马铃薯未传粉子房离体培养诱导双单倍体植株

以MS为基本培养基,附加不同水平的生长素,培养未传粉马铃薯子房,经三年试验,从两个品种中获得了双单倍体的绿色小植株,(2n=2x=24);从分化绿苗力很强的球状愈伤组织中,又不断地分化出许多绿色小植株。绝大多数品种,都可以诱导出愈伤组织,一般诱导率为70％左右。品种的基因型和培养基中的生长素种类与水平在愈伤组织分化绿苗中起着重要作用。通过扦插和试管微型薯培养,可以大量繁殖试管苗,这为马铃薯单倍体育种提供了较有利的条件。     

Somatic embryogenesis and organogenesis in Dendrocalamus hamiltonii

In this study, mature zygotic embryos, plant growth regulators, and various media were tested with the aim of developing an efficient regeneration system for plantlets of the bamboo species Dendrocalamus hamiltonii. Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 1.0–3.0 mg/l 2,4-dichlorophenoxyacetic acid. Optimal shoot differentiation and subsequent shoot growth were also obtained in MS medium supplemented with 2 mg/l benzyladenine, 1 mg/l kinetin, and 1 mg/l naphthaleneacetic acid. Root induction was enhanced by the addition of 5 mg/l indole-3-butyric acid to the culture medium. Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus initiation, shoot differentiation, and the development of plantlets from the mature zygotic embryos. Our data provide a useful basis for developing culture protocols for the regeneration of bamboo plants.     

In vitro propagation and rhizome formation in Curcuma longa Linn

Turmeric (Curcuma longa Linn.) which is cultivated by underground rhizomes is a slow propagating species. Multiplication and callus induction starting from the rhizome buds and shoot tips of C. longa in MS medium was carried out. A combination of naphthalene acetic acid (NAA; 1.0 mg/l) with kinetin (Kn; 1.0 mg/l) or NAA (1.0 mg/l) with 6-benzylaminopurine (BAP; 2.0 mg/l) was optimum for rapid clonal propagation of turmeric. A concentration of 2.5-3.0 mg/l of 2,4-dichlorophenoxy-acetic acid (2,4-D) was found to be optimum for callus induction. Regeneration of plantlets from a callus was successfully conducted in MS medium supplemented with standard growth hormones for multiplication at 25 +/- 2 degrees C under a 16 h photoperiod. These plantlets were successfully transferred to the field. Plantlets (4-month-old) were incubated in a medium containing different concentrations of sucrose supplemented with NAA (0.1 mg/l) and Kn (1.0 mg/l) at 27 +/- 2 degrees C under an 8 h photoperiod for induction of rhizomes. In vitro rhizome formation was observed in media containing 6 and 8% sucrose.     

Genetic transformation of Leymus chinensis with the PAT gene through microprojectile bombardment to improve resistance to the herbicide Basta

Chinese leymus [Leymus chinensis (Trin.) Tzvel.] is a perennial grass (tribe Gramineae) that is widely distributed throughout northern China and Mongolia where it is produced as a forage product. Severe production losses due to weed growth have serious economic consequences, and as non-selective herbicides not only kill the weeds but are also harmful to this forage grass, the introduction of a foreign gene for resistance to the herbicide Basta is necessary since this species lacks herbicide resistance. We have investigated the transformation of a gene for phosphinothricin acetyltransferase (PAT) through microprojectile bombardment in Chinese leymus. Calli from immature inflorescences cultured on N6 medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/l of glutamine were bombarded. The bombarded calli survived on selection medium with 1.0 mg/l of phosphinothricin (PPT). Twenty-three plantlets regenerated from resistant calli on differentiation medium supplemented with 1.0 mg/l 6-benzylaminopurine, 1.0 mg/l kinetin, and 1.0 mg/l PPT, and five of these regenerated plantlets survived on rooting medium with 1.0 mg/l of PPT. PCR and Southern blotting analyses indicated that the PAT gene had been integrated into the genomes of two Chinese leymus plantlets and that the gene was stably transferred to its clonal offsprings. There were no other phenotypic effects associated with transgene expression during vegetative growth except tolerance to the herbicide Basta.The Biotechnology of Pasture Plant Program is funded by the Key Project of the Chinese Academy of Sciences (KSCX1-08)     

