Somatic cell culture of rice cultivars with different grain types: Somaclonal variation in some grain and quality characters

Variations in some grain and quality characters in progenies of regenerated rice plants were studied. Grain length and weight decreased significantly, yet gel consistency increased. Variations in these quantitative characters of all cultivars studied were consistent, showing the tendencies of the variations. Grain protein contents of the somaclones were higher in one cultivar. Variability of most traits was increased by combining low-dosage radiation and tissue culture.     

Micropropagation of selected somaclones ofBegonia andSaintpaulia

Begonia x elatior plantlets which regenerated from leaf disk callus showed variations in plant morphology, number of flowers per plant, and flower size. Variations in flowering period, number of flowers per plant, and flower morphology were observed in Saintpaulia ionantha L. plants directly regenerated from leaf disk explants. The cytokinins, benzylaminopurine and zeatin, tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. Micropropagation of selected somaclones having the desirable trait of high number of flowers per plant was stable in the MV2 and MV3 generations.     

Comparison of carbonic anhydrase activity among various species of plantlets

During plant tissue culture, the culture container is small and sealed; the concentration of CO₂ in the microenvironment is relatively low. The plantlet growth is restrained for the shortage of CO₂ in the culture container. Carbonic anhydrase is a zinc-containing metalloenzyme that catalyzes the reversible conversion of bicarbonate to CO₂. The determination of carbonic anhydrase of leaves from Atractylodes lancea (thunb.) DC, Orychophragmus violaceus (L.) O.E. Schulz, Brassica juncea (L.) Czern.et Coss. cv. Luzhousileng, Brassica campestris L. cv. Chuanyou No.8, Brassica napus L cv. Oro, Brassica carinata Braun, Raphanus sativa L. var. raphanistroides Makino and their plantlets indicates that the carbonic anhydrase activity of leaves from both plantlets and fields varies from plant species to plant species, the carbonic anhydrase activity of leaves of Atractylodes lancea (thunb.) DC is the lowest among those plants, and the leaves of all plantlets are lower in carbonic anhydrase activity than the same species of plants from fields. The comparison of the growth rates of those plantlets shows that their relative growth rates are significantly different, plantlets of Atractylodes lancea have the slowest relative growth rate among those plants, and plantlets of Brassica juncea have the greatest relative growth rate. The relationship between RGR of plantlets and their CA activities is a significant linear function. It seems that there was certain correlation between carbonic anhydrase activities of plants and their growth rates. It suggests that in vitro, the greater the carbonic anhydrase activity of plantlet is, the higher its net photosynthetic rate, and the faster its growth rate. Those results offer a foundation to a rational medium choice in plant tissue culture.     

小冰麦异附加系的体细胞无性系建立及其变异的研究

从7种小冰麦异附加系的幼叶和成熟胚诱导出愈伤组织,建立了体细胞无性系,获得大量试管苗,并移栽成活。实验设计了适于小冰麦异附加系组织培养的 WG 培养基。愈伤组织诱导采用二次诱导方法。第一诱导培养基为 WG_2附加4mg/1 2,4-D、1mg/1 NAA。第二诱导培养基为 WG_2附加2m//1 2,4-D、0.5mg/1 NAA,和0.25mg/1KT。分化培养基为 WG_3附加0.5mg/1 KT、1mg/1 NAA 和100mg/1 Ad。再生植株的染色体检查表明,异附加系无性系的染色体数变异明显。保持2n=44的再生植株只有34.4%,而且变异植株中回复到2n=42的植株较多。再生植株中约有1/2发生了形态变异。在变异植株的花粉母细胞中观察到染色体的交换、易位等结构变化。特别在愈伤组织细胞中观察到多条染色体融合成多着丝点染色体和体细胞的染色体交叉,说明无性系中发生了染色体的交换和易位。     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.     

Culture and Sesquiterpene Analyses of Cells and Regenerated Plantlets of Pogostemon cablin Benth. (Patchouli)

Culture of cells of Pogostemon cablin Benth. on both solid andliquid media is described. In these cultures no free, volatilesesquiterpenes typical of the parent plant were detected bycapillary gas liquid chromatography and combined gas liquidchromatography-mass spectrometry using methods sensitive to4 x 10^–10 g sesquiterpene per mg fresh weight of planttissue. Under the culture conditions used, cultures freshlyinitiated from explants regenerated shoot-like structures. Thesewere developed into autotrophic plantlets. The morphology ofthese plantlets was unlike that of the parent plant, althoughglandular trichomes were present as in the parent plant. Onlyunidentifiable trace amounts of mass fragmentation patternstypical of sesquiterpenes could be detected in these plantlets,even when grown under similar conditions to the parent plants.     

