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Reichert, N.A. and B.S. Baldwin. 1996. Potential for kenaf improvement via somaclonal variation. p. 408-411. In: J. Janick (ed.), Progress in new crops. ASHS Press, Arlington, VA.

Potential for Kenaf Improvement via Somaclonal Variation

Nancy A. Reichert and Brian S. Baldwin

  1. METHODOLOGY
  2. RESULTS
    1. Adventitious Regeneration
    2. R0 and R1 Analyses for 'E41', 'G45' and 'G48'
    3. R1 and R2 Analyses for 'E41'
  3. REFERENCES
  4. Table 1
  5. Table 2
Kenaf (Hibiscus cannabinus L., Malvaceae) is a potential fiber crop adapted to the southern United States. As part of the kenaf improvement program at Mississippi State University, tissue culture is being employed as a means for introduction of new or altered traits into kenaf. Regenerants often display altered phenotypes, termed somaclonal variation (Larkin and Scowcroft, 1981). The exploitation of heritable somaclonal variants has been used in various plant improvement strategies (Larkin and Scowcroft 1981; Evans 1989; Larkin et al. 1989; Phillips et al. 1994) but success has been sporadic.

Our objectives were to: (1) develop an adventitious regeneration protocol for kenaf and, (2) analyze the regenerants (R0) and subsequent progeny for trait differences. Various traits were assessed over the course of two to three generations (R1 and R2) and compared to control plants.

METHODOLOGY

Internodal stem sections were obtained from field-grown plants of 'Everglades 41' ('E41'), surface-disinfested, then placed on a medium containing Murashige and Skoog (1962) salts, supplemented with (per liter): 1.0 mg thiamine-HCl, 0.5 mg pyridoxine-HCl, 0.5 mg nicotinic acid, and 100 mg myo-inositol, 3% sucrose, 0.8% phytagar, plant growth regulators [0.1 mg 1-naphthaleneacetic acid (NAA) plus 3.0 mg thidiazuron (TDZ)] with pH 5.8 for shoot initiation (Reichert and Liu 1994). Shoots were rooted on MS medium containing no plant growth regulators and transplanted into peat cubes for acclimatization. 'E41' R0 regenerants were transferred to the greenhouse for one week, then transplanted into the field at the Plant Science Research Center (PSRC), at Mississippi State Univ. in 1993. All plants were allowed to self-pollinate. R1 seeds were harvested by plant, then germinated in peat plugs in the greenhouse and transplanted into the field at the PSRC in 1994. R1 seedlings were compared to 'E41' seedlings produced in the same year.

In 1994, the regeneration protocol was found to be more efficient using leaf sections. Seeds of various kenaf cultivars were germinated aseptically and maintained in vitro via nodal sections on hormone free medium. These are referred to as seed clonal lines (SCL). Leaf sections were explanted on MS-based shoot induction medium containing (per liter) 0.1 mg NAA plus 3.5 mg TDZ (Reichert and Liu 1994). We obtained 150 R0 regenerants from 'E41', 'Guatemala 45' ('G45') and 'G48' for field testing at the PSRC in June-July, 1994. In addition, 20 clonal control plants (nodal section-propagated) from each SCL were transplanted into the field concurrently. These clonal plants were used to determine: (1) the normal range of variation within a clonal line as a result of environmental influences and (2) potential heterozygosity and subsequent segregation in the original seed. (No segregation was observed.) Both checks were necessary to determine altered phenotypes were actually due to the leaf regeneration protocol. Additional regenerants produced after July 1st were maintained in the greenhouse for the production of R1 seed.

In 1995, R2 seeds harvested from individual 'E41' R1 plants (generated in 1994; from R0s planted in 1993) as well as R1 seeds harvested from each R0 regenerant (field planted in 1994) were germinated in peat plugs in the greenhouse. Eight days later, each line was evaluated for altered seedling phenotypes then, 15 seedlings from each line were space planted in sib rows at the PSRC. Selfed seedlings obtained from SCL lines were space planted adjacent as controls. Plant height, stem girth, stem color, leaf shape, days to first flower bud, days to first open flower, and flower shape/morphology were determined in the field in 1994 and 1995.

RESULTS

Adventitious Regeneration

The leaf regeneration protocol initially developed for 'E41' was successful on six other cultivars: E71, G4, G45, G48, G51, and Tainung 1. Adventitious shoots were not obtained from 'Cuba 2032', 'Cubano', or 'GR2563'. However, sufficient numbers of plantlets for field trials were generated only from E41, G45, and G48.

