TISSUE CULTURE OF CALLUS FROM SEEDLING AND ADULT STAGES OF HEDERA HELIX

Callus from stem pieces of the juvenile (seedling) and adult stages of Hedera helix L. were grown for up to 17 passages on a medium containing mineral salts, vitamins, sucrose, and coconut water and for fewer passages on media which lacked coconut water. Seedling callus differed in its growth in culture from adult. It was less stable than the adult; i.e., it developed vigorous variants earlier and more freely than the adult callus. The tendency of seedling callus to adapt to unfavorable media was greater. On low salt media root formation by callus was infrequent. Adult calluses formed roots more freely on high salt media than seedling calluses did.     

Paraquat Resistant Tobacco Calluses with Enhanced Superoxide Dismutase Activity

Seven paraquat resistant calluses of tobacco (Nicotiana tabacumL. cv. Samsun) were obtained by three successive screeningsof protoplast-derived calluses on a paraquat containing medium.Superoxide dismutase (SOD) activity of the resistant calluseswas 14- to 159-fold that of the leaf cells on protein basis.Paraquat-resistant calluses, however, showed little increasein catalase and peroxidase activities. More than 90% of SODactivity in the resistant calluses was inhibited by KCN, aswas the SOD activity in leaves, indicating that the major SODin the callus appears to be the Cu, Zn containing enzyme. Thecallus cells, however, expressed the immunologically distinguishedSOD isozyme from the enzyme in the leaves. (Received April 23, 1984; Accepted August 6, 1984)     

Selection of NaCI Tolerant Calli and Regeneration of Plants in Maize

The embryonic calli produced from immature embryos of inbred “Huangzhao-4” of maize, that had been maintained for half a year, were transferred to media supplemented with different NaC1 concentrations (5, 10, 15, 20, 25, 30g/L) for callus selection. NaCl tolerant calli were established through three generations of selections. The growth and frequency of survival calli were affected significantly by NaCl concentration. The proliferetion of NaCl-tolerant calli was relatively good on medium containing of 10g/L NaC1. From these calli, plant lets could be produced on differentiation medium. On medium supplemented with 10g/L of NaC1 the plantlets could normally grow to transplantation. In NaCl-tolerant calli cultured on medium containing 10g/L of NaC1, the contents of free amino acids, free proline, Na+, K+ were 18.0%,87.3%,661.9%,25.5% respectively higher than those in un-selected calli grown on subculture medium, but Ca2+ content decreased significantly. On medium containing 10g/L of NaC1, cells and their organelles in NaCl-tolerant calli had normal morphology and structure, and vigorous metabolism, but in un-selected calli, the majority of cells turned to wards dying. Although tolerant plants regenerated and their filial ones had grown in non-salted soil, their progenies retained the property tolerance, but showed segregation of the degrees of tolerance. In 10g/L NaC1 solution, the seeds of progenies from one plant regenerated could germinate normally, and grow into healthy seedlings. Therefore, the NaCl-tolerant calli and plantlets that we have obtained NaCl-tolerant variants.     

冬凌草离体培养体系的建立及主要次生代谢产物的测定

以冬凌草叶片为外植体,研究不同浓度激素组合对冬凌草愈伤组织诱导及植株再生的影响,并对不同外植体(茎、叶)诱导愈伤、芽的分化能力及再生植株内主要次生代谢产物的含量进行了比较研究。结果表明:在MS 2.0 mg/L 6-BA 1.0 mg/L NAA培养基上诱导愈伤组织效果较好;在MS 2.0 mg/L 6-BA的培养基上诱导芽的效果较好;叶片和茎段在愈伤诱导培养基上均能产生大量的愈伤组织,但其再分化能力以茎段最好;再生苗生根培养基以0.3 mg/L IBA最好;以叶为外植体诱导的再生植株中冬凌草甲素、迷迭香酸的含量均高于以茎为外植体诱导的再生植株。     

利用组织培养技术选育玉米抗小叶斑病突变体

大叶斑病和小叶斑病是玉米最严重的病害,选育抗大、小叶斑病优良自交系及单交种是提高玉米产量的重要措施。Ｇｅｎｇｅｎｂａｃｈ等以玉米Ａ６１９ｃｍｓＴ愈伤组织为材料,筛选出对小叶斑病毒素具有抗性的愈伤组织与再生植株［１,２］,开辟了玉米抗病育种的新途径。...     

