Reversibility of the effects of cadmium on the growth and nitrogen metabolism in the tomato(Lycopersicon esculentum)

In order to better understand the effects of heavy metals on the growth of plants, we decided to perform recovering experiments by following both chemical and physiological parameters in cadmium pre-stressed tomato seedlings after cadmium had been removed from the nutrient solution. The work shows that cadmium suppression results in resumption of growth activity. The biomass of leaves and stems rose steadily. The increase in root biomass exceeded those of leaves and stems. At the same time, nitrate content was increased to reach the level obtained with unstressed controls. In all the organs studied, the activities of the enzymes involved in the anabolic nitrogen primary assimilation pathways (nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) soared after that cadmium had been removed. While NAD(+)-dependent glutamate dehydrogenase (GDH-NAD+) activity also rose progressively during the recovering time, the cognate NADH-dependent glutamate dehydrogenase (GDH-NADH) activity decreased. This result allows us to propose that the ammonia produced by the stress-induced protein catabolism is detoxified and re-assimilated by the GDH-NADH isoenzyme. On the basis of these results, we will discuss the ability of the plant to dilute the effects of pollutants during the recovering period. An important outcome of this work is that a transient contamination of the culture medium by pollutants is not necessarily followed by a significant depreciation in product yield or quality.     

Somatic hybridization between Lycopersicon esculentum and Lycopersicon pennellii

Summary Selection and screening methods were devised which resulted in the identification of a number of somatic hybrid callus clones following fusion of Lycopersicon esculentum protoplasts and L. pennellii suspension culture protoplasts. Visual selection for callus morphology combined with a high fusion frequency and irradiation of one parental protoplast type (¹³⁷Cs source, 1.5 Krads) resulted in selection of a callus clone population containing a high proportion of somatic hybrids. Analysis of a dimeric isozyme for the presence of a heterodimeric form was found to be satisfactory for distinguishing parental-type calli, somatic hybrid calli, and mixed calli derived from both types of unfused parental cells. No somatic hybrid calli produced shoots, although the sexual hybrid between L. esculentum and L. pennellii regenerated well under the culture conditions employed. This result suggests that the non-regenerable growth habit of the L. pennellii suspension culture was dominant in the somatic hybrid. The culture conditions described here are suitable for obtaining regenerated plants from L. esculentum mesophyll protoplasts. L. esculentum protoplast calli from fusion cultures gave rise to shoots with L. esculentum phenotype at higher frequency than calli from control unfused L. esculentum mesophyll protoplast cultures. The use of probes for species-specific organelle DNA fragments allowed identification of organelle DNA restriction fragments in digests of total DNA from small samples of individual callus clones. The callus clones analyzed either carried predominantly one parental plastid DNA type or mixtures of both types. Use of a mitochondrial DNA (mtDNA) probe which distinguishes two parental mtDNA fragments revealed that the L. pennellii-specific fragment was present in all clones examined, but the L. esculentum fragment was absent or in low proportion.     

Growth and photosynthesis of Lycopersicon esculentum (L.) plants as affected by nitrogen deficiency

Fully expanded leaves of tomato (Lycopersicon esculentum) growing with either complete or nitrogen-deficient nutrient solution were analysed for leaf water status, gas exchange and chlorophyll fluorescence during the vegetative and reproductive phases. N-deficiency did not affect leaf water relations but did decrease light saturated photosynthetic rate as well as stomatal conductance in the vegetative stage. A lower variable to maximum fluorescence ratio (Fv/Fm) was found in N-limited plants which also showed an increase in leaf starch content and in starch to sucrose ratio. The inhibition of photosynthesis and the alteration of photosynthates partitioning were responsible for the growth reduction in N-stressed plants. During the reproductive phase the limitation of photosynthesis may be due to a large accumulation of starch which determines both a decrease in the carbon demand from the sinks and a decrease in CO2 conductance in the mesophyll. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of chilling on photosynthesis and activities of some enzymes of sucrose metabolism in Lycopersicon esculentum Mill

