Evaluation of carbon sources,gelling agents,growth hormones and additives for efficient callus induction and plant regeneration in Indian wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) genotypes using mature embryos

Various factors affecting in vitro regeneration like different carbon sources, different gelling agents, and growth additives were assessed comprehensively for callus induction and plant regeneration for five Indian wheat cultivars using mature embryos as the explants for the first time. The tissue culture responses of cultivars WH-1105, HD-2967, and PBW-343 have not been reported earlier. Besides, the effect of different concentrations of the cytokinin, zeatin has also been optimized. Using the optimized factors, the efficiency of five different varieties, i.e., HD 2967, C 306, RAJ 3765, WH 1105, and PBW 343 was evaluated for regeneration. Modified MS basal medium containing dicamba reduced precocious germination of the embryo and induced embryogenic callus more efficiently. Removal of embryogenic calli from non-regenerable structures during early callus phase improved plant regeneration. These calli on zeatin (1.0 mgl^-1) and dicamba (0.1 mgl^-1) containing medium showed the highest regeneration frequency (98%) with a maximum of 8-9 shoots per calli. Maltose had the maximum callusing and regeneration percentage than other carbon sources. Various gelling agents did not have any significant difference on the regeneration. Of all the varieties, C-306 and HD-2967 were found to be more regenerative and can be used in transformation experiments.     

Genetics of androgenesis in winter and spring wheat genotypes

Androgenetic response and its inheritance was studied in nine winter type and two spring type wheat genotypes by crossing them in line × tester fashion. The 18 F₁ and 11 parental genotypes were raised in completely randomized block design with three replications and subjected to anther culture. Data were recorded with respect to per cent calli induction, percent regeneration and per cent green plant development. Analysis of variance revealed considerable genetic variation for all the three component traits of androgenesis. Both additive and non-additive genetic variances were involved in the androgenetic response with the preponderance of non-additive gene action in case of calli induction and additive gene action for regeneration and green plant development. Low estimates of heritability (narrow sense) for calli induction and moderate for regeneration and green plantlet development were obtained. The genotypes, Henies VII, Pnfjoumee, Saptdhara, Sentry, Purthi of winter wheat and HPW147 of spring wheat were identified as good general combiners for almost all the androgenetic parameters and have potential use in future haploidy breeding programmes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. I. Ability of 1B—-1R wheat-rye translocation forms for androgenesis 45 winter wheat varieties or F₁ hybrids, F₂ populations and lines with 1B—1R wheat-rye translocation were tested for their anther culture ability. A total of 48058 anthers was cultured on Potato-2 medium. When averaged over all genotypes and the two experimental years frequencies of embryoid formation of 5.4—6.8 per 100 anthers were observed. Plant regeneration efficiency from embryoids ranged from 5.3—9.1 % or a mean of 4—5 green plants per 1000 anthers plated. The results confirmed the preferential regeneration frequency of gametes with the 1BL—1RS chromosome compared to the gametes with the 1BL—IBS chromosome. Multiple peroxidase were used as marker. The effect of cold pretreatment or of media on the androgenetic response and productivity was not important. On the contrary the variability between the anther response from single ears of the same genotype was noticeable. Examples are presented for the transferability of the androgenetic ability to breeding material. Most green plants obtained were haploid or spontaneous doubled haploid. By cloning it was guaranteed, that progenies were obtained from most of the haploids after colchicine treatment.     

Anther callus induction and green plant regeneration at high frequencies from an interspecific rice hybrid Oryza sativa Linn. x O. rufipogon Griff

Summary Callus induction and green plant regeneration at high frequencies from an interspefic hybrid, Oryza sativa L. x O. rufipogon Griff. has been achieved by simply coordinating the growth regulators in the induction medium. The study was conducted with two different basal media (Potato-2 and N₆) and seven different combinations of growth regulators 2,4-D, NAA and kinetin. Synergistic effects of the two auxins in enhanced anther response to callus induction and subsequent green plant regeneration were observed in both media. The highest frequency of callus induction was obtained on Potato-2 medium supplemented with 1 mg/12,4-D, 2 mg/l NAA and 1 mg/l kinetin. The same combination of growth regulators which yielded higher frequencies of callus induction also induced higher mean number of calli per anther. Although the calli formed on N₆ medium showed high regenerability, there was a concomitant increase in the number of albinos among the regenerants. The auxins in the induction media had considerable influence on the regeneration capacity of the calli. The regeneration frequencies were higher from calli formed in the presence of both auxins in the induction media. The levels of growth regulator combinations seem to influence the green plant regeneration especially for calli induced on Potato-2 medium. Among the pollen grain derived plants the majority were either haploids or double haploids and very few were chromosomal variants.     