The Effect of Plant Growth Regulators and Sucrose on the Micropropagation and Microtuberization of <Emphasis Type="Italic">Dioscorea nipponica</Emphasis> Makino

The effects of sucrose, plant growth regulators, MS (Murashige and Skoog), and ½MS salt media formulations were investigated for the development of shoot cultures, microtuber induction, and plantlet regeneration in Dioscorea nipponica. The cytokinin N-benzyladenine (BA) in the range of 0.5–2.0 mg/l showed strong enhancing effects on microtuber induction only when used in conjunction with the auxin alpha-naphthalene acetic acid (NAA), with the effect that NAA increased from 0.5 to 2.0 mg/l. Murashige and Skoog salt media supplemented with sucrose at 3% (w/v) gave the highest frequencies of shoot induction (86%) when BA was present at 2.0 mg/l and NAA at 1.0 mg/l. Sucrose at 7% (w/v) was the single most significant medium constituent for microtuber growth. The heaviest microtubers were formed on media containing 1.0 mg/l BA and 2.0 mg/l (0.073 g), especially with 7% sucrose (3.46 g). With media containing ½MS, 2% sucrose, and 0.1% (w/v) activated charcoal, the percentage of rooting was maximal when supplemented with 1.0 mg/l BA and 0.5 mg/l NAA for the in vitro produced shoots (95%) and BA and NAA both at 0.5 mg/l for the microtubers (100%). When removed from culture flasks and transferred into sterilized soil in a greenhouse, most of the hardened plantlets survived (over 91% after 1 week), and they were suitable for field planting after 1 month.     

Virus elimination and pathogen-free plantlets regeneration in Cucurbita pepo L.

An efficient in vitro protocol was established for developing pathogen-free plantlets in Cucurbita pepo through meristem culture. Meristems of about 0.3–0.5 mm in size were isolated from shoot tips of 25–30 day old in vitro grown plants. For primary establishment of isolated apical meristem, MS liquid medium supplemented with 2.0 mgl KIN and 0.5 mg/l GA₃ was found to be most effective in both cultivars. MS semisolid medium containing 2.0 mg/l BAP were found to be most effective for shoot development from primarily established meristem in both cultivars. A good number of shoots were not concomitant with good rooting. The best root induction was found in media having 1.0 mg/l IBA in cv. Bulum. It was found that cv. Bulum was better than cv. Rumbo in all stages of meristem culture. The presence of virus in plantlets was achieved by DAS-ELISA test, where 68–81% plantlets have been proved to be virus free among the studied viruses. Healthy growth and vigour was observed in meristem derived plants over their source plants after cultivation under natural conditions.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

Somatic embryogenesis and plant regeneration from leaf callus of Ocimum basilicum L

A effective protocol for complete plant regeneration via somatic embryogenesis has been developed for Ocimum basilicum L. Callus was initiated from leaf explant of young plant on supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 mg l(-1), 3% sucrose and 0.9% agar. The calli showed differentiation of globular structure embryos when transferred to MS medium containing 2,4-D 0.5 mg l(-1) and BAP 1.0 mg l(-1). The maximum globular structure embryos were further enlarged and produced somatic embryos in MS basal medium supplemented with BAP 1.0 mg l(-1)+NAA 1.0 mg l(-1) + KN 0.5 mg l(-1). Continued formation of globular embryo and germination of embryos occurred in this medium. Complete plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.     