The effects of chilling stress on leaf photosynthesis and sucrose metabolism were investigated in tomato plants (Lycopersicon esculentum Mill. cultivar Marmande). Twenty-one-day-old seedlings were grown in a growth chamber at 25/23 °C (day/night) (control) and at 10/8 °C (day/night) (chilled) for 7 days. The most evident effect of chilling was the marked reduction of plant growth and of CO₂ assimilation as measured after 7 days, the latter being associated with a decrease in stomatal closure and an increase in Ci. The inhibition in photosynthetic rate was also related to an impairment of photochemistry of photosystem II (PSII), as seen from the slight, but significant change in the ratio of F_v/F_m. The capacity of chilled leaves to maintain higher q_P values with respect to the controls suggests that some protection mechanism prevented excess reduction of PSII acceptors. The results of the determination of starch and soluble sugar content could show that chilling impaired sucrose translocation. The activity of leaf invertase increased significantly in chilled plants, while that of other sucrose-metabolizing enzymes was not affected by growing temperature. Furthermore, the increase in invertase (neutral and acid) activity, which is typical of senescent tissue characterized by reduced growth, seems to confirm that tomato is a plant which is not a plant genetically adapted to low temperatures.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were obtained by fusion of leaf protoplasts from both species after irradiation of protoplasts or leaf tissue of L. peruvianum with 50, 300, or 1,000 Gy of gamma-rays. These radiation doses were sufficient to abolish the growth of the L. peruvianum protoplasts. The hybrids were selected for their ability to regenerate plants; this regeneration capacity derived from L. peruvianum. All asymmetric hybrid plants were aneuploid. The ploidy level, the morphology, and the regeneration rate were analyzed in relation to the radiation dose applied to L. peruvianum. After a low dose (50 Gy), most hybrids had near-triploid chromosome numbers, whereas after a high dose (300 or 1,000 Gy), most hybrids had near-pentaploid numbers. The morphology of the asymmetric hybrids was intermediate between that of L. esculentum and symmetric somatic hybrids of both species (obtained without irradiation treatment), and approached the morphology of L. esculentum to a greater extent after a high dose of irradiation. The asymmetric hybrids regenerated more slowly than the symmetric hybrids and regeneration proceeded more slowly after a high dose than after a low dose of irradiation. The high-dose hybrids also grew more slowly, flowered less, and set fruits less than the low-dose hybrids. No seeds could be obtained from any asymmetric hybrid.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were analysed for the retention of genes and alleles specific for L. peruvianum. The hybrids were obtained by fusion of protoplasts from L. esculentum with those of L. peruvianum (the donor), the latter having been irradiated before fusion with 50, 300 or 1,000 Gy of gamma-rays. The retention of three different types of genes or alleles was analysed. (1) The gene coding for kanamycin resistance, which is dominant and had been introduced in most of the L. peruvianum donor plants by transformation. It was present at one locus in 16 L. peruvianum donor plants and at two loci in one donor plant. (2) The genes coding for acid phosphatase, locus Aps-1, and glutamate oxaloacetate transaminase (GOT); different alleles of these genes are co-dominant and were detected by isozyme analysis. (3) Eighteen single gene morphological markers for which most of the L. esculentum genotypes used were homozygous recessive. Kanamycin resistance from donor plants with one locus was retained in about 50% of the asymmetric 30H-hybrids (the donor was irradiated with 300 Gy). L. peruvianum specific alleles of Aps-1 and GOT were present in at least 70% of the hybrids; the retention of donor alleles was lower in 30H- than in 5H-hybrids (donor irradiated with 50 Gy). On average, 73% of the L. peruvianum-specific alleles (one or both) of the morphological markers were detected in the 30H-hybrids. Several of the L. esculentum genotypes used were homozygous recessive for two morphological markers on the same chromosome; in 43% of the 30H-hybrids derived from them, only one of these markers was complemented by the L. peruvianum allele. This is an indication of frequent breakage of the L. peruvianum chromosomes. Several hybrid calli regenerated genotypically different shoots. On the whole, this analyses confirms the conclusion drawn from the cytogenetic and morphological analysis of these asymmetric hybrids, namely that irradiation prior to fusion eliminates the L. peruvianum genome to only a limited extent.     