Optimization of mature embryo-based high frequency callus induction and plant regeneration from elite wheat cultivars grown in China

We have developed an efficient procedure for plant regeneration of elite wheat cultivars using mature embryos. Firstly, we established the optimal combination of basal media, inoculation method and pretreatment method using biostatistical methods. The results indicated that the combination of MS medium and longitudinally bisected mature embryos showed the highest culture efficiency, whereas the pretreatment method had no significant effects on callus induction or plant regeneration. A 70% primary callus induction rate was achieved on MS medium containing 2 mg/l 2,4‐d for all tested cultivars. Primary calli were then transferred onto the subculture medium to initiate embryogenic calli. Supplementation of the subculture medium with the appropriate combination of phytohormones (2.0 mg/l 2,4‐d , 0.5 mg/l BA and 0.1 mg/l NAA) significantly enhanced embryogenic callus production. The addition of AgNO₃ (10 mg/l) in regeneration medium promoted plant regeneration, whereas CuSO₄ stimulated root formation. The use of this protocol achieved successful plant regeneration in eight tested cultivars. The culture efficiency ranged from 15.3% to 36.8%, suggesting this regeneration system may be an effective alternative for wheat genetic transformation.     

Evidence for genetic control and media effect on haploid regeneration in the anther culture of hexaploid wheat (Triticum aestivum L.)

Crosses were made between seven hexaploid wheat genotypes. Twenty-one F₁ hybrids and their parents were grown in a greenhouse with 16 h day/8 h night at 25°C and 15°C, respectively. The experiment was a complete randomized block design with three replications. Each replication consisted of one pot with three plants. Anther culture was performed in two different induction media (CHB and W14) and androgenetic traits were studied. Statistical analysis was carried out separately for each induction medium. Genetic variation was highly significant for androgenetic traits and the best parent (IBPT 19) produced 68 embryos and 9.3 green plants per 100 anthers in CHB medium. Genetic components were affected by induction media and some components were significant in one medium and non significant in the other. General combining ability (GCA) was significant for all androgenetic traits, except for albino plant regeneration in both media and total plant regeneration in CHB medium, whereas specific combining ability (SCA) was not significant for the traits studied. Narrow sense heritability was high for embryo induction frequency and green and total plant regeneration. All our results indicate that androgenetic parameters can be improved in hexaploid wheat by genetic means.     

基因型和环境条件对小麦花药培养效果的影响

为进一步提高小麦花培育种效率,明确花药培养力的遗传控制基础,以11个小麦品种及其组配的20个F₁杂种为材料,探讨了基因型、培养基和环境条件对愈伤组织诱导率的影响。在W14D、W14gD、W14GD培养基上,Alondra、Verry、石4185、新春9号和百农3217的花药易被诱导产生愈伤组织,诱导率为25.3%~51.9%,其中石4185是目前公认的花培育种优良亲本,新春9号为新发现的优良花培基因型。以宁春4号配制的部分F₁杂种的愈伤组织诱导率较高,大多数组合高于10.0%,表明宁春4号与供试品种间具有较高的花药培养配合力。小麦花培育种技术要求亲本之一具有较高的花药愈伤组织诱导率或较高的花药培养配合力。小麦花药培养力的遗传控制复杂,表现为数量性状遗传,亲本花药培养力很高,其F₁组合花药培养力不一定很高,这与双亲配合力有关。小麦花药培养中,供体植株生长和愈伤组织诱导的适宜条件为较长的营养生长期、适宜的前期(分蘖期)温度和较高的中期(拔节后期)温度。在添加低浓度生长素和葡萄糖的液体培养基中发现小麦花药直接成苗现象,2,4-D诱导花药直接成苗效果优于Dicamba。随着年度间气候升高的影响,相同基因型花药愈伤组织诱导率呈现增加趋势。     

Phenylacetic acid stimulation of direct shoot formation in anther and somatic tissue cultures of rice (Oryza saliva L.)