Progeny tests of barley,wheat, and potato regenerated from cell cultures after in vitro selection for disease resistance

Summary Because plant cells cultured in vitro express genetic variability and since they can be regenerated into functional plants, procedures have been designed to use this system for the production of plants with new important agronomic characteristics, particularly for disease resistance. For barley, wheat, and potato somaclones have been found that were less susceptible to a toxin of Helminthosporium, fusaric acid, Fusarium coeruleum, F. sulphureum, or Phytophthora infestans, when screened in the first in-vitro-derived generation. Here the progeny of such somaclones is evaluated after natural and artificial infection, using greenhouse-grown or field material. The progenies of the same somaclones did not express detectable differences, which indicated that no heterozygous mutations occurred. Most lines and clones differed in their level of susceptibility to the pathogen compared to the level of the starting material, but these data were in no instance significant. It is discussed here whether this lack of significance is due to a lack of genetic differences or whether the test procedures are in adequate for detecting and securing the slight, probably quantitative, alterations.     

Organ-specific and ploidy-dependent somaclonal variation; a new tool in breeding

The organ-specific somaclonal variation means the differences between the variability of somaclones originated from different somatic tissue of plant. Significant differences in some agronomical characters were achieved among somaclones of seed and plumule meristem origin. The ploidy-dependent somaclonal variation means the differences between the variability of somaclones originated from different ploidy-level tissue. Increased variation among regenerated plants was postulated by origin from cultured cells of reduced ploidy level. The comparison of somaclonal variation in the progenies of diploid plants regenerated from callus of haploid and diploid origin supported the ploidy dependent theory. The pollenhaploid somaclone method (PHS-method) was developed and tested for utilization somaclonal variation in rice breeding. The PHS-method comprises the two well-known and widely applied in vitro methods which are the androgenesis (another culture) and genetic instability of cultured haploid somatic cells (callus cultures). Developmental varieties produced by this breeding sheme are under certification in Hungary.     

Genetic integrity of somaclonal variants in tea (Camellia sinensis (L.) O Kuntze) as revealed by inter simple sequence repeats

Adoption of inter simple sequence repeats (ISSR) technique to analyze the genetic variability of somatic embryo derived tea plants was evaluated. Morphological characterisation of the field grown plants revealed no identical character aligning with the parent, UPASI-10. Out of 40 primers, 15 exhibited concurrent polymorphism were selected for the study. Genetic variability of somaclones derived from single line cotyledonary culture ranged from 33.0 to 55.0%. A unique fragment of 1.2Kb was visible in majority of the accessions whereas the fragments below the length of 0.6Kb were noticed only in 50% of the variants. Out of 120 interactions attempted using Pearson's coefficient correlation, only 9.2% of somaclones exhibited significant similarity at genetic level. Dendrogram constructed based on simple matching coefficient revealed a distance of 2.257-3.317 between the final clusters. This strengthens the existence of wide genetic variation among the somaclones.     

Development and Evaluation of a Laboratory-Based Technique for Screening Somaclonal Regenerants of Watercress (Rorippanasturtium-aquatium) for Resistance to Crook Root Disease (Spongospora subterranea f. sp. nasturtii)

A laboratory based method has been developed for infecting watercress with the crook root fungus ( Spongospora subterranea f.sp. nasturtii ). This utilised plastic trays of nutrient solution on which watercress shoots, supported by floats, were grown for a pre-inoculation period of 14 days before an inoculum consisting of 1 g of crook roots was added. Seven watercress someclones derived from plantlets regenerated from callus of a parental commercial control line (clone A) were tested onthree separate occasions. No variation within the parental control plants was seen but significant variation both between and within the individual somaclones over successive tests was observed. Two somaclones showed increased resistance to crook root disease compared with parental control plants.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

Physiological and Biochemical Analysis of Somaclones Derived from Leaf Explants of Lathyrus sativus

Low ODAP somaclones have been evaluated for physiological and biochemical parameters especially in relation to attributes that lead to increased biomass production. All the somaclones during development had substantially lower ODAP content in leaves as compared to parent P24. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. Somaclone Bio L12 had the highest whereas parent P24 and Bi0164 had the least leaf area. Harvest index was the highest and biomass production was the lowest in the Bio 164. Bio L08 gave the highest seed yield. Photosynthetic rates were also higher in Bio L12, although no significant positive correlation was observed in leaf photosynthesis and seed yield. The differences in physiolpgical and biochemical parameters indicate the possibility of development of high yielding genotypes. The results in present investigation show differences in photosynthetic rate, leaf characteristics, seed yield and ODAP content among somalones and parent. Somaclones with extremely low ODAP content with variability in leaf morphology and photosynthetic rate is indicative of variation induced during plant tissue culture.     

