Micropropagation of a mature colchicine-polyploid and irradiation-mutant of Betula pendula Roth

Tetraploid plantlets were regenerated from cultured apical and axillary buds of a 23-year-old colchicine-polyploid and irradiation-mutant Betula pendula Roth tree. Bud explants were grown on modified Murashige and Skoog medium supplemented with 2.0 mg l(-1) benzylaminopurine (BAP) and 0.01 mg l(-1) 1-naphthaleneacetic acid (NAA). The medium allowed both induction of adventitious buds and development of shoots. The cut ends of new shoots produced new buds and shoots during a 4-week culture period. The micropropagated shoots were rooted on modified Murashige and Skoog medium containing 0.1 mg l(-1) NAA as the sole growth regulator. Plantlets were transferred to a peat/soil mixture (1:1) in the greenhouse, acclimated and then transplanted to a cold frame. The regenerated plantlets had a tetraploid chromosome set (4n = 56) and an altered leaf morphology typical of colchicine-polyploid birches. The leaves were hypertrophied and asymmetrical, with curly leaf margins. The mutant nature of the parent tree was also evident in the light-green color of the leaves of the plantlets.     

Rapid Micropropagation of Edible Bamboo Dendrocalamus asper

Abstract

Micropropagation protocols for Dendrocalamus asper using nodal shoots and seeds culture are described. Multiple shoots were induced through forced axillary branching. Ninety-five percent of the nodal shoot explants taken from juvenile primary and lateral branches, produced multiple shoots through axillary buds activation within 2 to 8 weeks on Murashige & Skoog's (MS) medium supplemented with 0.1-15 mg/l benzyladenine (BA). The cultured seeds also produced multiple shoots (1-20) within 6 weeks on this medium. The multiple shoot differentiation was influenced by the concentration of BA in the medium. The in vitro generated shoots were excised and subculture on MS + 3.0 mg/l BAP for further shoot multiplication. Fifteen to 20 fold rate of shoot multiplication was achieved by regular subculturing. These shoots were multiplied for more than 3 years without loss of vigor. Ninety-five percent of the shoots were rooted, when propagules (each consisting of cluster of 3 shoots) were transferred on to MS medium with 3.0 mg/l NAA or 10 mg/l indole-3-butyric acid (IBA).

To date, 18,000 plants (through axillary bud initiated from nodal ex-plants) and 6,000 plants from seed culture have been hardened and acclimatized. 12,000 plants have been field transferred.     

核桃离体培养中外植体褐化的研究

对核桃离体培养中外植体的褐变进行了初步研究，结果表明：4月份取嫩枝茎段进行培养，效果最差，褐变死亡率高，取6月份半木质化茎段进行培养，效果较好，休眠芽次之。液体状态的培养基可以减轻外植体褐变。培养基中附加激素6-BA和IAA、GA时，外植体的褐变程度较低，外植体腋芽萌发与生长分化较好。培养基中加入硫代硫酸钠对防止外植体褐变。提高外植体萌芽率和有效新梢率有促进作用。     

Photosynthesis of oak leaves under water stress: maintenance of high photochemical efficiency of photosystem II and occurrence of non-uniform CO(2) assimilation

Shoot cultures of Quercus rubra (L.) were established from both juvenile and adult plant material. Initial explants from epicormic shoots formed on the basal zone of the trunks had a greater capacity for in vitro establishment than explants from crown branches. The growth of vigorous axillary shoots was obtained by culturing decapitated shoots horizontally on Woody Plant Medium supplemented with 0.2 mg l(-1) of 6-benzylaminopurine. After 3 weeks of culture the shoots were transferred to fresh medium for two more weeks, giving a 5-week multiplication cycle. Efficient shoot production was achieved by combining three treatments favoring the growth of lateral buds: excision of the apex, horizontal culture and cytokinin treatment. The addition of indoleacetic acid or indolebutyric acid to the multiplication medium did not improve shoot proliferation rates, and naphthaleneacetic acid was detrimental. Recycling the same explant for several successive subcultures improved the efficiency of the propagation procedure. Using the optimal multiplication procedures, nine clones (six of juvenile origin and three from adult trees) were tested in vitro and it was found that genotype and age affected performance.     