The effect of phenylacetic acid (PAA) on rice (Oryza saliva L.) anther culture was investigated with six genotypes, using 2,4-D as control. In the two-step culture protocol, replacing 2, 4-D with PAA in the induction medium did not influence callus induction but significantly improved the shoot differentiation from callus, particularly in the indica cultivar Teqing. The anther-derived calli of all genotypes regenerated shoots directly on the callus induction medium containing PAA. Most of the directly-regenerated plantlets had well-developed root systems and were therefore readily transplanted into soil. The improved shoot differentiation potential and the frequency of direct regeneration depended on genotype, basal medium and PAA concentration. The one-step green shoot regeneration frequencies obtained were 1.98% with the indica cultivar ‘129’, 1.5% with the indica × japonica hybrid ‘Teqing/02428’ (F₁), and 1.98% with the indica × indica hybrid ‘Waiyin 2/C.B.’ (F₁). The PAA-based one-step method was most effective on the anther culture of indica genotypes. Three DH populations have been constructed from hybrids (F₁) via one-step culture. PAA also enhanced the one-step plantlet formation in rice somatic tissue culture.     

新陆早32号、33号的体细胞胚胎发生和植株再生比较研究

以新疆主栽品种新陆早32号、33号和对照YZ1为研究材料,通过不同浓度的激素组合成功诱导获得了体细胞胚,并进一步发育成苗.研究发现,所用的12种激素组合均能有效诱导愈伤组织.虽然IBA+KT组合有利于诱导YZ1和新陆早33号的快速分化,但在增殖生长过程中体细胞胚容易褐化、死亡:在2,4-D+KT组合中,0.1 mg·L...     

NAA或潮霉素对小麦成熟胚愈伤组织分化的影响

以5个栽培小麦品种诱导而来的成熟胚愈伤组织为外植体,研究了在分化培养基中加入NAA对小麦成熟胚愈伤组织分化的效果以及潮霉素浓度对不同阶段成熟胚愈伤组织分化的影响。研究结果表明,在对照的分化培养基中(含5mg/L激动素)加入0.1mg/L萘乙酸(NAA,1-naphthlcetic acid)形成的分化培养基,可显著提高4个供试小麦基因型的成熟胚愈伤组织的分化率,提高幅度达到12%～32%。不同的潮霉素浓度在"花培1号"愈伤组织的诱导阶段进行抗性筛选的结果表明,即使是在潮霉素浓度达到250mg/L时,仍有24%左右的愈伤组织存活率,这些存活的愈伤组织仍可能分化形成绿点甚至分化成苗,说明在"花培1号"愈伤组织的诱导阶段进行潮霉素抗性筛选是不适合的。5个不同小麦品种分化阶段进行潮霉素筛选具有明显的效果,但不同品种之间存在一定差异,其中:宁麦13的适宜潮霉素浓度为20mg/L,花培1号和扬麦158的适宜潮霉素浓度约为30mg/L,宁麦9和宁麦16的适宜潮霉素浓度在30～40mg/L左右。因此,我们认为,在分化阶段进行潮霉素抗性筛选是理想的筛选时期,在分化培养基中添加20～40mg/L浓度的潮霉素即可完全抑制小麦成熟胚愈伤组织的分化。本研究结果将有助于改进和完善普通小麦成熟胚遗传转化体系。     

Studies on somaclonal variants for resistance to scab in bread wheat (Triticum aestivum L.) through in vitro selection for tolerance to deoxynivalenol

This study was conducted to develop an efficient in vitro selection system for scab resistance by using in vitro screening for tolerance to deoxynivalenol (DON). Immature embryos of two wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11, and their reciprocal F₁ hybrids were cultured on MS medium supplemented with 2,4-D 2 mg/l and 0.6 × 10^-4 M DON for callus induction. The responses of callus induction and plant regeneration to 0.6 × 10^-4 M DON differed significantly between resistant and susceptible varieties, according to observed scab resistance levels at the plant level in the field. The percentage of callus formation of resistant variety Sumai 3 on induction medium containing DON was higher than that of susceptible variety Mianyang 11. Regeneration of DON-tolerant calli on DON-containing differentiation medium differed significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was decreased 3-fold on DON-containing medium. By an inoculation test with conidiospores of Fusarium graminearum Schw, we obtained several resistant lines from progenies of regenerated plants from DON-tolerant calli. These somaclonal lines had lower disease scoring (reaction index, infected spikelets and disease incidence), shorter plants and better yield components than Sumai 3, a famous Chinese resistant variety. This revised version was published online in July 2006 with corrections to the Cover Date.     

