Embryo induction via anther culture in papaya and sex analysis of the derived plantlets

We investigated the embryo induction of papaya by anther culture, and identified the sex of plantlets derived from embryos using a sex-diagnostic PCR. Anthers, containing approximately 80% uninucleate pollen, were collected from 10 to 14 mm long male flower buds. They were pre-treated on agar (0.8%) or in liquid medium for 1–5 days at 25 or 35 °C, then transferred to agar medium with 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Agar and liquid media used for the pre-treatment contained water only or MS nutrients with or without sucrose (2.0%). On the agar medium, no embryos were induced at any pre-treatment temperature. In the liquid medium at 25 °C, embryos were induced at about 1.0% (rate of anthers forming embryos) in MS medium with sucrose for 3 or 5 days. At 35 °C, embryo induction rate tended to increase up to about 4.0% when anthers were treated in water for 1 day or MS medium with sucrose for 3 or 5 days. The sex of plantlets established through anther culture was analyzed using a sex-diagnostic PCR. All plantlets were determined as female. From these results, we suggest that all plantlets established through anther culture were of microspore origin, and that the anther culture technique is useful for the breeding of female papaya.     

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ Kn, which contained only half concentration of NH₄NO₃. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm²) was transferred to solid media (MS + 0.2 mg l⁻¹ 2,4-D + 5.0 mg l⁻¹ BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.     

In vitro induction of tetraploid ginger (Zingiber officinale Roscoe) and its pollen fertility and germinability

In vitro induction of tetraploid ginger (Zingiber officinale Roscoe) and its pollen fertility and germinability were investigated. The growth of shoot tip cultures on agar MS medium containing 2.0 mg l⁻¹ BA was greater than that of similar cultures in liquid MS medium with the same BA concentration. In liquid medium, the combinations of 0.5, 1.0, or 2.0 mg l⁻¹ BA with 0.05 mg l⁻¹ NAA tended to enhance the growth of the cultures. The efficiency of tetraploid induction was assessed by treating shoot tip explants on agar or in liquid MS medium containing 2.0 mg l⁻¹ BA, 0.05 mg l⁻¹ NAA, and 0.2% (w/v) colchicine for 4, 8, 12, and 14 days. The total number of tetraploids induced on solid medium was 18, but only five in liquid medium. On both media, the colchicine treatment for 8 days gave the maximum level of tetraploid induction. Therefore, it was found that the treatment of shoot tip explants on agar medium containing 2.0 mg l⁻¹ BA, 0.05 mg l⁻¹ NAA, and 0.2% (w/v) colchicine for 8 days was the most efficient way of inducing tetraploid ginger. Induced tetraploid strains, ‘4× Kintoki’, ‘4× Sanshu’, and ‘4× Philippine cebu 1’, had higher pollen fertility and germinability than the diploid counterparts, i.e., in the diploid strains, pollen fertility ranged from 0.3 to 6.2% and the germination rate from 0.0 to 0.1%, while in the tetraploid strains, pollen fertility ranged from 27.4 to 74.2% and the germination rate from 4.8 to 12.9%.     

Prohexadione-calcium affects growth and flowering of petunia and impatiens grown under photoselective films

The response of petunia (Petunia x hybrida Vilm.-Andr. ‘Countdown Burgundy’) and impatiens (Impatiens wallerana Hook ‘Accent Orange Tempo’) to Prohexadione-calcium was evaluated under a clear and a far-red light absorbing greenhouse (A_FR) film to investigate the dosage effect of Prohexadione-Ca and to determine if it can overcome the flowering delay under FR deficient greenhouse environments. Prohexadione-Ca reduced stem elongation of petunia and impatiens under A_FR and clear films when applied 3 weeks after germination. Late applications were less effective. In both crops, main stem length decreased in a quadratic pattern as the concentration of Prohexadione-Ca increased. Under both films, 50–100 mg l⁻¹ Prohexadione-Ca resulted in ≈30% shorter petunia plants. Greater concentrations (500 and 1000 mg l⁻¹) resulted in excessively short plants (over 70%). Prohexadione-Ca at 100 mg l⁻¹ delayed anthesis of petunia by 8 and 3 days under the clear film and the A_FR film, respectively during less inductive photoperiods but had no effect during inductive photoperiods. In impatiens, Prohexadione-Ca at 100 mg l⁻¹ delayed anthesis over 10 days under clear or A_FR film. Greater concentrations (200 and 300 mg l⁻¹) inhibited flowering of impatiens. Prohexadione-Ca treatments significantly affected flower color development. Untreated petunia plants had dark burgundy flowers. Prohexadione-Ca treatment increased L*, a*, and C* values and decreased hue angle indicating that the flowers were faded. Flowers of untreated impatiens plants were bright orange color. Prohexadione-Ca at 100 mg l⁻¹ increased L* value and decreased a*, b*, and C* values indicating that significant petal fading had occurred. Flowers of treated plants were nearly white under both films. Although effective in height control, loss of color would be a major limitation to the use of Prohexadione-Ca on flowering crops.     

