金属离子对霍山石斛类原球茎增殖及植株再生的影响

 采用单因素浓度试验和正交试验研究了Mg²⁺ 、Ca²⁺ 、Fe²⁺ 、Mn²⁺和Zn²⁺离子对液体培养霍山石斛类原球茎增殖的影响。结果表明, 5种金属离子对类原球茎增殖影响的主次顺序为Fe²⁺ >Mn²⁺ >Mg²⁺ >Ca²⁺ > Zn^{2 +} , 促进类原球茎增殖的优化组合为Mg²⁺ 1.5 mmol·L ^-1、Ca²⁺ 4.5 mmol·L ^-1、Fe²⁺ 0.1 mmol·L ^-1、Mn²⁺ 0.5 mmol·L ^-1、Zn²⁺ 0.06 mmol·L ^-1。经优化的液体培养基增殖的类原球茎能保持植株再生能力, 平均每克类原球茎增殖后可再生植株1 985.2株, 是相同时间内对照类原球茎再生植株数的4.2倍。     

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.     

春石斛兰类原球茎分化过程中抗氧化酶活性和可溶性蛋白的变化

以春石斛兰(Dendrobium nobileSw.)为试材,研究了类原球茎在分化芽和根系发生过程中抗氧化酶系统中多种酶活性及可溶性蛋白的变化规律。结果表明,在分化过程中抗坏血酸过氧化物酶(APX)、愈创木酚过氧化物酶(POD)、多酚氧化酶(PPO)和过氧化氢酶(CAT)的活性和可溶性蛋白含量均呈现先降后升的趋势;超氧化物歧化酶(SOD)活性则呈现先升后降的变化。对过氧化物酶同工酶和可溶性蛋白进行SDS-PAGE电泳发现,在分化过程中均有新谱带的产生或原有谱带的消失。     

金钗石斛类原球茎原生质体的分离

以具有较多生长点的金钗石斛PLBs为材料,研究通过酶解法获得原生质体的适宜条件。结果表明,酶液配比为φ=1%纤维素酶（Cellulase Onozuka R10）＋φ=0.4%果胶酶（Macerozyme Onozuka R10）＋0.65mol·L-1甘露醇,pH5.7;黑暗条件下,（25±2）℃,60r.min-1振荡酶解6h,800r.min-1离心分离4min,获得的原生质体质量较好。经计数和伊凡蓝染色法检测,用此法获得的金钗石斛PLBs原生质体鲜质量产量为（8.25±0.17）×105个.g-1,活性为（90.24±1.84）。（蓝光（470～75nm）激发活力检测发现较小的分生细胞具有较强的活力。     

金钗石斛类原球茎诱导及增殖的正交试验

通过正交试验,筛选了金钗石斛Dendrobiumnobile类原球茎(protocrom likebodies,PLBs)诱导及增殖适宜的培养基.以1/2MS附加8g·L-1琼脂为基本培养基,诱导以NAA、TDZ(噻二唑苯基脲,thidiazuron)、CPPU[N(2氯4吡啶基)N1苯脲]做三因子四水平试验,增殖则以NAA、BA、蔗糖、pH值做四因子四水平试验,根据L16(45)正交表设计,分别以PLBs诱导率和增质量率为指标,筛选了适宜于PLBs诱导和增殖的培养基.结果显示:1/2MS+30g·L-1蔗糖+8g·L-1琼脂+0.01mg·L-1TDZ+0.005mg·L-1CPPU,pH5.6的培养基适宜于PLBs诱导,1/2MS+20g·L-1蔗糖+8g·L-1琼脂+20g·L-1香蕉汁+1.0mg·L-1NAA+0.2mg·L-1BA,pH5.6的培养基增殖效果较好;诱导率和增质量率分别为46.00%和756.74%.在各因子中,NAA对PLBs诱导率的影响最大,并对PLBs的诱导起抑制作用;蔗糖质量浓度对PLBs增质量率影响最大,且较低的质量浓度更为适宜;筛选出的PLBs诱导和增殖培养基经重复试验,结果相对稳定.     

