed. A histological study of the explants at the sites of nodule formation indicated that the shoots developed from meristematic zones initiated within small outgrowths of tissue similar to those occuring in adventive organogenesis but the presence of shoot and root meristems associated with the same axis suggests the formation of somatic embryos.     

紫罗兰愈伤组织诱导及植株再生的研究

以紫罗兰幼苗子叶、子叶柄、下胚轴作外植体接种MS附加不同激素的培养基诱导愈伤组织，并进一步诱导分化出芽及再生植株。子叶和下胚轴外植体在MS＋0.1mg/LNAA培养基上愈伤组织发生率达100％，下胚轴愈伤组织转移MS 0.1mg/L 6-BA培养基上易诱导分化出小苗，分化频率达67％，再生小苗在1／2MSA＋0.2mg/L IBA培养基上生根率达86％。     

In vitro propagation and regeneration of an industrial plant kenaf (Hibiscus cannabinus L.)

The present study report a protocol for the efficient in vitro propagation of kenaf (Hibiscus cannabinus L., an industrial crop having high cellulosic fiber content) on hormone free MS medium using the shoot apex and nodal explants. Shoot tips and nodes were isolated from 15 days old seedlings cultivated on MS medium. Different combinations and concentrations of auxin/cytokinin were used and added to the MS medium to assess the shoot and root induction of theses explants. Several subcultures were drived in order to enhance the multiplication rate. Healthy and well developed in vitro propagated shoots were transferred for acclimatization under greenhouse conditions in pots filled with different substrates (sand + compost or perlite). Our results showed that shoots could elongate and root within 4-6 weeks on MS basal medium without any callus formation. However, addition of growth regulators to the MS medium leaded to a decrease in shoot and root induction rates. Indeed, the highest shoot regeneration frequency (90.5%) was obtained on MS control medium. Elongated shoots were transferred onto the same hormone free MS medium using five subcultures where the multiplication rate reached the highest value (3.66) at the fifth and last step. The in vitro rooted plantlets were acclimatized in greenhouse and successfully transplanted to natural conditions with 70% survival.     

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.     

Extracts from Black Carrot Tissue Culture as Potent Anticancer Agents

Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38–46 % at 6.25 μg/ml concentration for all calli and natural extracts. However, a significantly high IC₅₀ value of 170.13 μg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.     

园艺植物体胚发生及植株再生技术研究

通过无患子科荔枝,龙眼,天南星科白鹤芋,火鹤芋,花叶芋,芭蕉科香蕉,葫芦科西瓜甜瓜甜兰科卡特兰,大花蕙兰,石竹科满天星,康乃馨等５０多个品种品系体细胞胚胎发生的诱导技术研究,探索出园艺植物获得体细胞发生的两种。途径。第一途径由外植体先诱导出愈伤组织然后产生胚状体,此途径的技术关键在于前期必需在附加高浓度２,４－Ｄ的培养基上作激发培养,如荔枝,天星,康乃馨,火鹤芋,香芋等可由引途径产生体胚。第二种途     

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.     

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。     

花生幼叶芽诱导和植株再生研究

从萌发9－10d的花生幼嫩叶片上切取中段作外植体，接种MS＋BA 3mg/L NAA 0.8mg/L AgNO3 2mg/L诱芽培养基，12－14d后产生丛生芽点，芽诱导率达78．9％，平均每外植体产生9个丛生芽。4周后转至MS BA 3mg/L AgNO3 2mg/L诱导芽伸长，诱导生根后获得再生植株。