改良菜心离体培养植株再生体系的研究（简报）

This investigation has developed an efficient and fast method for plant regeneration from petiole of cotyledon explants of Brassica campestris L. subsp. chinensis Makino var. parachinensis Tsen et Lee. A medium was designed for B. campestris subsp. chinensis var. parachinensis to obtain the high frequency of shoot regeneration, which contained BAP 2 mg/L, NAA 0.75-1.0 mgL and 7.5 mg/L AgNO3 solution to the half of NH4+ concentration's MS basic medium. 60 mL/L coconut milk were added to all of media. In this method, frequency of shoot regeneration of "youqing caixin" reached as high as 91.2% and the number of shoots per explant reached as high as 4.7 plants. The result showed that there was a positive correlation between frequency of shoot regeneration and number of shoots per explant. The little shoots could be observed five days after inoculation and were formed directly. The inducing rate of roots of the shoots reached as high as 100% and the rate of viability of transferred mature plant reached higher than 95%. The regeneration period from petiole with cotyledon to a seedling was shorten to about 49 days. Factors influencing in vitro explant regeneration were studied.     

改良菜心离体培养植株再生体系的研究(简报)

菜心(Brassica campestris L.subsp.chinensis Makino var.parachinensis Tsen et Lee)为十字花科芸薹属芸薹种中国白菜亚种中的一个变种,又名“菜薹”。它是我国南方特产蔬菜之一,在蔬菜的周年供应上有重要地位。目前迫切需要培育抗病虫、抗逆和具有其他优良农艺性状的新品种,以提高菜心的     

白菜高效再生系统的建立及其不定芽发生的组织学观察（简报）

白菜(Brassica campestris Lssp．chinensis Makino)起源于欧洲的野生芸薹,有许多变种和类型,是我国尤其是长江流域及南方各省普遍栽培的重要蔬菜种类之一,在农业生产中占有重要的地位。由于白菜的植株再生频率同其他芸薹属作物相比较低,因此,影响了基因工程技术在白菜品种改良上的应用。虽     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

彩色大白菜子叶的不定芽再生

以彩色大白菜子叶为外植体,研究不同激素配比和AgNO3对不定芽再生的影响。结果表明:单独附加细胞分裂素(6-BA或TDZ)的MS培养基,不能诱导子叶不定芽分化;而同时附加生长素(NAA)和细胞分裂素(6-BA或TDZ),不定芽的再生频率提高,最高为15%;AgNO3与细胞分裂素及生长素配合使用,能大幅度提高子叶不定芽的再生频率,提高率最高达42.5%。与6-BA相比,TDZ对不定芽再生的效果更好。当TDZ浓度为0.05mg/L、NAA为0.3mg/L、AgNO3为8mg/L时,产生丛状芽数目最多,再生率最高,达50%。     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.     

Use of protocorm-like bodies for studying alkaloid metabolism in <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.     

提高榨菜离体培养植株再生频率

采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg／L6．BA 0．2mg／L2,4-D的组合较为适宜,其不定芽再生频率可达50％,且外植体以下胚轴为好：而CPPU和2,4-D的适宜组合为1．5mg／L 0．2mg／L,其不定芽再生频率高达66．67％,最适外植体为带柄子叶。同时,研究结果显示,添加0．25～1mg／L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。     

An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO₃, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO₃. Moreover, the application of AgNO₃ promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO₃ from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing.