弗吉尼亚栎不定芽增殖及试管苗再生影响因子研究

[目的]探析弗吉尼亚栎组织培养中不定芽增殖和生根的关键影响因子,建立弗吉尼亚栎试管苗再生体系,为弗吉尼亚栎无性系选育提供技术支撑。[方法]以弗吉尼亚栎带芽茎段为外植体,采用不定芽增殖途径,研究弗吉尼亚栎组织培养过程中外植体选择、褐化控制以及培养基选择、激素配比对不定芽增殖和生根的影响。[结果]表明:向培养基中添加抗坏血酸、硫代硫酸钠和活性炭均显著降低外植体的褐化半径(p<0.05),其中,外植体在含有3.00、5.00 g·L^-1活性碳的培养基中褐化半径最小。基本培养基筛选试验表明:以1/4MS或WPM为基本培养基时,平均芽长和芽数明显优于MS培养基。不定芽增殖最佳培养基为1/4MS+1.20 mg·L^-1 6-BA,培养周期40 d,增殖倍数可达6.6。最佳生根培养基为1/4MS+0.50 mg·L^-1 IBA+0.50 mg·L^-1NAA,生根率达53.33%,且根粗壮,木质化程度较高。组培苗移植到灭菌河沙中,平均成活率达57.78%。[结论]培养基成分和激素配比是影响弗吉尼亚栎不定芽增殖和试管苗再生的主要因子,低盐培养基(1/4MS或WPM)不仅可以促进不定芽增殖,且能够减轻外植体褐化;还发现野外取材的外植体极易褐化,而来源于无菌苗的外植体,褐化程度明显低于野外取材的外植体。     

Plantlet formation in pitch pine (Pinus rigida mill.) by tissue culture methods

Management of forest lands includes reforestation, ideally with superior propagules. Tissue culture technology, one approach to obtaining superior clones, is now being applied to conifers. Excised embryos and young seedling parts of Pinus rigida were induced to form multiple shoots when cultured on defined media containing a cytokinin. The age of the explants and the composition of the culture medium significantly influenced the frequency of adventitious bud formation. Temperature and photoperiod during culture also strongly influenced the differentiation and development of the buds. The elongation of the buds was achieved by elimination of cytokinin, reducing the concentration of salts and carbohydrate and addition of activated charcoal to the medium. Rooting was induced by treating the shoots with indole-3-butyric acid and naphthalene acetic acid for 5 days before transfer to auxin-free medium. The regenerated plantlets were then transferred to soil under non-sterile conditions for further development.     

MICROPROPAGATION IN TISSUE CULTURE OFDAHURIAN LARCH

Dormant buds of Larix gmelinii(4-30years old)were cultured in vitro.Axillary budsgrew on the explants,and60％-65％ of the explants’axillary buds,a differentiation rate of 60％ wasobtained on the explants collected from the 30-year-old trees.The maximum number of axillarybuds was 26 in one induction per initial explant.Bud clusters were separated into individual budsand most of them elongated into shoots.A few roots grew on the shoots.The MS(Murashige andskoog)and SH(Schenk and Hildebrandt)were more efficient media than the WPM.(Woody PlantMedium).The best hormone Combinations for the axillary bud inductions were BA1+NAA0.01 andBA2+NAA0.2(mg/L).The procedure was as follows:(1)Apical buds were explanted on theno—hormone basal agar medium and grown for 1 or 2 weeks;(2)Explants were transferred onto thebud—inducing medium and grown for 2 weeks and then(3)Cultured on the basal medium withouthormones for axillary bud elongation;(4)Bud clusters were separated and cultured continuously toa minimum height of1     

窄冠白杨优良无性系“HN-1”快繁技术体系建立

以窄冠白杨优良无性系"HN-1"的幼嫩茎段为外植体,探索了丛生芽增殖、生根及移栽适宜条件,建立起组织快繁技术体系,研究结果表明:MS培养基可作为HN-1丛生芽增殖的基本培养基,细胞分裂素6-BA浓度为0.3mg/L时对丛生芽增殖的作用显著;适宜的增殖培养基为MS+6-BA 0.3mg/L+NAA 0.1mg/L+IBA 0.1mg/L+IAA 0.15mg/L;适宜的生根培养基为1/2MS+NAA 0.1mg/L+IBA 1.0mg/L;培养条件采用正常光照生根效果最好,生根率达71.1%;适宜的移栽基质为草炭、黄土、细沙容积比1∶1∶1,移栽苗成活率达93.8%。     

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^-1 6-benzyladenine and 0.2 mg L^-1 a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^-1 NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.     

色木槭组织培养技术研究

以色木槭带芽茎段为外植体进行色木槭组织培养技术研究,通过对不同植物生长调节剂组合的筛选,诱导出丛生芽,获得了再生植株。结果表明：色木槭茎芽最佳灭菌效果是用0．2％ HgCl2灭菌时间12 min ,存活率为65％;茎芽在MS培养基上添加0.2 mg ·L -1 NAA作为启动培养基,添加3．0 mg ·L -16-BA时可以实现增殖,增殖系数最大,为5．0;以1/2MS为基本培养基,取培养后健壮的芽苗,接种到含有0．1 mg ·L -1 NAA的培养基上进行生根培养,建立色木槭的组培体系。     

樟脑型樟树芽器官离体培养技术

以樟脑含量＞70％的樟脑型樟树优株的嫩枝为外植体,进行芽器官离体培养技术研究.结果表明：每年的1～4月外植体灭菌消毒接种,无菌活体获取率比较高;适宜樟脑型樟树组培的基本培养基：M2 （MS＋Ca （NO3）2.4H2O 300 mg/L）;芽诱导培养激素组合：6-BA4 mg/L＋NAA1.5 mg/L＋KT 0.5 mg/L;芽增殖培养激素组合：6-BA3.0 mg/L ＋NAA0.5 mg/L ＋KT0.2 mg/L;在培养基中添加L-半光氨酸10 mg/L抑制芽褐化效果良好.     