Ultrastructure and lipid alterations induced by cadmium in tomato (Lycopersicon esculentum) chloroplast membranes

The effects of cadmium (Cd) uptake on ultrastructure and lipid composition of chloroplasts were investigated in 28-day-old tomato plants (Lycopersicon esculentum var. Ibiza F1) grown for 10 days in the presence of various concentrations of CdCl2. Different growth parameters, lipid and fatty acid composition, lipid peroxidation, and lipoxygenase activity were measured in the leaves in order to assess the involvement of this metal in the generation of oxidative stress. We first observed that the accumulation of Cd increased with external metal concentration, and was considerably higher in roots than in leaves. Cadmium induced a significant inhibition of growth in both plant organs, as well as a reduction in the chlorophyll and carotenoid contents in the leaves. Ultrastructural investigations revealed that cadmium induced disorganization in leaf structure, essentially marked by a lowered mesophyll cell size, reduced intercellular spaces, as well as severe alterations in chloroplast fine structure, which exhibits disturbed shape and dilation of thylakoid membranes. High cadmium concentrations also affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the fatty acid content and a shift in the composition of fatty acids, resulting in a lower degree of fatty acid unsaturation in chloroplast membranes. The level of lipid peroxides and the activity of lipoxygenase were also significantly enhanced at high Cd concentrations. These biochemical and ultrastructural changes suggest that cadmium, through its effects on membrane structure and composition, induces premature senescence of leaves.     

Pre-exposure of calli to ozone promotes tolerance of regenerated Lycopersicon esculentum cv. PKM1 plantlets against acute ozone stress

Studies were performed to evaluate the effects of pre-exposure of calli to ozone in promoting tolerance of the regenerated Lycopersicon esculentum cv. PKM1 (tomato) plantlets against acute ozone stress (AOS). Calli induced from tomato leaf explants were subjected to pre-treatment with ozone: T(1)=100ppb, T(2)=200ppb and T(3)=300ppb. For the control (C) calli, charcoal-filtered air was supplied to test differential sensitivity of regenerated plantlets to acute ozone stress. All treated calli were subsequently transferred to shooting, rooting medium and acclimatized. The plantlets regenerated from the respective ozone (T(1), T(2), T(3))-treated calli are referred to here as T(1), T(2), T(3) plantlets and the plantlets regenerated from control calli are referred to as control plantlets. The frequencies of regeneration of tomato plantlets from the calli were T(1)=86%, T(2)=82% and T(3)=67%, and 92% regeneration was obtained from control calli. In order to evaluate the ozone tolerance, all the regenerated plantlets were exposed to the acute ozone exposure (AOE). After AOE, the T(2) plantlets endured remarkably well by experiencing reduced ozone stress, which was evident from the lower level of hydrogen peroxide and oxidative stress-related enzymes such as ascorbate peroxidase (EC 1.11.1.11) and superoxide dismutase (EC 1.15.1.1) activities relative to T(3), T(1) and C plantlets. All T(2) plantlets showed enhanced tolerance against AOE by upholding enhanced soluble phenol content, a higher level of foliar and apoplastic ascorbic acid, elevated dehydroascorbate reductase (EC 1.8.5.1) and glutathione content. The present study reveals that the calli pre-exposed to T(2) ozone treatment resulted in an increase in the level of antioxidants and provided the plants greater protection against acute ozone stress.     

Salinity effects on growth and carbon balance in Lycopersicon esculentum and L. pennellii

The effects of salinity on growth and carbon balance in the cultivated tomato Lycopersicon esculentum (L.) Mill. cv. VF 234 and in a wild, salt-tolerant relative, L. pennellii (Correll) D'Arcy accession Atico, were compared. The level of salinity that significantly reduced growth and affected morphology was lower for the cultivated than for the wild species.
Net CO₂ fixation, stomatal conductance and the specific activity of ribulose bisphosphate carboxylase (EC 4.1.1.39) were more decreased by salinity in the wild tomato than in the cultivated. In both species, the salinity-associated decrease in assimilation was related to a decrease in net photosynthesis and to carbon reallocation favoring heterotrophic organs.     

