Callus induction and plant regeneration from lateral shoots of herbaceous bamboo Mniochloa abersend

Callus induction and plant regeneration of Mniochloa abersend via lateral shoots were conducted in this study. Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L naphthaleneacetic acid (NAA) was effective for compact callus induction. Remarkably, calli on the MS medium with 0.1 mg/L 2,4-D yielded the highest folds of proliferation (8.01), and showed a high potential capacity to differentiate 1 year after subculture. In addition, the compact calli possessed 100% differentiation rate and generated more shoots that were green and strong in 1.0 mg/L kinetin and 1.0 mg/L NAA. Vigorous roots were generated in the 1/2MS supplemented with 0.5 mg/L indole-3-butyric acid, and the resultant plantlets exhibited 90% survival rate after they were hardened and transplanted. The established regeneration system of M. abersend provides a promising platform for bamboo gene function study.     

An Experiment on the Winter-Killing of Vegetable Crops in Market Gardens

Summary

The development of an efficient methodology for the genetic transformation of orchids is needed in order to support thegenetic engineering of orchids. It is therefore important to identify those factors affecting the transformation process.Previously, we reported a convenient method for the transformation of Phalaenopsis amabilis using Agrobacterium tumefaciens, in which intact protocorms were used. We also found that embryos cultured on a medium containing tomato extract grew more rapidly than those cultured on a medium with coconut water. When we used protocorms grown on a medium containing tomato extract, we obtained regenerated shoots that had been transformed with a kanamycin resistance gene at relatively high frequencies (7 – 17%). These results suggest that the rate of growth of pre-cultured protocorms may be important for the successful regeneration of transformed shoots. We also obtained regenerated shoots that had been transformed with the green fluorescent protein (GFP) gene at a high frequency (10 – 14%). Both the presence and expression of these transgenes were confirmed in transformed plants by molecular analyses and by the detection of green fluorescence following excitation with blue light.     

Introduction of Xa21, a Xanthomonas-resistance gene from rice,into ‘Hamlin’ sweet orange [Citrus sinensis (L.) Osbeck] using protoplast-GFP co-transformation or single plasmid transformation

Summary

Plasmid DNA (pARS108) containing the non-destructive selectable marker Green Fluorescent Protein (GFP) gene, and a plasmid containing a cDNA of the Xa21 gene from rice (pXa21-mtaq) were co-transformed into ‘Hamlin’ orange protoplasts using polyethylene glycol (PEG). Alternatively, plasmid DNA (pAO3), containing both genes (GFP and Xa21) was directly transformed into ‘Hamlin’ orange protoplasts. Over 1,000 transgenic plantlets were regenerated from approx. 80 independent transformation events. The transgenic plants showed normal growth and stable GFP expression over more than 2 years in the greenhouse. This is the first report of a large population of transgenic ‘Hamlin’ sweet orange plants containing one or more target gene(s), using a protoplast-GFP transformation system. Polymerase chain reaction (PCR) revealed the presence of the Xa21 cDNA and the GFP genes in all single plasmid transformed plants, and in 35% of the co-transformed plants. Southern blot analysis showed the integration of the cDNA into one-to-five different sites per plant.Western blot analysis showed the accumulation of the rice XA21 protein in the transgenic sweet orange plants. This is the first time that a gene from rice has been stably integrated and expressed in sweet orange plants. Using the protoplast-GFP transformation system, it is possible to avoid the use of Agrobacterium, antibiotic resistance genes, and destructive assay systems.     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

Integration of the rolA gene into the genome of the vigorous apple rootstock A2 reduced plant height and shortened internodes

Summary

The aim of this work was to dwarf the vigorous apple rootstock A2 by insertion of the rolA gene. To optimize conditions for a successful transformation, regeneration tests were carried out. The use of sucrose in the regeneration medium gave higher regeneration frequency than sorbitol in some cases and the shoot number per regenerated leaf was higher at 10 |j.M TDZ compared with 2.5 |j.M TDZ on the sucrose medium. Two transgenic clones, verified by PCR and Southern analysis, have been obtained on the sucrose medium together with 2.5 |j.M TDZ and 1.0 or 2.5 |j.M NAA and wounding by forceps. The two clones, named LAI and LA2, contained both the rolA and nptll genes. The results of in vitro rooting showed that LAI had a lower rooting percentage and a reduced root number per rooted shoot than the untransformed control shoots and the clone LA2 on the rooting medium containing 5 |JLM IBA. Growth analysis revealed that both transgenic clones had a reduced plant height and a shortened internode length compared with the control plants. However, the node number and the stem diameter were significantly larger for clone LAI than clone LA2 and the control plants.     