     

药用植物金荞麦愈伤组织诱导与植株再生

目前转基因技术已成为植物定向遗传改良的重要手段,而建立稳定高频的离体再生系统是实现遗传转化的基础和前提.本试验以25 ～30 d苗龄的金养麦(Fagopyrum dibotrys)无菌苗叶片、茎节间、叶柄为外植体进行愈伤组织诱导与植株再生研究.结果表明:叶片在MS +2,4-D 4.0 mg/L +6-BA 1.0 mg/L培养基上愈伤组织诱导率达到89％.茎节间在MS +2,4-D 2.0 mg/L +6-BA 2.0 mg/L培养基上愈伤组织诱导率为87％.叶柄在MS +2,4-D 4.0 mg/L +6-BA 2.0 mg/L+ IBA 0.2 mg/L培养基上的最高诱导率仅为54％.愈伤组织分化不定芽的适宜培养基为MS +6- BA2.0 mg/L +TDZ0.2 mg/L +NAA0.2 mg/L;金荞麦不定芽在1/2 MS +NAA 0.5 mg/L的培养基上生根效果最好.组培再生植株经炼苗后移栽到田间成活率达80％以上,且生长表现正常.高频完整再生体系的建立,为金荞麦进一步遗传操作和扩大药材资源奠定了基础.     

杉木再生系统的比较研究

通过对杉木(Cunninghamia lanceolata Hook)再生系统的比较研究,建立了子叶、下胚轴、茎段和针叶等四个再生体系。子叶在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率为93.7%±0.45%,平均芽数为3.76±0.25;下胚轴在附含2mg/L BA和0.2mg/L NAA的DCR培养基中再生频率为96.2%±0.35%,平均芽数为17.4±0.18;茎段在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达100%,平均芽数为3.28±0.11;针叶在附含1.5mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达84.5%±0.45%,平均芽数仅为1.42±0.08。不定芽在附含0.2mg/L BA和0.02mg/L NAA的DCR培养基中有效伸长。芽苗经预处理后在附含0.3mg/L IBA的1/2 DCR培养基中生根效果最佳。     

Influence of genotype and explant source on the in vitro regeneration ability of different melon varieties

Nine genotypes of melon (Cucumis melo L.) were selected for the investigation of regeneration. Most of the tested varieties showed regeneration ability on medium containing 0.5 mg l?1 or 1 mg l?1 BA, but following the appearance of shoot buds, only six varieties produced leafy shoots. The effect of combinations of BA with different auxins (IAA, NA, 2,4-D) and ABA in the culture medium on shoot regeneration was tested on cotyledon explants of 'Hógolyó' and 'Hale's Best'. To establish optimal conditions for the adventitious shoot induction six types of seedling-derived explants were prepared from seedlings of four different ages. The best results for shoot forming capacity were achieved with cotyledons followed by decapitated seedlings and hypocotyls derived from 4-day-old seedlings. Cotyledon segments of 'Hógolyó' and 'Hale's Best' were also cultivated on media with different concentrations of IAA and BA supplemented with 0.26 mg l?1 ABA. The highest number of well-formed plantlets was counted for 'Hógolyó' on the medium supplemented with 0.9 mg l?1 BA+ 0.6 mg l?1 IAA+ 0.26 mg l?1 ABA. This is the first report on the in vitro regeneration of 'Hógolyó' from decapitated seedling and hypocotyl explants and of 'Javított Zentai', 'Muskotály', 'Hógolyó', 'Tétényi csereshéjú' and 'Magyar Kincs' from cotyledon explants.     

梨叶柄再生不定芽的研究

研究获得了梨品种八月红和水晶的叶柄再生不定芽,不定芽由愈伤组织分化形成。诱导叶柄再生不定芽的适宜培养基为NN69＋IBA0.5mg/L（八月红）或IAA0.5mg/L(水晶）＋TDZ1.0mg/L＋蔗糖30g/L＋琼脂6.0g.。AgNO3浓度在0.1-1.5mg/L范围对八月红梨叶柄再生有促进作用,培养基中附加AgNO30.5mg/L八月红梨叶柄再生效率最高。     

Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants

Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10^–7 M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10^–7 M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.Abbreviations ABA Abscisic acid - BA 6-Benzylaminopurine - GD Gresshoff and Doy (1972) nutrient medium - NAA -Naphthaleneacetic acid.     