Interaction of Auxin-TIBA in Lycopersicon esculentum

[5-³H] indoleacetic acid (IAA) was applied to the apical budof young tomato plants (Lycopersicon esculentum Mill.) treatedor not with 2,3,5-triiodobenzoic acid (TIBA). Chromatographicanalysis showed that treatment with TIBA increased the quantityof breakdown products in the apical 1.5 mm segment of the stem.Autoradiographs of ultra-thin sections of this segment wereprepared after treatment with glutaraldehyde. The inhibitionof polar auxin transport by TIBA caused an increase in the densityof labelling of all the cellular types in the shoot apex. TheTIBA treatment provoked a modification of the structure of theshoot apex in which the greatest increases in density of labellingwere detected in the superficial tunical layer and in the pithmeristem. The increase in radioactivity of the superficial tunicallayer could be explained by the presence of polyphenols in thevacuoles. For the pith meristem, the TIBA treatment intensifiedthe high labelling previously observed at this level in thecontrol plants. This could indicate a role for auxin in cellularelongation. It could also point to the importance of the pithmeristem in the functioning of the shoot apex. Key words: [³H] IAA, Lycopersicon esculentum, TIBA     

双酚A与镉对植物光合作用的复合微生态效应

近年来,国内外学者研究了双酚A (BPA)及重金属镉(Cd)单一因子对植物光合作用的影响.但在自然背景下,污染多呈复合型,因此研究BPA和Cd对二者叠加的空间内植物光合作用的复合影响意义重大.本文综述了BPA对植物光合作用的影响及机制,及Cd对植物光合作用的影响及其机制,其中单一BPA和Cd对植物光合作用的影响均表现为:低浓度促进光合作用,高浓度反之.根据相关研究推测了植物在受到复合胁迫时的生态响应,相应的生理生化反应,及光合过程的响应机制.最后提出了BPA与Cd对植物复合胁迫可能开展的相关研究方向.     

Organelle analysis of symmetric and asymmetric hybrids between Lycopersicon peruvianum and Lycopersicon esculentum

Summary The organelles of somatic hybrids obtained from symmetric and asymmetric fusions between the Lycopersicon species L. peruvianum and L. esculentum were analyzed by DNA hybridization methods. In the asymmetric fusions the L. peruvianum protoplasts were gamma-irradiated at a dose of 50, 300 and 1,000 Gy. The organelles were characterized using the Petunia chloroplast probe pPCY64 and the mitochondrial EcoRI-SalI fragment of the Pcf gene. In all symmetric and asymmetric hybrid plants, a total of 73 being analyzed, only one of the parental chloroplast genomes was present, except for one hybrid plant which harbored both parental chloroplast genomes. No recombination and/or rearrangement in the chloroplast genome could be identified with the pPCY64 probe. Irradiation of the L. peruvianum protoplasts did not significantly reduce the fraction of asymmetric hybrids with L. peruvianum chloroplasts. A novel mitochondrial restriction pattern was present in 5 out of 24 hybrids tested. In 9 hybrids novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

Influence of rooting-medium temperature on growth of Lycopersicon esculentum

The influence of rooting-medium temperatures (maintained constant for 54 days from sowing) on tomato seedling growth was exerted primarily on development during the first 2 weeks after sowing, when an optimum emerged at 25 °C. This influence was attributable to an effect of temperature on the partitioning of dry matter between root and shoot during a few days before and after the start of photosynthesis. The adverse effect of suboptimal (but not supra-optimal) temperature was also associated with a reduction in the phosphorus content of the tissue, there being little effect on either nitrogen or potassium content.     

