An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.)

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3–5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).     

提高籼稻愈伤组织再生频率的研究

为了提高籼稻愈伤组织的植株再生频率,研究了影响再生的各种因素,如：在诱导培养基或继代培养基中加细胞分裂素和萘乙酸（ＫＴ、ＢＡＰ、玉米素或Ｚｉｐ１毫克／升）,或加Ｔｈｉｄｉａｚｕｒｏｎ（０．５毫克／升）,以及愈及组织的部分干燥处理等。这些措施明显地提高籼稻愈伤组织的再生频率。结合使用这些处理可使ＴＮ１、ＩＲ７２和ＩＲ６４的愈伤组织再生植株频率较对照提高５－１４倍。     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

A comparison of callus induction and plant regeneration from different embryo explants of triticale (x Triticosecale wittmack)

Immature, mature and endosperm-supported mature embryos of six triticale cultivars (BDMT-98-8S, Melez-2001, Mikham-2002, Presto, Tacettin Bey and Tatlicak-97) were cultured in vitro to compare the levels of callus induction and plant regeneration. Immature embryos, 15-18 days after anthesis, were aseptically excised and placed with the scutellum upwards on a callus culture medium consisting of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised from the imbibed seeds and placed scutellum up on MS medium supplement with 2 mg l(-1) 2,4-D. Endosperm-supported mature embryos were moved slightly in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l(-1) 2,4-D for callus induction. The developed calli and regenerated plants were maintained on hormone-free MS medium. Variability among the genotypes was observed for all the types of embryo culture. Immature embryos from "Presto" and endosperm-supported mature embryos from "Mikham 2002" had excellent regeneration capacities (92.0% and 97.3%, respectively) and the highest number of plants regenerated growing in soil (9 and 13, respectively). A comparison of the responses of the three explants used indicated that the endosperm-supported mature embryo was the most useful explant for plant regeneration in triticale.     

Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL^–1 and 26 mgL^–1 ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MS Murashige and Skoog - NAA napthaleneacetic acid - Trp tryptophan - 2,4-D 2,4-dichlorophenoxyacetic acid     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

Totipotency of coleoptile tissue in indica rice (Oryza sativa L. cv. ch 1039)

Embryogenic and non-embryogenic calluses were induced from 3,4,5 and 7d old coleoptile segments of indica rice (Oryza sativa L. cv. CH 1039). Compact, globular, yellow and creamy embryogenic and white friable non-embryogenic callus arose from the cut end and entire length of the coleoptile segments. Murashige and Skoog's (MS) medium supplemented with 2.5mg/1 2,4-D was used as callus induction medium. Plant regeneration from coleoptile segments occurred with the transfer of embryogenic callus to MS basal medium supplemented with 2.0mg/1 BAP and 0.5mg/1 NAA in combination. Average number of regenerated plants from one coleoptile ranged from9.1 to 14.0.Four day old coleoptiles showed the highest frequency of plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) - NAA 1-naphthalene acetic acid     

High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex

Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.     

Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture

The effect of genotype, growth regulators and preconditioning of donor plants on callus induction in anther culture of flax was investigated. Anthers were cultured on modified MS medium supplemented with five different combinations of plant growth regulators. The results suggested that specific combinations of growth regulators must be designed for each genotype. Major differences between the present results and previous reports are discussed. The influence of sucrose concentration was also investigated. For flax cultivar, 'Mikael', callus induction was higher in medium supplemented with 1 mg l(-1) BAP and 2 mg l(-1) 2,4D containing 6% sucrose, while this combination of growth regulators significantly increased callogenesis in cultivars 'Lirina', 'Barbara' and 'Szaphir' when supplemented with 9% or 12% sucrose. The preconditioning of donor plants influenced callogenesis in subsequently isolated anthers. Anthers from donor plants grown at a lower temperature (18/14 degrees C) significantly increased callus induction over those from plants grown at a higher temperature (22/18 degrees C), although each genotype still required optimization of growth regulator combinations in the induction medium. Only 'Mikael' regenerated shoots when the callus was from induction medium supplemented with 2 mg I(-1) BAP and 1 mg l(-1) NAA.     