鱼腥草体细胞胚胎发生和植株再生

目的:利用鱼腥草的叶片和叶柄为材料,进行体细胞胚胎诱导及植株再生研究。方法:运用正交设计试验,考察在改良的MS固体培养基上添加不同种类、不同浓度的植物生长物质组合及其配比对鱼腥草愈伤组织诱导、体细胞胚胎发生及植株再生的影响。结果:鱼腥草无菌苗叶片在含有2,4-D 1.0 mg/L 6-BA 0.5 mg/L的改良MS培养基上能诱导出胚性愈伤组织;胚性愈伤组织在含有6-BA 1.0 mg/L的改良的MS培养基上诱导体细胞胚的发生;叶柄在含有6-BA 1.0 mg/L改良MS培养基上直接产生体细胞胚。体细胞胚在改良的MS NAA0.1 mg/L 6-BA 1.0 mg/L的培养基上能够快速繁殖,形成大量不定芽,在不加任何激素的MS培养基上就可以萌发出不定根,发育为成完整植株,在MS IBA 1.0 mg/L的固体培养基上能够形成大量的根。结论:建立了鱼腥草体细胞胚胎发生及植株再生的体系。     

‘香槟’月季的组织培养和试管开花诱导

本文对‘香槟’月季（80sachinensis‘Xiangbin’）的组织培养技术和诱导试管开花进行了研究。结果表明：以茎段为外植体能诱导获得无菌苗,适宜的启动培养基为MS＋6-BA1．0mg-L-1＋IBA0．1mg·L-1,幼芽继代增殖的最佳培养基是MS＋6．BA1．0mg·L-1。＋IBA0．1～0．2mg·L-1,诱导生根的适宜培养基为1／2MS＋NAA0．3mg·L-1,生根率达80．0％。诱导试管开花的适宜培养基为MS＋6．BA0．5mg·L-1＋NAA0．1mg·L-1最适宜的诱导试管开花的蔗糖含量是30g·L-1;在三角瓶中培养,试管花可以正常开放,在培养瓶中培养花芽不能正常开放;MS培养基中增加2倍磷的含量,可以提高花芽诱导率,为25．O％;诱导试管开花的最适培养条件为温度21℃,光照强度80～100μmol·m-2．s-1,光照时间16h—d-1。     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

An efficient regeneration system and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese upland rice cultivar Handao297

A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants. Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency were investigated with Agrobacterium strain AGL1 containing the plasmid pCAMBIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots by five to tenfold in comparison to medium without TDZ.     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.     

血叶兰的组织培养与快速繁殖

以血叶兰的嫩茎段为外植体, 建立了血叶兰的组培快繁体系。结果表明： 芽诱导最适宜的培养基是MS＋6-BA 5.0 mg·L-1; 最适芽增殖培养基为MS＋6-BA 5.0 mg·L-1＋NAA 0.5 mg·L-1＋TDZ 0.3 mg·L-1, 芽增殖率达4.92倍; 最佳壮苗培养基为MS＋NAA 0.3 mg·L-1＋GA3 1.0 mg·L-1＋15%椰汁, 芽苗高度达到4.33 cm, 芽粗为0.61 cm; 生根最适培养基为1/2MS＋IBA 1.0 mg·L-1＋15%香蕉＋0.5 g·L-1活性炭, 生根率在93.0%以上; 炼苗后, 移栽在泥炭土＋珍珠岩＋松树皮（3：1：1, V/V/V）混合基质中, 存活率高于87.0%。     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM₃ medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM₃ medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM₃ medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5?±?2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA₃ + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80?±?0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.     

荷兰鸢尾(Iris xiphium L. var. hybridum)的组织培养

取荷兰鸢尾（ＩｒｉｓｘｉｐｈｉｕｍＬ．ｖａｒ．ｈｙｂｒｉｄｕｍ）鳞茎片不同部位外植体块,接种于附加不同激素配比的基本培养基上,其中取自鳞茎片基部外植体块２ｍｍ×２ｍｍ×２ｍｍ,培养基为ＭＳ＋ＢＡ１．０ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ的不定芽诱导率为最高（７０％）;最理想的增殖培养基为ＭＳ＋ＢＡ２．０ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ。不定芽直径４～５ｍｍ,培养基为ＭＳ＋ＢＡ０．２ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ有利于不定根的发生,诱导生根率达８３３％。试管苗不经练苗可直接出瓶,移栽于泥炭∶田土∶河沙＝１∶１∶１（Ｖ／Ｖ）的基质中。     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.