Somatic hybridization between Solanum ochranthum and Lycopersicon esculentum

Incorporation of genes from wild species has been a major contributor to tomato improvement in recent years. Solanum ochranthum, a woody vine-like tomato relative, is a potential source of resistance against tomato diseases and insect pests but is genetically isolated from tomato. Somatic hybridization methods were developed to facilitate the use of S. ochranthum for tomato germplasm improvement. Leaf mesophyll protoplasts of S. ochranthum and selected Lycopersicon esculentum genotypes were chemically fused with polyethylene glycol. The protoplasts were initially cultured in Shepard's CL, a Murashige and Skoog-based medium, containing 1 mg l^-1 NAA, 0.5 mg l^-1 N⁶-benzyladenine and 0.5 mg l^-1 2,4-dichlorophenony-acetic acid. Tetraploid and hexaploid hybrid regenerants and regenerants of an L. esculentum parent were recovered; S. ochranthum did not regenerate. Hybridity was established by morphological characters, peroxidase isozyme and RAPD markers.Abbreviations MS Murashige and Skoog (1962) medium - CL Shepard (1980) cell layer medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP N⁶-benzyladenine - MES 2-N-morpholinoethanesulfonic acid - PEG polyethylene glycol - RAPD randomly amplified polymorphic DNA - PPM potato propagation medium - TPM tomato propagation medium - OM modified Murashige and Skoog (1962) medium - OM + AC modified Murashige & Skoog (1962) medium + activated charcoal     

Endophytic fungi in leaves of Lycopersicon esculentum Mill

Endophytic fungi were isolated from healthy tomato leaves from field-grown plants. Several surface-sterilization techniques were assayed for removing and killing epiphytes on tomato leaves. Surface-sterilization with an undiluted commercial solution of sodium hypochlorite was employed. To determine the spectrum of endophytes, samples were collected in February 1998 and 1999 from tomato plants (cultivar Tommy) cultivated in the experimental fields of Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Buenos Aires, Argentina. In general, different spectra of species were isolated in 1998 and 1999.Alternaria alternata was the fungus most frequently isolated from tomato leaves in 1999, but it was the second most common species in 1998. In contrast, Colletotrichum gloeosporioides was the fungus most frequently isolated in 1998, but it was not found in 1999. Species of other genera, such as Cladosporium and Penicillium, were isolated in both years.     

Epicuticular Lipid Accumulation on the Leaves of Lycopersicon pennellii (Corr.) D'Arcy and Lycopersicon esculentum Mill

A comparison was made of epicuticular lipid accumulation on leaves of Lycopersicon pennellii and Lycopersicon esculentum Mill. cv VF36 from 5 to 16 weeks of age. Epicuticular lipids were a small fraction of the leaf dry weight (0.16%) of 5-week-old `VF36', and increased to only 0.96% of the leaf dry weight after an additional 12 weeks of growth. In contrast, leaves from 5-week-old and 17-week-old L. pennellii plants had, respectively, 0.94% and 19.9% of their total dry weight in epicuticular lipid. Lipid accumulation was not affected by drought stress. Leaf position appears to influence the amount of lipid on the leaf surface. A glycolipid appears to be exuded from the terminal cell of glandular trichomes found on the leaves, stems, peduncles, calyxes, and fruits of L. pennellii.     

Somatic hybrid plants between Lycopersicon esculentum and Solanum lycopersicoides

Summary Leaf mesophyll protoplasts of Lycopersicon esculentum (2n=2x=24) were fused with suspension culture-derived protoplasts of Solanum lycopersicoides (2n=2x=24) and intergeneric somatic hybrid plants were regenerated following selective conditions. A two phase selection system was based on the inability of S. lycopersicoides protoplasts to divide in culture in modified medium 8E and the partial inhibition of L. esculentum protoplasts by the PEG/DMSO fusion solution. At the p-calli stage, putative hybrids were visually selected based on their hybrid vigor and lime-green coloration in contrast to slower growing parental calli characterized by a watery, whitish-brown coloration. Early identification of the eight hybrid plants studied was facilitated by isozyme analysis of leaf tissue samples taken from plants in vitro at the rooting stage. Regenerated plants growing in planting medium were further verified for hybridity by 5 isozymes marking 7 loci on 5 chromosomes in tomato. These included Skdh-1 mapped to chromosome 1 of tomato, Pgm-2 on chromosome 4, Got-2 and Got-3 on chromosome 7, Got-4 on chromosome 8, and Pgi-1 and Pgdh-2 both on chromosome 12. Fraction I protein small subunits further confirmed the hybrid nature of the plants with bands of both parents expressed in all hybrids. The parental chloroplasts could not be differentiated by the isoelectric points of the large subunit. Seven of the eight somatic hybrids had a chromosome number ranging from the expected 2n=4x=48 to 2n=68. Mixoploid root-tip cells containing 48, 53, 54 or 55 chromosomes for two of the hybrids were also observed.Michigan Agricultural Experiment Station Journal Article No. 11736. Supported by Grant No. I-751-84R from BARD — The United States — Israel Binational Agricultural Research and Development Fund     