Induction of pollen plantlets in rice by spikelet culture

Induction of pollen callus and subsequent regeneration of plantlets from the callus have been achieved from rice spikelets cultured in a liquid medium containing sucrose, -naphthalene-acetic acid and kinetin. When spikelets are cultured in a medium containing 6% sucrose, calluses are released into the medium where they continue to increase in size without undergoing organogenesis. On the other hand, in a medium containing 2% sucrose, calluses are retained within the anther locule where they differentiate into plantlets. Cytological studies have shown that calluses have their origin either in the vegetative cell of asymmetrically dividing pollen grains or in both cells of pollen grains which divide more or less equally.     

Cycloheximide resistance in carrot culture: a differentiated function

Cultured carrot cells grow as unorganized callus tissue in medium containing auxin. Upon removal of the auxin from the medium, they grow in an organized manner and differentiate into embryos. In the normal cell line, W001C, the callus growth can be inhibited by cycloheximide, but the embryonic growth cannot. A variant cell line, WCH105, whose callus growth is resistant to cycloheximide, was isolated. The mechanism of cycloheximide resistance in embryos of both lines and in WCH105 callus was found to be cycloheximide inactivation. In addition to auxin, bromodeoxyuridine can also promote callus growth in carrot culture. Callus cultures maintained by bromodeoxyuridine behave the same as do those maintained by auxin. WCH105 callus is resistant, whereas W001C callus is sensitive to cycloheximide inhibition. Except for the onset of embryogenesis, cycloheximide inactivation is expressed throughout the embryo developmental stages up to the plantlets. These results suggest that cycloheximide inactivation is a function expressed in the differentiated, but not in the undifferentiated, tissues.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

发根农杆菌LBA9402Bin19转化红豆草及再生转基因植株

Hypocotyl segments of Onobrychis viciaefolia were transformed by Agrobacterium rhizogenes LBA9402 which harboured pBin19 and pRi1855. Seedling age and preculture time of hypocotyl segments influenced the transformation frequency. Paper electrophoresis revealed that 70% of single hairy root cultures could synthesize agropine. Calli were induced from hairy root segments on MS medium containing 0-9.05 mumol/L 2,4-D and 0-2.22 mumol/L 6-BA at first, then they were transferred onto MS0 medium without kanamycin for regeneration. Constitution and concentration of phytohormones in callus induction media affected subsequent regeneration of calluses on MS0 medium remarkably. Regeneration frequency and shoot number per callus declined when 2,4-D concentration in callus induction media increased from 4.52 to 9.05 mumol/L, while they ascended when 6-BA in callus induction media increased from 0 to 2.22 mumol/L. On MS medium supplemented with 4.52 mumol/L 2,4-D and 2.22 mumol/L 6-BA, only 14.2% hairy root segments could produce calluses, but the regeneration frequency reached 58.1% and the shoot number per callus was 37.2. In 32 analysed plants regenerated from 8 kanamycin resistant hairy root lines, 25 were nptII positive and showed different copy numbers.     

Aluminum borate whisker-mediated production of transgenic tobacco plants

An aluminum borate whiskers-mediated transformation system for calluses of tobacco (Nicotiana tabacum, cv. SR-1) has been developed. A total of 50 small pieces of calluses were vigorously agitated in a liquid medium containing aluminum borate whiskers, pBI221 plasmid carrying the -glucuronidase (GUS) gene, and pBI222 plasmid carrying the hygromycin phosphotransferase (HPT) gene. After treatment, calluses were cultured to select for hygromycin resistance, and three resistant calluses were obtained. Adventitious shoots were produced from each hygromycin-resistant callus and were transferred to rooting medium. A total of three plantlets obtained from each hygromycin-resistant callus were acclimatized and established in soil. Polymerase chain reaction analysis revealed that all the plantlets were cotransformed with both the GUS and HPT genes. Detached leaves of transgenic individuals showed clear hygromycin resistance when cultured in liquid medium. Histochemical assay for GUS revealed that one of these transgenic plants expressed the GUS gene, indicating coexpression of foreign genes.     

Sodium chloride resistant cell line from haploidDatura innoxia mill

Summary A cell line resistant to sodium chloride was selected from callus cultures of haploidDatura innoxia by cloning under selective pressure. Cells of the resistant cell line retained their resistance even after subculture in absence of NaCl. Plantlets could be regenerated from resistant cells in the presence as well as absence of NaCl. In contrast, regeneration of plantlets was not possible from normal cells in the presence of NaCl, although regeneration readily occurred in the absence of NaCl.To examine the stability of the resistance in the long-term, callus cultures were initiated in presence of NaCl from stem expiants of the differentiated plantlets. All expiants of plantlets derived from resistant cells showed callus formation. This callus, derived from resistant explants, retained the trait of resistance upon subculture.     

