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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Shoot induction and plant regeneration from receptacle tissues of Lilium longiflorum

An efficient system has been developed for the in vitro plant regeneration of Lilium longiflorum Thunb. by culturing receptacle sections from flower buds. The sections were cultured on one-half MS medium plus 30 g l⁻¹ sucrose, 8 g l⁻¹ agar, 5.4 μM NAA or 4.9 μM IBA plus 2.2 μM BAP. A section size of 3–4 mm was found to be optimal. After 60 days an average of 41 shoots were formed per explant. More vigorous shoots were obtained by subculturing on hormone-free medium with 20 g l⁻¹ sucrose. Rooting occurred on one-half MS medium with 1.1 μM NAA. Rooted plants were hardened-off in a greenhouse for two months, and normal flowering plants were produced.     

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.     

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.     

Microsporogenesis and anther culture in carob tree (Ceratonia siliqua L.)

An in vivo study was made on male flowers of carob tree (Ceratonia siliqua L.), in order to establish a correlation between the flower and anther development, and microsporogenesis. In addition studies were conducted to find which phase is more appropriate for anther culture and haploid production. During the development of male flowers, six stages were identified. The male gametophytic cycle begins when flowers are in developmental phase 0, with the formation of the epidermis, endothecium, primary sporogeneous tissue, primary parietal cells and pollen mother cells. During developmental phase I we observed the formation of pollen mother cells, the microspore tetrads, and uni- and binucleate pollen grains. At developmental phase II, uni- and binucleate microspores, and completely formed pollen grains were observed. In developmental phase III we could observe mature pollen grains ready to be released from the anthers as single binucleate pollen grains. Anthers from flowers at developmental phases I and II, with microspores at late uninucleate to early binucleate stage, were cultured in semi-solid Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) combined with one of the citokinins: N⁶-benzyladenine (BA), kinetin (Kin), zeatin (Zea) and thidiazuron (TDZ). To obtain embryogenic calli anthers should be collected from flowers in developmental phase I. High frequencies of callogenesis were obtained, and the best medium for calli induction was MS supplemented with 0.5 mg l⁻¹ 2,4-D + 4 mg l⁻¹ TDZ. The frequency of haploid cells was found to be 17.2%.     

Fruit set,seed development and embryo germination in interploid crosses of citrus

Interploid crosses of diploid Kinnow mandarin, Succari sweet orange and Sweet lime with tetraploid Kinnow were made reciprocally for the production of triploid plants. Low fruit set and occurrence of underdeveloped or empty seeds was common in all interploid crosses. The hybrid fruits harvested after 12–14 weeks or 7 months after pollination produced many underdeveloped and few developed seeds; however, tetraploid Kinnow as seed parent yielded more developed seeds per fruit. The under developed seeds were devoid of endosperm. Nucellar embryony was higher in the diploid than in the tetraploid strain of Kinnow. Immature embryos of seeds harvested 12–14 weeks after pollination were cultured in vitro on MS media supplemented with adenine sulphate (20 mg l⁻¹) and malt extract (500 mg l⁻¹). Maximum germination (75%) of hybrid embryos from underdeveloped seeds was obtained in 4× × 2× cross of Kinnow strains. The germinated plantlets were transferred into pots and noted 59–89.3% survival rate in various crosses.     

Daylength and photon flux density influence the growth regulator effects on morphogenesis in epicotyl segments of Troyer citrange

The optimal growth regulator addenda for adventitious shoot regeneration in epicotyl cuttings of Troyer citrange (Citrus sinensis×Poncirus trifoliata) varied with the conditions of illumination. The response to illumination and to growth regulators differed for the direct and the indirect (through callusing) pathways of regeneration. Shoot formation through the direct organogenic pathway decreased as the concentration of benzyladenine (BA) in the medium was increased in the range 2.2–22 μM, when the explants were incubated in the dark or under an 8 h daylength. For explants incubated under a 16 h daylength, the number of shoots formed increased with BA concentration. Optimal conditions of incubation for shoot formation through the direct pathway were either an 8 h daylength with a photon flux density of 74 μmol m⁻² s⁻¹ in the presence of 2.2 μM BA, or a 16 h daylength with a photon flux density of 37 μmol m⁻² s⁻¹ in the presence of 22 μM BA. Irrespective of the conditions of incubation, shoot formation through the indirect organogenic pathway was suppressed by the addition of 22 μM BA to the medium. Optimal conditions for shoot formation through this pathway were incubation under an 8 h daylength at a photon flux density of 74 μmol m⁻² s⁻¹ in the presence of 2.2 μM BA. At the optimal conditions indicated, the addition of the synthetic auxin naphtaleneacetic acid (0.54 μM) reduced shoot formation. Irrespective of the pathway of regeneration, the number of shoots formed decreased markedly with the distance of the cutting from the cotyledonary node.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

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.     

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.     

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.     

Optimization of somatic embryogenesis in suspension cultures of horsegram [Macrotyloma uniflorum (Lam.) Verdc.]—A hardy grain legume

Cell suspension cultures were established from immature cotyledon derived calli from drought tolerant legume horsegram [Macrotyloma uniflorum (Lam.) Verdc.]. Embryogenic callus could be originated from cut slices of the immature cotyledons on MS solid medium [Murashige, T. Skoog, K., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497] augmented with 1.0 μM zeatin and 4.5 μM NAA. Numerous somatic embryoids (26.4%) appeared on MS liquid basal nutrient medium with 5.6 μM NAA and with absence of zeatin during 3 weeks culture. Sustained cell division resulted in the formation of cell aggregates, and then progressed to globular, heart and further if they differentiate properly to torpedo and cotyledonary stages within 5 weeks. Transfer of individual embryos on to a fresh MS basal medium with no plant growth regulators was able to achieve complete maturation. Only a relatively few number of embryos developed into root/shoot when transferred to 0.9 μM GA₃, 15 g/l⁻¹ sucrose and 2.4 g/l⁻¹ gelrite containing medium. Substitution of sucrose associated with the use of l-glutamine gave, in the range of concentrations tested, the strongest enhancement of the embryo growth and development. About 5% of somatic embryos were converted into true-to-type fertile plants.