新陆早42号体细胞胚发生和植株再生

 以新陆早33号为对照研究新疆主栽品种新陆早42号的体细胞胚胎发生。研究表明,所用4种激素组合均能有效诱导愈伤组织,但二者仅在经过IBA 1.0 mg·L^-1+KT 0.5 mg·L^-1和2,4-D 0.1 mg·L^-1+KT 0.1 mg·L^-1两种激素组合诱导初生愈伤后,才能胚胎发生。新陆早42号在IBA 1.0 mg·L^-1+KT 0.5 mg·L^-1培养基上46 d就开始胚胎发生;新陆早33号则不能直接胚胎发生,需继代到MSBP培养基上培养48 d才有胚胎发生。经2,4-D 0.1 mg·L^-1+KT 0.1 mg·L^-1组合培养的愈伤均需要继代到MSBP培养基上培养才能胚胎发生,新陆早42号和新陆早33号胚胎发生的最早时间分别是71 d和81 d。胚性愈伤在铺有滤纸的MSBF培养基上分化成胚并发育成再生植株。新陆早42号在140 d有根系发育良好的能嫁接植株（株高 7～8 cm）,而新陆早33需180 d。即成功建立了新陆早42号的再生体系,且其胚胎发生能力和再生能力均优于对照。     

Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Production of doubled haploids (DHs) through androgenesis induction is an important biotechnological tool for plant breeding. In some species, DHs are efficiently obtained through embryogenesis from isolated microspore cultures. In eggplant, however, this process is still at its infancy, despite the economic relevance of this important agricultural crop. To date, only two studies have focused previously on this process, suggesting that in eggplant microspore cultures, the only morphogenic response is callus formation. Given the notable lack of studies on eggplant microspore cultures, in this work we explored this process with different experimental approaches. We studied the response of different cultivars and characterized the development of microspores induced to divide and proliferate. We demonstrated that microspore-derived embryos (MDEs) can be produced in eggplant; however, MDEs stopped at the globular stage, to turn into euploid and principally mixoploid calli. From these calli, 60 % of DH plants could be regenerated. In order to promote microspore induction we evaluated the effect of polyethylene glycol (PEG) and mannitol. PEG, but not mannitol, significantly increased induction of microspore embryogenesis. We also tested the ability of eight different media compositions to promote efficient plant regeneration from calli. In order to test it in a genotype-independent manner, we previously developed a method to generate clonal callus populations derived from single microspore-derived calli. Together, the results presented hereby constitute an efficient way to produce eggplant DHs through microspore culture. In addition, they contribute significant insights into the knowledge of the particularities of androgenesis induction in this species.     

冬小麦遗传再生体系及根癌农杆菌介导的转化研究

摘要:以冬小麦品种临优145的幼胚为外植体,消毒后用解剖刀挑取幼胚,盾片朝上接种于MS+2,4-D 2 mg/L+肌醇100 mg/L＋MES 400 mg/L＋CH 100 mg/L[7]＋40g/L麦芽糖＋8g/L琼脂的培养基中诱导愈伤组织, 每两周继代一次,将诱导出的淡黄色、颗粒状Ⅱ型胚性愈伤组织接种于分化培养基（MS+ZT 1 mg/L+IAA 1mg/L+ MES 400 mg/L＋CH 100 mg/L+麦芽糖40g/L+gelrite 2.6g/L,PH5.8）上培养2－3周,然后转接到再生培养基(1/10MS+NAA 0.5mg/L+KT 0.5mg/L+蔗糖30g/L+ gelrite 2.6g/L,PH5.8)中进行再生成苗。接种1229块幼胚,得到987块愈伤组织, Hyg抗性愈伤组织123块,抗性再生植株34株。在建立了再生体系的基础上,用根癌农杆菌介导法将GUS基因导入幼胚愈伤组织,抗性植株的PCR检测呈阳性。X－gluc染色表明, 少部分愈伤组织出现肉眼可见的蓝色晕斑,说明GUS基因已经在小麦幼胚愈伤组织中表达。     

Callus induction and plant regeneration from anther and inflorescence culture of Sorghum

