Tissue culture study of unit activity of the rat cerebral cortex

Spontaneous unit activity and morphological characteristics of visual cortical neurons from young rats aged 1 and 2 days were investigated during long-term culture (up to 34 days) of explants in vitro. Three types of spontaneous unit activity were found: single spikes, volleys, and grouped discharges. The types of spontaneous activity were found to depend on the duration of cultivation of the brain tissue. The discharge of single spikes, characteristic of neurons during the first 7–15 days in culture, subsequently was replaced by grouped discharges or volleys of spikes. The changes in unit activity were shown to coincide in time with morphological maturation of the synapses. In experiments in which strychnine (10 µg/ml) was added to the culture medium, a marked increase was observed in the mean frequency of spontaneous unit activity, accompanied by the appearance of discrete series of volley-type discharges. The genesis of spontaneous cortical unit activity is discussed on the basis of these findings.     

Tissue culture in Haworthia

Summary Plants regenerated on two different media (NK and I) from the calluses of simple or cloned subcultures, which were originated from a single stock callus of Haworthia setata derived from its flower bud, were observed for eight characters, i.e., somatic chromosome number in root tips, growth vigor, leaf shape, leaf color, number of stomata per unit leaf area, esterase zymogram, chromosome association at meiotic metaphase I in pollen mother cells, and pollen fertility. From these regenerates plants with different characters from those of the parental plant were obtained. With regards to chromosomal aberrations, tetraploids, aneuploids, plants with a part of the chromosome segment deleted, with reciprocal and non-reciprocal translocations, or with paracentric inversions and those showing sub-chromatid aberrations at meiosis were obtained. The NK medium tended to regenerate more tetraploids and less plants carrying translocation than the I medium.Chromosome variabilities in somatic cells of the regenerates correlated with those of the calluses, from which they regenerated, while they did not correlate with either the meiotic irregularities (chromosome association at MI) or pollen fertility of the regenerates. From these facts, it was concluded that a rather large number of callus cells participate in the regeneration of an individual plant, although, however, only a few limited types of the cells form its germ line.Polyploidy affected growth vigor, leaf shape, stomata number and chromosome association at MI, but its effects were not detected on other characters. Chromosomal aberrations at the diploid level produced no clear changes in the regenerate's phenotype except in meiotic chromosome configuration and pollen fertility.Most chromosomal variants obtained in the present study are already reported in plants collected from wild populations, but plants with the deletion of a whole chromosome (karyotype 7L+6S) or chromosome segment (7L+1M+6S and 14L+2M+12S) have never been reported: this fact suggests that tissue culture is a powerful tool for producing plants with novel karyotypes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 436     

Tissue culture and the use of transgenic plants to study plant development

Summary Recent advances in plant molecular biology have depended largely on the development of efficient methods of introducing foreign DNA into plant cells. Gene transfer into plant cells can be achieved by either direct uptake of DNA or the natural process of gene transfer carried out by the soil bacteriumAgrobacterium. Although both of these processes allow the generation of stably transformed plants, the former offers the advantage of allowing the study of transient expression of gene constructs in protoplasts cultured in vitro. In addition to the potential application of transgenic plants in agriculture and biotechnology they can be used to study the expression of foreign DNA, to carry out the functional analysis of plant DNA sequences, to investigate the mechanism of viral DNA replication and cell-to-cell spread, as well as to study transposition. Moreover, the versatility of the gene transfer vectors is such that they may be used to isolate genes unamenable to isolation using conventional protocols. Presented in the Formal Symposium Frontiers in Cell Biotechnology at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Tissue culture inHaworthia

Effects of three different auxins and kinetin in various combinations on greening and redifferentiation of the callus ofHaworthia setata were investigated. All auxins at the concentration of 50 mg/l inhibited callus greening. NAA in combination with kinetin promoted both callus greening and production of redifferentiated shoots. Low concentrations of IAA without kinetin promoted redifferentiation of shoots, but not callus greening. Addition of 2,4-D completely inhibited both greening and redifferentiation regardless of the level of kinetin except for the effects on shoot formation in the medium with 0.1 mg/l 2,4-D added. The calluses with the highest chlorophyll content were observed in the medium containing 2.0 mg/l kinetin without any auxins or with 0.1 mg/l NAA added. Most frequent shoot redifferentiation was observed in the medium containing 0.1 mg/l IAA without kinetin (redifferentiation rate; 67%), followed by the medium containing 10 mg/l NAA with 2.0 mg/l kinetin (44%), and 0.1 mg/l 2,4-D with 0.2 mg/l kinetin (33%). Generally, higher degrees of greening were associated with better growth. However, the auxins (IAA, NAA and 2,4-D) given at concentrations optimal for growth did not exhibit the highest degree of callus greening. Differences of the three auxins in their actions and interaction with kinetin were disclosed. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 423     