李砧木Marianna离体叶片再生体系优化研究

以李砧木‘Marianna’试管苗新梢顶端第1片叶为外植体,研究激素组合、基本培养基种类及外植体类型等对不定芽再生的影响。结果表明:1/2 MS基本培养基和WPM培养基再生率显著高于MS和SH培养基;叶片附带叶柄的外植体再生率和再生不定芽数显著高于叶柄和切除叶柄的叶片外植体;最佳再生培养基为1/2MS+2.0mg/L TDZ+0.1 mg/L IBA+0.25%琼脂+3.0%蔗糖,最高再生率和再生不定芽数分别为81.7%和7.46±1.38个;最佳生根培养基为1/2MS+0.5~1.0 mg/L IBA,能获得96.7%生根率、较高的生根数和根长。     

向日葵离体再生体系的建立

为了建立高效的向日葵离体再生体系,从基因差异、外植体取材、生长素和细胞分裂素浓度、附加物的添加等方面出发,对向日葵愈伤诱导、分化、生根等过程进行了系统优化。结果表明：杂交材料相对于自交材料更容易实现再生;最佳外植体是生长4 d的子叶;最佳愈伤诱导培养基是MS培养基 (MS) +2.0 mg/L 6-苄基腺嘌呤 (6-BA)+0.5 mg/L奈乙酸 (NAA)+1.0 mg/L激动素 (KT),诱导率最高可达100％;最佳分化培养基是MS+0.2 mg/L 6-BA+0.5 mg/L NAA+0.3 mg/L KT+0.3 mg/L硝酸银 (AgNO3)+0.2 g/L活性炭 (AC),芽分化率可达71％;最佳生根培养基是1/2 MS+0.6 mg/L吲哆丁酸 (IBA),生根率最高为77％。方差分析表明,材料基因型、外植体生长时间、激素、AgNO3、AC对向日葵再生呈现显著性影响。     

芥蓝下胚轴离体培养及高频率植株的再生

三个芥蓝品种下胚轴离体植株再生的条件的研究结果表明：下胚轴切口处可直接诱导出芽,诱导“早花尖叶芥蓝”、“中花尖叶芥蓝”和“迟花尖叶芥蓝”直接出芽的最佳激素组合分别为2．0mgL-1BA,0．3mgL-1NAA＋2．0mgL-1BA,0．5mgL-1NAA＋2．0mgL-1BA,其相应的芽发生频率分别为84．6％,86．7％,93．3％。诱导芽发生的最适蔗糖浓度是1％。培养基中加入4．0mgL-1AgNO3和500mgL-1MES可显著提高芽再生频率。再生芽在MS附加0．1mgL-1NAA的培养基上诱导生根形成完整植株。离体再生苗与种子萌发实生苗田间生长外形差别不大,但长势稍慢。     

Organogenesis and Plantlet Regeneration in Vitro of Populus euphratica

The research of organogenesis and in vitro plantlet regeneration of Populus euphratica Oliver was carried out using the tender shoots from mature tree as initial explants and MS medium as the basic medium. The effects of plant growth regulators (PGR) on the regeneration were compared. The results showed that the concentration of PGR was not strictly required for the organogenesis of the excised organs and callus, but the ratio of BA to NAA was important. Calli could be induced from the excised leaves and stems cultured on the medium with 0.5 mg/L BA and 0.5 mg/L NAA. The embryonic callus could be multiplied in dark on the medium supplemented with 0.25 mg/L BA and 0.5 mg/L NAA. For the adventitious bud regeneration of the leaf and callus, supplement with 0.5 mg/L BA and 0.1 mg/L NAA was appropriate, giving a regeneration frequency of 82.9% and 100%, respectively. The suitable level of BA and NAA for the excised stem's was 0.1 mg/L and 0.01 mg/L respectively, yielding a regeneration frequency of 83 %. Rooting occurred on the MS medium with half strength of macronutrient and addition of 0.015 mg/L NAA, and the rooting rate could reach up to 86.2%. The techniques of somatic cell cloning of P. euphratica was established in vitro. The problems of deterioration of the subcultured shoots were also discussed.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.