Induction of Endosperm Calluses and Regeneration of Endosperm Plantlets of Asparagus officinalis

The endosperm callus has been induced from the young endosperm of Asparagus officinalis L. on the MS supplemented with auxin. The induction frequency of callus amounts to 65.9%–83.1%. When the callus was transferred to the medium supplemented with lower concentration of NAA 0.1 ppm or containing BA 1 ppm and NAA 0.5 ppm, the differentiation of shoots, roots and a few embryoids began to occur. A few calluses and embryoids can develop into plantlets. The chromosome number in the cells from the same root tip and shoot apex of endosperm plantlet is very unstable. They can be euploids (n=10, 2n=20, 3n=30, 4n=40). or aneupl0ids (n=6, 7, 17, 25, 53).     

Effect of physical desiccation on plant regeneration efficiency in rice (Oryza sativa L.) variety super basmati

This experiment assessed the effect of partial physical desiccation on plant regeneration efficiency in scutellum-derived embryogenic calluses of rice (Oryza sativa L.) variety Super basmati. A number of callusing cultures were developed, and efficient callus induction was observed on MS (Murashige and Skoog) basal medium supplemented with 2.0 mg/L 2,4-dichlorophenoxy acetic acid. The calluses were proliferated on the same medium for 3 weeks and then shifted to dehydration desiccation treatment for 72 h. The desiccated calluses were cultured on different media for somatic embryogenesis and plant regeneration. A medium with 2.0 mg/L α-napthaleneacetic acid, 10.0 mg/L abscisic acid , 2.0 mg/L kinetin was best for somatic embryogenesis only, but not for further plant development. After 10 d, differentiated calluses were sub-cultured on medium with various concentrations and types of carbohydrates (carbon source) in ¹MS_2j medium. A large number of plantlets (14.51±2.81 and 8.56±2.90 plants/callus) were regenerated via chemical desiccation, on MS with 3% maltose+3% sorbitol and 6% sucrose, respectively. Under dehydration on only simple MS (3% sucrose), 11.23±3.22 plants/callus were developed. Under conditions of dehydration and chemical desiccation, plant regeneration rates were higher than the calluses cultured on simple MS medium in the presence of plant growth regulator. After somatic embryogenesis, >25% plants were sterile. The protocol used here may allow maximum regeneration of normal and fertile plantlets of super basmati rice within 3 months.     

菘蓝花药培养及单倍体的诱导

探讨菘蓝花药处于单核晚期的形态指标,并以适宜发育时期的花药为外植体,进行花药培养及单倍体诱导。实验结果表明,4℃低温处理2d后,在含有6-BA0．5mg·L-1和NAA1．0mg·L-1。的Ms培养基上,花药愈伤组织的诱导率为23．35％;将其转接到Ms附加6．BA1．0mg·L-1,NAA0．5mg·L-1的分化培养基上,80．00％以上的愈伤组织可以诱导产生不定芽;再将分化出的试管苗转接到1／2MS＋NAA1．0mg·L-1的生根培养基上,3d左右即可获得完整植株。经叶边缘压片检查染色体数目,花药培养所得的弱小绿苗为单倍体植株。     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

Protocorm-like body (PLB) formation and plant regeneration from the callus culture of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall <Emphasis Type="Italic">ex</Emphasis> Lindl.

An efficient system was established for a higher frequency of protocorm-like body (PLB) formation from the callus of Dendrobium candidum Wall ex Lindl. The calluses were induced from longitudinally bisected segments of protocorms and subcultured two times every 40d on Murashige and Skoog medium with macronutrients at half strength, micronutrients at full strength, 2% sucrose, and with 8.8μM 6-Benzylaminopurine. PLB formation was achieved when calluses were transferred onto the same basal medium without any plant growth regulators. PLBs developed into intact plantlets about 2cm in height and with four roots when on basal medium with 2.7μM 1-naphthaleneacetic acid. Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Histological observations proved that globular somatic embryos could be produced from the inside and surface of the embryogenic callus during PLB formation.     