Heterologous expression of the multi-functional cellulase gene (mfc) from the mollusc Ampullaria crossean,in Volvariella volvacea

Volvariella volvacea, or straw mushroom, is a widely cultivated and important edible fungus. In this paper, we used straw mushroom as a host to express an exogenous mfc gene encoding a multi-functional cellulase (MFC) cloned from the mollusc Ampullaria crossean. The purpose was to increase expression of MFC in the new host, as well as to improve cellulose degradation and increase future yields of V. volvacea. MFC is an enzyme with exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-xylanase activities. The A. crossean mfc gene was expressed in V. volvacea under the control of an endogenous promoter (gpd-Vvs) using the expression vector pgVvs-mfc, constructed by ligating the gpd-Vvs promoter with the mfc gene. The expression plasmid, pgVvs-afp, containing an afp gene encoding an anti-freeze protein (AFP) cloned from spruce budworm as the selective marker gene, was co-transformed into protoplasts of V. volvacea along with the vector pgVvs-mfc using polyethylene glycol (PEG)-mediated transformation. Putative transformants of V. volvacea were obtained by exposing the mycelia to regeneration medium (200 g l^?1 potato extract, 20 g l^?1 dextrose, 20 g l^?1 agar, and 0.8 M D-mannitol) at 0°C. PCR and Southern blotting were used to identify positive transformants. The results indicated that the mfc gene had been integrated into the genome of V. volvacea. The total cellulose activity, based on filter paper degradation, and carboxymethyl cellulase and xylanase activities in the transformants were 3.92, 4.75, and 58.99 Units ml^?1, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed an approx. 46 kDa band, which was equivalent to the expected size of the MFC protein of A. crossean. Thus, the mfc gene appeared to have been expressed heterologously in V. volvacea.     

梨叶片培养与转基因研究进展

建立稳定、高效的叶片不定梢再生体系,是梨转基因成功的关键环节。影响叶片不定芽形成的主要因素有基因型、培养基、植物生长调节剂、碳源和氮源等。而基因型、抗菌肽的活性、稳定性、农杆菌的侵染力、抗生素的种类、浓度及标记基因的选择等则影响基因的转化。目前已有多种目的基因转入梨中,但受体再生率较低,转化方法单一,转化品种有限。综合有关文献综述了国内外梨叶片培养和遗传转化研究所取得的进展,并结合存在的问题对今后的工作重点提出了几点建议。     

蝶形花亚科8种槐树的组织培养及再生能力的基因型效应

 对国槐等8种(亚种) 蝶形花亚科植物组织培养再生能力的研究结果表明: 8种槐树叶片的再生能力相差较大, 不定芽分化率由高到低排序为金叶槐( 79.03% ) 、黄金槐( 41.46% ) 、香花槐(36.2% ) 、刺槐(34.2% ) 、黄花槐(25% ) 、国槐(22.7% ) 、四倍体刺槐(21% ) 、红花槐(18% ) ; 愈伤组织诱导率、芽分化率及试管苗的生根率排列次序与叶片芽分化率基本相同。从本试验结果可得出8种槐树基因型对组织培养再生能力具有如下调控效应及特点: 属间、种间及亚种间基因型对培养基种类及植物生长调节剂的要求, 对脱分化与再分化能力的调控, 对不同组织、器官的再生能力的调控均具有同一性;不同基因型、甚至同一基因型的不同组织、器官还具有一定的特异性。     