R0 and R1 Analyses for 'E41', 'G45' and 'G48'

R0 kenaf regenerated adventitiously from leaves displayed somaclonal variation for stem color based on a five point color scale. Stems were classified in discrete classes based upon the relative amount of anthocyanins present. Individual plants fell into one of the following color classes: green, light pink, dark pink, red, or burgundy. 'G45' regenerants displayed greater variation in stem color than the SCL control plants. The SCL controls displayed stem colors varying from light pink to dark pink due to environmental effects. 'G45' R0 plants displayed stem colors spanning from green all the way to red; displaying less or more anthocyanins in the stem than the SCL controls. 'E41' R0 plants displayed greater amounts of anthocyanins in the stem (dark pink to red) than the corresponding SCL controls (light pink to dark pink). No differences were detected in 'G48' between the R0 and SCL plants for stem color.

R0 flowers were observed for differences in flower shape/morphology. Normal kenaf flowers have a crimson eye with five very wide light yellow flower petals which overlap slightly. Most R0 plants displayed normal flowers, but there were some notable exceptions. In 'E41', flowers containing narrow, non-overlapping petals were observed. Most were composed of five petals, but one plant consistently displayed flowers which contained six and seven petals and one 'G48' regenerant displayed flowers with 10 petals. One 'E41' R0 regenerant had flowers that were consistently male sterile (vestigial anthers) and one 'G48' R0 regenerant produced flowers with indehiscent anthers. One R1 plant from a 'G45' R0 line was observed to produce flowers that were pink due to "bleeding" of the crimson eye into the petals.

R1 and R2 Analyses for 'E41'

R1 seedlings of all R0 plants of 'E41' displayed variation in seedling characteristics and when mature expressed variation in days to flower as compared with 'E41' controls. Germination of the R1 seed revealed abnormal seedling traits. These characteristics were compared to 'E41' seedlings generated from control plants grown in the field, adjacent to the R0 during the same season (Table 1). There was a higher percentage of abnormal seedlings from the R1 plants than from the 'E41' control seedlings. An albino (chlorophyll deficient) mutation was displayed in 5% of the R1 seedlings, but was not observed in the controls and was never detected in the millions of seeds planted in the kenaf cultivar trial conducted in 1994 at the PSRC.

At maturity 'E41' (R1) plants were not compared to controls for stem color or leaf shape since the controls were not SCL. Therefore, environmental influences as well as potential outcrossing which could contribute to variation in stem color could not be ruled out. Flowering dates for R1 plants were compared to normal field grown 'E41' plants (Table 2). The first flower bud appeared four weeks earlier and the first open flower appeared more than three weeks earlier in the R1 population versus field-derived 'E41' plants. The dates when 50% of the plants had flower buds or open flowers occurred approximately two weeks earlier in the R1 (Table 2). No differences were detected between R1 and field-derived 'E41' plants for plant height, stem girth, and flower shape. R2 seed harvested from one 'E41' R1 plant displayed the albino (chlorophyll deficient) trait upon germination at a frequency of 3%.

R0, R1, and initial R2 data confirm that the adventitious regeneration protocol developed for kenaf results in regenerants displaying new or altered phenotypes. Some visible traits that appeared in kenaf regenerants and subsequent progeny (albino, crinkled cotyledons, different colored reproductive structures, male sterility) were similar to those previously observed as single gene mutations in progeny of maize (Zea mays L.) somaclones (Phillips et al. 1994). Early flowering date is probably the most important variant phenotype to come from this work. However, altered flower color, polypetally, and male sterility are also desirable characteristics for the kenaf breeding program. All of these altered phenotypes are indicators that heritable changes can be made in the genome of kenaf via this leaf regeneration protocol. This protocol has proven to be successful on seven cultivars to date. Continued screenings will determine if any traits will be useful to the kenaf breeding program.

REFERENCES

Table 1. Seedling traits in control and R1 kenaf ('E41').

Distribution (%)
Treatment n Normal seedling Empty seed Crinkled cotyledon Chlorophyll deficient Single cotyledon
Controlz 250 93 5 2 0 0
R1y 176 57 27 10 5 1
zControl seeds were from a commerial seedlot grown under the same conditions.
ySeeds harvested from R0 plants in 1993 and pooled.

Table 2. Days to flower for control vs R1 kenaf ('E41').

Days after plantingz
Treatment n First flower bud 50% plants w/ flower buds First open flower 50% plants open flower
Controly 135 122 130±6x 136 149±5
R1w 139 94 106±5 112 125±7
zJune 3 was designated as day 1.
yControl plants were initiated from seed in the field on May 19.
xNumbers ±1 SD, where applicable.
wPlants were initiated from seed on May 23 and transplanted to the field on June 3.

Last update June 17, 1997 aw