In vitro regeneration of Salix nigra from adventitious shoots

Black willow (Salix nigra Marsh.) is the largest and only commercially important willow species in North America. It is a candidate for phytoremediation of polluted soils because it is fast-growing and thrives on floodplains throughout eastern USA. Our objective was to develop a protocol for the in vitro regeneration of black willow plants that could serve as target material for gene transformation. Unexpanded inflorescence explants were excised from dormant buds collected from three source trees and cultured on woody plant medium (WPM) supplemented with one of: (1) 0.1 mg l(-1) thidiazuron (TDZ); (2) 0.5 mg l(-1) 6-benzoaminopurine (BAP); or (3) 1 mg l(-1) BAP. All plant growth regulator (PGR) treatments induced direct adventitious bud formation from the genotypes. The percentage of explants producing buds ranged from 20 to 92%, depending on genotype and treatment. Although most of the TDZ-treated inflorescences produced buds, these buds failed to elongate into shoots. Buds on explants treated with BAP elongated into shoots that were easily rooted in vitro and further established in potting mix in high humidity. The PGR treatments significantly affected shoot regeneration frequency (P < 0.01). The highest shoot regeneration frequency (36%) was achieved with Genotype 3 cultured on 0.5 mg l(-1) BAP. Mean number of shoots per explant varied from one to five. The ability of black willow inflorescences to produce adventitious shoots makes them potential targets for Agrobacterium-mediated transformation with heavy-metal-resistant genes for phytoremediation.     

4PU、NAA、IBA在中华猴猕桃微繁殖技术中的应用研究

研究结果表明:1.使用新型的苯基脲衍生物4pU与生长素配合,可明显提高茎段和离体叶片的分化,其活性约为6苄基氨基嘌呤、6-BA)、玉米素(ZT)的10倍。2.在附加4pU(1毫克/升)的培养基上,分化不定芽和茎段丛生芽,个体发育较弱,须经在低浓度4pU(0.3～0.5毫克╱升)和6-BA(1毫克/升)培养基中再培养,方可获得健壮的试管苗。3.中华猕猴桃试管苗在1/2MS+NAA_(0.5)+IBA_(0.5)的培养基中培养10天后,移栽成活率可达95％左右。     

色木槭芽直接增殖途径的培养条件

为建立色木槭芽直接增殖的诱导培养体系,以色木槭野生树木休眠芽萌发枝条为材料,进行芽增殖的培养条件研究。结果表明,4月份是适宜休眠芽培养的取材时期。嫩茎在2%次氯酸钠浸泡20 min是最佳消毒方案。NAA浓度对休眠芽萌发和嫩茎生长的影响具有显著差异。MS+0.1 mg/L NAA+20 g/L蔗糖(pH5.8)是适合休眠芽萌发和嫩茎伸长的培养基。培养30天时,腋芽萌发率可达63.3%,嫩茎平均高度可达15.9 mm。6-BA对芽直接增殖的促进效果好于KT。不同激素组合中,IBA与6-BA组合对芽增殖和丛生芽生长的效果好于NAA与6-BA组合、NAA与KT组合、IBA与KT组合。MS+0.1 mg/L IBA+1 mg/L 6-BA+20 g/L蔗糖是适合芽增殖和丛生芽生长的培养基。培养30天时,芽增殖率可达90%,增殖倍数可达3.19,且茎芽生长正常。     

Micropropagation of valley oak shoots from seedling explants

Main and interactive effects of basal medium and cytokinin concentration on bud and shoot development of micropropagated valley oak explants were examined. Stem segments with axillary buds were placed on BTM (Broad Leaved Tree Medium), GD (Gresshoff-Doy) and WPM (Woody Plant Medium) media supplemented with 0.3, 0.7 and 1.5 mg 1^–1 of BAT (6-benzylaminopurine). Overall, BTM and GD media were superior for the micropropagation of this species, and both media promoted development of vigorous and relatively abundant shoots. Conversely, fewer shoots were initiated on WPM medium and those produced were frequently chlorotic. Irrespective of basal medium, the quantity of shoots produced after nine weeks generally increased as the concentration of BAP increased, but rosettes with short internodes and numerous leaves predominated on media supplemented with 0.7 or 1.5 mg 1^–1 of BAR Shoots on media with 0.3 mg 1^–1 of BAP, however, exhibited the long internodes and two or three leaves per shoot characteristic of typical valley oak sprouts, and were most suitable for subsequent micropropagation procedures.     