外源抗坏血酸对离体苹果叶片衰老的影响

 以苹果(Malus domestica Borkh. ) 品种‘嘎拉’秋梢成熟叶片为试材, 在5 mmol·L ^{- 1}抗坏血酸(AsA) 培养条件下, 研究了外源AsA 对离体苹果叶片衰老过程( 0 ～72 h) 中抗坏血酸—谷胱甘肽(AsA - GSH) 循环各组分和AsA合成关键酶L - 半乳糖酸- 1, 4 - 内酯脱氢酶(GalLDH) 的影响。结果表明, 外源AsA部分抑制了离体苹果叶片衰老过程中膜脂过氧化, 降低了H2O2含量, 但对表示膜损伤的相对膜透性影响不大。对AsA - GSH循环各组分来说, 外源AsA维持了叶片衰老过程中抗坏血酸过氧化物酶(APX) 和脱氢抗坏血酸还原酶(DAHR) 的高活性, 部分抑制了单脱氢抗坏血酸还原酶(MDHAR) 活性的下降, 同时降低了GSH的氧化程度, 从而提高了AsA含量和再生能力。外源AsA 在衰老前期抑制了GalLDH活性, 降低了AsA的合成能力, 而后期维持了GalLDH的高活性。     

Cloning,characterisation, and expression analysis of an oleosin gene in coconut (Cocos nucifera L.) pulp

Summary

Oleosins are structural proteins found in oil bodies, organelles found in the cells of plant tissues with a high oil content that undergo extreme desiccation as part of their maturation process. Oleosins stabilise oil bodies. In this paper, a full-length cDNA sequence homologous to oleosin, a seed-storage oil-body protein, from coconut (Cocos nucifera L.) was identified from cDNA libraries during fruit development and characterised. The gene, termed Coco-Ole, contained an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The predicted amino acid sequence of coconut oleosin had a molecular mass of 13.0 kDa, and showed 92% (AAF76238.1) and 67% (AAC02239.1) sequence similarity to oil palm (Elaeis guineensis Jacq.) and rice (Oryza sativa) oleosin proteins, respectively. The amino acid sequence clustered in the same branch as oil palm in the cladogram, but was distant from other species. The results of semi-quantitative RT-PCR indicated that the Coco-Ole gene was expressed only in the pulp, and its expression increased significantly during pulp development. Compared with fluctuations in oil content, expression of the Coco-Ole gene was consistent with the anabolism of oil during pulp development. The cloning and sequencing of the Coco-ole gene provides a new marker for studies on oil body biogenesis and fruit development in coconut.     

In vitro regeneration and Agrobacterium mediated transformation in gladiolus

Summary

In vitro regeneration and transformation studies were conducted on two cultivars of gladiolus. Cormels of 1.0 to 1.5 cm diameter cut into 2–3 mm thick slices of top, middle and bottom, and in vitro derived bisected shoot tips were used as explants on MS medium supplemented with 18.6 μM kinetin for multiple shoot induction. Amongst the cormel slices, the top slice gave better shoot induction response of 89% with an average of 2.4 shoots per explant over both cultivars. In vitro derived bisected shoot tips were inoculated on the medium oriented cut-side up, cut-side down and vertically both with and without the cormel base attached. Bisected shoot tips without attached cormel base and inoculated in the cut-side down orientation showed an average of 90% shooting response. In vitro derived shoot tips were used as explants for transformation. Explants were wounded by scalpel and particle bombardment with 1.6 μm naked gold particles by the biolistic delivery system. The wounded explants, after 3 d of recovery period, were co-cultivated with Agrobacterium strain LBA4404 harbouring the binary vectors pBI141 and pTOK233 which contained gus reporter gene with rice actin and 35S promoters respectively. GUS expression frequencies of 5.3% and 23% was obtained from scalpel and particle bombardment wounded explants, respectively. Particle wounded explants showed an average of 63 and 103 GUS spots when co-cultivated with pBI141 and pTOK233 binary vectors respectively. Explants co-cultivated with pBI141, after three weeks of selection on antibiotic containing medium showed blue streaks of GUS expression. It was concluded that Agrobacterium could infect the monocot gladiolus and transform the tissue eficiently when tissues were prewounded with naked gold particles delivered by particle gun.     

Propagation of Bambusa vulgaris (yellow bamboo) through nodal bud culture

Summary

Bambusa vulgaris (yellow bamboo), is the most commonly cultivated and used bamboo species in many countries. With the increased demand for bamboo, the importance of bamboo plantations has been realized. This would require large quantities of planting material continuously for which tissue culture techniques offer a solution. The propagation of B. vulgaris through nodal-bud culture was studied. Single nodal segments were tested for bud-break and shoot growth on basic Murashige and Skoog (1962) medium (MS) supplemented with different combinations and concentrations of growth regulators. Results suggest that cytokinin is important for bud-break. Gibberellic acid enhances multiple shoot production in this species. The position of the node on the culm appears to affect bud-break and multiplication, middle nodes are the most suitable. Also, removal of prophylls enhances bud-break. The shoots developed from axillary buds could be rooted on MS basic medium at 50% macro and IBA (0.25 μ). Upon transfer to the field (after four weeks in the rooting medium), the shoots developed into true-to-type plants.     