番茄种子及其萌发期化感作用分析

采用种子发芽生物测试法,以发芽率、发芽势、发芽指数和活力指数为指标,对番茄(Lycopersicon esculentum Mill.)种子及其萌发期对大白菜[Brassica campestris ssp.pekinensis(Lour.)G.Olsson]、萝卜(Raphanus sativus Linn.)、黄瓜(Cucumis sativus Linn.)和番茄种子萌发的化感效应进行测定,并采用化感效应指数(RI)和化感综合效应(SE)对化感作用进行评价.结果表明:用30 mg·mL-1番茄种子浸提液培养大白菜、萝卜和黄瓜种子,各项发芽指标均低于对照;番茄种子萌发可导致套种的大白菜、萝卜和黄瓜种子的各项发芽指标降低;番茄幼苗分泌物对大白菜、萝卜和黄瓜种子的萌发也均有抑制作用;在4个发芽指标中,对种子活力指数的抑制效应最大.与对照相比,0.5和1.0g番茄幼苗模拟腐解物对大白菜、萝卜和黄瓜种子的发芽率有一定的促进作用,但均导致种子发芽指数和活力指数的降低;而1.5g番茄幼苗模拟腐解物对3种蔬菜种子各项发芽指标的抑制作用最大;随模拟腐解物质量的增加,番茄种子的发芽指标值也逐渐降低.8、16和24 mg·mL-1番茄幼苗浸提液对大白菜、萝卜、黄瓜和番茄种子的萌发有明显抑制作用,随浸提液浓度提高,受试蔬菜种子的各项发芽指标均逐渐降低.总体上看,在番茄种子萌发过程中对受试蔬菜种子萌发的RI值多数为负值、SE值均为负值.研究结果证实:番茄种子含有化感物质,在其萌发过程中分泌与释放化感物质并抑制胚根生长,且除抑制其他植物种子的萌发外还具有自毒作用.     

Salinity effects on water relations in Lycopersicon esculentum and its wild salt-tolerant relative species L. pennellii

Cultivated tomato Lycopersicon esculentum (L.) Mill. cv. P-73 and its wild salt tolerant relative L. pennellii (Correll) D'Arcy accession PE-47, were grown during spring-summer 1989 under unheated plastic greenhouse conditions. Plants were submitted to two different salt treatments using 0 and 140 mM NaCI irrigation water. In both tomato species, salinity caused a proportionally larger reduction in leaf area than in leaf weight and, in L. esculentum , a proportionally larger decrease in stem weight than in leaf weight. Daily variations in leaf water potential (Ψ₁) were fundamentally due to changes in the evaporative demand of the atmosphere. Reductions in Ψ₁ due to salinity were consistent only in L. esculentum . In all the conditions studied, leaf turgor was maintained. Leaf conductance (g₁)was higher in L. esculentum than in L. pennellii .Salinity induced a clear reduction in g₁ levels in L. esculentum whereas, in L. pennellii , this reduction was noted only in May. In both species the Ψ^o_s (leaf osmotic potential at full turgor) levels were reduced by salinity. The bulk modulus of elasticity (E) and relative water content at turgor loss point (RWC_tlp) were not affected by salinity. The RWC_tlp values in L. pennellii seem to be controlled by E values.     

Development and differentiation of haploid Lycopersicon esculentum (tomato)

Summary Haploid callus cultures of selected races of Lycopersicon (tomato) species can be obtained from anther culture. This is a further demonstration of a proposed general method of haploid culture developed with Arabidopsis thaliana. Differentiation of haploid callus of Lycopersicon esculentum can be controlled both in the dark and the light by hormones added to defined minimal media. Development to plantlets is achieved only in the light. Callus cells can be induced to develop into seedless pseudo-fruits. Chromosome counts on callus cells or root-tip cells establishes haploidy (n=12).Haploidy can be maintained in culture on defined minimal media for at least one year.