High frequency plant regeneration from immature embryos of an elite barley cultivar (Hordeum vulgare L. cv. Morex)

An efficient plant regeneration system was developed for Hordeum vulgare L. 'Morex' barley, an important United States malting cultivar. The protocol was based on a series of experiments involving the sizes of immature embryos and the culture media. We found that the embryo size is critical for the establishment of embryogenic callus. Smaller embryos (0.5-1.5 mm) showed a much higher ability to produce embryogenic callus capable of regenerating green plants with fewer albinos than did the larger embryos (1.6-3.0 mm). Either 3 mg/l 2,4-dichlorophenoxyacetic acid or dicamba in modified Murashige and Skoog's (MS) medium was optimum for the induction of embryogenic callus. The embryogenic callus maintained high regeneration during six subcultures in the callus induction medium. Efficient shoot regeneration was obtained on modified MS medium containing 0.5-1.0 mg/l 6-benzylaminopurine (BA). Regenerated shoots were rooted on half-strength MS medium containing 0.2 mg/l IBA. Plants were successfully transferred to soil and grown to maturity in the greenhouse. This efficient plant regeneration system provides a foundation for generating transgenic plants of this important barley cultivar.     

Regeneration of plantlets from mature embryo calli of Western Ghats land race cultivar of rice, Oryza sativa L

The Malnad region located in the Western Ghats of Karnataka is known for the cultivation of indigenous rain fed land race cultivar of rice. The present study was to investigate the callogenic and caulogenic potentialities of the two indigenous rice cultivar namely Karimundaga and Kanadatumba using dehusked mature embryo explants. For callus and shoot bud differentiation, the explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-D (1-3 mg/L), IAA (1-2 mg/L), Kn (1-4 mg/L) and BAP (1-4 mg/L). The morphogenic potentialities of the two rice cultivar differed in texture of callus. In both the cultivar callogenic frequency was optimized at 1 mg/L 2,4-D concentration, it was 94% in Karimundaga and 58% in Kanadatumba. Supplementation of IAA either alone (1-2 mg/L) or in combination with Kn or BAP at 1 to 4 mg/L concentration of each induces shoot bud differentiation from the calli. In the cultivar Karimundaga caulogenic frequency was highest (10.60 +/- 2.55) at 1.0 mg/L IAA and 4.0 mg/L BAP concentration. While in the cultivar Kanadatumba highest number of shoot buds (7.90 +/- 2.69) was differentiated at 1.0 mg/L IAA and 4.0 mg/L Kn concentration. The calli derived regenerants were successfully acclimatized in the greenhouse and agro-morphological variations were evaluated. The growth characteristics and yield related parameters exhibited by in vitro plants were lower than the in vivo plants.     

Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis> Sm. using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective transformation and direct regeneration of E. tereticornis Sm.     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile

Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS?+?1 mg/l BAP?+?1 mg/l NAA, while indirect regeneration from callus was obtained on MS?+?1 mg/l BAP?+?2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling.     

Callus induction and high frequency plant regeneration in Italian millet (Setaria italica)

Callus was induced from mature seeds of two cultivars of Setaria italica (L.) on Murashige and Skoog's medium (1962) supplemented with 2mg/l 2,4-D and 0.5 mg/l KN. Regenerating ability of the callus was better in the cultivar 315 compared to 212. Organogenesis was influenced not only by cytokinin, but also by the sucrose concentration in the medium. High frequency (80%) plant regeneration was achieved and quantified on the basis of callus fresh weight. The ability of the callus (cultivar 212) to regenerate whole plants was retained until the 5th passage, but during the 6th passage it declined considerably.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN Kinetin - BAP 6-benzylaminopurine - MS Murashige and Skoog basal medium     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.