Influence of the antimitotic agents colchicine and oryzalin on in vitro regeneration and chromosome doubling of diploid bananas (Musa spp.)

Summary

In vitro cultures of the four diploid banana cultivars, Sannachenkadali (AA), Anaikomban (AA), Kunnan (AB) and Thattillakunnan (AB) were treated with two antimitotic agents, colchicine (C₂₂H₂₅NO₆) and oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) to induce ploidy alterations, particularly the induction of tetraploids, which would serve as useful breeding material for creating newer, improved triploids. Both antimitotic agents, but particularly colchicine, had a negative effect on the in vitro regeneration of the four cultivars. This was reflected in terms of delay in regeneration, reduced multiple shoot regeneration rates, regeneration of smaller microshoots with lower fresh weights, and reduced response to rhizogenesis. Colchicine, as expected, had a negative effect on the number of multiple shoots regenerated. However, oryzalin at lower concentrations (10 mM and 20 mM) resulted in the regeneration of more microshoots per culture than the untreated control. The chromosome doubling capacity of colchicine was equal to that of oryzalin only at 125–200 times higher concentrations. The cultivars exhibited a genome-related response.     

Influence of 1-methylcyclopropene on pectic enzyme activities,gene expression,and cell wall modification in papaya (Carica papaya cv. ‘Sunrise’) fruit

SUMMARY

‘Sunrise’ papaya fruit harvested at two stages of maturity [colour break (< 10% yellow peel colour) and 25% yellow peel colour] were treated with 100 nl l^–1 1-methylcyclopropene (1-MCP) to determine its effects on ripening, on the activities and levels of gene expression of polygalacturonase (PG), pectin methyl esterase (PME), and βgalactosidase ( βGal), and on the degradation of cell wall components. 1-MCP delayed ripening and the onset of the climacteric, although the peak in the respiration rate was almost the same as that in untreated control fruit. Colour-break fruit treated with 1-MCP exhibited a continuous increase in ethylene production, but at a lower rate than in control fruit. Consequently, 1-MCP-treated fruit ripened with a concomitant reduction in firmness, which was accompanied by an increase in PG and βGal enzyme activities and gene expression. On the other hand, fruit treated with 1-MCP at the 25% yellow stage exhibited lower levels of ethylene production and developed pulp with a rubbery texture at the ripe stage which was attributed to reduced PG, βGal, and PME enzyme activities and gene expression. This was consistent with the higher level of cell wall polysaccharides measured in 1-MCP-treated fruit. The above results indicated that ‘Sunrise’ papaya fruit can be treated with 1-MCP at the colour break stage since they have a greater capacity to recover from the effects of 1-MCP than fruit treated at the 25% yellow stage.     

虾青素合成关键酶基因bkt 在‘Brookfield Gala’苹果中的遗传转化及表达

 虾青素是一种氧化型酮式红色类胡萝卜素,具有更强的抗光氧化能力。将β–胡萝卜素酮化酶（虾青素生物合成的关键酶）基因bkt构建入表达载体pCAMBIA1301中,获得植物表达载体p1301-bkt,转化根癌农杆菌EHA105,获得工程菌,以黄肉苹果‘Brookfield Gala’无菌试管苗叶片为受体,进行遗传转化。筛选压确定结果表明：‘Brookfield Gala’对潮霉素（Hyg）很敏感,叶片再生最佳Hyg选择压为3 mg · L^-1,试管苗增殖为4 mg · L^-1,生根为2 mg · L^-1;头孢霉素（Cef）浓度≤400 mg · L^-1时对叶片再生芽数的影响不明显。GUS染色、PCR和RT-PCR检测结果表明,有8株转基因植株整合bkt基因并获得了表达,其表型有红色产生;转基因植株类胡萝卜素的HPLC测定显示,虾青素和角黄素在叶片中的积累量达到2.85和1.79 μg · g^-1。本研究结果显示有望通过调控代谢途径,在苹果中合成虾青素,提高果实自身的抗光氧化能力,防止日灼。     