Tissue culture inHaworthia

Effects of three auxins and kinetin on growth of the calluses of two species ofHaworthia, H. aristala andH. setata, were investigated. Stock calluses derived from the flower buds of these species were maintained for two years on RM-1964 agar medium containing 5 mg/l NAA. Small pieces of the stock calluses were transferred to the basal medium containing either auxin, IAA, NAA or 2,4-D in six different concentrations (0.1–50 mg/l) combined with three concentrations (0–2 mg/l) of kinetin; in total, 54 kinds of media were used. Fresh weight of the calluses was measured 0 to 30 days from transfer and transformed to the natural logarithm. The linearity of their growth curves against the culture period was tested. The growth curves of theH. aristata calluses grown in dark and under continuous light and that of theH. setata callus grown in dark gave similar regression coefficients of 0.07 to 0.11, indicating that the doubling time of the callus mass was about 6.3 to 10.1 days. After 42 to 50 days from inoculation, the fresh weight of each individual callus was recorded, and the data were statistically analyzed. All auxins at the concentration of 50 mg/l significantly inhibited callus growth. Kinetin did not affect growth of theH. aristata callus in dark, while its effect on theH. setata callus was detected under light. Interaction of kinetin was found with IAA and 2,4-D inH. aristata and with IAA and NAA inH. setata. REsponses of theH. aristata callus to auxins and kinetin, when grown in dark, were different in several points from those of theH. setata callus grown under light. The best callus growth was observed in the following media; 0.2 mg/l kinetin supplemented with 1 mg/l IAA, or 0.5 mg/l 2,4-D, and 2 mg/l kinetin with 0.5 mg/l NAA inH. aristata, and 0.2 mg/l kinetin supplemented with 1 mg/l IAA, 5 mg/l NAA or 0.1 mg/l 2,4-D inH. setata. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 413.     

Tissue culture: the unrealized potential

Lack of differentiated functions of the tissue of origin in tissue culture thought to be due to dedifferentiation was shown to be due to selective overgrowth of fibroblasts. Enrichment culture techniques, (alternate animal and culture passage), designed to give the functionally differentiated cells selective advantage over the fibroblasts resulted in a large number of functionally differentiated clonal strains. Thus the dogma of dedifferentiation was destroyed. It is proposed to substitute the dedifferentiation hypothesis with the hypothesis that cells in culture accurately represent cells in vivo without the complex in vivo environment. With the development of hormonally defined media, combined with functionally differentiated clonal cell lines, the potential of tissue culture studies is greatly augmented. Hormonal responses and dependencies can be discovered in culture and the discovery of dependencies of cancer cells has led to a new rationale for therapy.     

Tissue culture: the unlimited potential

Lack of tissue-specific differentiated functions of cells in tissue culture, once thought to be due to “dedifferentiation”, was shown to be due to selective overgrowth of fibroblasts by a series of simple experiments that challenged the prevailing dogma. Following this insight, enrichment culture techniques (alternate animal and culture passage) were designed to give functionally differentiated tumor cells selective advantage over the fibroblasts. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types, providing the final point of destruction of the dogma of “dedifferentiation.” Instead, the hypothesis was proposed that cells in culture accurately represent cells in vivo, but without the complex in vivo environment. With the development of hormonally defined media and its combination with functionally differentiated clonal cell lines, this concept has been strengthened and the potential of tissue culture studies has been greatly augmented. Hormonally defined media allow the culture of cell types that cannot be grown in conventional, serum-supplemented media. These approaches demonstrate that hormonal responses and dependencies can be discovered in culture. Following this thinking and the discovery of hormonal dependencies of cancer cells has led to a new rationale for therapy. Tissue culture and cell technology continue to play an important role in solving human health problems.     

仙人掌果的组织培养

以仙人掌果的植株顶端幼嫩茎段为外植体,在添加NAA 0.5mg/L+6-BA 5.0mg/L的MS培养基中诱导产生丛生芽,在此培养基上继代培养,丛生芽数不断增加。在添加NAA 0.2mg/L+IBA 1.0mg/L的MS培养基上可正常长根。     

皂质芦荟的组织培养

将皂质芦荟的茎段及幼苗作为外植体进行组织培养,试验结果表明：皂质芦荟的茎段经40d左右可诱导形成愈伤组织,再经20d萌生再生芽：幼苗培养需要30d左右基部直接分化再生芽。同时经试验筛选出愈伤组织形成、再生芽分化和生根的最适培养基为：MS＋6－BA 2.0mg/L NAA 0.1mg/L、MS 6-BA 3.0mg/L NAA 0.1mg/L和MS NAA 0.3mg/L。     

兰州百合的组织培养(简报)

以兰州百合植株地下部幼嫩鳞片作为外植体,在MS 6—BA0．5mg/L NAA0．5mg/L的培养基上诱导产生不定芽效果最佳。靠近鳞茎盘部位的外植体诱导出的芽数量较多、质量较好。在添加NAA0．15mg/L的1／2MS生根培养基中可正常发根。