Micropropagation of field tested superior Eucalyptus grandis hybrids

Three superior clones of Eucalyptus grandis hybrids were micropropagated through several steps. Five-year-old trees were girdled to induce juvenile sprouts. Cultures were attempted from mature branches and sprouts. Branches from mature trees were 100% contaminated while sprouts were only 40% contaminated. Pre-initiation hormone free medium and dark environment were used to screen for contaminants and to reduce production of phenolic compounds. Initiation of auxillary buds was achieved with modified MS plus 0.05 mg/l NAA and 0.5 mg/l BAR High multiplication rates were obtained on auxin-free medium with 0.6 mg/1 BAR Elongation of shoots was best on media with high auxin (2.5 mg/l of IBA) and cytokinin (1–1.5 mg/l of zeatin). Continual subculture on the multiplication medium improved rooting significantly. Up to 98% rooting was achieved on 1/4 MS with 2 mg/l IBA. Rooted propagules were successfully transferred to a mist greenhouse with 82% survival, and then to greenhouse conditions.     

口红花组培与细胞诱变试验效果

以MS为基本培养基,在不同的激素成份下,培养口红花(AeschynanthuspulcherG.Don)叶片,得出适合诱导其愈伤组织培养基为MS+2,4-D0-4mg·L-1+BA0-3mg·L-1,诱导率为60%,适合分化不定芽的培养基为BA0-5mg·L-1和NAA0-3mg·L-1,其分化芽可达3～4倍。采用γ射线辐射其幼芽,辐射剂量为1-72Gy·min-1,总剂量为40～45Gy,诱变率达6%,同时对其壮苗进行生根培养,选用1/2MS基本培养基附加IBA0-3mg·L-1和NAA0-5～0-6mg·L-1,生根率100%,生根时间40d。     

Organogenesis and terpenoid synthesis in Mentha arvensis

Leaf discs obtained from field grown plants of Mentha arvensis were used to initiate multiple shoots on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (5 mg l(-1)) and naphthaleneacetic acid (0.5 mg l(-1)). Profuse rooting was achieved when the well-grown shoots were cultured on half strength MS medium supplemented with indole-3-acetic acid (2 mg l(-1)). The regenerated plantlets were hardened and successfully transferred to soil and grown to maturity. Tissues at different stages of differentiation were analyzed for their essential oil content and characteristic monoterpene pattern. Tissue culture raised plants show the same essential oil profile as that of the parent plant. However, tissues at early stages of growth show distinct changes in oil composition, such as high levels of pulegone in shoot cultures.     

灯盏细辛离体叶片再生植株的研究

以灯盏细辛（Erigeron breiscapus）叶片为外植体,研究不同培养基配方对灯盏细辛离体培养过程中愈伤组织、不定芽形成,壮苗培养和离体植株生根等方面的影响。研究结果表明,低浓度的细胞分裂素与生长素配比可诱导灯盏细辛愈伤组织产生,其中MS＋BA1．00mg／L＋NAA0．50mg／L培养基配方,愈伤组织诱导率达到99．22％;不定芽诱导需要较高浓度的细胞分裂素,适宜培养基MS＋KT4．00mg／L＋IBA0．50mg／L的不定芽诱导率为38．51％,诱导系数为0．49;MS＋BA0．50mg／L＋NAA0．50mg／L＋水解酪蛋白1000．00mg／L＋PVP1000．00mg／L＋GA0．50mg／L培养基可促进不定芽分化生长,形成离体植株;离体植株生根需要高浓度生长素,适宜培养基为1／2MS＋BA0．50mg／L＋NAA3．00mg／L＋IBA3．00mg／L＋活性炭0．30％。     

离体培养条件下核桃器官发生和体细胞胚胎发生

采自幼树、成年树及成年树伐根萌条的嫩梢腋芽,在改良DKW培养基+6-BA1.0mg/L中可伸长生长;在加有6-BA1.0mg/L+IBA 0.01mg/L的继代培养基中,可保持腋芽的生长并形成不定芽。芽增殖率为每月600％左右。芽苗经5.0mg/L IBA处理7天,然后在含活性炭的无激素培养基中培养20天,有54％可生根成苗。5月中旬至6月上旬的幼胚,在改良DKW培养基+6-BA1.0 mg/L中,黑暗培养30天左右可产生体细胞胚,并可连续多代保持胚性分生能力。体胚在无激素的发芽培养基中黑暗培养7天左右可发芽成苗。胚性愈伤组织在悬浮振荡培养中可保持分生能力。