大花蕙兰原球茎的诱导、增殖及其解剖结构研究

通过诱导根状茎上的休眠芽萌发,以其茎尖和幼茎段材料为外植体进行组织培养,建立了大花蕙兰(Cymbidium hynridum)的无菌繁殖体系,并筛选出大花蕙兰无菌繁殖的最佳培养基组成。原球茎诱导的较适宜的培养基为MS 1.5 mg.L-16-BA 0.1 mg.L-1KT 0.05 mg.L-1NAA,诱导率达15%;原球茎增殖培养基在诱导培养的基础上添加150 g.L-1香蕉泥,其增殖系数1个月内可达15~35。原球茎是由顶端分生组织后面的薄壁细胞脱分化,形成胚性细胞,经球状胚发育而来。     

农杆菌介导的DnMADS2基因转化金钗石斛初步研究

为建立稳定有效的金钗石斛转化体系,进一步研究花发育相关基因的功能,为石斛兰分子育种提供基础,本研究以金钗石斛类原球茎（PLBs）为转化受体,通过农杆菌介导的方法将DnMADS2基因转入金钗石斛。结果表明：用20 mg/L潮霉素筛选4个月后获得抗性PLBs,对其进行HPT基因和DnMADS2基因的PCR检测表明,目的基因已成功整合到PLBs中。获得的转化PLBs在抗性培养基上分化为不定芽后,再经生根壮苗培养获得转化植株。对转化植株进行Southern检测,结果表明,DnMADS2基因已整合到5株金钗石斛转化植株基因组中。据此建立的稳定高效的金钗石斛转化体系和获得的转基因植株,将为DnMADS2基因功能的进一步研究和金钗石斛转基因育种奠定基础。     

Production of protocorm-like bodies with bioreactor and regeneration in vitro of Oncidium ‘Sugar Sweet’

To produce mass propagules of Oncidium ‘Sugar Sweet’, we tested the feasibility of producing protocorm-like bodies (PLBs) using 5 l balloon type air-lift bioreactors, and selected an optimal culture medium for shooting and rooting in vitro. The results showed that liquid bioreactor cultures were more efficient for PLBs proliferation when compared to solid- and liquid-agitated flask cultures. The maximum PLBs biomass (326.3 g per bioreactor, FW) was obtained in the immersion bioreactor culture, with the growth ratio reaching 10.9 after 50 days of culture. This was obviously higher than the ebb and flood bioreactor culture. During bioreactor culture, sucrose and electric conductivity (EC) in the culture medium were negatively correlated with PLBs growth; the highest PLBs fresh and dry biomass was obtained 40 days after culture. An inoculation density of 20 g (FW) was optimal for PLBs growth in a 3 l working volume of 5 l bioreactor. Furthermore, MS medium supplemented with 2.0 mg l⁻¹ BA for shooting and 0.5 mg l⁻¹ IBA for rooting was optimal during in vitro culture, the plantlets were successfully established in the potting substrate.     

Micropropagation of Cymbidium Sleeping Nymph through protocorm-like bodies production by thin cell layer culture

Transverse thin cell layers (tTCLs) of protocorm-like bodies of two stages of PLBs (30 d and 60 d old) of Cymbidium Sleeping Nymph were used as explants to induce PLBs by using coconut water (CW) as a natural additive. 5% (v/v) CW supplemented to KC medium induced an average of 5 PLBs per responding tTCL of 30 d old PLBs with 83% of responding tTCLs. A low percentage of responding tTCLs were observed in 60 d old PLBs’ tTCLs. Anatomical and confocal microscopic studies traced the origin of PLBs to subepidermal layers of the tTCL. A significantly high percentage of shoot regeneration was obtained from PLBs formed on 1–10% (v/v) CW from tTCLs of 30 d old PLBs in comparison to PLBs induced on control after first subculture on KC medium (without CW). The induced PLBs regenerated into plantlets with velamenous roots and these plantlets were transferred to greenhouse conditions on cocopeat:perlite (9:1) with nearly 100% survival. Post-transfer performance of the plantlets was monitored. The results suggest tTCLs as potential explants (with respect to economy of precious hybrid materials) which can overcome the slow growth of hybrid PLBs and coconut water as a single natural additive for the mass multiplication of commercially important orchids.     

环草石斛愈伤组织和拟原球茎中生物碱和多糖含量的研究

用苯酚-浓硫酸法和溴甲酚绿酸性染料比色法测定了不同培养条件下得到的环草石斛愈伤组织和拟原球茎中总生物碱和多糖的含量.结果表明,愈伤组织和拟原球茎具备了代替原植物作为药用和提取原药成分的可能性;悬浮培养的愈伤组织中总生物碱和多糖的含量最高.该研究结果为从环草石斛愈伤组织中直接提取原药成分的研究提供了参考.