Effects of IAA and BA on development of globular,heart- and torpedo-stage embryos from cell suspensions of three grape genotypes

In order to initiate cell and embryo suspensions, embryogenic calluses derived on NN solid medium with 2,4-D and BA from petioles of in vitro grown plants of three interspecific grapevine hybrids were cultured in three versions of liquid NN medium: (1) without growth regulators, (2) 0.1 mg l⁻¹ IAA and (3) 0.5 mg l⁻¹ BA. Cell and embryo suspensions were incubated two and three times in these versions of liquid media in various combinations. Incubating suspensions two times in hormone-free media and/or with 0.1 mg l⁻¹ IAA led to formation of globular embryos in the three cultivars studied and small numbers of heart-stage embryos in ‘Podarok Magaracha’ and ‘Intervitis Magaracha’. Numerous heart-stage embryos developed in ‘Intervitis Magaracha’ and ‘Podarok Magaracha’ when the suspensions had been initiated in medium with 0.5 mg l⁻¹ BA, and in ‘Bianca’ this was achieved after two incubations in the above medium. Torpedo-stage embryos were formed after subculturing heart-stage embryo suspensions in medium with 0.1 mg l⁻¹ IAA in all study cultivars. If only small numbers of embryos of a certain developmental heart- or torpedo- stage were formed, such cell and embryo suspensions need to be repeatedly subcultured in liquid medium with specific growth regulators to enable this process.     

Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing

Multiple shoots were produced from node explants of lime (Citrus aurantifolia (Christm.) Swing) on MS medium supplemented with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin) and α-naphthaleneacetic acid (NAA). The highest number of shoots, nine shoots per node, were produced on a medium containing 2 mg l⁻¹ BAP (8.8 μM), 1 mg l⁻¹ kinetin (4.6 μM) and 1 mg l⁻¹ NAA (5.4 μM). Depending on the concentration of BAP and kinetin, NAA either inhibited, stimulated or did not affect shoot multiplication, which also depended on the cytokinin level. Maximum shoot length was obtained from treatments containing 0.5 mg l⁻¹ BAP (2.2 μM) combined with 1 mg l⁻¹ kinetin (4.6 μM) and 0.5 mg l⁻¹ NAA (2.7 μM). The largest leaves of resultant shoots were produced on a medium containing 0.5 mg l⁻¹ each of kinetin (2.3 μM) and NAA (2.7 μM). Transferring in vitro shoots to rooting media containing indole-3-butyric acid (IBA) and NAA produced complete plantlets. The highest rooting percentage was obtained on a medium containing either 1 mg l⁻¹ NAA (5.4 μM) alone or 0.5 mg l⁻¹ NAA (2.7 μM) combined with 2 mg l⁻¹ IBA (9.6 μM), whereas the highest number of roots were produced on a treatment containing both 2 mg l⁻¹ NAA (10.8 μM) and 2 mg l⁻¹ IBA (9.6 μM). Roots elongated most on treatments containing 0.5 mg l⁻¹ of either NAA (2.7 μM) or IBA (2.4 μM). Shoot growth associated with the rooting phase was the highest in response to 2 mg l⁻¹ IBA (9.6 μM) or 0.5 mg l⁻¹ NAA (2.7 μM). Plantlets that survived acclimatization, 82%, exhibited normal growth in soil under greenhouse conditions.     

Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.)