In vitro induction of tetraploid garlic with trifluralin

Garlic (Allium sativum) is propagated asexually. Since sexual cross breeding is almost impossible, means for effective breeding are not currently available and the available production cultivars are seriously aged and degenerated. A possible alternative for breeding is chemical induction. Trifluralin, a type of herbicide, has been reported to provoke chromosome doubling. However, this chemical had not been tested on garlic. We tested various trifluralin concentrations and treatment durations for efficiency in the induction of tetraploid garlic. A clove base of garlic with a stem cv. Gailiang was used as the ex-plant to induce calluses on Murashige and Skoog (MS) medium; the calluses were then inoculated onto MS medium containing different levels of trifluralin and cultured to induce chromosome number variation in vitro. Garlic calluses were effectively induced via the ex-plant and both shoots and roots differentiated well on MS medium containing 6-benzylaminopurine at 3.0 mg/L and indole-3-acetic acid at 0.1 mg/L. However, increases in trifluralin concentration and treatment duration reduced the survival rate and differentiation rate of calluses. Garlic callus cultured for 15 days on medium containing 100 μM trifluralin gave the highest rate of chromosome doubling. Through observation of chromosome number in the root apical cells and the morphology of guard cells on the leaf epidermis of the regenerated plantlets, it was clear that chromosome number variation was induced and tetraploids were produced in vitro by trifluralin treatment.     

The effects of level of 2,4-D and time in culture on regeneration rate and chromosome numbers of regenerants from calli of the hybrid Triticum aestivum cv. Chinese Spring ph1b x Thinopyrum ponticum (2n = 10x = 70).

Thinopyrum ponticum (Podp.) Barkworth &D. R. Dewey (2n = 10x = 70) has excellent resistance for both leaf and stem rusts. Long-term callus cultures were established from the immature embryos of a hybrid between Triticum aestivum L. (2n = 6x = 42) x Th. ponticum. They were maintained in culture for over 2 years and continued to grow and have organogenetic capacity. With increasing time on a maintenance medium, the plant regeneration rates of the hybrid calli decreased when transferred to regeneration media containing 0.1, 0.2, or 0.5 mg/L 2,4-D, but the rate of decrease was much higher at 0.5 mg/L than at either 0.1 or 0.2 mg/L 2,4-D. After 3 months of subculture, the highest plant regeneration rate was obtained on the medium containing 0.5 mg/L 2,4-D (1.11 plantlets/callus), while on the 24th month of subculture the highest plant regeneration rate was obtained on the medium containing 0.1 mg/L 2,4-D (0.20 plantlets/callus). Thus, it was shown that as the calli aged it was important to reduce the level of 2,4-D in the regeneration medium. Over 2 years, a total of 667 regenerants were successfully transferred and grown to maturity. Chromosome numbers in root-tip cells were determined for 539 regenerants and ranged from 36 to 70. Telocentric chromosomes were frequent. A fertile plant was found among the regenerants after 15 months of subculture. It had 56 chromosomes with 2.15 (1-6) univalents, 22.76 (17-26) closed bivalents, 3.55 (1-9) open bivalents, and 0.41 (0-3) trivalents and was highly resistant to stem rust race 15B-1. Callus culture of wide hybrids can be used to introgress characters from alien species into wheat.     

苦丁茶愈伤组织的诱导与褐变抑制

以叶片为材料,对苦丁茶愈伤组织的诱导,继代培养及褐变调控的研究表明：（１）苦丁茶愈伤组织的诱导及继代培养均以ＭＳ附加ＢＡ ２．０ｍｇＬ＾－１和ＮＡＡ ４．０ｍｇＬ＾－１的培养基效果最好;（２）０．１％的植酸可明显促进愈伤组织生长、抑制褐变,而硫代硫酸钠效果最差;（３）连续培养４０－５０ｄ愈伤组织增长倍数达到最大值;（４）继代２７次以后愈伤组织生长速度开始下降。这些条件为下一步细胞培养生产苦丁茶甙等     

黄连体细胞胚胎发生的研究

黄连(Coptis chinensis)叶片外植体在 MS 2,4-D 1 ppm 培养基上很容易产生愈伤组织。愈伤组织在转入分化培养基 MS 6-BA 0.5ppm NAA 1ppm 培养基上以后,能产生大量胚状体。胚状体可经过球形、心形、鱼雷形及子叶期等诸阶段发育成小植株。对胚状体用4%的藻酸钠和2%的氯化钙进行人工种皮包埋后,在无菌条件下,胚状体转变成苗。愈伤组织在分化培养基上经几次继代后,整个愈伤组织可转变为胚性愈伤组织并形成一个个胚性细胞团。胚状体可从其表面或愈伤组织内的任一细胞团产生。这一研究结果为获得大量分散的单个胚状体及人工种子的研制提供了良好的实验系统。