Summary Twenty-five inbred lines, including grain and forage types from the USA and China, two hybrids, one Sorghum almum, and one Parasorghum (S. versicolor) were tested for their response to anther culture. Three nutrient media were effective in inducing anther calli from six cultivars (Xin White, TX 403-TSB, DDY Sommer Milo, TX 2779, Brawley, and Spur Federal) and one was effective for plant regeneration for one cultivar, Xin White. Averaged over media, callus induction frequency (number of calli per 100 anthers) was highest in cultivars Xin White and TX 403-TSB (6.7 and 3.9%, respectively). The means of cultivars for media C17-2 and Ms-t-z-2, 4.3 and 3.2%, respectively, were superior to that for medium 85D3-2 (0.1%). Expressed as an average of the six cultivars and three media the mean calli induction frequency was 2.6%; however, differential responses of genotype and medium were noted. Among the 10 regeneration media tested, medium MS-d-4 containing Murashige and Skoog basal components plus 2.0 mg/l indole-3-acetic acid (IAA) and 2.5 mg/l kinetin was the most effective for plant regeneration. Numbers of albino plants and calli developing only roots increased directly with callus-induction time, whereas the frequency of plant regeneration decreased. Regenerated plants had varied numbers of chromosomes in root tip cells: 10, 15, 20, 40, and 60. The 29 regenerated plants that reached maturity, however, were highly fertile and contained only 10 bivalents in pollen mother cells. Normal chromosome number and behavior for the regenerated plants suggest that induced calli originated from cells other than microspores. However, spontaneous chromosome doubling in microspore-derived haploids may occur. The appearance of albinos also implies that haploids may have been produced from anther culture.Joint contribution of the Dept. of Agronomy and USDA-ARS, Kansas Agricultural Experiment Station, Manhattan, KS 66506-5501, USA. Contribution no. 88-566-J.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

石栗树茎段组织培养的研究

建立石栗组织培养的技术体系,筛选出适合诱导愈伤组织和愈伤组织分化的培养基。从外植体消毒,激素配比,培养基筛选等方面对石栗组织培养进行了探索。结果表明诱导愈伤组织的主要因素是细胞分裂素和生长素的比值。TDZ对愈伤组织诱导丛生芽有抑制作用。诱导愈伤组织的最适培养基为：1/2MS+ZT 2 mg/L+NAA 0.5 mg/L+氯化胆碱2 mg/L,诱导愈伤组织丛生芽的最适培养基为1/2MS+6-BA 1 mg/L +IBA 0.1 mg/L。影响诱导愈伤组织的主要因素是细胞分裂素和生长素的比值。     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of inbreeding and growth regulators on the in vitro androgenesis of wheatgrass, Agropyron glaucum

The capacity of haploid production by anther culture has been analysed in 42 crossbred genotypes (wild type) and in 32 genotypes from inbreeding generations (I₂-I₃) of wheatgrass, Agropyron glaucum. On potato-2 medium, about 50% of the genotypes investigated were capable of embryoid and callus induction. For the regeneration of haploid plants, five media containing various growth regulators were used. The crossbred genotypes were superior to the inbred lines in all the androgenesis parameters examined.     

Influence of winter and spring wheat genetic backgrounds on haploid induction parameters and trait correlations in the wheat × maize system

The interactive influence of winter and/or spring wheat genetic background on haploid induction parameters and trait correlation was studied by hybridizing five elite and diverse genotypes each of winter and spring wheat and their F₁s (winter × winter, spring × spring, and winter × spring, generated in a diallel design excluding reciprocals) with a single genotype of maize. Data were recorded with respect to per cent seed formation, embryo formation, and regeneration. High genetic variability was present among the wheat genotypes (parents + F₁s) for the three haploid induction parameters. Significant differences were obtained within and between different groups viz., spring wheats, winter wheats, spring × spring wheats, winter × winter wheats, and winter × spring wheats with respect to the three haploid induction parameters based on ANOVA. The winter genotypes (winter parents and winter × winter wheat hybrids) responded better than the spring groups (spring wheat parents, spring × spring and winter × spring wheat hybrids) with respect to embryo formation and winter × spring wheat hybrids yielded significantly the highest numbers of regenerants. Correlation studies amongst the haploid induction parameters indicated that the genes controlling seed formation and haploid plantlet regeneration are negatively correlated when the genetic backgrounds of both ecotypes are combined in winter × spring hybrids. Haploid embryo formation had no association with seed formation and regeneration in all genetic backgrounds, suggesting independent inheritance.