Studies were conducted to improve adventitious bud regeneration in roses (Rosa hybrida L.), specifically to extend the protocol to different genotypes and to initiate production of multiple shoots per explant. The best results were obtained by using a two-stage procedure where excised leaflets were incubated on Murashige and Skoog (MS) (1962) induction medium with 6.8 μM TDZ plus 0.49 μM IBA in the dark for 7 days and subsequently transferred to an MS-based regeneration medium with 2.22 μM BA plus 0.049 μM IBA exposed to a PPFD of 15 μmol m⁻² s⁻¹ PAR. Bud formation capacity was also significantly affected by the genotype and the environment, such as the use of bottom cooling creating a lower RH in the vessel. The addition of silver nitrate to the induction medium also significantly improved the percentage of regeneration in three genotypes tested. Regenerated shoots failed to elongate when transferred to MS proliferation medium containing 0.5 mg l⁻¹ BA, however maximum bud development and elongation were achieved when kinetin in the range 1–2 mg l⁻¹ was used. Elongated shoots were excised and rooted best on zero growth regulator half-strength MS modified medium. Rooted plantlets were acclimatized under greenhouse conditions for evaluation of somaclonal variation.     

Factors affecting the shoot multiplication of Persian walnut (Juglans regia L.)

A number of experiments were conducted to identify suitable procedure for in vitro shoot multiplication of Persian walnut (Juglans regia L.). Three different nutrient media (DKW, MS and WPM) and three different gelling agents (Phytagel, Difco Bacto agar and a mixture of Phytagel and Difco Bacto agar) were studied in the first experiment. Driver and Kuniyuki walnut (DKW) medium solidified with 2.2 g l⁻¹ Phytagel was found optimum. Performance of explants was better on DKW medium than on MS and WPM. The DKW and MS media were not significantly different from each other, but both of them were significantly better than WPM, which was a very poor medium for this species. Phytagel alone was significantly better than Difco Bacto agar or Phytagel combined with Difco Bacto agar. In another experiment different concentrations of BA were studied. Medium containing 1.0 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was the best, although medium containing 0.6 and 0.8 mg l⁻¹ BA were also successful, and utilisation of 0.4 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was optimum for shoot elongation. Application of different kinds of auxins (IAA, IBA and NAA at 0.01 or 0.1 mg l⁻¹) with 1.0 mg l⁻¹ BA were also studied. Media containing IBA were significantly better than media containing IAA for shoot fresh weight, but neither of them was significantly different from media containing NAA. Application of 0.01 mg l⁻¹ or 0.1 mg l⁻¹ auxin, with 1.0 mg l⁻¹ BA, was not significantly different for shoot multiplication of Persian walnut. The morphology of shoots on media containing 0.01 mg l⁻¹ IBA was the best.     

GA4+7 plus benzyladenine reduce foliar chlorosis of Lilium longiflorum

The effectiveness of two commercial formulations of gibberellin (GA) and benzyladenine (BA) for reducing foliar chlorosis on Easter lily (Lilium longiflorum Thunb.) was compared. On a per liter basis, plants were sprayed with 0, 100, 200, or 400 mg (BA equivalent) of Accel (GA₄₊₇:BA of 1:10) or Promalin (GA₄₊₇:BA of 1:1) when the crop leaf area index (LAI)=3. One group of plants was sprayed with 100 mg of Accel or Promalin (BA equivalent) per liter twice: once at LAI=3 and again 3 weeks later. Plants were harvested when the largest flower bud on each plant measured 13 cm in length, stored for 0 or 3 weeks at 2.5°C in the dark, and then moved into a post-harvest evaluation room at 21°C, where foliar chlorosis was monitored for 3 weeks. Senescence of some lower leaves on plants in every treatment was evident at harvest, and incidence of senescence increased during the 21 days of post-harvest evaluation. Cold storage increased the number of leaves senescing during the subsequent evaluation period. Application of Promalin or Accel significantly reduced leaf senescence compared to that of untreated plants. At harvest, 21% of the leaves on untreated plants were senescent, while plants treated with Promalin or Accel averaged 3 or 9% senescent leaves, respectively. Following 7 days of post-harvest evaluation, Promalin was more effective in preventing chlorosis than Accel at the 400 mg l⁻¹ (BA equivalent) level. Following 14 or 21 days of post-harvest evaluation, Promalin was more effective than Accel for the 100 mg l⁻¹ 2× and 400 mg l⁻¹ (BA equivalent) treatments.Plants in all Promalin and Accel treatments were taller than untreated plants 1 week after sprays were applied. At harvest, plants sprayed with Promalin were between 6 and 14 cm taller than untreated plants, but those treated with Accel were the same height as untreated plants.Neither Promalin nor Accel influenced the occurrence of malformed or aborted flowers in this study. However, cold storage significantly increased the number of plants with aborted buds and malformed flowers. Unstored plants averaged 0.16 aborted buds and 0.02 malformed flowers each, while those stored 3 weeks averaged 0.51 aborted buds and 0.18 malformed flowers each.     

Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)

Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 μM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 μM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 μM 2,4-D and 1 μM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 μM 2,4-D and 1 μM BA and subsequently transferred to media containing 2–4 μM 2,4-D and 0 or 0.5 μM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 μM 2,4-D and 1 μM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 μM 2,4-D and 0.5 μM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks.     

The effect of mycorrhizal symbiosis on the development of micropropagated artichokes

In this work, microrosettes of Cynara cardunculus L. var. scolymus Fiori of the “catanese” type were subcultured in a medium supplemented with 6-benzylaminopurine (BAP) (0.05 mg l⁻¹). For root induction, indoleacetic acid (IAA), α-naphthalene acetic acid (NAA) and indole-3-butyrric acid (IBA) were used at three concentrations: 2, 5 and 10 mg l⁻¹. The highest percentage of rooted shoots was aided by the presence of 10 mg l⁻¹ IAA.Once transplanted in pots, the plantlets were inoculated with 10 g Glomus viscosum strain A6 (AM fungus). Acclimatisation was clearly facilitated by the addition of the AM fungus. Indeed, the mycorrhizal plantlets registered a survival of between 90 and 95% for the rooting shoots and 60% for the non-rooting shoots.The botanical characterization of the material produced was carried out in field and was based on several morphological and productive parameters. Data collected confirm the characteristics of the original cultivar, the efficiency of the in vitro propagation material and the possibility of using this technique in early types of artichoke.     

An investigation on the effect of different plant growth regulating compounds in in vitro shoot tip and node culture of lemon seedlings

The possible application of some less commonly used in vitro growth regulating compounds is outlined. A number of treatments were applied to determine the best way of inducing in vitro shoot proliferation and rooting on a modified Driver–Kuniyuki [HortScience 19 (1984) 507] basal medium of lemon (Citrus limon (L.) Burm, f. cv. Interdonato) seedlings. 6-Benzyladenine (BA) alone (1, 2 and 4 mg l⁻¹) and in combination with either orange juice (10%, v/v), silver nitrate (3 mg l⁻¹), gibberellic acid (GA₃) (0.1 mg l⁻¹ at the establishment stage and 0.5 mg l⁻¹ at all combinations during the proliferation stage) or abscisic acid (ABA) (0.2 mg l⁻¹ only at the establishment stage) were used to stimulate shoot formation during the establishment and the proliferation stage. The combination of BA with ABA gave a high rate of shoot formation, while GA₃ and silver nitrate enhanced shoot elongation. When these shoots were transferred to the rooting stage, the effect of application of two different auxins (indole-3-butyric acid (IBA) and α-napthaleneacetic acid) was examined, as well as different methods of application (auxin added to the basal medium and auxin application by dipping the base of the explant in auxin solution). Dipping the base of the explants in a 50% ethanol solution of IBA at 1000 mg l⁻¹ for 5 s resulted in 80% rooting with subsequent 90% survival of these explants, during acclimatization under mist.     

Micropropagation of Karonda (Carissa carandas) through shoot multiplication

Shoot tips from field grown, mature plants of Carissa carandas cv. Pant Sudarshan were cultured on Murashige and Skoog’s (MS) basal medium supplemented with benzyladenine (BA) and indolebutyric acid (IBA) during different seasons. The maximum sprouting rate was obtained with 1.5 cm long explant collected in spring season (February–March) followed by those collected in summer season (April–June). Shoot proliferation was highest on MS basal media supplemented with 3.0 mg l⁻¹ BA. Rooting of microshoots was noted to be the best in 1/2 MS plus 0.8 mg l⁻¹ IBA and 0.2 mg l⁻¹ naphthalene acetic acid (NAA). The rooted plantlets were successfully acclimatized in vermiculite:sand:soil (1:1:1) potting mixture.     

Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear

This study aimed to improve in vitro shoot proliferation efficiency without inducing hyperhydricity in Japanese pear. The shoot number increased at 2.5–10.0 mg l⁻¹ benzyladenine (BA), while shoot fresh mass increased at 1.0 and 2.5 mg l⁻¹ BA. Different macroelement formulation did not affect shoot proliferation, but adding activated charcoal (AC) to the medium inhibited markedly the production of axillary shoots and biomass and many shoots were hyperhydric. Different carbon sources (CS) significantly increased the shoot number and fresh mass, with the best results for shoot proliferation at 20–30 g l⁻¹ sorbitol. With gelling agents, the shoot number increased at 0.4 and 0.6% agar and 0.3% gellan gum, while fresh mass increased at 0.4% agar. The hyperhydric explants were more than 30% at 0.4–0.6% agar and at any concentration of gellan gum. The improved culture (woody plant (WP) supplemented with 20 g l⁻¹ sorbitol, 0.1 mg l⁻¹ 3-indolyl-butyric acid (IBA), 2.5 mg l⁻¹ BA and 0.8% agar) and double-phase culture (the same medium using a double-phase liquid-gelling agent solidified culture system) produced a higher number of axillary shoots than the conventional culture (1/2MS supplemented with 0.1 mg l⁻¹ IBA, 1.0 mg l⁻¹ BA, 30 g l⁻¹ sucrose and 0.8% agar), moreover, double-phase culture had a higher fresh mass than the other cultures.     

Methyl jasmonate and CMN-pyrazole applied alone and in combination can cause mature orange abscission

Methyl jasmonate (MJ) and CMN-pyrazole alone and in combination were applied to ‘Hamlin’ and ‘Valencia’ orange trees (Citrus sinensis (L.) Osbeck) in seven separate experiments in the 1998/1999 and 1999/2000 growing seasons to induce abscission of mature fruit for mechanical harvesting. MJ alone significantly reduced fruit detachment force (FDF) of ‘Hamlin’ oranges at concentrations of 10 mM and higher. However, MJ at 20 and 100 mM caused significant defoliation. For ‘Hamlin’ oranges, the most effective treatments were those combining 10 mM MJ and 50 mg l⁻¹ CMN-pyrazole, and 20 mM MJ and 25 mg l⁻¹ CMN-pyrazole. ‘Valencia’ oranges did not respond to 10 mM MJ treatments alone or in combination with CMN-pyrazole, and required MJ at 20 mM in combination with CMN-pyrazole to loosen this late-maturing variety to below 50 N, but excessive defoliation occurred.     

Foliar treatment of Mn deficient ‘Washington navel’ orange trees with two Mn sources

The objective of this study was the comparison of the effect of two Mn sources (MnSO₄·H₂O, MnEDTA) which were applied at various concentrations (0, 200, 400, 800, and 1200 mg Mn l⁻¹) to the leaves of ‘Washington navel’ orange trees in order to correct Mn deficiency.One hundred and seventy days after the foliar application of Mn solutions, the mean Mn concentrations in the leaves treated with MnSO₄·H₂O (200, 400, 800 or 1200 mg Mn l⁻¹) or MnEDTA (400, 800 or 1200 mg Mn l⁻¹) were significantly higher than those of the control leaves. Manganese sulfate (MnSO₄·H₂O) was more effective than MnEDTA regarding the improvement of the leaf Mn concentrations of the trees, when applied at equal Mn concentrations. Finally, the leaf Mn concentrations were in the sufficiency range (>25 mg kg⁻¹ d.w.), only after the application of 800 or 1200 mg Mn l⁻¹ as MnSO₄·H₂O.     

Arsenic as a factor affecting virus infection in tomato plants: changes in plant growth,peroxidase activity and chloroplast pigments

The influence of arsenic and Cucumber mosaic virus (CMV), applied separately and simultaneously on young tomato plants was studied. The plants were cultivated in containers under glasshouse conditions. Four main variants were arranged. The first one was without additional As pollution of soil, named as a control, and the other three variants, with As added at 25, 50 and 100 mg kg⁻¹ to dry soil respectively. Half of the plants in each experimental container were inoculated with CMV and the rest uninoculated. A clear response in plant behavior under the conditions of biotic and abiotic stress was estimated. Both arsenic and virus infection had a negative effect on tomato plants by limiting the growth of their roots and above growth parts. The changes in roots were more significant than of stems. Virus infection was a stronger stress factor than arsenic applied at levels of 25 and 50 mg kg⁻¹. The effect of each stress factor applied separately was enhanced in cases of their simultaneous application. The strongest negative effect was manifested in the infected plants, treated with excess arsenic of 100 mg kg⁻¹. It was established that the infection, caused by CMV in tomatoes, was affected by the presence of arsenic in the soil and concentration of the latter. Doses of 25 and 50 mg kg⁻¹ were favorable for infection development, while the dose of 100 mg kg⁻¹ was an inhibitor.Virus infection induced stronger specific peroxidase activity (SPOA) than As treatment. The combination of both stress factors reduced the positive peroxidase response caused by virus infection. Arsenic at rate 50 and 100 mg kg⁻¹, virus infection and the combination of both stress factors at 25 mg kg⁻¹ reduced chlorophyll a, chlorophyll b and carotenoid content The virus infection in cases of the higher arsenic doses reduced the As effect. There was an interaction between the two effects of biotic and abiotic stress. When arsenic and virus infection were applied simultaneously, they caused modification of the effect of each stress on the plants, when applied separately.     