Regeneration and transformation of the premier UK apple (Malus × pumila Mill.) cultivar Queen Cox

Summary

A number of factors were assessed for their effects on in vitro shoot proliferation and adventitious shoot regeneration. More in vitro leaves of a quality suitable for use in regeneration and transformation experiments were obtained from shoots on DKW proliferation medium compared with MS medium, and also on MS and DKW media containing phloroglucinol. Compared with MS medium, shoot proliferation was greater on MS with halved levels of NH₄NO₃ and KNO₃. Adventitious shoots were hyperhydric on MS-based but not on DKW-based regeneration medium. More adventitious shoots regenerated on media solidified with Sigma Agargel than on media with Sigma Phytagel or Gelcarin (FMC). Viable transformed shoots were recovered on Sigma Agar or Agargel but not Phytagel. Wounding of leaf explants by stabbing with needles, and stabbing combined with scoring with a scalpel, increased the number of calli regenerating, and these methods, as well as solely scoring with a scalpel, increased the number of calli regenerating shoots compared with the control. Combined stabbing and scoring resulted in more calli producing shoots than solely scoring or stabbing. Vortexing leaf explants with silicon carbide whiskers increased the percentage of subsequently formed calli that regenerated shoots compared with the control. Transformed shoots were regenerated following co-cultivation with Agrobacterium tumefaciens EHA101 harbouring the binary vector pSCV1.6 (with selectable marker gene npt II and GUS reporter gene uid A). The number of transformed shoots as a percentage of explants varied from 0.5% to 2.2%. Molecular analysis of the four extant transformed lines confirmed integration of the transgene and indicated that in three lines there was one integration site, and in one line there were four sites of integration.     

Propagation of Vaccinium in Vitro

Abstract

Micropropagation techniques are important for clonal multiplication, germplasm improvement, and gene conservation of three commercially cultivated and medicinally important Vaccinium species: cranberries, blueberries, and lingonberries. The in vitro propagation of Vaccinium species using axillary bud proliferation and adventitious shoot regeneration has been investigated in a number of previous studies. The morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, which is again genotype specific. This review presents the progress in-depth of various aspects of Vaccinium propagation in vitro for its commercial production. It also discusses the issues that still need to be addressed to utilize the full potential of plant tissue culture techniques in mass propagation of Vaccinium species.     

Effect of cytokinin content of the regeneration media on in vitro rooting ability of adventitious apple shoots

Rooting ability of ‘Royal Gala’ shoots regenerated on media with different cytokinin content (thidiazurone, benzyladenine, benzyladenine-riboside and meta-topolin-riboside) was observed directly after regeneration and compared after subculture on hormone-free medium (A), or on medium with decreased cytokinin content (B), or elevated gibberellic acid (GA₃) content (C) for a week, or after a subculture on proliferation media (1.0 mg l⁻¹ benzyladenine-riboside) for four weeks (treatment D). Rooting and acclimatization of regenerants after directly regeneration was not successful. Subculture of shoots on hormone-free medium did not improve the rooting ability of shoots, while their subculture on B and C media resulted in up to 36% rooting rate depending on regeneration media from which the shoots originated. The best rooting rate (up to 76%) was achieved in shoots regenerated on medium with benzyladenine-riboside after treatment D similarly to rooting ability of micropropagated shoots (80%). The rooting capacity of shoots depended on both the cytokinin content of the regeneration media and different subculture media used. All rooted shoots survived after acclimatization.     

草莓主栽品种再生和转化的研究

 建立草莓主栽品种高效、稳定的离体再生体系和遗传转化体系, 获得了转基因植株。‘弗吉尼亚’和‘森嘎拉’的叶片离体再生芽频率达到100 %。试管苗叶片与农杆菌菌株EHA105 共培养3 d。共培养后的叶片在附加卡那霉素40 mg/L 的再生培养基上选择培养4周后, 外植体再生出转化芽, 弗吉尼亚的转化芽再生频率可达6. 8 %。采用组织化学染色法对随机选取的10 个GUS 基因转化植株进行基因表达测定, 结果5 个植株强烈表达GUS 活性。转bar 基因植株在附加除草剂草丁膦10 mg/L 的培养基上能够正常分化, 在田间对草丁膦表现出强烈抗性。转基因植株开花、结果正常。