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B₅ medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R₁ generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R₂ generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.     

RAPD analysis of blast resistant somaclones from upland rice cultivar IAC 47 for genetic divergence

Seventeen somaclones of upland rice cultivar IAC 47 showing different plant types, and either resistance or susceptibility to leaf blast, were utilized for random amplified polymorphic DNA (RAPD) analysis. Somaclones exhibited differences in reaction to isolates of Pyricularia grisea. Two somaclones (SC02 and SC04) were resistant to all three field isolates of somaclones, while the cultivar IAC 47 was susceptible. The inheritance study of two distinct plant types, one with erect bright green leaves and the other with droopy yellow green leaves, showed that a single possibly different, dominant gene governs each plant type. Of 32 random decamer primers utilized, OPA02 and OPD02 detected polymorphisms between somaclones showing erect bright green leaves and droopy yellow green leaves. Reliable grouping exhibiting 80% similarity was achieved with 17 primers. Leaf blast resistance to race IC-2 of P. grisea was associated with the plant type of erect bright green leaves.     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

In vitro induction, screening and detection of high essential oil yielding somaclones in turmeric (Curcuma longa L.)

Leaf of turmeric contains an essential oil used extensively in perfumery, pharmaceuticals and aromatherapy. Five somaclones were induced in turmeric on MS media with varying amounts of plant growth regulators. All somaclones were subsequently transferred to the field. Essential oil was extracted from leaves of in vitro and ex vitro grown plants and subjected to quantitative and qualitative evaluation. A positive correlation was established between the leaf oil content and oil constituent of in vitro grown and field transferred somaclones. Somaclones (C2, C4, C5) containing 0.16–0.18 % oil in vitro retained normal oil content (0.48–0.5 %) in the field. Similarly in vitro grown somaclones C3 and C7 with 0.36 and 0.25 % oil content retained proportionately increased oil yields of 1 % and 0.76 under ex vitro condition. GC–MS analysis of the oil revealed similar spectrum of constituents both among in vitro and ex vitro grown plants with alpha-phellandrene as major one. Thus the novel method of in vitro screening could be applied for rapid identification of high essential oil yielding turmeric genotypes thereby reducing labour, cost and time required in conventional ex vitro screening of somaclones.     

组培增殖方式对网纹草嵌合性状稳定性的影响

以一种具有良好的谱系细胞标记的周缘嵌合体网纹草(Fittonia albivenis)为研究材料,对不同增殖方式下其组培苗嵌合性状的稳定性进行了观察分析。结果表明,以茎段诱导腋芽增殖时网纹草的稳定性很高,但以丛生芽增殖时其嵌合性状发生了变异,变异率为21.32%;叶片组织解剖学观察结果显示,母本嵌合体茎尖分生组织中红和绿2种细胞谱系的细胞层排布在丛生芽再生过程中发生了改变,这可能是导致新类型嵌合体产生的原因。研究结果不仅对植物嵌合体种苗的组培生产具有重要的指导意义,而且为嵌合体的种质创新工作提供了新方法。     

Somaclonal variation in celery: screening for resistance to Fusarium oxysporum f. sp. apii

Summary Two methods were used to screen putative Fusarium-resistant celery (Apium graveolens L.) plantlets from cell culture: placing plantlets on a mycelial mat for one month or planting them directly in Fusarium-infested soil. Resistant phenotypes were identified with both methods, but the plants grown on the mycelial mat died before they reached reproductive maturity. Four plants, K, T-2, T-3, and R-R₁ from the soil screen, survived and produced viable seed. Tests of self-pollinated progeny, in field and greenhouse conditions, showed that T-2, T-3, and R-R₁ were superior to the original cultivar, 5270R, with respect to disease resistance, as measured by vascular discoloration and plant height. Chi-square analysis of progeny scores for root and crown decay showed that the new variation was heritable and appeared to be conditioned by more than one locus.     

Molecular analysis of micropropagated neem plants using aflp markers for ascertaining clonal fidelity

Summary The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time.