Influence of nickel-contaminated soils on fenugreek (Trigonella corniculata L.) growth and mineral composition

Leafy vegetables accumulate higher amount of heavy metals like nickel (Ni) due to their more leafy vegetative growth. Therefore, a screenhouse experiment was conducted using an alkaline sandy loam soil equilibrated with graded levels of Ni (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 and 300 mg kg⁻¹ soil) to assess the Ni accumulation pattern and its influence on growth and micronutrient distribution in fenugreek plants. Green as well as the dry matter yields of fenugreek increased slightly up to 20 g Ni kg⁻¹ soil but decreased significantly with the application ≥40 mg Ni kg⁻¹ soil. Crops showed characteristic toxicity symptoms of interveinal chlorosis in pots receiving ≥40 mg Ni kg⁻¹ soil. While the total content of Ni in the plant tissues increased consistently with increasing rates of applied Ni, the roots accumulated much higher amount of this element compared to the shoot. The content of Fe in plants showed an increase whereas that of Cu and Zn experienced a decrease with the rise in the applied Ni.     

A comparative study of chlorophyll loss and its related mechanism during fruit maturation in the pericarp of fast- and slow-degreening litchi pericarp

Changes in the levels of chlorophyll a and b and their derivatives, and chlorophyllase activities were compared in the pericarp of ‘Feizixiao’ (slow-degreening) and ‘Nuomici’ (fast-degreening) litchis (Litchi chinensis Sonn.). Cluster bagging and growth regulators were used to regulate the degreening process of ‘Feizixiao’. HPLC analysis revealed the presence of chlorophyllide a, pheophorbide a and pheophytin a as chlorophyll derivatives in litchi pericarp. Loss of chlorophylls in ‘Feizixiao’ pericarp was slower than that in ‘Nuomici’ pericarp. The concentrations of chlorophyll a and b in the pericarp of ‘Feizixiao’ were significantly higher than those in ‘Nuomici’ during fruit maturation, whereas the chlorophyll derivatives in the former were noticeably lower than in the latter. Chlorophyllase activity increased as chlorophylls diminished. Chlorophyllase activity peaked around the colour-break in ‘Nuomici’, being much higher than that in ‘Feizixiao’. Both cluster bagging and treatment with abscisic acid (ABA, 200 mg l⁻¹) or jasmonic acid (50 mg l⁻¹) significantly decreased the chlorophyll content and increased the chlorophyllase activity and the anthocyanin level. In fruit treated with 6-benzyl aminopurine (6-BA, 100 mg l⁻¹) the degradation of chlorophylls was delayed, with concomitant decrease of chlorophyllase activity and anthocyanin level. These results imply that chlorophyllase is the crucial factor in the regulation of the chlorophyll loss in the pericarp.     

Micropropagation of Vitis thunbergii Sieb. et Zucc., a medicinal herb,through high-frequency shoot tip culture

This study describes a protocol for rapid and large-scale in vitro propagation of the valuable medicinal herb Vitis thunbergii Sieb. et Zucc.. Culture conditions influencing shoot proliferation and rooting of the two clones (three- and five-lobed) were examined. Three medium formulations, Murashige-Skoog (MS), Woody Plant Medium (WPM) and Nitsch and Nitsch (NN) medium, were tested for growth of shoot tip culture, and WPM was found to have a superior proliferation rate. The chlorophyll content of leaves was highest in those cultured on NN, followed by those on MS and WPM medium. WPM medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BA) displayed the highest proliferation rate (15–19 nodes or 3–4 shoots per explant). The rooting was optimized using MS medium supplemented with 0.5 mg l⁻¹ napthaleneacetic (NAA) with eight roots, 3 cm long after 1 month of culture. High frequency callus formation was observed in the basal end of explants cultured on NAA-containing medium. Following acclimatization, rooting plantlets were transferred to the plastic house with a 95% survival rate.